Plant Diversity 39 (2017) 80e88
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Degeneration of photosynthetic capacity in mixotrophic plants,
Chimaphila japonica and Pyrola decorata (Ericaceae)
Jiaojun Yu a, b, c, Chaobo Wang a, b, c, Xun Gong a, b, c, *
a
Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China
Yunnan Key Laboratory for Wild Plant Resources, Kunming 650201, PR China
c
Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Kunming, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2016
Received in revised form
17 November 2016
Accepted 21 November 2016
Available online 12 January 2017
The evolution of photosynthesis is an important feature of mixotrophic plants. Previous inferences
proposed that mixotrophic taxa tend to retain most genes relating to photosynthetic functions but vary
in plastid gene content. However, no sequence data are available to test this hypothesis in Ericaceae. To
investigate changes in plastid genomes that may result from a transition from autotrophy to mixotrophy,
the plastomes of two mixotrophic plants, Pyrola decorata and Chimaphila japonica, were sequenced at
Illumina's Genome Analyzer and compared to the published plastome of the autotrophic plant Rhododendron simsii, which also belongs to Ericaceae. The greatest discrepancy between mixotrophic and
autotrophic plants was that ndh genes for both P. decorata and C. japonica plastomes have nearly all
become pseudogenes. P. decorata and C. japonica also retained all genes directly involved in photosynthesis under strong selection. The calculated rate of nonsynonymous nucleotide substitutions and synonymous substitutions of protein-coding genes (dN/dS) showed that substitution rates in shade plants
were apparently higher than those in sunlight plants. The two mixotrophic plastomes were generally
very similar to that of non-parasitic plants, although ndh genes were largely pseudogenized. Photosynthesis genes under strong selection were retained in the two mixotrophs, however, with greatly
increased substitution rates. Further research is needed to gain a clearer understanding of the evolution
of autotrophy and mixotrophy in Ericaceae.
Copyright © 2017 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by
Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
(Editor: Xuewen wang)
Keywords:
Chimaphila japonica
Pyrola decorata
Mixotroph
Plastid genome
1. Introduction
Plant photosynthesis, which occurs in plastids, is the primary
energy source for life on Earth. However, some plants have partially
lost their photosynthetic ability and become mixotrophic (MX),
able to use both autotrophic and heterotrophic strategies to derive
water and nutrients. Among angiosperms, mixotrophs exist in two
groups: semiparasitic (e.g. genus Cuscuta) and partial mycoheterotrophic (MH) plants. The MH plants obtain carbon not only
from their photosynthetic activity, but also from mycorrhizal fungi.
This is a plant-fungus relationship which has evolved convergently
in Orchidaceae and Ericaceae (Selosse and Roy, 2009; Tedersoo
* Corresponding author. Key Laboratory for Plant Diversity and Biogeography of
East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming
650201, PR China.
E-mail address: [email protected] (X. Gong).
Peer review under responsibility of Editorial Office of Plant Diversity.
et al., 2007). Mixotrophs deploy a dual nutritional strategy which
may have evolved repeatedly from various autotrophic (AH) plant
lineages. However, the detailed evolutionary processes that lead to
MH remain unclear (Bidartondo, 2005; Cameron et al., 2006; Julou
et al., 2005; Tedersoo et al., 2007; Zimmer et al., 2007). Motomura
et al. (2010) suggested that MH evolved after the establishment of
MX rather than through direct shifts from AH to MH.
Heterotrophs have highly divergent morphology and DNA sequences (Barkman et al., 2007; Delannoy et al., 2011; Lemaire et al.,
2011; Moore et al., 2010; Nickrent et al., 1998), thus precise
phylogenetic relationships of heterotrophic plants to their respective autotrophic relatives have been notoriously difficult to ascertain (Stefanovi
c and Olmstead, 2004). Plastid genome (plastome) of
flowering plants is generally conserved in size, structure, gene
content, and synteny (Gao et al., 2010; Wicke et al., 2011). However,
owing to relaxation of strong functional constraints normally
associated with photosynthesis, plastid genomes of heterotrophs
exhibit a wide range of evolutionary degradation (Gao et al., 2010;
http://dx.doi.org/10.1016/j.pld.2016.11.005
2468-2659/Copyright © 2017 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This
is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Yu et al. / Plant Diversity 39 (2017) 80e88
Li et al., 2013; Wicke et al., 2011, 2013). To date, only several MH
plant plastomes have been entirely sequenced (Delannoy et al.,
2011; Li et al., 2013; Logacheva et al., 2011; Wicke et al., 2013;
Wickett et al., 2008). In contrast to the highly reduced genome of
fully MH plants (Gruzdev et al., 2016; Li et al., 2013; Wicke et al.,
2013), the plastid genomes of Neottia Guett. and Aneura Dumort.
have retained many genes related to photosynthesis. This may be
attributed to a presumably recent switch of this species to mycoheterotrophy (Wickett et al., 2008).
Ericaceae is known to have formed symbiotic relationships with
fungi. The tribe Pyroleae consists of mixotrophic species (with the
exception of the fully MH Pyrola aphylla), while tribes Pteroporeae
and Monotropeae consist of fully MH plants (Kron et al., 2002). All
other tribes of the Ericaceae are, as far as is known, fully autotrophic. Although analysis of chloroplast DNA has contributed significantly to our understanding of Ericaceae evolution, most of the
conclusions drawn thus far are based on nucleotide sequence
segments (Kron, 1997; Kron et al., 2002). Extensive sampling and
southern hybridization analysis across Ericaceae showed that
mixotrophic taxa tend to retain most plastid genes relating to
photosynthetic functions but vary regarding the gene content
(Braukmann and Stefanovic, 2012). In general, the plastome has a
low rate of nucleotide substitution, which facilitates comparisons
of variation in a wide range of plant taxa (Drouin et al., 2008; Gaut
et al., 2011). Therefore, comparative analyses of the plastomes of
autotrophs, mixotrophs and full heterotrophs, would shed new
light on the evolution of plastome genomes in Ericaceae. With the
loss of photosynthetic ability, genes required for the photosynthetic
apparatus are predicted to undergo random mutations under
relaxed natural selection (Funk et al., 2007; Logacheva et al., 2011;
McNeal et al., 2007; Wolfe et al., 1992). Although some chloroplast
genes have been retained and remain functional in several holoparasitic plants (Bungard, 2004), many holoparasites have
reduced plastid genomes due to excessive gene loss and increased
substitution rates in the remaining genes (Gruzdev et al., 2016;
Li et al., 2013; Molina et al., 2014; Wicke et al., 2013; Wolfe et al.,
1992). Examining nonsynonymous over synonymous substitution
(dN/dS) ratios can be used to determine whether genes are evolving
under purifying selection (Logacheva et al., 2011; McNeal et al.,
2007).
