Cjb-2020-0054.Pdf

Botany

What are the genomic consequences for plastids in a mixotrophic orchid (Epipactis helleborine)?

Journal: Botany

Manuscript ID cjb-2020-0054.R1

Manuscript Type: Article

Date Submitted by the 06-Aug-2020 Author:

Complete List of Authors: Valencia-D., Janice; Southern Illinois University Carbondale, School of Biological Science Whitten, William; Florida Museum of Natural History Neubig, Kurt; Southern Illinois University Carbondale, School of Biological ScienceDraft Albinism, Epipactis helleborine, mixotrophy, mycoheterotrophy, Keyword: plastome

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

What are the genomic consequences for plastids in a mixotrophic orchid (Epipactis

helleborine)?

Janice Valencia-D.¹,3, W. Mark Whitten²†, Kurt Neubig¹

¹School of Biological Sciences, Southern Illinois University, 1125 Lincoln Dr., Carbondale,

Illinois, 62901, USA; [email protected], [email protected]

²Florida Museum of Natural History, University of Florida, 1659 Museum Rd., Gainesville,

FL 32611, USA

3Corresponding author

†Author deceased

Corresponding author: Draft

Janice Valencia-D.1

1125 Lincoln Dr, Carbondale, Illinois, 62901-6509, USA

Phone: 618-203-2724, Fax: 618-453-3441

Email address: [email protected]

Abstract

The chloroplast (plastid) controls carbon uptake, so its DNA sequence and function are highly conserved throughout the land plants. But for those that have alternative carbon supplies, the plastid genome is susceptible to suffer mutations in the photosynthetic genes and overall size reduction. Fully mycoheterotrophic plants receive organic carbon from their fungi partner, do not photosynthesize and also do not exhibit green coloration (or produce substantial quantities of chlorophyll). Epipactis helleborine (L.) Crantz exhibits all trophic modes from autotrophy to full mycoheterotrophy. Albinism is a stable condition in individuals of this species and does not prevent them from producing flowers and fruits. Here we assemble and compare the plastid genome of green and albino individuals. Our results show that there is still strong selective pressure in the plastid genome. Therefore, the few punctual differences among them, to ourDraft knowledge, do not affect any normal photosynthetic capability in the albino plant. These findings suggest that mutations or other genetically controlled processes in other genomes, or environmental conditions, are responsible for the phenotype.

Albinism, Epipactis helleborine, mixotrophy, mycoheterotrophy, plastome

Introduction

About 23,000 species of land plants derive their resources from fungi at least during some

part of their lives, and the vast majority of them are orchids (Merckx 2013). This way of

living, known as mycoheterotrophism, provides part or all the carbon supplies that the plants

need (Leake 1994; Freudenstein and Barrett 2010; Merckx 2013; Selosse et al. 2016;

Lallemand 2018). Based on the dependency of plants to the fungi, they can be ‘initially

mycoheterotrophic’ if they only have a strong association during early developmental stages,

‘partially mycoheterotrophic’ if they retain their relationship with the fungi and develop

photosynthetic capabilities (also called mixotrophic, Gebauer and Meyer 2003; Selosse and

Roy 2009), or ‘fully mycoheterotrophic’ if they only rely on the organic carbon provided by

the fungus (Merckx 2013; Wicke and Naumann 2018; Jacquemyn and Merckx 2019). During

the initial stages of seed germination andDraft protocorm development, orchids depend on fungal

partners for nutrition. Later on, they become self-sufficient, but retain a close relationship

with their mycorrhizal partner (reviewed in Dearnaley et al. 2012). Scattered through the

family, fully mycoheterotrophic taxa have arisen at least 35 times in the Orchidaceae family

based on phylogenetic reconstructions (Freudenstein and Barrett 2010; Barrett et al. 2019)

and they comprise around 235 species in 43 genera (Merckx 2013).

Fully mycoheterotrophic taxa lose their photosynthetic capabilities in a process that is

correlated with the reduction of the plastid (cp) genome (e.g., Delannoy et al. 2011; Wicke et

al. 2013; Feng et al. 2016; Lam et al. 2018). The common steps in the cp degradation across

parasitic and heterotrophic land plants have been described in a model consisting of five

stages (Barrett and Davis 2012; Barrett et al. 2014; Wicke et al. 2016); slightly modified by

(Wicke and Naumann 2018). The first stage is the loss or degradation of ndh genes, which is

a recurrent event observed in orchids and is not always related with the transition to

mycoheterotrophic conditions (e.g., Kim et al. 2015, 2018; Wicke and Naumann 2018). The second is the degradation of primary photosynthetic genes (subunits of pet, psa, psb genes) and the plastid-encoded polymerase gene (rpo). Once the plant becomes fully heterotrophic, during the third step, it loses the atp and rbcL genes and then housekeeping genes (tRNAs, rRNAs and plastid ribosomal proteins). During the fourth stage, genes associated with other metabolic functions (accD, clpP, ycf1, ycf2) are lost. Finally, there can be a complete loss of the cp-genome as exemplified in Rafflesia (Molina et al. 2014).

Mutations in the cp- genome can lead to an achlorophyllous phenotype. Although some fully mycoheterotrophic plants produce small amounts of chlorophyll (Cummings and

Welschmeyer 1998; Barrett et al. 2014), most exhibit a variety of colors such as red, purple, brown, or white. Achlorophyllous plantsDraft can also be produced artificially in the lab by altering plastid genes, as has been done in tobacco (i.e., Santis‐Maciossek et al. 1999;

Fleischmann et al. 2011), wheat (Xia et al. 2012) and several other taxa (Kumari et al. 2009 and references therein). In these cases, alterations of photosynthesis-related genes cause an albino phenotype in normally green (i.e., autotrophic) species.

Among Neottieae, albinism has been best described in members of Cephalanthera (Renner

1938; Salmia 1989; Julou et al. 2005; Abadie et al. 2006; Roy et al. 2013) and Epipactis

(Salmia 1989; Selosse et al. 2004; Jakubska and Schmidt 2005; Lallemand 2018). In

Epipactis helleborine, albino individuals are not abundant in natural populations, but individuals produce white shoots every growing season which make the albinism a permanent condition (Salmia 1989). To date, no studies have addressed the question of if the albino phenotype is heritable and linked to cp-genome modifications. To determine if

achlorophyllous individuals of E. helleborine have alterations in their photosynthesis-related

genes, we sequenced and assembled cp-genomes for green and albino plants.

