Unprecedented Parallel Photosynthetic Losses in a Heterotrophic Orchid Craig F. Barrett,*,1 Brandon T. Sinn,1 and Aaron H. Kennedy2 1Department of Biology, West Virginia University, Morgantown, WV 2Mycology and Nematology Genetic Diversity and Biology Laboratory, USDA-APHIS, Beltsville, MD *Corresponding author: E-mail: [email protected]. Associate editor: Tal Pupko

Abstract Heterotrophic are evolutionary experiments in genomic, morphological, and physiological change. Yet, genomic sampling gaps exist among independently derived heterotrophic lineages, leaving unanswered questions about the process of genome modification. Here, we have sequenced complete plastid genomes for all species of the leafless orchid genus , including multiple individuals for most, and leafy relatives Basiphyllaea and Bletia.Ourobjectivesare to determine the number of independent losses of photosynthesis and to test hypotheses on the process of genome degradation as a result of relaxed selection. We demonstrate four to five independent losses of photosynthesis in Hexalectris based on degradation of the photosynthetic apparatus, with all but two species displaying evidence of losses, and variation in gene loss extending below the species level. Degradation in the atp complex is advanced in Hexalectris warnockii, whereas only minimal degradation (i.e., physical loss) has occurred among some “housekeeping” genes. We find genomic rearrangements, shifts in Inverted Repeat boundaries including complete loss in one accession of H. arizonica, and correlations among substitutional and genomic attributes. Our unprecedented finding of multiple, independent transitions to a fully mycoheterotrophic lifestyle in a single genus reveals that the number of such transitions among land plants is likely underestimated. This study underscores the importance of dense taxon sampling, which is highly informative for advancing models of genome evolution in heterotrophs. Mycoheterotrophs such as Hexalectris provide forward-genetic opportunities to study the consequences of radical genome evolution beyond what is possible with mutational studies in model organisms alone. Key words: heterotroph, phylogenomics, plastome, pseudogene, selection, chloroplast, deletion, evolution.

Introduction and contain the all four of the highly conserved, plastid- encoded ribosomal RNA species (e.g., Zhu et al. 2016). The Heterotrophic plants present prime examples of genomic sequencing of complete plastid genomes (plastomes) has be- Article modification due to relaxed selective constraints on photo- come more feasible because of high-throughput sequencing synthetic function. They are highly useful as “evolutionary technology and assembly algorithms, and thus plastomes experiments” in that they manage to survive and reproduce from throughout the tree of life are being published at a rapid under natural conditions despite drastic modifications, in pace (see Gitzendanner et al. 2018). contrast to many induced mutants in model systems which Plastid genome evolution in heterotrophic plants has re- would almost certainly go extinct in the wild. Thus, hetero- ceived much attention in recent years (e.g., reviewed by trophs can increase our knowledge on how whole-organismal Graham et al. [2017], Hadariova et al. [2018], and Wicke systems adjust to such radical changes. Plastid genomes are of and Naumann [2018]), yet only a handful of studies have particular interest because they are small, nonrecombining included taxon sampling of sufficient density to apply phylo- (i.e., in the sense of swapping material among homologous genetic, comparative approaches (Wicke et al. 2013, 2016; chromosomes as in nuclear DNA), and uniparentally inher- Barrett et al. 2014, 2018; Feng et al. 2016; Braukmann et al. ited; occur in relatively high copy number per cell compared 2017; Schneider et al. 2018). Many studies have focused on with nuclear and (typically) mitochondrial genomes; and are single plastomes, which may fail to capture several key aspects conserved in gene order, content, and, overall genome struc- of the process of plastid genome evolution, especially in cases ture, with a large and small single copy region bisected by a of rapidly accumulated genotypic and phenotypic change large Inverted Repeat (IR; typically 20–30 kb) across angio- due to relaxed selection and increased rates of substitution. sperms (see Ruhlman and Jansen 2014). The genes and Recent studies have begun to incorporate dense sampling of spacers of the IR are among the most conserved regions of plastomes within clades, for example, at or near the genus the plastome in terms of gene order and substitution rates and species level (Wicke et al. 2013; Barrett et al. 2014, 2018; and contribute to overall structural stability of the chromo- Feng et al. 2016; Braukmann et al. 2017; Schneider et al. 2018). some, multimeric structure among plastid chromosomes, This approach is important in that it allows plastome