In order to test if photosynthetic genes remain the most highly
conserved in the plastid genome and whether relaxation of functional constraint precedes gene losses both before and after the
evolution of heterotrophy in Ericaceae, we sequenced two plastomes of partial MH plants in the tribe Pyroleae (Pyrola decorata
and Chimaphila japonica) which may be highly modified. The goal of
this study was to provide a broad picture of chloroplast gene
diversification in Pyroleae, and an understanding of the role of
mixotrophs in the long history of Ericaceae evolution.
2. Methods
Plant material from P. decorata and C. japonica were collected
from Qiongzhusi of Kunming (25 00 4500 N, 102 420 0400 E; altitude
2100 m), Yunnan Province, China. The plastid DNA was isolated
from 100 g of fresh leaves using an improved extraction method
that includes high ionic strength buffer at low pH (3.6) buffer
(Triboush et al., 1998). Chloroplast DNA was sequenced with Illumina's Genome Analyzer at Beijing Genomics Institute (BGI) in
Shenzhen, China. Subsequently, SOAP de novo were used to
assemble the sequence reads of chloroplast genomes (Li et al.,
2009). Eight small gaps were closed by PCR using Qiagen Taq Polymerase. Both plastid DNA and genomic DNA were used for PCR.
Primers used for PCR were designed using available information
from both ends of the gap. All primers were ordered from Sangon
81
Biotech (Shanghai, China). PCR products were analyzed by electrophoresis on 1% agarose gel and then sequenced using standard
Sanger protocols on ABI 3730 instruments. The thermal cycling
program was as follows: 80 C for 5 min; then 33 cycles of (95 C for
45 s; 42e52 C for 45 s; 65 C, 1 min), followed by 65 C for 5 min.
Annotation of the sequenced genome was performed using
DOGMA (Wyman et al., 2004), complemented with manual
adjustment for start and stop codons and for intron/exon boundaries. Pairwise estimates of nonsynonymous substitution (dN) and
synonymous substitution (dS) rates (dN/dS) were calculated for P.
decorata, C. japonica, Rhododendron simsii (Moore et al., 2010), as
well as Nicotiana tabacum L., when necessary (Kunnimalaiyaan and
Nielsen, 1997), with the ratio calculated relative to Panax ginseng
(Kim and Lee, 2004) as the outgroup. Non-triplet indels of pseudogenes were also adjusted and calculated. Plastomes from 41 seed
plants were chosen and downloaded from GenBank (Supplement
Table 1). The 43 taxa were divided into three groups: fully heterotrophic plants (Group I), partial heterotrophic plants (Group II,
including two newly sequenced plastomes in this study) and
autotrophic plants (Group III) (Supplementary Table 1). All 79
protein-coding chloroplast genes (excluding ycf15, function presently unknown) were extracted from each genome and translated
into amino acid sequences. Subsequently, they were classified in
ten functional gene groups (Supplementary Table 2). Amino acid
sequences for each gene were aligned by MUSCLE using MEGA 5
(Tamura et al., 2011) with default settings, verified by visual inspection, and used as guides to align the nucleotide sequences.
For dN/dS calculations, all protein-coding plastid genes were
included in the analysis. The amino acids were only used in the
alignment process; all analyses were done on nucleotide sequences. RNA editing sites were not considered. Standard errors
were computed under the Kumar method using MEGA 5 (Tamura
et al., 2011) for each gene and for classes of genes that together
encode subunits of larger proteins. Wilcoxon rank sum tests performed with Kruskal Wallis Test method to estimate p-values between gene groups using SPSS software.
3. Results
3.1. Structure and gene content of the plastome
Illumina paired end (85 bp) sequencing produced 366 Mb of
data for C. japonica and 470 Mb for P. decorata (Table 1). We aligned
paired-end reads of each species to the published plastome of P.
ginseng (Kim and Lee, 2004), which was chosen as a reference
Table 1
Assembling and annotation of two Chloroplast genomes.
Raw data (Mb)
Total reads (90 bp per read)
Aligned reads
Aligned %
Number of contigs
Average contig length (bp)
Size of largest contig (bp)
Contig N50 (bp)
Number of scaffolds
Average scaffold length (bp)
Size of largest scaffold (bp)
Scaffold N50 (bp)
Overall average read depth (estimated value)
Number of gaps
Average gap length (bp)
Size of largest gap (bp)
Sequence GC%
Chimaphila
Pyrola
470.45
3188238
527015
16.53
42
3061
15751
11365
37
3478
18085
15350
329.38x
17
1123
1452
35.94
366.35
3188238
413293
12.96
94
203
640
221
52
1769
16373
3039
295.2x
118
402
2237
36.21
82
J. Yu et al. / Plant Diversity 39 (2017) 80e88
genome. In total, 527,015 and 413,293 paired-end reads were
mapped to the reference genome for C. japonica and P. decorata,
respectively. After de novo assembly with minor changes, we obtained more than 80% of the whole plastome. Gaps in the two
plastomes were then filled by PCR sequencing. At last, we obtained
97% of the C. japonica plastome containing three gaps, and 94% of
the P. decorata plastome with 17 gaps. Though less than 2000 bp
(except ycf1 region), the unfinished gaps contain repeated sequences and lower GC content, so they were difficult to sequence
using normal procedures. Although incomplete, the sequence data
were sufficient for evolutionary analysis.