Epipactis helleborine belongs taxonomically to Epipactis section Epipactis, a clade in which

the species are difficult to recognize taxonomically and genetically (Tyteca and Dufrene

1994; Squirrell et al. 2001; Ehlers et al. 2002; Brzosko et al. 2004; Sramkó et al. 2019). Most

of the species belonging to the E. helleborine alliance originate from a few populations that

are restricted to small regions (with the exception of a paraphyletic group forming E.

helleborine) (Sramkó et al. 2019). To understand the variation in the cp-genome composition

across these closely related species, we compared our findings with other Epipactis species

for which the plastid genome was available through GenBank. Draft

Materials and Methods

Collection, extraction, sequencing

Green and albino forms of E. helleborine (W. Mark Whitten 4891 and 4890, respectively)

were freshly collected in Virginia, at the Mountain Lake Biological Station in an Appalachian

montane Quercus-Tsuga forest. Vouchers were deposited at the University of Florida

Herbarium (FLAS) and leaf tissues were preserved in silica-gel.

Total DNA was extracted from each sample using a CTAB method (Doyle and Doyle 1987),

followed by a silica purification column step (Neubig et al. 2014). The samples were

quantified using Qubit dsDNA BR assay (Thermo Fisher, Waltham, MA, USA) and

concentrations adjusted approximately to the same value for both (Whitten 4890: 57.4ng/µL,

Whitten 4891: 57.6ng/µL). Library preparation, barcoding and sequencing were conducted at

Rapid Genomics, LLC (Gainesville, FL, USA). Pooled fragments of 350-450 bp were sequenced on an Illumina HiSeq X platform to get 150 bp paired-end reads.

Plastid genome assembly

The paired-end reads were trimmed for quality at 0.05 probability and assembled in Geneious

10.2.3 (https://www.geneious.com) with a combination of reference and de novo assemblies.

Epipactis mairei (KU551264) and E. veratrifolia (KU551267) produced by Feng et al. (2016) were used as references.

Polymerase Chain Reaction (PCR)

Primers were designed for the amplification of the 32,000-32,400 bp region in the trnC(CAC) intron, which could not be confidentlyDraft recovered from the Illumina data. We designed two primers for each flank in selected regions with good Illumina data coverage (depth >30).

Then the four possible combinations of the four primers were amplified per sample in order to ensure complete length coverage. Designed forward primers were 5’-

CTTCCGCCTTGACAGGGCGG-3’ and 5’-CTTCCGCCTTGACAGGGCGG, and the reverse primers were 5’-TTCTGTCGGACTAATCACTCCC-3’ and 5’-

ACTACGGGGAAGACTCCTCC-3’. PCR was performed in 25µL reactions consisting of:

1.0 µL template DNA (approximately 50ng), 5.0µL of 5× GoTaq Buffer (Promega, Madison,

Wisconsin), 2.0 µL of 25mM MgCl2, 0.5 µL of 10µM dNTPS, 0.5µL each of 10uM primers,

0.5 units of Taq polymerase and volume was adjusted with water. The PCR program consisted of 2 min of denaturation (98ºC), followed by 36 cycles of 10 s denaturation (98ºC),

15 s annealing (55ºC), 1.5 min elongation (72ºC), with a final 3 min elongation (72ºC).

The PCR products were purified using ExoSAP (USB Corporation, Cleveland, OH, USA)

following the manufacturer's protocols. Cycle sequencing was performed in cleaned products

using the parameters 90°C for 1 min, followed by 40 cycles of 15 s at 96°C, 10 s at 50°C, 3

min at 60°C with a ramp of 1º per s with Big Dye dideoxy terminator v3.0 (Applied

Biosystems, Waltham, MA, USA). Sequencing was performed with both amplifying primers

per PCR reaction on an ABI 3130XL Gene Analyzer (Applied Biosystems).

Final assembly and annotations

Completed cp-genomes were generated in Geneious through de novo assembly of contigs

obtained with Illumina reads and Sanger sequences from PCR. In order to obtain the

definitive coverage count and other stats, the Illumina reads of each sample were mapped to

their corresponding cp-genome in Geneious.Draft The assembly was done in three iterations,

allowing 10% mismatch per read and allowing gaps with a maximum of 5% per read and 10

nt of size. Annotations of coding-regions, rRNAs and tRNAs were performed in GeSeq

(https://chlorobox.mpimp-golm.mpg.de/geseq.html) and then manually curated. OGDRAW

(Lohse et al. 2013) was used to generate the circular view genomes. The sequences were

prepared for submission to GenBank using GB2sequin (Lehwark and Greiner 2019) and then

manually revised and adjusted. The plastid genomes were submitted to GenBank and are

available as the following accessions: MK593537 (Whitten 4891) and MK608776 (Whitten

4890).

Comparative genome analysis

In order to identify hotspots of polymorphisms in the cp-sequence, seven E. helleborine

alliance plastid genomes were compared. Sequences of E. albensis Nováková & Rydlo

(MH590348), E. atrorubens (Hoffm. ex Bernh.) Besser (MH590349), a European E.

helleborine (MH590351), E. microphylla Sieber ex Nyman (MH590352) and E. purpurata

Sm. (MH590354) were downloaded from GenBank and aligned in Geneious using the

MAFFT plugin (Katoh and Standley 2013). Nucleotide diversity Pi (π) (Nei 1987) among the genomes was calculated using DnaSP 6.12.03 (Rozas et al. 2017). The calculation omits gaps and missing data in the alignment.

Phylogenetic relationships among the alliance species was performed for the entire cp- genome (LSC, IR and SSC) using E. veratrifolia Boiss. & Hohen. (KU551267) as outgroup.

A Maximum Likelihood analysis was conducted in the IQTree v1.0 (Nguyen et al., 2015).