ß The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Open Access 1884 Mol. Biol. Evol. 36(9):1884–1901 doi:10.1093/molbev/msz111 Advance Access publication May 6, 2019 Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus . doi:10.1093/molbev/msz111 MBE evolution to be interpreted on a finer scale—and more im- Transitions to full mycoheterotrophy are most likely facili- portantly, from a phylogenetic-comparative perspective— tated by the condition of “initial mycoheterotrophy” in revealing details on the types of changes that occur and their orchids, all of which begin their lives as nonphotosynthetic, mechanistic causes, not merely the result after several millions mycoparasitic seedlings (protocorms) that eventually be- of years of evolution on a single plastome. come photosynthetic with the development of leaves or Species-level sampling in the leafless orchid genus other organs capable of photosynthesis (Rasmussen 1995; Corallorhiza (plastome sequencing) and parasitic vine Bidartondo 2005; Leake 2005). Full mycoheterotrophy can Cuscuta (family Convolvulaceae; DNA probe blotting) has be interpreted as a pedomorphic shift that has occurred revealed multiple independent shifts to full heterotrophy many times in separate orchid lineages, and initial mycohe- within single genera, and thus it is likely that the number of terotrophy can be viewed as a preadaptation to such shifts, hypothesized transitions to complete heterotrophy and loss for example, with the characteristic mycotrophic coralloid of photosynthesis have been underestimated in other groups rhizome structure found in heterotrophic species represent- (Revill et al. 2005; Funk et al. 2007; McNeal, Arumugunathan, ing a protracted, juvenile protocorm stage (Rasmussen 1995; et al. 2007; McNeal, Kuehl, et al. 2007; Braukmann et al. 2013; Freudenstein and Barrett 2010; Merckx, Freudenstein, et al. Barrett et al. 2014, 2018). Further, it remains to be determined 2013). These changes are often accompanied by fungal host if photosynthetic loss and subsequent plastome degradation shifts from “typical” orchid mycorrhizal fungal partners— can be detected below the species level. saprotrophic taxa termed rhizoctonias (teleomorphic What types of genomic changes occur in heterotrophic families Ceratobasidiaceae, Tulasnellaceae, and plants? Much attention has been given to the evolutionary Sebacinaceae)—to ectomycorrhizal or even arbuscular my- fates of plastid-encoded gene systems coinciding with or fol- corrhizal fungi, though some of the former are known to lowing the evolution of heterotrophy (e.g., dePamphilis and form ectomycorrhizas (Taylor and Bruns 1997; Taylor et al. Palmer 1990; Wolfe et al. 1992; Barbrook et al. 2006; Krause 2003; Barrett et al. 2010; Kennedy et al. 2011). 2008, 2011, 2012; Wicke et al. 2011, 2013). Barrett and Davis Mycoheterotrophs in clades throughout the orchids ex- (2012) and subsequently Barrett et al. (2014) proposed a gen- hibit several convergent morphoanatomical characteristics eral conceptual model, in which plastid-encoded gene sys- during the transition from autotrophy to heterotrophy. tems are lost in a more-or-less linear, irreversible pattern (but These changes have resulted in modifications to several allowing exceptions), following the general sequence of 1) organs, including reduction or loss of roots and leaves, prev- ndh genes, encoding a chloroplast NAD(P)H dehydrogenase; alence of cleistogamy (closed-flowers), autonomous self- 2) psa, psb, rbcL, ccsA, cemA, ycf3,andycf4, encoding various pollination, reduction or loss of stomata, and reduction in complexes involved directly in photosynthesis (e.g., photo- vascular tissues (Leake 1994; Leake et al. 2008; Merckx and systems I and II, RuBisCO); 3) rpo, a plastid-encoded RNA Freudenstein 2010). polymerase; 4) atp, encoding a thylakoid ATP synthase; and 5) The leafless orchid genus Hexalectris is composed of nine “housekeeping” genes involved in basic organellar functions species, all of which have been presumed to be full mycohe- such as intron splicing and translation (e.g., matK, rpl, rps, rrn, terotrophs, distributed throughout Western North America and trn). Barrett et al. (2014) combined stages 2 and 3, as the and Mexico (Catling 2004; Kennedy and Watson 2010), with a rpo complex primarily transcribes photosynthesis-related single species, H. spicata, extending into the eastern USA genes, and thus these two gene classes typically experience (Catling and Engel 1993). Hexalectris is a member of the or- concomitant degradation in heterotrophic lineages. Further chid subtribe (tribe , subfamily slight modifications of these conceptual models are proposed ), which contains four genera based on the by Naumann et al. (2016) and Graham et al. (2017),basedon recent phylogenetic analysis and classification (Chase et al. availability of additional heterotrophic plastomes. The con- 2015; Freudenstein and Chase 2015). Bletiinae represents an sensus among all the aforementioned conceptual models is apt case study in the transition to obligate parastitism, as its that stage 2 (i.e., loss of genes involved directly in photosyn- four genera encompass different growth forms, including 1) thesis) represents whole-organismal loss of photosynthetic theleafy,green,epiphyticChysis; 2) the leafy, green, terrestrial function, which thus is a major transitory event in both phys- Bletia; 3) the green, terrestrial Basiphyllaea, with 1–2 highly iology and genome evolution of these plants. reduced or basal leaves; and 4) the leafless, terrestrial, myco- Orchids, with >28,000 accepted species ( List 2013), heterotrophic Hexalectris, which at least superficially have experienced a greater number of independent transi- appears to lack visible green tissue (supplementary fig. S1, tions to obligate heterotrophy and the resulting losses of Supplementary Material online). photosynthesis than any other major clade, making this the Although convergent morphological reduction is ap- ideal family for testing hypotheses associated with drastic parent in Bletiinae, especially in the leafless Hexalectris,it evolutionary changes in the plastid genome (Merckx and is unclear whether these changes have occurred in parallel Freudenstein 2010). The loss of photosynthesis is hypothe- with the loss of photosynthesis and resulting plastome sized to have occurred at least 30 times in the orchids alone, degradation. For example, the orchid Corallorhiza representing 2/3 of all such transitions to fully mycoheter- includes leafless photosynthetic and nonphotosynthetic otrophic lifestyles in plants (Freudenstein and Barrett 2010). species, meaning that multiple, independent losses of This form of obligate heterotrophy involves fungus-to- photosynthesis can occur within a single genus after the orchid transfer of carbon and essential nutrients. loss of leaves (Zimmer et al. 2008; Cameron et al. 2009;