The plastomes of both MX plants are about 140 kb, which is
larger than most published holoparasites, including Cistanche
deserticola Ma (~102 kb) (Li et al., 2013), Cuscuta reflexa Roxb. and C.
exaltata Engelm. (~121 kb and 125 kb, respectively) (Funk et al.,
2007; McNeal et al., 2007), as well as Monotropa hypopitys L.
(34.8 kb) (Gruzdev et al., 2016), but smaller than the complete
plastome of the autotroph N. tabacum (Kunnimalaiyaan and
Nielsen, 1997). Similar results were reported for Corallorhiza Gagnebin (Barrett et al., 2014) and Orobanchaceae (Wicke et al., 2013).
The plastome of P. decorata contains 105 genes, while Chimaphila
japonia contains 102 genes (Table 2). Both contain all 37 photosystem genes, 30 tRNA genes, and 4 rRNA genes. The majority of the
variation in plastome genes comes from photorespiration genes
(ndh complex). The entire ndh gene complex suffered gene loss and
pseudogenization. In addition to ndhJ, which has shown diminished
hybridization signals for all green Ericaceae, i.e., for both autotrophic and mixotrophic members of this family (Braukmann and
Stefanovi
c, 2012), we found that ndhF has been completely lost in
both plants, while ndhI has also been lost in C. japonica. The
remaining genes of the ndh complex in both plastomes are pseudogenes. This finding differs from hybridization signal data reported previously (Braukmann and Stefanovi
c, 2012). The gene
rpoA RNA polymerase (rpo) has also been lost from both plastomes.
Furthermore, both plastomes lack a functional ycf2 gene, which has
become a pseudogene with only about 100 bp left. Three essential
genes were not detected: clpP, accD and ycf1. Previous studies reported that the hybridization signal for clpP was diminished in most
Ericaceae (Braukmann and Stefanovi
c, 2012), so we hypothesized
that these genes might actually be lost rather than not detected.
Ribosome protein genes also appeared to be absent in both P. decorata (rp123 and rps15) and C. japonica (rps18). The absence of hybridization signal in Pyroleae for rpl23 is unique within Ericaceae
and distinguishes this tribe from the rest of the family (Braukmann
and Stefanovi
c, 2012). Our results were consistent with both predictions and previous research on plastomes of semiparasitic plants
(Jansen et al., 2007; Wicke et al., 2013).
3.2. Pairwise dN/dS ratios for protein-encoding genes
Table 2
Plastome gene contents of Pyrola decorata and Chimaphila japonia.
Photosystem
Pd
Cj
Photorespiration
Pd
Cj
tRNA
Pd
Cj
atpA
atpB
atpE
atpF
atpH
atpI
ccsA
cemA
petA
petB
petD
petG
petL
petN
psaA
psaB
psaC
psaI
psaJ
psbA
psbB
psbC
psbD
psbE
psbF
psbH
psbI
psbJ
psbK
psbL
psbM
psbN
psbT
psbZ
rbcL
ycf3
ycf4
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
ndhA
ndhB
ndhC
ndhD
ndhE
ndhF
ndhG
ndhH
ndhI
ndhJ
ndhK
ribisome
infA
rpl2
rpl14
rpl16
rpl20
rpl22
rpl23
rpl32
rpl33
rpl36
rps2
rps3
rps4
rps7
rps8
rps11
rps12
rps14
rps15
rps16
rps18
rps19
other
clpP
accD
ycf1
ycf2
ycf15
j
j
j
j
j
j
j
j
j
j
trnA-ugc
trnC-gca
trnD-guc
trnE-uuc
trnF-gaa
trnfM-cau
trnG-gcc
trnG-ucc
trnH-gug
trnI-cau
trnI-gau
trnK-uuu
trnL-caa
trnL-uaa
trnL-uag
trnM-cau
trnN-guu
trnP-ugg
trnQ-uug
trnR-acg
trnR-ucu
trnS-gcu
trnS-gga
trnS-uga
trnT-ggu
trnT-ugu
trnV-gac
trnV-uac
trnW-cca
trnY-gua
rRNA
rrn16
rrn23
rrn4.5
rrn5
rpo
matK
rpoA
rpoB
rpoC1
rpoC2
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Pd
þ
þ
þ
þ
Pd
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Cj
þ
þ
þ
þ
Cj
þ
þ
þ
þ
j
j
j
j
j
j
j
j
Pd
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
N
þ
þ
þ
Pd
N
N
N
Cj
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
N
þ
Cj
N
N
N
j
j
þ
þ
Note: Pd, P. decorata; Cj, C. japonia; j, pseudogene; þ, present; -, missing; N,
undetected.
Strength of selection is commonly measured by calculating the
ratio of the rates of nonsynonymous substitution over synonymous
substitutions, dN/dS (Muse and Gaut, 1997). To determine whether
the cpDNA of plants at different heterotrophic levels experienced
differing selective pressures, we calculated the dN/dS values to
measure the strength of selection for each species (Figs. 1e5). The
estimated d, dN, dS and dN/dS rates of the protein-coding chloroplast genes shared between C. japonica, P. decorata and their
autotrophic relatives (R. simsii) are shown in Fig. 1. Overall, the
three Ericaceae species, C. japonica, P. decorata and R. simsii, have
higher d, dN, dS and dN/dS rates than N. tabacum. Within Ericaceae,
the MX C. japonica and P. decorata have higher d, dN, dS rates but
lower dN/dS ratios than autotrophic (AH) R. simsii, which suggests
that MX plants collectively accumulate more dS and dN than AH
plants.