The model used was K3Pu+F+I and was selected by the BIC using ModelFinder

(Kalyaanamoorthy et al. 2017). UFBoot was performed to get Bootstrap approximation values with 1000 pseudoreplicates (HoangDraft et al. 2018). The phylogenetic tree was modified in FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Results

A total of 4,018,520 and 5,862,246 pair-end reads were obtained for the green and albino plants, respectively. The amount of paired-end reads that mapped to the cp-genomes was

111,749 (2.78%) with a mean coverage of 103.68× for the green individual and 159,795

(2.72%) with a mean coverage of 167.39× for the albino individual. The plastid genome of both forms of E. helleborine is 159,795 bp and consists of a single circular, double-stranded

DNA sequence showing the typical quadripartite structure (Fig. 1). It includes a pair of inverted repeats (IR=26,893 bp), one small single copy region (SSC=18,785bp), and one long single copy region (LSC=87,224 bp) (Table 1). There are 86 protein-coding regions, 32 tRNAs and 8 rRNAs and no genes are pseudogenized in either of the genomes (Table 2).

The cp-genomes of the green and albino plants are 99.99% similar. There are six

substitutions located in the SSC between the ndhF and rpl32 genes. To our knowledge, these

substitutions do not affect any normal photosynthetic function in the cells.

The seven plastid genomes of the E. helleborine alliance are 159,236 - 159,824 bp (Std Dev:

201.2) and are 99.7% identical. Their alignment is 160,194 bp long and holds 3,293

insertions /deletions (indels). The longest indels are 310 bp long (psbM-trnD(GUC)), 172 bp

long (clpP-psbB) and 68 bp (petA-psbJ). There are 239 polymorphic sites (excluding gaps

and missing data) and the nucleotide diversity =0.00053. A sliding window approach shows

four highly variable spots located in the intergenic spacers atpH-I, trnC(GCA)-petN, petA-psbJ

and ndhF-rpl32 (Fig. 2). Thirty-one genesDraft present 28 synonymous (dS) and 35 non-

synonymous (dN) substitutions within the helleborine alliance. Most of that variation is

unique to E. microphylla and E atrorubens. The Virginian E. helleborine cp-genomes do not

exhibit any substitutions in their coding genes with respect to the consensus. Genes with

more substitutions are ycf1 (7 dN, 2 dS), petA (3 dN, 2 dS), accD (2 dN, 2 dS) and ycf2 (2

dN, 2 dS); other genes have two or less substitutions. The substitutions do not affect the

reading frames of these species, except for E. microphylla, where five genes reduced or

increased the original frame size.

There were 88 phylogenetically informative characters among the seven studied samples. The

phylogenetic relationships based on plastid data show two well supported clades (Fig. 3): one

with E. atrorubens and E. microphylla species and the other with the poorly differentiated E.

albensis, E. helleborine and E. purpurata species. The Virginian samples of E. helleborine

were sister but were not most closely related to the European sample, which appears as the first divergent branch in the E. helleborine alliance clade.

Discussion

Plastid genomes of green and albino plants

The cp-genome of green and albino E. helleborine contains all gene regions expected in autotrophic orchids. The length of the sequences did not vary from the expectations based on other plastid genomes of species in the same clade. We did not find evidence of mutations in coding regions including all ndh genes that appear in full and in reading frame. In the closely related species Epipactis microphylla, the loss of reading frame in ndhD, ndhE, ndhF and ndhH in partially mycoheterotrophic greenDraft individuals has been reported (Lallemand et al.

2019a). To our knowledge, this is the first evidence of a fully mycoheterotrophic individual that retained all the plastid encoded genes, even in the subunits of ndh. The percentage of plastid Illumina reads was similar among the green and albino individuals (2.78% vs. 2.72%, respectively). The contig coverage (depth) of the green and albino plastid genomes is not very different (103.68× vs. 167.39×, respectively). Taking the percentage as an indicator of the amount of cp-DNA in the sampled tissue, the results indicate that there is no reduction in the amount of cp-genomes produced and kept in the leaf cells of the albino plant. These findings are in agreement with studies of albino tobacco plants, where there was no reduction in the amount of plastids per cell (Bae 2001). Also, Salmia (1989) did not find any reduction in the amount of plastids in the leaves of albino E. helleborine individuals. Instead, the differences appeared in the development of the thylakoid membranes as would be expected in non- chloroplast plastids (e.g., amyloplasts).

The lack of differences among the plastid genomes of the green and albino individuals

indicates that there are no mutations in the cp-genome during early stages of albinism that

affect photosynthetic genes. An intact plastid genome implies that albino individuals could

contribute to the genetic pool of photosynthetically functional plastids and are not isolated

dead-ends on the way to being obligately mycoheterotrophic. Studying albinos in

Cephalanthera, Tranchida-Lombardo et al. (2010) found that the levels of genetic diversity

were the same in albino vs. green individuals and that the albino individuals do not form an

independent genetic lineage since there was no significant clustering among individuals

based on the phenotype. Even though there is no photosynthetic activity in the albino plants

(Suetsugu et al. 2017; Lallemand et al. 2019b), the plastid gene regions are transcribed

(Lallemand et al. 2019b) and then mechanisms that prevent the degradation of the DNA

should be present. Suetsugu et al. (2017)Draft pointed out that the albino plants and its fungal

partner are under greater oxidative stress. Consequently, genes involved in biotic stress and

the ones that encode antioxidant function are upregulated in the achlorophyllous individuals

(Suetsugu et al. 2017; Lallemand et al. 2019b).

Albinism

Several studies have investigated the differences between green and albino plants. They have

focused on morphology and anatomy (Salmia 1989), shifts in the mycorrhizal community

(Julou et al. 2005; Abadie et al. 2006; Ogura-Tsujita and Yukawa 2008), on fitness (Roy et

al. 2013; Shefferson et al. 2016), and on physiology (Gonneau et al. 2014; Suetsugu et al.

2017; Lallemand et al. 2019b), but there is no clear consensus on how the albino phenotype

emerges.

Achlorophyllous individuals retain their albino condition through time based on 2-14 years of field observations (Selosse et al. 2004; Abadie et al. 2006; Tranchida-Lombardo et al. 2010).

Thus, the cause of the albino phenotype whether genetic, environmental, or a combination of both, probably acts during the early stages of plant development. A genetic cause of the albino phenotype cannot be discarded. As we demonstrated, albinism is not caused by modifications in the cp-genome, so potential mutations should be searched in other genomes.