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Table 1. Details of Accessions Included in This Study.

Coll.# Genus Species Loc Length IR LSC SSC GC x-cov #Reads Mapped %Plastid Reads Baco1 FL 158,368 26,910 86,711 17,873 37.3 60.8 13,219,124 139,287 1.05 32 Bletia roezlii JA 158,720 26,860 87,177 17,823 37.2 598.0 14,792,462 940,676 6.36 47 Hexalectris arizonica TX 109,543 4,475 90,507 10,086 35.9 169.8 11,912,444 184,256 1.55 271 Hexalectris arizonica TX 105,192 n/a n/a n/a 35.5 485.3 46,290,820 507,602 1.10 377 Hexalectris arizonica AZ 109,461 4,306 90,957 9,892 35.9 415.7 87,738,174 451,735 0.52 36 Hexalectris brevicaulis JA 138,101 31,225 68,147 7,504 37.1 443.0 69,828,048 608,415 0.87 41 Hexalectris brevicaulis JA 137,846 31,263 67,707 7,399 37.1 193.1 26,559,816 170,483 0.64 166 Hexalectris colemanii AZ 117,252 4,865 97,471 10,051 36.2 36.6 6,253,740 37,771 0.60 64 Hexalectris grandiflora TX 152,460 27,090 83,614 14,669 36.8 551.0 49,121,014 834,057 1.70 48 Hexalectris nitida TX 111,720 15,147 71,963 9,063 37.2 72.6 12,620,482 88,569 0.70 79 Hexalectris nitida TX 111,592 15,075 72,400 9,042 37.1 100.4 12,461,696 110,985 0.89 270 Hexalectris nitida TX 111,519 15,148 72,181 9,042 37.1 146.7 13,268,698 161,991 1.22 85 Hexalectris parviflora JA 136,661 27,129 71,757 10,508 37 398.8 46,935,030 540,450 1.15 500 Hexalectris parviflora CH 134,807 26,814 70,342 10,837 37.1 112.1 12,089,618 149,663 1.24 320 Hexalectris revoluta TX 145,345 25,555 83,813 10,422 36.9 206.3 14,764,714 297,143 2.01 44 Hexalectris spicata NC 120,704 23,681 63,566 9,776 37.2 761.3 35,558,544 911,263 2.56 50 Hexalectris spicata TX 120,185 23,582 62,242 10,779 37.2 1137.5 47,730,484 1,348,183 2.83 267 Hexalectris spicata OK 121,646 23,681 63,674 10,610 37.2 932.7 61,605,466 1,125,190 1.83 276 Hexalectris spicata IN 120,027 23,564 62,140 10,759 37.3 45.6 19,701,640 55,164 0.28 49 Hexalectris warnockii TX 119,057 17,332 66,903 17,490 36.9 703.5 38,633,900 829,431 2.15 69 Hexalectris warnockii TX 119,058 17,339 66,938 17,442 36.9 115.3 14,329,294 135,738 0.95

NOTE.—Coll.#, collection number; Loc, locality (US or Mexican state); length, plastome size in bp; IR, length of the inverted repeat; LSC, large single copy region; SSC, small single copy region; GC, GC content of the plastome excluding one IR copy; x-cov, mean coverage depth of the plastome; #reads, total number of reads generated; mapped, the number of reads mapping to the completed plastome; %plastid reads, percentage of the total read pool mapping to the plastome; and GenBank, NCBI accession number.