Fig. 1. Deletions (d), synonymous deletions (dS), nonsynonymous deletions (dN), and the
ratio of nonsynonymous to synonymous deletions (dN/dS) for 80 genes in four species
related to Panax ginseng (NCBI Reference Sequence: NC_006290.1, GI: 52220789).
J. Yu et al. / Plant Diversity 39 (2017) 80e88
83
Fig. 2. Comparisons of dN/dS ratio of ten gene sets between mixotrophic plants (P. decorata and C. japonica) and autotrophic plants (R. simsii) in Ericaceae.
Fig. 3. Comparisons of dN/dS ratio of the ndh gene set between mixotrophic plants (P. decorata and C. japonica) and autotrophic plants (R. simsii) in Ericaceae. * indicates missing
data.
The variations in nonsynonymous substitution rates may reflect
different selective constraints on plastid gene products in different
evolutionary lineages. Using the two newly obtained plastid
genome sequences, we tested whether substantial changes have
occurred in the selective pressure on genes. The 79 chloroplast
protein-coding genes were classified in ten groups according to
their function (excluding ycf15, function presently unknown)
(Supplementary Table 2). The calculated corrected distances of
nonsynonymous nucleotide substitution per nonsynonymous sites
(dN) and synonymous substitution per synonymous sites (dS) for C.
japonica and P. decorata relative to a common-used outgroup of
Panax demonstrates an interesting trend toward relaxed selection
in the genome of the fully autotrophic Rododendron (Fig. 2). Of 79
protein-coding genes shared between the two taxa, 56 genes
(72.7%) have higher dN/dS value in Rododendron than in Nicotiana
(Supplementary Table 3). Furthermore, 12 out of 13 functionally lost
genes in C. japonica and P. decorata had higher dN/dS in Rododendron, indicating these genes may have already been under relaxed
selection prior to the evolution of heterotrophy in Ericaceae. These
findings are consistent with previous studies on Convolvulaceae
(McNeal et al., 2007). Analysis of the combined set of ndh genes
revealed the ratio of nonsynonymous substitution rates to
synonymous substitution rates. We also tested whether ratios of
overall selection between classes of genes remaining in two MX
plants were similar to autotrophic taxa. These tests showed how
patterns of synonymous and nonsynonymous substitution vary
between sampled Ericaceae taxa relative to Panax or the various
classes of genes in the plastome (Fig. 3). While there were minor
changes in synonymous rates between different gene classes,
relative ratio tests of synonymous rates showed no significant differences. However, nonsynonymous rate values for psa and psb
genes were significantly different from both atp, rpl and rps genes,
and there were lower nonsynonymous rates for pet and psa and psb
genes in all pairwise comparisons performed (Fig. 2,
Supplementary Table 3). Photosynthetic genes showed low nonsynonymous substitution rates, and the dN/dS ratios were higher
for photosynthetic genes relative to other gene groups. This suggests that photosynthetic genes have been under strong purifying
selection during the evolution of heterotrophic plants. Of the
photosynthetic genes, the ndh and pho genes tended to have high
dN/dS ratios. Mean dN was higher in Group I and Group II than in
Group III, and Wilcoxon rank sum tests showed that the values of
dN are significantly different in Group I and II relative to Group III
(P < 0.001; Fig. 4); also, the values of dS were significantly different
84
J. Yu et al. / Plant Diversity 39 (2017) 80e88
Fig. 4. Boxplots of the value of dS (A), dN (B) and dN/dS (C) for ten groups of genes. Wilcoxon rank sum tests were used to show that dN values for Group II were extremely
significantly higher than for other angiosperms (P < 0.001), whereas values of dS were significantly different (P ¼ 0.024). The horizontal line is the mean value of dN or dS. For each
gene group, the box represents values between quartiles, dotted lines extend to minimum and maximum values, outliers are shown as circles, and the thick, black lines show
median values. Small dots show values for gene groups that are statistically different than the values for same gene group in other angiosperms. Boxes include 50% of the distributions. Note: *P < 0.05, **P ¼ 0.05e0.001, ***P < 0.001.
J. Yu et al. / Plant Diversity 39 (2017) 80e88
85
Fig. 5. Comparisons of genes with dN/dS>1 between mixotrophic plants (P. decorata and C. japonica) and autotrophic plants (R. simsii) in Ericaceae. * indicates missing data.
relative to dN and dN/dS (P ¼ 0.024; Fig. 4). Rates for each gene
group were compared to reveal patterns of substitutions (Fig. 4). In
general, values of dN were higher in Group II in comparison to
Group III, and comparisons revealed that ribosomal protein and
RNA polymerase genes were the most significantly different relative to genes involved in photosynthesis. Values of dS exhibited
similar yet weaker patterns of variation. Large and small subunit
ribosomal protein genes (rpl and rps, respectively) and psb genes
were the most significantly different (both P < 0.001). Exceptionally, psbI and psaC had the highest values of dN and dS for photosystem genes (Supplementary Table 3). Nucleotide substitutions
were compared and revealed a strong lineage-specific pattern of
increases in dN and dS for different heterotrophic levels. However,
these results also showed that values of dN were greater overall for
Group II relative to other groups.
4. Discussions
4.1. Gene loss and pseudogenization of mixotrophic plastomes
Plastid gene loss is a general trend in heterotrophic taxa and
depends largely on selective pressure on photosynthetic function.