On the other hand, among the possible extrinsic causes, the mixotrophic condition merits further study due to its recurrent presence in the Epipactis helleborine alliance. A few additional examples of albino orchids have been reported outside of the Neottieae tribe, such as in Goodyera repens (Cameron et al. 2006; Suetsugu et al. 2019) and Platanthera minor

(Yagame et al. 2012; Lallemand 2018). In both cases, the presence of green, partially mycoheterotrophic individuals might beDraft an exaptation in the evolution of albinism. Selosse et al. (2004) proposed that the acquisition of ectomycorrhizal partners leads to a predisposition to achlorophylly in Neottieae orchids. Five of the six genera in this tribe include mixotrophic representatives. Cephalanthera, Epipactis and Limodorum have some species with green and albino individuals, and Neottia includes some fully mycoheterotrophic species. Moreover, mixotrophic and fully mycoheterotrophic species exhibit ectomycorrhizal fungal symbionts, in some cases even truffles (Ascomycota), in their mycorrhizal communities (Selosse et al

2004; Jacquemyn et al. 2015; Jacquemyn et al. 2017; Suetsugu et al. 2017) and in higher percentages than the common orchid endomycorrhizae (typically from Basidiomycota). Thus, there is correlation among the symbiotic relationships between ectomycorrhizal fungi and albinism in orchids. Furthermore, in transcriptomes of E. helleborine, Lallemand et al.

(2019b) and Suetsugu et al. (2017) did not find differences in gene expression related to albinism giving no further explanations of the causes of albinism.

Mycoheterotrophy

In terms of the mycorrhizal associations, it is still not clear how much the fungi contribute to

the development of different degrees of dependency in mycoheterotrophic plants. In

Orchidaceae, few clades show transition among different trophic modes as clearly as

Neottieae (Selosse et al. 2004; Feng et al. 2016; Zhou and Jin 2018; Lallemand et al. 2019a),

where the full spectrum from autotrophy to fully mycoheterotrophy can be found. Recently, it

was demonstrated that E. helleborine is capable of using all of them, since it can live

autotrophically under in vitro conditions without its ectomycorrhizal partner (May et al.

2020). The ability to grow autotrophically, or as partially and fully mycoheterotrophic may

improve the plant’s probability of survival and likely, contribute to its rapid range expansion

in North America since its introduction before 1880 (Squirrell et al. 2001). Classified as an

agricultural and environmental weed byDraft the Global Compendium of Weeds (www.hear.org),

E. helleborine exhibits low specificity in its habitat and it can grow in anthropogenically

disturbed areas as well as in shady woodlands. Green individuals display different levels of

mycoheterotrophy depending on light availability (Preiss et al. 2010; Gonneau et al. 2014).

Those individuals in open areas are autotrophic or partially mycoheterotrophic and those in

forested areas are fully mycoheterotrophic (Renner 1938).

The fungal community varies with the plant trophic modes, so that most autotrophic orchids

are associated with endomycorrhizal (Rhizoctonia) fungi and mycoheterotrophic orchids with

ectomycorrhizal partners (Jacquemyn et al. 2017; Lallemand et al. 2019a). However, instead

of a shift between endo- and ectomycorrhizal partners in species of Epipactis, there are

changes in the abundance of those different types of mycorrhizal partners through time

(Jacquemyn et al. 2016, 2017). Nonetheless, these transitions have not been detected in E.

helleborine among the green and albino plants (Abadie et al. 2006), possibly because studies

have focused on the most abundant fungi rather than on community assembly. Whether the interactions or replacements of the fungal community during early stages of development have any effects on the manifestation of the albino phenotype or not remain to be tested.

Other possible causes of albinism

Some genetic causes of the albinism are mutations in the nuclear DNA or nuclear-plastid genome incompatibility that affect chloroplast biogenesis or chlorophyll synthesis (Kumari et al. 2009). Nuclear genes that induce albinism have been found in several crops (i.e., Maize:

Asakura et al. 2004; Cotton: Ladygin 2007; Rice: Jiang et al. 2007). To date, possible nuclear mutations associated with the phenomenon have not been found in orchids.

Epipactis helleborine alliance Draft

Potential differences in the plastid genome below the species level has been poorly studied in plants. In mixotrophic groups, plastid genome comparisons have shown unexpected differences, either within the same population or among them. Barrett et al. (2019) suggests that in fully mycoheterotrophic orchids cp-genome degradation might be an ongoing process among populations within species. In Gastrodia elata, differences in published plastid genomes are considerably high for a relatively small cp-genome (35,180 bp, 670 insertions/deletions and 457 of single nucleotide polymorphisms) (Park et al. 2020).

Similarly, at the population level, four species of Hexalectris (H. arizonica, H. brevicaulis,

H. parviflora and H. warnockii) (Barrett et al. 2019) and two species of Corallorhiza (C. maculata and C. striata) (Barrett et al. 2014) exhibit differences in presence/absence or pseudogenization of coding-sequence genes. These studies demonstrate that the exploration of cp-genomes within species could facilitate the discovery of new genomic processes.

Contrary to what happens in fully mycoheterotrophic species, the cp-genome of the albino

individual did not show any important modifications. These findings are consistent with the

idea that there is an increase in purifying selection of some photosynthetic genes in Neottieae

(plastid encoded polymerase genes and rbcL) in order to keep the cp-genome intact. The

comparison of the E. helleborine alliance plastid sequences showed a nucleotide diversity of

=0.00053, a small number that reflects the poor degree of differentiation among the species

in the alliance.

Our data show that the North American E. helleborine samples are not most closely related to

the European one. Various reasons may explain this, such as a limited amount of taxon

sampling, but nonmonophyly is likely explained by a complex evolutionary history. Sramkó

et al. (2019) found that E. helleborine Draftwas deeply paraphyletic relative to many other

recognized species (i.e., E. albensis, E. greuteri, E. muelleri, E. pontica). A second

possibility is that the entity that is known as E. helleborine in North America has been

misidentified and represents another species in the group. Because of the high similarity

between species in this alliance, it may have been incorrectly applied by North American

botanists.

One anomalous aspect of the analysis is the high amount of molecular substitutions in the

autogamous E. microphylla (Fig. 3, represented by a long branch in the tree) compared with

the other species that are allogamous. The same pattern of branch length heterogeneity was

found by Sramkó et al. (2019) using RAD-Seq data among autogamous and allogamous

species.