Barrett and Davis 2012; Barrett et al. 2014, 2018). Here, we Phylogenetic Analyses sampled all species of Hexalectris (including multiple Relationships accessions for most), plus representatives of the related, We used likelihood, Bayesian, and parsimony to analyze leafy Bletia and Basiphyllaea using high-throughput concatenated, filtered locally collinear blocks (LCBs; i.e., the Illumina sequencing, and assembled complete plastomes. “LCB” matrix, based on all syntenic regions concatenated) We undertake a phylogenetic, comparative approach to and a combined matrix of all protein-coding regions (“CDS” elucidate the broad- and fine-scale processes of plastome matrix) to resolve and provide support for relationships modification associated with the transition to full myco- among Hexalectris plastomes. Based on the LCB matrix, we heterotrophy. Specifically, we address the following ques- found strong support and posterior probability (pp) for a tions: 1) Do complete plastomes provide resolution and single topology, with no conflicting relationships among re- support for relationships among members of Beltiinae, construction methods (fig. 1A). Basiphyllaea is sister to and specifically within Hexalectris?2)Whatarethemajor Hexalectris,andwithinHexalectris, H. warnockii is sister to patterns of plastid genome modification across subtribe all other accessions. Three accessions of H. arizonica are sup- Bletiinae, particularly within the leafless Hexalectris, both ported as sister to H. colemanii, which collectively is sister to among and within species? 3) Is there genomic evidence two accessions of H. parviflora. This entire clade is sister to a for loss or retention of photosynthesis in members of the single accession of H. revoluta. Four accessions of H. spicata leafless Hexalectris, and further, is there evidence for a form a clade, within which accessions follow a topology of single loss or multiple losses of photosynthesis in the ge- ([{276 Indiana, 44 North Carolina}, 267 Oklahoma], 50 Texas). nus? 4) How do substitution rates and patterns of selec- Accessions of H. spicata are sister to three accessions of H. tion vary among species that have lost photosynthetic nitida, all from Texas, USA. The H. nitida-spicata clade is sister apparatus compared with those in which it is retained? to a H. arizonica-colemanii-parviflora-revoluta clade, and this entire clade is sister to H. brevicaulis. All relationships are Results supported at >99% bootstrap/jackknife (or >0.99 pp), with the exception of H. spicata accessions44(NC)and276(IN). Plastid Genome Data Relationships are nearly identical for the “CDS” matrix based Plastome Data, Coverage Depth, Percent Plastid DNA on gene-partitioned analysis of coding regions (fig. 1B), except The mean number of paired end reads generated per library for the placement of H. revoluta, which instead is supported was 28,172,786 with a standard deviation of 17,282,194.6 as sister to two accessions of H. parviflora. (range ¼ 6,253,740–87,738,174) (table 1). Coverage depth ranged from 36.6 to 1,137.5, and plastid DNA content per Plastid Genome Structural Evolution read pool ranged from 0.28% to 6.36%. Percent similarity for Plastome Size allaccessionsisgiveninsupplementary table S1, We compared sizes across representatives of the Bletiinae in Supplementary Material online. order to quantify the overall degree of sequence loss (or gain).

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Bletia roezlii JA 32 Basiphyllaea corallicola FL Baco1 TX 49 * Hexalectris warnockii H. warnockii TX 69 * H. grandiflora TX 64 JA 36 * H. brevicaulis * Hex brevicaulis JA 41 H. nitida TX 48 * TX 79 * H. nitida * * H. nitida TX 270 H. spicata TX 50 * H. spicata OK 267 * * H. spicata NC 44 1/73/54 H. spicata IN 276 H. revoluta TX 320 0.003 substitutions per site JA 85 * * H. parviflora H. parviflora CH 500 AZ 166 1/99/99 H. colemanii * H. arizonica AZ 377 * TX 47 Bletia roezlii JA 32 H. arizonica AZ 271 Basiphyllaea corallicola FL Baco1 1/100/99 H. arizonica Hexalectris warnockii TX 49 * H. warnockii TX 69 TX 64 * H. grandiflora H. brevicaulis JA 36 * Hex brevicaulis JA 41 * H. nitida TX 48 * H. nitida TX 79 TX 270 * * 1/99/99 H. nitida H. spicata TX 50 * H. spicata OK 267 * H. spicata NC 44 * * H. spicata IN 276 H. revoluta TX 320 * H. parviflora JA 85 0.003 substitutions per site * H. parviflora CH 500 1/100/99 H. colemanii AZ 166 * H. arizonica AZ 377 * H. arizonica TX 47 * H. arizonica AZ 271

FIG.1.Phylogenetic trees for LCB (above) and CDS matrices (below). * ¼ pp of 1, ML bootstrap value of 100%, and Parsimony jackknife value of 100%; otherwise, values are indicated respectively. Scale bar and branch lengths ¼ substitutions per site from Bayesian trees.