As shown in Table 2, none of the genes involved directly in the
photosynthetic pathway have been lost in either MX Ericaceae in
our study (Table 2), which indicates that plastid-encoded genes for
the photosynthetic apparatus are highly conserved in land plant
plastomes and evolved under strong selection for maintaining
genes directly involved in photosynthesis in mixotrophic taxa
(except ndh genes). Gene loss or pseudogenization have only been
reported in heterotrophic plants (Wickett et al., 2008). Although
MX plants have reduced photosynthetic activity, photosyntheticrelated genes are retained as a whole. This is similar to hemiparasitic Cuscuta, in which genes in the photosynthetic pathways
have generally been retained, except for the loss of psaI (Funk et al.,
2007; McNeal et al., 2007). It has been previously hypothesized that
rbcL potentially has a function unrelated to photosynthesis
(Bungard, 2004). The retention of a rbcL open reading frame has
been observed in other fully heterotrophic taxa and further investigation is required to elucidate its role outside of photosynthesis
(Krause, 2008). Values of dN/dS for photosystem I (psa genes group)
and photosystem II genes (psb genes group) were not significantly
different among the three groups studied here; but for group atp,
pet and ndh, it was extremely significantly different (P < 0.001). This
indicates that photosystem genes have been undergoing stronger
selective pressure.
4.1.1. ndh complex
The ndh complex may be involved in chlororespiration and
electron recycling (Blazier et al., 2011; Claude and Palmer, 1990)
and is presumed to consist of the first genes lost in the transition to
heterotrophy. They have been pseudogenized or entirely lost
several times during land plant evolution (Martín and Sabater,
2010; McNeal et al., 2007). The loss of the ndh complex is correlated with a decreased reliance on photosynthesis and it is thought
to be dispensable in mild environments when maintaining photosynthetic rigor is no longer essential for survival (Martín and
Sabater, 2010). This is particularly true for mixotrophic plants
living under forest canopies in which the ability to exploit neighboring hosts improves the ability to survive low light conditions
(Selosse and Roy, 2009). If the ndh complex is dispensable under
these conditions, then we can predict parallel loss of this complex
in other lineages of mixotrophic plants with an otherwise preserved photosynthetic apparatus analogous to that in MX species.
Within mixotrophic Pyroleae, all the ndh genes have been pseudogenized. The tendency to lose all ndh genes provides an opportunity to investigate the loss of these genes from the plastome
(Fig. 3). Throughout land plants, the presence of mycorrhizae and
ndh loss appear to be only imperfectly correlated (Braukmann and
Stefanovic, 2012). Our results here suggest there is strong evidence
that ndh gene loss in MX plants is inevitable.
4.1.2. Plastid-encoded polymerase
The plastid-encoded RNA polymerase (PEP) is a multi-subunit
enzyme and is encoded by four plastid genes: rpoA, rpoB, rpoC1,
and rpoC2. A loss of the PEP subunits encoded by the plastid rpo
genes would therefore deprive plastids of the possibility of transcribing genetic information, making these genes essential for life
(Wicke et al., 2011). We found that three of four rpo genes were
retained in the plastomes of C. japonica and P. decorata. The absence
of plastid-encoded rpoA genes in two MX plants may suggest a shift
86
J. Yu et al. / Plant Diversity 39 (2017) 80e88
from PEP to nuclear-encoded plastid RNA polymerase (NEP) for the
remaining transcriptional units (Krause, 2008). Similar transitions
have been observed in Epifagus Nutt. and Cuscuta and these transitions are presumed to precede loss of photosynthetic capacity
(Delannoy et al., 2011; McNeal et al., 2007; Wolfe et al., 1992). To
date, the only exceptions are the plastomes of Custuca gronovii and
C. obtusiflora, which were the first and only plastomes with a
confirmed loss of the entire rpo gene set in a plastome where
photosynthesis genes are not only presented (Funk et al., 2007),
but, moreover, actively expressed (Berg et al., 2003). This indicates
that rpoA gene loss may be the beginning of all rpo gene loss in
Ericaceae MX taxa.
4.1.3. Large and small ribosomal protein (rpl and rps) genes
The two MX Ericaceae plants also exhibited some losses of large
and small ribosomal protein (rpl and rps) genes, which is more
extensive than those observed in Rhizanthella R.S. Rogers, Cuscuta
and Epifagus (Delannoy et al., 2011; McNeal et al., 2007; Wolfe et al.,
1992). Most genes coding for ribosomal subunit proteins have been
transferred to the nuclear genome. However, land plant plastomes
commonly encode twelve proteins for the small ribosomal subunits
(rps genes) and nine large ribosomal subunit proteins (rpl genes).
Loss of rps and rpl genes from plastomes is rare, but has been
detected in rosids (Jansen et al., 2007, 2011; Magee et al., 2010) and
a variety of non-photosynthetic or minimally photosynthetic
angiosperm. The loss of rpl23 in P. decorata and C. japonica was
confirmed, but whether rps15 and rps18 were lost or simply undetected is unclear. Loss of rps and rpl genes from plastomes does
not affect the expression of ribosomal protein, so these losses
indicate a greater reliance on nuclear encoded polymerases and
ribosomal proteins to translate the remaining plastid genes
(Braukmann and Stefanovi
c, 2012).
4.1.4. ycf1, clpP and accD genes were undetected
One interesting result in this study is that ycf1, clpP and accD were
undetected in both P. decorata and C. japonica. These genes have
functions outside of photosynthesis and were expected to be
retained in heterotrophic plants, as previously hypothesized
(Barbrook et al., 2006; Bungard, 2004). Chloroplast-encoded clpP
has been reported to be involved in multiple processes of chloroplast development, including a housekeeping function that is
indispensable for cell survival (Shikanai et al., 2001). Nevertheless, it
is known that clpP and accD can be functionally transferred to the
nucleus and these loci have been lost multiple times from the
plastids of flowering plants (Jansen et al., 2007). Parsimony analyses
reconstructing unambiguous changes in gene content among plants
revealed that the gene ycf1 was gained in a common ancestor of
several green algae and land plants (Maul et al., 2002). Therefore,
our detection of a pseudogene copy of ycf2 may provide evidence
that those four divergent genes have been transferred to the nucleus
in MX Ericaceae. Functions of ycf1 and ycf2 have been hypothesized
to involve a number of processes: ATPase-related activities,
chaperone-function, cell division, and structural remodeling and/or
linkage of plastid chromosomes to protein and/or membrane
structures (Wicke et al., 2011). Complete loss of both ycf1 and ycf2
from the plastomes of some, but not all, derived monocot lineages
have been reported previously (Guisinger et al., 2010; Maier et al.,
1995; Yang et al., 2010). It is therefore possible that these genes
may now be less essential in MX Ericaceae plastomes.