Conclusions

It has been proposed that mixotrophic plants depict an intermediate stage between partial and fully mycoheterotrophic modes (Salmia 1986; Selosse et al. 2004; Julou et al. 2005; Abadie et al. 2006; Stöckel et al. 2011). In this study, we tested the presence of mutations in the cp- genome of an albino E. helleborine which physiologically acts as fully mycoheterotrophic.

No differences between green and albino cp-genomes were found in any coding sequence, leading us to conclude that the causes of the albinism might be controlled by nuclear or mitochondrial mutations, or by environmental changes (likely based on fungal relationships) during early stages of plant development. These findings show additional evidence that strong purifying selection is acting on the cp-genome to keep it intact, consistent with

Lallemand et al. (2019a), likely as a strategy to increase the survival chances of the species under all trophic modes. However, further studies are needed to understand variation in the cp-genome and the changes that occurDraft below the species level, especially in isolated autogamous populations.

Acknowledgments

This work was supported by startup funds from SIU to Neubig, NSF DUE-1564969 to

Neubig, and support for Whitten by NSF DEB-1442280. The first author is supported by a

Fulbright-Colciencias scholarship. We also thank Kent Perkins and the University of Florida

Herbarium for curation of vouchers. In the course of preparing this study, our friend and colleague W. Mark Whitten passed away; we thank him for his contributions to this study and years of devoted support for science and nature.

References

Abadie, J.-C., Püttsepp, Ü., Gebauer, G., Faccio, A., Bonfante, P., and Selosse, M.-A. 2006.

Cephalanthera longifolia (Neottieae, Orchidaceae) is mixotrophic: a comparative

study between green and nonphotosynthetic individuals. Botany, 84(9): 1462–1477.

Asakura, Y., Hirohashi, T., Kikuchi, S., Belcher, S., Osborne, E., Yano, S., et al. 2004. Maize

mutants lacking chloroplast FtsY exhibit pleiotropic defects in the biogenesis of

thylakoid membranes. Plant Cell, 16(1): 201–214.

Bae, C. 2001. Regulation of chloroplast gene expression is affected in ali, a novel Tobacco

albino mutant. Ann. Bot. 88(4): 545–553. doi:10.1006/anbo.2001.1495.

Barrett, C.F., and Davis, J.I. 2012. The plastid genome of the mycoheterotrophic

Corallorhiza striata (Orchidaceae) is in the relatively early stages of degradation.

Am. J. Bot. 99(9): 1513–1523.Draft

Barrett, C.F., Freudenstein, J.V., Li, J., Mayfield-Jones, D.R., Perez, L., Pires, J.C., and

Santos, C. 2014. Investigating the path of plastid genome degradation in an early-

transitional clade of heterotrophic orchids, and implications for heterotrophic

angiosperms. Mol. Biol. Evol. 31(12): 3095–3112.

Barrett, C.F., Sinn, B.T., and Kennedy, A.H. 2019. Unprecedented parallel photosynthetic

losses in a heterotrophic orchid genus. Mol. Biol. Evol. 36(9): 1884–1901.

Brzosko, E., Wróblewska, A., and Talalaj, I. 2004. Genetic variation and genotypic diversity

in Epipactis helleborine populations from NE Poland. Plant Syst. Evol. 248(1–4): 57–

69.

Cameron, D.D., Leake, J.R., and Read, D.J. 2006. Mutualistic mycorrhiza in orchids:

evidence from plant–fungus carbon and nitrogen transfers in the green-leaved

terrestrial orchid Goodyera repens. New Phytol. 171(2): 405–416.

doi:10.1111/j.1469-8137.2006.01767.x.

Cummings, M.P., and Welschmeyer, N.A. 1998. Pigment composition of putatively

achlorophyllous angiosperms. Plant Syst. Evol. 210(1–2): 105–111.

Dearnaley, J.D.W., Martos, F., and Selosse, M.-A. 2012. 12 Orchid mycorrhizas: molecular

ecology, physiology, evolution and conservation aspects. In Fungal Associations.

Edited by B. Hock. Springer, Berlin, Heidelberg. pp. 207–230. doi:10.1007/978-3-

642-30826-0_12.

Delannoy, E., Fujii, S., Colas des Francs-Small, C., Brundrett, M., and Small, I. 2011.

Rampant gene loss in the underground orchid Rhizanthella gardneri highlights

evolutionary constraints on plastid genomes. Mol. Biol. Evol. 28(7): 2077–2086.

Doyle, J., and Doyle, J. 1987. Genomic plant DNA preparation from fresh tissue-CTAB

method. Phytochem. Bull. 19(11): 11–15.

Ehlers, B.K., Olesen, J.M., and Agren,Draft J. 2002. Floral morphology and reproductive success

in the orchid Epipactis helleborine: regional and local across-habitat variation. Plant

Syst. Evol. 236(1–2): 19–32.

Feng, Y.-L., Wicke, S., Li, J.-W., Han, Y., Lin, C.-S., Li, D.-Z., et al. 2016. Lineage-specific

reductions of plastid genomes in an orchid tribe with partially and fully

mycoheterotrophic species. Genome Biol. Evol. 8(7): 2164–2175.

doi:10.1093/gbe/evw144.

Fleischmann, T.T., Scharff, L.B., Alkatib, S., Hasdorf, S., Schöttler, M.A., and Bock, R.

2011. Nonessential plastid-encoded ribosomal proteins in Tobacco: A developmental

role for plastid translation and implications for reductive genome evolution. Plant

Cell, 23(9): 3137–3155. doi:10.1105/tpc.111.088906.

Freudenstein, J.V., and Barrett, C.F. 2010. Mycoheterotrophy and diversity in Orchidaceae.

In Diversity, phylogeny and evolution in the monocotyledons, the proceedings of the

Fourth International Conference on Monocot Systematics, 25–37. Aarhus University

Press, Copenhagen, Denmark.

Gebauer, G., and Meyer, M. 2003. 15N and 13C natural abundance of autotrophic and myco-

heterotrophic orchids provides insight into nitrogen and carbon gain from fungal

association. New Phytol. 160(1): 209–223.

Gonneau, C., Jersáková, J., de Tredern, E., Till-Bottraud, I., Saarinen, K., Sauve, M., et al.

2014. Photosynthesis in perennial mixotrophic Epipactis spp.(Orchidaceae)

contributes more to shoot and fruit biomass than to hypogeous survival. J. Ecol.