Plastome length varies little between the leafy Bletia roezlii to the IR in two other accessions is 587.5 compared with and the reduced-leaved Basiphyllaea corallicola at 158,720 584.5—or roughly equivalent—for the rest of the plas- and 158,368 bp, respectively. Plastome length varies consider- tome. By contrast, H. arizonica accession 47, which has an ably, however, in the leafless Hexalectris, from a maximum of IR, has a mean coverage depth of 275.8 in the IR and 152,460 bp in H. grandiflora to a minimum of 105,192 bp in an 182.6 outside the IR. Further, the IR junctions and cor- accession of H. arizonica (accession 271, table 1; fig. 2), repre- responding regions in accession 271 (which lacks an IR) senting approximately a 1.5-fold difference in total plastome are covered by paired end information, verifying contigu- length. ity across regions. The single accession of H. colemanii included here has an IR length of 4,865, similar to that IR Variation. We compared the size, structure, and gene in H. arizonica. Thus, aside from the loss of the IR in one content in the IR region among members of Bletiinae, to accession of H. arizonica, the net percent change in the quantify its contribution to overall change in genome size of the IR is þ16% in H. brevicaulis,and84% in H. evolutionary structural dynamics. The length of the IR arizoniza.Onaverage,theIRregionsofHexalectris species in the photosynthetic Bletia and Basiphyllaea is 26,860 are 30% smaller than that of the leafy Bletia and and 26,910 bp, respectively, whereas the IR ranges from Basiphyllaea. Variation in IR length is predominantly 31,225 and 31,263 bp in both accessions of Hexalectris due to boundary shifts relative to insertion/deletion. brevicaulis,to4,475and4,306intwoofthethreeacces- sions of H. arizonica. A third accession of H. arizonica has no detectable IR region (table 1, fig. 2, and supplementary Locally Collinear Blocks fig. S2, Supplementary Material online). Evidence for this We analyzed LCBs in MAUVE in order to characterize the IR loss comes from analysis of coverage depth: mean cov- degree of synteny among regions of the plastid genome in erage depth for accession 271 in the region corresponding Hexalectris relative to leafy, photosynthetic relatives. MAUVE

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0 kb 20 40 60 80 100 120 140 160

32 Bletia roezlii JA Baco1 Basiphyllaea corallicola 64 Hexalectris grandiflora TX 320 Hexalectris revoluta TX 36 Hexalectris brevicaulis JA 41 Hexalectris brevicaulis JA 85 Hexalectris parviflora JA 500 Hexalectris parviflora CH 267 Hexalectris spicata OK 144 Hexalectris spicata NC 50 Hexalectris spicata TX 276 Hexalectris spicata IN 69 Hexalectris warnockii TX 49 Hexalectris warnockii TX 166 Hexalectris colemanii AZ 79 Hexalectris nitida TX 270 Hexalectris nitida TX 48 Hexalectris nitida TX 47 Hexalectris arizonica TX 377 Hexalectris arizonica AZ 271 Hexalectris arizonica AZ