4.2. Selective constraint on plastid genes in mixotrophy
Nonsynonymous substitutions tend to have larger influences on
gene function than synonymous substitutions, leading to autotrophic plant group experiencing stronger evolutionary constraints on
their cpDNA than heterotrophic plant group. Nonsynonymous
mutations are purified through selection to maintain an efficient
photosynthetic system. The dN/dS ratios varied among the different
genes, implying that different genes accumulated different
amounts of deleterious mutations (Fig. 4). Autotrophic plants have
lower dN/dS ratios in their cpDNA than heterotrophic species. Thus,
the cpDNA of autotrophic plants has experienced stronger purifying
selection to maintain efficient photosynthetic function. In heterotrophic plants, the demand for photosynthetic products can be
derived from symbiotic, parasitic or other organisms. Because of
this heterotrophic individuals that with lower metabolic efficiency
are more likely to survive and reproduce in a population than
autotrophic individuals under similar circumstances. Taken
together, differing constraints on photosynthetic function, as well
as the influence of effective population size, explain differences in
selective patterns in cpDNA of plants with different photosynthetic
capacities.
To assess the selection constraint on plastid genes in P. decorata
and C. japonica, we analyzed synonymous and nonsynonymous
substitutions in protein-coding genes. Rhododendron, an autotrophic Ericaceae plant, was also included in this analysis. The examination of dN/dS ratio for individual genes that are shared
between them demonstrated that most genes in both MX species
are evolving under purifying selection (Fig. 2). There were two
genes with dN/dS ratios greater than 1: Rhododendron rp123 and C.
japonica rps7. The sequence of rpl23 is lost in P. decorata and C.
japonica, while rps7 differs by a single substitution (nonsynonymous) (Fig. 5). rpl23 is a pseudogene in several photosynthetic plants (Logacheva et al., 2008). High dN/dS in this gene may
indicate the relaxation of selection constraint as an initial stage of
pseudogenization. However, in rps14 and rpl33, dN/dS have been
found to be increased for short genes (Haddrill et al., 2007), and
therefore may not be biologically relevant.
To gain a general view of substitution rates in P. decorata and C.
japonica, we calculated substitution values for all genes combined
in one sequence. This demonstrated that dN/dS ratios are very
similar between two photosynthetic species and between photosynthetic and nonphotosynthetic ones. In contrast, the number of
both synonymous and nonsynonymous substitutions differed
greatly. The apparent effect of higher substitution rate is often
observed in sunshade plants when compared with sunlight plants
(Mathews et al., 2003). This implies that this effect is related to
heterotrophy. It also suggests that plastid genes in MX Ericaceae
plants have higher mutation rates but retain their function.
Rhododendron has a much higher dN/dS ratio than P. decorata and C.
japonica, indicating that autotrophic Ericaceae plant plastomes are
undergoing a degrading stage that may be caused by their relationship with mycorriza (Kerley and Read, 1995, 1997; Stribley and
Read, 1980). All classes of genes appear to have evolved under
strong negative selection with dN/dS ratios much lower than 1,
with photosystem and pet genes remaining the most highly
conserved, while the AH plant Rhododendron has a slightly higher
dN/dS ratio. Our results suggest that during the evolutionary history of Pyroleae, there was a tendency to increase heterotrophy in
the MX group, which is consistent with carbon nutrition research
(Matsuda et al., 2012).
5. Conclusions
By sequencing the plastomes of two MX Ericaceae plants, we
have been able to gain a better understanding of the plastome of
mixotrophs as dependence on photosynthesis for carbohydrate
production decreases. Plastomes of the two mixotrophic plants, P.
decorata and C. japonica, are generally very similar to that of nonparasitic plants, although have a greater degree of ndh gene
J. Yu et al. / Plant Diversity 39 (2017) 80e88
pseudogenization. Genes directly involved in photosynthesis and
under strong selection are retained in the two mixotrophs, but with
greatly increased overall nucleotide substitution. By studying the
similarities and the differences between the two MX and AH plants,
we have gained insights into which changes might occur in the
plastid genome during the transition from autotrophy to heterotrophy and identified future research directions. Because of
unclosed plastome sequences based on present data assembly,
further research is also required to gain a clear understanding of the
evolution of autotrophs and mixotrophs in Ericaceae.
Acknowledgments
We gratefully acknowledge four anonymous reviewers for useful comments on the manuscript.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.pld.2016.11.005.
References
Barbrook, A.C., Howe, C.J., Purton, S., 2006. Why are plastid genomes retained in
non-photosynthetic organisms? Trends Plant Sci. 11, 101e108.
Barkman, T.J., McNeal, J.R., Lim, S.-H., et al., 2007. Mitochondrial DNA suggests at
least 11 origins of parasitism in angiosperms and reveals genomic chimerism in
parasitic plants. BMC Evol. Biol. 7, 248.
Barrett, C.F., Freudenstein, J.V., Li, J., et al., 2014. Investigating the path of plastid
genome degradation in an early-transitional clade of heterotrophic orchids, and
implications for heterotrophic angiosperms. msu252 Mol. Biol. Evol. 31 (12),
3095e3112.
Berg, S., Krupinska, K., Krause, K., 2003. Plastids of three Cuscuta species differing in
plastid coding capacity have a common parasite-specific RNA composition.
Planta 218, 135e142.