102(5): 1183–1194.

Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q., and Le, S.V. 2018. UFBoot2:

Improving the Ultrafast Bootstrap Approximation. Mol. Biol. Evol. 35(2):518-522.

doi: 10.1093/molbev/msx281. Draft

Jacquemyn, H., Brys, R., Waud, M., Busschaert, P., and Lievens, B. 2015. Mycorrhizal

networks and coexistence in species-rich orchid communities. New Phytol. 206(3):

1127–1134.

Jacquemyn, H., and Merckx, V.S. 2019. Mycorrhizal symbioses and the evolution of trophic

modes in plants. J. Ecol. 107(4): 1567–1581.

Jacquemyn, H., Waud, M., Brys, R., Lallemand, F., Courty, P.-E., Robionek, A., and Selosse,

M.-A. 2017. Mycorrhizal associations and trophic modes in coexisting orchids: an

ecological continuum between auto-and mixotrophy. Front. Plant Sci. 8: 1497.

Jacquemyn, H., Waud, M., Lievens, B., and Brys, R. 2016. Differences in mycorrhizal

communities between Epipactis palustris, E. helleborine and its presumed sister

species E. neerlandica. Ann. Bot. 118(1): 105–114.

Jakubska, A., and Schmidt, I. 2005. Chlorophyll-free form of Epipactis albensis Nováková et

Rydlo (Orchidaceae, Neottieae) in the ‘Skarpa Storczyków’nature reserve near Orsk

(Lower Silesia, Poland). Acta Bot. Silesiaca, 2: 151–154.

Jiang, H., Li, M., Liang, N., Yan, H., Wei, Y., Xu, X., et al. 2007. Molecular cloning and

function analysis of the stay green gene in rice. Plant J. 52(2): 197–209.

doi:10.1111/j.1365-313X.2007.03221.x.

Julou, T., Burghardt, B., Gebauer, G., Berveiller, D., Damesin, C., and Selosse, M.-A. 2005.

Mixotrophy in orchids: insights from a comparative study of green individuals and

nonphotosynthetic individuals of Cephalanthera damasonium. New Phytol. 166(2):

639–653.

Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K., von Haeseler, A., and Jermiin, L.S. 2017.

ModelFinder: fast model selectionDraft for accurate phylogenetic estimates. Nat. Methods,

14(6): 587.

Katoh, K., and Standley, D.M. 2013. MAFFT Multiple Sequence Alignment Software

Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 30(4): 772–

780. doi:10.1093/molbev/mst010.

Kim, H.T., Kim, J.S., Moore, M.J., Neubig, K.M., Williams, N.H., Whitten, W.M., and Kim,

J.-H. 2015. Seven new complete plastome sequences reveal rampant independent loss

of the ndh gene family across orchids and associated instability of the inverted

repeat/Small Single-Copy region boundaries. PLOS ONE, 10(11): e0142215.

doi:10.1371/journal.pone.0142215.

Kim, H.T., Shin, C.-H., Sun, H., and Kim, J.-H. 2018. Sequencing of the plastome in the

leafless green mycoheterotroph Cymbidium macrorhizon helps us to understand an

early stage of fully mycoheterotrophic plastome structure. Plant Syst. Evol. 304(2):

245–258.

Kumari, M., Clarke, H.J., Small, I., and Siddique, K.H. 2009. Albinism in plants: a major

bottleneck in wide hybridization, androgenesis and doubled haploid culture. Crit. Rev.

Plant Sci. 28(6): 393–409.

Ladygin, V.G. 2007. Disturbed structure and function of chloroplasts during blocked

biosynthesis of 5-aminolevulinic acid in the light. Biol. Bull. 34(3): 248–258.

Lallemand, F. 2018. Evolution des interactions mycorhiziennes et de la mycohétérotrophie

chez les orchidées. PhD Thesis Muséum national d’Histoire naturelle, Paris. France.

Lallemand, F., Logacheva, M., Le Clainche, I., Bérard, A., Zheleznaia, E., May, M., et al.

2019a. Thirteen new plastid genomes from mixotrophic and autotrophic species

provide insights into heterotrophy evolution in Neottieae orchids. Genome Biol. Evol.

11(9): 2457–2467. doi:10.1093/gbe/evz170.

Lallemand, F., Martin-Magniette, M.-L.,Draft Gilard, F., Gakière, B., Launay-Avon, A.,

Delannoy, É., and Selosse, M.-A. 2019b. In situ transcriptomic and metabolomic

study of the loss of photosynthesis in the leaves of mixotrophic plants exploiting

fungi. Plant J. 98(5): 826–841.

Lam, V.K., Darby, H., Merckx, V.S., Lim, G., Yukawa, T., Neubig, K.M., et al. 2018.

Phylogenomic inference in extremis: A case study with mycoheterotroph plastomes.

Am. J. Bot. 105(3): 480–494.

Leake, J.R. 1994. The biology of myco-heterotrophic (‘saprophytic’) plants. New Phytol.

127(2): 171–216.

Lehwark, P., and Greiner, S. 2019. GB2sequin - A file converter preparing custom GenBank

files for database submission. Genomics, 111(4): 759–761.

doi:10.1016/j.ygeno.2018.05.003.

Lohse, M., Drechsel, O., Kahlau, S., and Bock, R. 2013. OrganellarGenomeDRAW—a suite

of tools for generating physical maps of plastid and mitochondrial genomes and

visualizing expression data sets. Nucleic Acids Res. 41(W1): W575–W581.

May, M., Jąkalski, M., Novotná, A., Dietel, J., Ayasse, M., Lallemand, F., et al. 2020. Three-

year pot culture of Epipactis helleborine reveals autotrophic survival, without

mycorrhizal networks, in a mixotrophic species. Mycorrhiza, 30(1): 51–61.

doi:10.1007/s00572-020-00932-4.

Merckx, V.S. 2013. Mycoheterotrophy: an introduction. In Mycoheterotrophy: The Biology

of Plants Living on Fungi. Edited by V. Merckx. Springer. pp. 1–17.

Molina, J., Hazzouri, K.M., Nickrent, D., Geisler, M., Meyer, R.S., Pentony, M.M., et al.