FIG.2.Scaled representation of plastid genomic regions in Bletia, Basiphyllaea, and Hexalectris, ranked by decreasing total plastome length. Black arrows ¼ IR copies and gray arrows ¼ genomic inversions relative to Bletia and other angiosperm plastomes. Thin black lines on the left ¼ LSC region and to the right ¼ small single copy region. Note that Hexalectris arizonica accession 271 lacks an IR. provides evidence for 12 syntenic regions, considering all 21 ycf1,andycf2) nor evidence of loss-of-function in any ribo- accessions. Both accessions of H. warnockii share a 29-kb in- somal RNA gene (rrn). version in the large single copy (LSC) region (fig. 2 and sup- We find evidence of intraspecific variation in functional plementary fig. S3, Supplementary Material online), gene content in four of the six species for which multiple containing the following genes: trnSGCU, trnGUCC, trnRUCU, individuals were sampled (fig. 3 and supplementary fig. S4, watpA, watpF, atpH, watpI, rps2, wrpoC2, wrpoC, wrpoB, Supplementary Material online). Overall, 8% of all functional trnCGCA, petN, psbM, trnDGUC, trnYGUA, trnEUUC, trnTGGU, and physical gene loss events happen within species of wpsbD, wpsbC, trnSCGA, psbZ, trnGUUC, trnfMCAU, rps14, Hexalectris, based on our sampling. Most gene losses within wpsaB, wpsaA, wycf3,andtrnSGGA. Both accessions of H. species occur to small, photosynthesis-related genes, with the brevicaulis share an 6.7-kb inversion in the LSC as well, exception of three independent functional or physical losses containing wpsbB (50 partial), clpP, rps12 (50 exon), rpl20, of rpo genes (in H. warnockii and H. parviflora; fig. 3). rpoB and rps18, rpl33, psaJ, trnPUGG, trnWCCA, petG, petL, psbE, psbF, psaJ have been lost in H. warnockii accession 49, whereas they psbL, psbJ,andpetA. The double cut and join (DCJ) distance remain as pseudogenes in accession 69. psbT and psbZ have is 1 for both H. warnockii and H. brevicaulis relative to all other been lost in accession 41 in H. brevicaulis,whereaspsbJ has accessions, corresponding to the number of major structural become a pseudogene in accession 377 of H. arizonica but not rearrangements of the plastome. in the other two accession of this species. In H. parviflora, psaI and psaJ are pseudogenes in accession 500, whereas petG has been lost and rpoC has become a pseudogene in accession 85. Physical and Functional Gene Losses Transfer RNA genes are largely conserved, with a few no- UGA We quantified the numbers of putatively functional coding table exceptions. trnS is missing from both accessions of GGU genes, pseudogenes, and wholescale gene losses to summarize H. warnockii,andtrnT is missing from all accessions of H. CAA the structural evolutionary consequences of relaxed selection arizonica. trnL presentsamoreambiguouscase(fig. 3 and pressure on each plastid gene system and genome structure supplementary fig. S5, Supplementary Material online), in overall. Figure 3 and supplementary figure S4, Supplementary that there has been a 3-bp deletion relative to all other Material online, summarize the number of putatively func- taxa in both accessions of H. warnockii, whereas there has tional genes, pseudogenes, and physical gene losses among been a 5-bp insertion in a single accession of H. parviflora Bletia, Basiphyllaea,andHexalectris. All plastid genes are pu- (accession 500 from Chihuahua, Mexico). Predicted second- tatively functional in Bletia and Basiphyllaea,asevidencedby ary structures vary in these accessions, but all length variation intact reading frames. Hexalectris grandiflora and H. revoluta is observed in the “variable loop” (V-loop) region. contain pseudogenes and have lost some members of the ndh complex but show no evidence of functional/physical Loss of Photosynthetic Capacity loss of genes essential for photosynthesis. All other accessions Topology Tests for a Single Loss of Photosynthesis of Hexalectris show extensive degradation of the ndh complex We used Bayes factor calculations based on stepping-stone and “photosynthesis-related” genes (e.g., psa/psb, pet,and sampling of the posterior distribution to quantify evidence for rpo). Hexalectris warnockii further displays evidence of pseu- a single or multiple losses of photosynthesis in leafless dogenes for subunits of the ATP synthase (atp)complex,with Hexalectris. Our analyses provide strong evidence against five of six atp genesaspseudogenes(allbutatpH). No single the topology in which all Hexalectris accessions displaying photosynthesis-related gene is present as an intact reading evidence of photosynthetic loss are constrained to be frame in all species of Hexalectris. There is neither evidence of monophyletic, as compared with a negative constraint in functional or physical loss in any of the protein-coding which they are nonmonophyletic (mean ln Lmonophyly ¼ “housekeeping” genes (e.g., infA, rps/rpl, accD, matK, clpP, 129,194.1, mean ln Lnonmonophyly ¼124,795.9, and Bayes 1888 Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus . doi:10.1093/molbev/msz111 MBE

NAD(P)H Dehydrogenase (ndh) petA petB petD petG psaA psaB psbA psbB psbC Photosynthesis related (e.g. psa/b, rbcL) 47 Hexalectris arizonica TX psbZ ycf3 ycf4 trnT-ggu rpoA rpoB rpoC1 rpoC2 Plastid-encoded RNA Polymerase (rpo) psbH psbN psbT rbcL 271 Hexalectris arizonica AZ ATP Synthase (atp) psbJ psbD psbM ‘Housekeeping’ (e.g. rRNA, petA petB petG psaA psaB psbA psbB psbC psbD 377 Hexalectris arizonica AZ tRNA, rpl/s) ndhA ndhH 166 Hexalectris colemanii AZ psaI = physical loss psaJ

psbE psbF psbH psbJ psbL rbcL ycf3 ycf4 rpoB rpoC1 500 Hexalectris parviflora CH

petG rpoC2 trnL- caa 85 Hexalectris parviflora JA ? 320 Hexalectris revoluta TX ndhE ndhI 44 Hexalectris spicata NC psbZ rbcL rpoB rpoC1