Bidartondo, M.I., 2005. The evolutionary ecology of myco-heterotrophy. New Phytol.
167, 335e352.
Blazier, J.C., Guisinger, M.M., Jansen, R.K., 2011. Recent loss of plastid-encoded ndh
genes within Erodium (Geraniaceae). Plant Mol. Biol. 76, 263e272.
Braukmann, T., Stefanovi
c, S., 2012. Plastid genome evolution in mycoheterotrophic
Ericaceae. Plant Mol. Biol. 79, 5e20.
Bungard, R.A., 2004. Photosynthetic evolution in parasitic plants: insight from the
chloroplast genome. Bioessays 26, 235e247.
Cameron, D.D., Coats, A.M., Seel, W.E., 2006. Differential resistance among host and
non-host species underlies the variable success of the hemi-parasitic plant
Rhinanthus minor. Ann. Bot. 98, 1289e1299.
Claude, W.d., Palmer, J.D., 1990. Loss of photosynthetic and chlororespiratory genes
from the plastid genome of a parasitic flowering plant. Nature 348, 337e339.
Delannoy, E., Fujii, S., des Francs-Small, C.C., et al., 2011. Rampant gene loss in the
underground orchid Rhizanthella gardneri highlights evolutionary constraints
on plastid genomes. Mol. Biol. Evol. 28, 2077e2086.
Drouin, G., Daoud, H., Xia, J., 2008. Relative rates of synonymous substitutions in the
mitochondrial, chloroplast and nuclear genomes of seed plants. Mol. Phylogenetics Evol. 49, 827e831.
Funk, H.T., Berg, S., Krupinska, K., et al., 2007. Complete DNA sequences of the
plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and
Cuscuta gronovii. BMC Plant Biol. 7, 45.
Gao, L., Su, Y.J., Wang, T., 2010. Plastid genome sequencing, comparative genomics,
and phylogenomics: current status and prospects. J. Syst. Evol. 48, 77e93.
Gaut, B., Yang, L., Takuno, S., et al., 2011. The patterns and causes of variation in plant
nucleotide substitution rates. Annu. Rev. Ecol. Evol. Syst. 42, 245e266.
Gruzdev, E.V., Mardanov, A.V., Beletsky, A.V., et al., 2016. The complete chloroplast
genome of parasitic flowering plant Monotropa hypopitys: extensive gene losses
and size reduction. Mitochondrial DNA Part B 1, 1e2.
Guisinger, M.M., Chumley, T.W., Kuehl, J.V., et al., 2010. Implications of the plastid
genome sequence of Typha (Typhaceae, Poales) for understanding genome
evolution in Poaceae. J. Mol. Evol. 70, 149e166.
Haddrill, P.R., Halligan, D.L., Tomaras, D., et al., 2007. Reduced efficacy of selection in
regions of the Drosophila genome that lack crossing over. Genome Biol. 8, R18.
Jansen, R.K., Cai, Z.Q., Raubeson, L.A., et al., 2007. Analysis of 81 genes from 64
plastid genomes resolves relationships in angiosperms and identifies genomescale evolutionary patterns. Proc. Natl. Acad. Sci. 104, 19369e19374.
Jansen, R.K., Saski, C., Lee, S.-B., et al., 2011. Complete plastid genome sequences of
three rosids (Castanea, Prunus, Theobroma): evidence for at least two independent transfers of rpl22 to the nucleus. Mol. Biol. Evol. 28, 835e847.
Julou, T., Burghardt, B., Gebauer, G., et al., 2005. Mixotrophy in orchids: insights
from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium. New Phytol. 166, 639e653.
87
Kerley, S.J., Read, D.J., 1995. The biology of mycorrhiza in the Ericaceae. New Phytol.
131, 369e375.
Kerley, S.J., Read, D.J., 1997. The biology of mycorrhiza in the Ericaceae. New Phytol.
136, 691e701.
Kim, K.J., Lee, H.L., 2004. Complete chloroplast genome sequences from Korean
ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution
among 17 vascular plants. DNA Res. 11, 247e261.
Krause, K., 2008. From chloroplasts to“ryptic” plastids: evolution of plastid genomes
in parasitic plants. Curr. Genet. 54, 111e121.
Kron, K.A., 1997. Phylogenetic relationships of Rhododendroideae (Ericaceae). Am. J.
Bot. 84, 973.
Kron, K.A., Judd, W.S., Stevens, P.F., et al., 2002. Phylogenetic classification of Ericaceae: molecular and morphological evidence. Botanical Rev. 68, 335e423.
Kunnimalaiyaan, M., Nielsen, B.L., 1997. Fine mapping of replication origins (oriA
and oriB) in Nicotiana tabacum chloroplast DNA. Nucleic Acids Res. 25,
3681e3686.
Lemaire, B., Huysmans, S., Smets, E., et al., 2011. Rate accelerations in nuclear 18S
rDNA of mycoheterotrophic and parasitic angiosperms. J. Plant Res. 124,
561e576.
Li, R.Q., Yu, C., Li, Y.R., et al., 2009. SOAP2: an improved ultrafast tool for short read
alignment. Bioinformatics 25, 1966e1967.
Li, X., Zhang, T.-C., Qiao, Q., et al., 2013. Complete chloroplast genome sequence of
holoparasite Cistanche deserticola (Orobanchaceae) reveals gene loss and horizontal gene transfer from its host Haloxylon ammodendron (Chenopodiaceae).
PLoS One 8, e58747.
Logacheva, M.D., Samigullin, T.H., Dhingra, A., et al., 2008. Comparative chloroplast
genomics and phylogenetics of Fagopyrum esculentum ssp. ancestrale-a wild
ancestor of cultivated buckwheat. BMC Plant Biol. 8, 59.
Logacheva, M.D., Schelkunov, M.I., Penin, A.A., 2011. Sequencing and analysis of
plastid genome in mycoheterotrophic orchid Neottia nidus-avis. Genome Biol.