2014. Possible loss of the chloroplast genome in the parasitic flowering plant

Rafflesia lagascae (Rafflesiaceae).Draft Mol. Biol. Evol. 31(4): 793–803.

doi:10.1093/molbev/msu051.

Neubig, K.M., Whitten, W.M., Abbot, J.R., Elliott, S., Soltis, D.E., and Soltis, P. 2014.

Variables affecting DNA preservation in archival plant specimens. In DNA Banking

for the 21st Century: Proceedings of the US Workshop on DNA Banking. Edited by

W. Applequist and L. Campbell. William L. Brown Center, Missouri Botanical

Garden, St. Louis, Miss. pp. 81–112.

Ogura-Tsujita, Y., and Yukawa, T. 2008. Epipactis helleborine shows strong mycorrhizal

preference towards ectomycorrhizal fungi with contrasting geographic distributions in

Japan. Mycorrhiza, 18(6–7): 331–338.

Park, J., Suh, Y., and Kim, S. 2020. A complete chloroplast genome sequence of Gastrodia

elata (Orchidaceae) represents high sequence variation in the species. Mitochondrial

DNA Part B, 5(1): 517–519. doi:10.1080/23802359.2019.1710588.

Preiss, K., Adam, I.K., and Gebauer, G. 2010. Irradiance governs exploitation of fungi: fine-

tuning of carbon gain by two partially myco-heterotrophic orchids. Proc. R. Soc. B

Biol. Sci. 277(1686): 1333–1336.

Renner, O. 1938. Über blasse, saprophytische Cephalanthera alba und Epipactis latifolia.

Flora Oder Allg. Bot. Ztg. 132(3): 225–233.

Roy, M., Gonneau, C., Rocheteau, A., Berveiller, D., Thomas, J.-C., Damesin, C., and

Selosse, M.-A. 2013. Why do mixotrophic plants stay green? A comparison between

green and achlorophyllous orchid individuals in situ. Ecol. Monogr. 83(1): 95–117.

Rozas, J., Ferrer-Mata, A., Sánchez-DelBarrio, J.C., Guirao-Rico, S., Librado, P., Ramos-

Onsins, S.E., and Sánchez-Gracia, A. 2017. DnaSP 6: DNA sequence polymorphism

analysis of large data sets. Mol. Biol. Evol. 34(12): 3299–3302.

Salmia, A. 1986. Chlorophyll-free formDraft of Epipactis helleborine (Orchidaceae) in SE

Finland. Ann. Bot. Fenn. 23(1):49–57.

Salmia, A. 1989. General morphology and anatomy of chlorophyll-free and green forms of

Epipactis helleborine (Orchidaceae). Ann. Bot. Fenn. 26(2): 95–105.

Santis‐Maciossek, G.D., Kofer, W., Bock, A., Schoch, S., Maier, R.M., Wanner, G., et al.

1999. Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1:

molecular biology, biochemistry and ultrastructure. Plant J. 18(5): 477–489.

doi:10.1046/j.1365-313X.1999.00473.x.

Selosse, M.-A., Bocayuva, M.F., Kasuya, M.C.M., and Courty, P.-E. 2016. Mixotrophy in

mycorrhizal plants: extracting carbon from mycorrhizal networks. Chapter 25. In

Molecular Mycorrhizal Symbiosis, First Edition. Edited by F. Martin. John Wiley and

Sons. pp. 451-471.

Selosse, M.-A., Faccio, A., Scappaticci, G., and Bonfante, P. 2004. Chlorophyllous and

achlorophyllous specimens of Epipactis microphylla (Neottieae, Orchidaceae) are

associated with ectomycorrhizal septomycetes, including truffles. Microb. Ecol.

47(4): 416–426.

Selosse, M.-A., and Roy, M. 2009. Green plants that feed on fungi: facts and questions about

mixotrophy. Trends Plant Sci. 14(2): 64–70.

Shefferson, R.P., Roy, M., Püttsepp, Ü., and Selosse, M.-A. 2016. Demographic shifts related

to mycoheterotrophy and their fitness impacts in two Cephalanthera species.

Ecology, 97(6): 1452–1462.

Squirrell, J., Hollingsworth, P.M., Bateman, R.M., Dickson, J.H., Light, M.H., MacConaill,

M., and Tebbitt, M.C. 2001. Partitioning and diversity of nuclear and organelle

markers in native and introduced populations of Epipactis helleborine (Orchidaceae).

Am. J. Bot. 88(8): 1409–1418.

Sramkó, G., Paun, O., Brandrud, M.K.,Draft Laczkó, L., Molnár, A., and Bateman, R.M. 2019.

Iterative allogamy–autogamy transitions drive actual and incipient speciation during

the ongoing evolutionary radiation within the orchid genus Epipactis (Orchidaceae).

Ann. Bot. 124(3): 481–497.

Stöckel, M., Meyer, C., and Gebauer, G. 2011. The degree of mycoheterotrophic carbon gain

in green, variegated and vegetative albino individuals of Cephalanthera damasonium

is related to leaf chlorophyll concentrations. New Phytol. 189(3): 790–796.

Suetsugu, K., Yamato, M., Matsubayashi, J., and Tayasu, I. 2019. Comparative study of

nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera

velutina, an orchid mainly utilizing saprotrophic rhizoctonia. Mol. Ecol. 28(18):

4290–4299. doi:10.1111/mec.15213.

Suetsugu, K., Yamato, M., Miura, C., Yamaguchi, K., Takahashi, K., Ida, Y., Shigenobu, S.,

and Kaminaka, H. 2017. Comparison of green and albino individuals of the partially

mycoheterotrophic orchid Epipactis helleborine on molecular identities of

mycorrhizal fungi, nutritional modes and gene expression in mycorrhizal roots. Mol.

Ecol. 26(6): 1652–1669. doi:10.1111/mec.14021.

Tranchida-Lombardo, V., Cafasso, D., Cristaudo, A., and Cozzolino, S. 2010.

Phylogeographic patterns, genetic affinities and morphological differentiation

between Epipactis helleborine and related lineages in a Mediterranean glacial

refugium. Ann. Bot. 107(3): 427–436.

Tyteca, D., and Dufrene, M. 1994. Biostatistical studies of Western European allogamous

populations of the Epipactis helleborine (L.) Crantz species group (Orchidaceae).

Syst. Bot. 19(3): 424–442.