petG psaI psbD psbK psbM 267 Hexalectris spicata OK ndhD 276 Hexalectris spicata IN petB petD psaA psaB psaC psaI petA 50 Hexalectris spicata TX 270 Hexalectris nitida TX petL psbA psbB psbH psbN psbT ycf3 rpoB rpoC1 ndhA ndhH ycf4 psbC psbE psbF psbJ psbL rpoA rpoC2 79 Hexalectris nitida TX ndhA ndhK 48 Hexalectris nitida TX ndhJ ndhA ndhB ndhC ndhD ndhE ndhF ndhG ndhH ndhI psbT psbZ 41 Hexalectris brevicaulis JA ndhF petB petD petG psaA psaB psaI psbA psbB psbC psbD psbE psbF psbH psbJ psbL psbN rbcL ycf4 rpoA rpoB rpoC1 rpoC2 36 Hexalectris brevicaulis JA 64 Hexalectris grandiflora TX

rpoB psbJ 69 Hexalectris warnockii TX ndhD ndhE ndhF ndhH ndhI petA petB petD petG petN psaA psaB psaC psaI psbA psbC psbD psbH psbN psbT rbcL ycf3 ycf4 rpoA rpoC1 rpoC2 trnS-uga trnL-caa atpA atpB atpE atpF atpI

? 49 Hexalectris warnockii TX rpoB psbJ Baco1 Basiphyllaea corallicola 32 Bletia roezlii JA

FIG.3.Functional gene content of Bletia, Basiphyllaea, and Hexalectris based on ancestral state reconstruction of each gene under a loss-only (Dollo) model of transition probabilities, using the “CDS” matrix. Filled rectangles ¼ pseudogenes and open rectangles ¼ physical gene losses. “?” for trnLCAA ¼ evidence of length variation in the “variable loop” region, which may be a precursor to pseudogene formation. factor ¼ 4,398.2). The same is true for the analysis in which H. Maximum likelihood reconstruction of photosynthetic spicata, H. nitida, H. parviflora,andH. revoluta are constrained loss under a model restricting photosynthetic gains (i.e., a to be monophyletic (as in Kennedy and Watson [2010]and loss-only model) reveals four to five losses of photosynthesis Sosa et al. [2016]; mean ln L ¼135,098.1, Bayes factor ¼ in Hexalectris (fig. 4). Ancestral states for photosynthetic 10,302.2 when compared with negative constraint model). losses in H. warnockii and H. brevicaulis are unequivocal at >0.99 for a photosynthetic ancestor for both the LCB and CDS matrices. The ancestral state for the H. spicata-nitida Ancestral Reconstruction of Photosynthetic Status complex is nonphotosynthetic, though with a probability of We used ancestral state reconstruction to characterize the 0.63 for the LCB tree and 0.61 for the CDS matrix. For the LCB evolutionary history of photosynthetic loss in leafless tree, H. revoluta is sister to a clade of (H. parviflora,[H. cole- Hexalectris. Parsimony analysis under a cost matrix weighting manii,{H. arizonica}]). The ancestral state for the latter clade, maximally against photosynthetic gains reveals four and five excluding H. revoluta, is 0.76 in favor of a photosynthetic independent losses of photosynthesis, based on the LCB and ancestor, raising the possibility that H. parviflora and the H. CDS matrices, respectively (supplementary fig. S6, colemanii-arizonica clade each separately lost photosynthesis. Supplementary Material online): 1) Hexalectris warnockii,2) Conservatively, reconstruction based on the LCB matrix pro- H. brevicaulis,3)theH. parviflora-colemanii-arizonica clade vides evidence for a minimum of four independent losses in (LCB matrix), 4) the H. nitida-spicata clade, and 5) in H. Hexalectris. The situation for the CDS matrix is less equivocal, parviflora (CDS matrix). An analysis in which all character in that the H. revoluta is sister to H. parviflora, thus providing state changes are equally weighted gives a most parsimonious evidence for at least five independent losses. resolution of three total state changes: two losses (one in H. warnockii and the clade containing all taxa excluding H. war- nockii and H. grandiflora); and a gain of photosynthesis in H. Testing among Discrete Trait Models of Photosynthetic Loss revoluta. Finally, an analysis in which all changes are con- We tested among alternative character state transition mod- strained to be gains by weighting against losses yields four els to quantify the number of putative losses and gains of independent gains of photosynthesis (Bletia, Basiphyllaea, H. photosynthetic function. The maximum likelihood “equal grandiflora,andH. revoluta). rates” (ER) and “gain-only” models—in the latter,

1889 Barrett et al. . doi:10.1093/molbev/msz111 MBE

47 Hexalectris arizonica TX A CDS matrix (0.80) 271 Hexalectris arizonica AZ 5 377 Hexalectris arizonica AZ 166 Hexalectris colemanii AZ photosynthetic (0) 4 500 Hexalectris parviflora CH non-photosynthetic (1) 85 Hexalectris parviflora JA 320 Hexalectris revoluta TX 44 Hexalectris spicata NC 267 Hexalectris spicata OK 276 Hexalectris spicata IN 50 Hexalectris spicata TX 3 (0.61) 270 Hexalectris nitida TX 79 Hexalectris nitida TX 48 Hexalectris nitida TX 2 41 Hexalectris brevicaulis JA 36 Hexalectris brevicaulis JA 64 Hexalectris grandiflora TX 1 69 Hexalectris warnockii TX 49 Hexalectris warnockii TX Baco1 Basiphyllaea corallicola 32 Bletia roezlii JA