Evol. 3, 1296e1303.
Magee, A.M., Aspinall, S., Rice, D.W., et al., 2010. Localized hypermutation and
associated gene losses in legume chloroplast genomes. Genome Res. 20,
1700e1710.
Maier, R.M., Neckermann, K., Igloi, G.L., et al., 1995. Complete sequence of the maize
chloroplast genome: gene content, hotspots of divergence and fine tuning of
genetic information by transcript editing. J. Mol. Biol. 251, 614e628.
Martín, M., Sabater, B., 2010. Plastid ndh genes in plant evolution. Plant Physiol.
Biochem. 48, 636e645.
Mathews, S., Burleigh, J.G., Donoghue, M.J., 2003. Adaptive evolution in the photosensory domain of phytochrome A in early angiosperms. Mol. Biol. Evol. 20,
1087e1097.
Matsuda, Y., Shimizu, S., Mori, M., et al., 2012. Seasonal and environmental changes
of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola
japonica (Ericaceae) growing under different light environments. Am. J. Bot. 99,
1177e1188.
Maul, J.E., Lilly, J.W., Cui, L., et al., 2002. The Chlamydomonas reinhardtii plastid
chromosome: islands of genes in a Sea of Repeats. Plant Cell Online 14,
2659e2679.
McNeal, J.R., Kuehl, J.V., Boore, J.L., et al., 2007. Complete plastid genome sequences
suggest strong selection for retention of photosynthetic genes in the parasitic
plant genus Cuscuta. BMC Plant Biol. 7, 57.
Molina, J., Hazzouri, K.M., Nickrent, D., et al., 2014. Possible loss of the chloroplast
genome in the parasitic flowering plant Rafflesia lagascae (Rafflesiaceae). Mol.
Biol. Evol. 31, 793e803.
Moore, M.J., Soltis, P.S., Bell, C.D., et al., 2010. Phylogenetic analysis of 83 plastid
genes further resolves the early diversification of eudicots. Proc. Natl. Acad. Sci.
107, 4623e4628.
Motomura, H., Selosse, M.A., Martos, F., et al., 2010. Mycoheterotrophy evolved from
mixotrophic ancestors: evidence in Cymbidium (Orchidaceae). Ann. Bot. 106,
573e581.
Muse, S.V., Gaut, B.S., 1997. Comparing patterns of nucleotide substitution rates
among chloroplast loci using the relative ratio test. Genetics 146, 393e399.
Nickrent, D.L., Duff, R.J., Colwell, A.E., et al., 1998. Molecular phylogenetic and
evolutionary studies of parasitic plants. In: Soltis, Douglas E., Doyle, J.J. (Eds.),
Molecular Systematics of Plants II. Springer, New York, pp. 211e241.
Selosse, M.A., Roy, M., 2009. Green plants that feed on fungi: facts and questions
about mixotrophy. Trends Plant Sci. 14, 64e70.
Shikanai, T., Shimizu, K., Ueda, K., et al., 2001. The chloroplast clpP gene, encoding a
proteolytic subunit of ATP-dependent protease, is indispensable for chloroplast
development in tobacco. Plant Cell Physiol. 42, 264e273.
Stefanovi
c, S., Olmstead, R.G., 2004. Testing the phylogenetic position of a parasitic
plant (Cuscuta, Convolvulaceae, Asteridae): Bayesian inference and the parametric bootstrap on data drawn from three genomes. Syst. Biol. 53, 384e399.
Stribley, D.P., Read, D.J., 1980. The biology of mycorrhiza in the Ericaceae. New
Phytol. 86, 365e371.
Tamura, K., Peterson, D., Peterson, N., et al., 2011. MEGA5: molecular evolutionary
genetics analysis using maximum likelihood, evolutionary distance, and
maximum parsimony methods. Mol. Biol. Evol. 28, 2731e2739.
Tedersoo, L., Pellet, P., Koljalg, U., et al., 2007. Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: ecological evidence for
mixotrophy in Pyroleae. Oecologia 151, 206e217.
Triboush, S.O., Danilenko, N.G., Davydenko, O.G., 1998. A method for isolation of
chloroplast DNA and mitochondrial DNA from sunflower. Plant Mol. Biol.
Report. 16, 183.
88
J. Yu et al. / Plant Diversity 39 (2017) 80e88
Wicke, S., Müller, K.F., de Pamphilis, C.W., et al., 2013. Mechanisms of
functional and physical genome reduction in photosynthetic and nonphotosynthetic parasitic plants of the broomrape family. Plant Cell. 25,
3711e3725.
Wicke, S., Schneeweiss, G.M., Müller, K.F., et al., 2011. The evolution of the plastid
chromosome in land plants: gene content, gene order, gene function. Plant Mol.
Biol. 76, 273e297.
Wickett, N.J., Zhang, Y., Hansen, S.K., et al., 2008. Functional gene losses occur with
minimal size reduction in the plastid genome of the parasitic liverwort Aneura
mirabilis. Mol. Biol. Evol. 25, 393e401.
Wolfe, K.H., Morden, C.W., Palmer, J.D., 1992. Function and evolution of a minimal
plastid genome from a nonphotosynthetic parasitic plant. Proc. Natl. Acad. Sci.
89, 10648e10652.
Wyman, S.K., Jansen, R.K., Boore, J.L., 2004. Automatic annotation of organellar
genomes with DOGMA. Bioinformatics 20, 3252e3255.
Yang, M., Zhang, X., Liu, G., et al., 2010. The complete chloroplast genome sequence
of date palm (Phoenix dactylifera L.). PLoS One 5, e12762.
Zimmer, K., Hynson, N.A., Gebauer, G., et al., 2007. Wide geographical and ecological
distribution of nitrogen and carbon gains from fungi in pyroloids and monotropoids (Ericaceae) and in orchids. New Phytol. 175, 166e175.