Wicke, S., Müller, K.F., dePamphilis, C.W., Quandt, D., Bellot, S., and Schneeweiss, G.M.

2016. Mechanistic model of evolutionary rate variation en route to a

nonphotosynthetic lifestyle in plants.Draft Proc. Natl. Acad. Sci. U.S.A. 113(32): 9045–

9050. doi:10.1073/pnas.1607576113.

Wicke, S., Müller, K.F., de Pamphilis, C.W., Quandt, D., Wickett, N.J., Zhang, Y., Renner,

S.S., and Schneeweiss, G.M. 2013. Mechanisms of functional and physical genome

reduction in photosynthetic and nonphotosynthetic parasitic plants of the Broomrape

family. Plant Cell, 25(10): 3711–3725. doi:10.1105/tpc.113.113373.

Wicke, S., and Naumann, J. 2018. Molecular Evolution of Plastid Genomes in Parasitic

Flowering Plants. In Chapter 11. Advances in Botanical Research. Vol. 85. Edited by

S-M Chaw and R.K.Jansen. Elsevier. pp. 315–347. doi:10.1016/bs.abr.2017.11.014.

Xia, J., Guo, H., Xie, Y., Zhao, L., Gu, J., Zhao, S., Li, J., and Liu, L. 2012. Differential

expression of chloroplast genes in chlorophyll-deficient wheat mutant Mt135 derived

from space mutagenesis. Acta Agron. Sin. 38(11): 2122–2130.

Yagame, T., Orihara, T., Selosse, M.-A., Yamato, M., and Iwase, K. 2012. Mixotrophy of

Platanthera minor, an orchid associated with ectomycorrhiza-forming

Ceratobasidiaceae fungi. New Phytol. 193(1): 178–187. doi:10.1111/j.1469-

8137.2011.03896.x.

Zhou, T., and Jin, X. 2018. Molecular systematics and the evolution of mycoheterotrophy of

tribe Neottieae (Orchidaceae, Epidendroideae). PhytoKeys, 94: 39-49.

Draft

Table 1. Length and GC content of the cp-genome of green and albino E. helleborine

sequenced in this study.

Total LSC SSC IR CDS rRNAs tRNAs

Length 159,795 87,224 18,785 26,893 6,873 2,807 105

GC content (%) 37.3 35.1 30.7 43.2 38.1 55.2 52.3

Draft

Table 2. Plastid genes found in E. helleborine

Category Groups of gene Name of genes

trnA-UGC(×2), trnC-ACA (×2), trnC-GCA (×2),

trnD-GUC (×3), trnE-UUC (×3), trnF-GAA

(×2), trnG-GCC, trnH-GUG (×2), trnI-AAU,

trnK-UUU, trnL-CAA(×2), trnL-CAG, trnL-

UAA, trnL-UAG, trnM-CAU (×4), trnN-AUU,

trnN-GUU(×2), trnP-UGG (×2), trnQ-CUG,

trnQ-UUG (×3), trnR-ACG(×2), trnR-UCU (×2),

trnS-CGA (×2),trnS-GCU, trnS-GGA, trnS-

Protein synthesis and UGA, trnT-GGU (×2), trnT-UGU, trnV-

DNA-replication Transfer RNAs GAC(×2), trnW-CCA (×2), trnY-GUA (×2) Ribosomal RNAs Draft rrn16(×2), rrn23(×2), rrn4.5(×2), rrn5(×2) rps2, rps3, rps4, rps7(×2), rps8, rps11,

Ribosomal protein small subunit rps12(×3), rps14, rps15, rps16, rps18, rps19

Ribosomal protein large rpl2(×2), rpl14, rpl16, rpl20, rpl22, rpl23(×2),

subunit rpl32, rpl33, rpl36

Subunits of RNA

polymerase rpoA, rpoB, rpoC1, rpoC2

Photosynthesis Photosystem I psaA, psaB, psaC, psaI, psaJ

Photosystem I assembly protein ycf4

psbA, psbB, psbC, psbD, psbE, psbF, psbH,

Photosystem II psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ

Cythochrome b/f complex petA, petB, petD, petG, petL petN

ATP synthase atpA, atpB, atpE, atpF, atpH, atpI

ndhA, ndhB(×2), ndhC, ndhD, ndhE, ndhF,

NADH-dehydrogenase ndhG, ndhH, ndhI, ndhJ, ndhK

Large subunit Rubisco rbcL

Miscellaneous group Acetyl-CoA carboxylase accD

Cytochrome c biogenesis ccsA

Inner membrane protein cemA

ATP-dependent protease clpP

Translation initiation factor infA

Maturase matK

Translocon at the inner membrane ycf1

Enhancing stress tolerance ycf3

Gene unknown

function ycf2(×2)

Draft

Fig. 1. Gene map of the cp-genome of E. helleborine. Genes located outside of the circle are

transcribed in a clockwise direction whereas genes inside are transcribed in

counterclockwise direction. The lighter grey inner circle indicates AT content, while

the darker grey area indicates GC content (See Supplemental Figure S1 for colour

version).

Fig. 2. DNA Polymorphism analysis of the cp-genome of seven representatives of the E.

helleborine alliance (window length: 600 bp, step size: 200 bp).

Fig. 3. Phylogenetic relationships of representatives of the E. helleborine alliance inferred

from full cp-genome alignment (only one IR) using maximum likelihood (ML).

UFBootstrap values are 100% except as indicated. New samples are shown in bold

(Whitten 4891: green, Whitten Draft4890: albino)

Draft

Gene map of the cp-genome of E. helleborine. Genes located outside of the circle are transcribed in a clockwise direction whereas genes inside are transcribed in a counterclockwise direction. The lighter grey inner circle indicates AT content, while the darker grey area indicates GC content. (See Supplemental Figure S1 for colour version)

379x385mm (300 x 300 DPI)

DNA Polymorphism analysis of the cp-genomeDraft of seven representatives of the E. helleborine alliance (window length: 600 bp, step size: 200 bp).

182x108mm (300 x 300 DPI)

Draft

Fig. 3. Phylogenetic relationships of representatives of the E. helleborine alliance inferred from 81 plastid coding regions using maximum likelihood (ML). Bootstrap values are 100% except as indicated. New samples are shown in bold (Whitten 4891: green, Whitten 4890: albino).

328x223mm (300 x 300 DPI)