47 Hexalectris arizonica TX B LCB matrix (0.74) 271 Hexalectris arizonica AZ 5? 377 Hexalectris arizonica AZ (0.76) 4? 166 Hexalectris colemanii AZ 500 Hexalectris parviflora CH 85 Hexalectris parviflora JA 320 Hexalectris revoluta TX 44 Hexalectris spicata NC 267 Hexalectris spicata OK 276 Hexalectris spicata IN 50 Hexalectris spicata TX 3 (0.63) 270 Hexalectris nitida TX 79 Hexalectris nitida TX 48 Hexalectris nitida TX 2 41 Hexalectris brevicaulis JA 36 Hexalectris brevicaulis JA 64 Hexalectris grandiflora TX 1 69 Hexalectris warnockii TX 49 Hexalectris warnockii TX Baco1 Basiphyllaea corallicola 32 Bletia roezlii JA

FIG.4.Trees and ancestral state reconstructions of photosynthetic capability based on (A) the “CDS” matrix and (B) the “LCB” matrix. Photosynthetic capability is represented as a binary character, under a model allowing only losses of photosynthesis and no gains. Green circles/pie charts represent the probability of a photosynthetic ancestor; red represents a nonphotosynthetic ancestor. Numbers in black next to nodes represent the likelihood fraction of the most likely ancestral state; all other nodes have likelihoods >0.95. Numbers in red indicate the inferred instances of photosynthetic loss. Arrows indicate the alternative position of Hexalectris revoluta based on each matrix.

1890 Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus . doi:10.1093/molbev/msz111 MBE

Table 2. Tests of Alternative Models of Transition Probabilities table S3, Supplementary Material online). dS is further corre- Between Photosynthetic Character States (0 ¼ Photosynthetic and lated with DCJ distance (i.e., rearrangements), whereas dN is 1 ¼ Nonphotosynthetic). further correlated with IR length. GC content is not signifi-

Model q-Matrix ln L fp AICc AICweight q01; q10 cantly correlated with any other trait. Total plastome length is positively correlated with the length of coding DNA, the ER, equal rates q01 5 q10 26.78896 1 15.58 0.42 0.71; 0.71 ARD, different q01 6¼ q10 26.78896 2 17.58 0.15 0.71; 0.72 number of functional genes, tandem repeats, and indels. A rates nearly identical set of correlations is found for the cumulative Gain-only q10 only 26.82156 1 15.64 0.41 0; 0.68 length of coding DNA as for total plastome length. The num- Loss-only q01 only 29.70410 1 21.41 0.02 1.14; 0 ber of functional genes is positively correlated with tandem NOTE.—“q-matrix” specifies the specific state transition constraints for each model; repeat content and negatively correlated with indel content. “ln L” is the likelihood of the model; fp, the number of free model parameters; DCJ distance is correlated with both tandem (negative) and “AICc” is the corrected Akaike information criterion; “AICweight” is the Akaike dispersed repeats (positive), as well as indel content (positive). weight for a particular model in favor of the alternatives; and “q01; q10” represent the estimated transition probabilities. Lastly, IR length is positively correlated with both tandem and dispersed repeat content. photosynthesis can only be gained and not lost (i.e., 1 ! 0 only)—had the highest Akaike weights (for the “ER” model, Discussion AW ¼ 0.42; for the “gain-only” model AW ¼ 0.41), thus providing the best two explanations among the four com- We present compelling evidence of four to five indepen- peting models of discrete character change (table 2). The dent losses of photosynthesis in a single genus of myco- “loss-only” model, which allows only photosynthetic losses heterotrophic orchid, which is unprecedented among and no gains (i.e., only 0 ! 1) had the lowest weight among studies published thus far on plastid genome evolution the four models compared (AW ¼ 0.02), and the “allowing in heterotrophic plants. This is particularly striking be- different rates” model had the second lowest weight (AW ¼ cause it indicates that independent loss of photosynthesis 0.15). has occurred within approximately half of the species in this genus. Our findings therefore indicate that the num- Evolutionary Rate Variation and Photosynthetic Loss ber of independent transitions to an obligately mycohe- terotrophic, nonphotosynthetic lifestyle has likely been Photosynthetic Capacity and Substitution Rate Associations underestimated in orchids, and also across land plants. We used TraitRateProp and RELAX to explore evidence of Our conclusions are based on evidence from 1) plastome different substitution rates and relaxed selection associated size reduction, photosynthetic gene loss, and pseudoge- with photosynthetic loss. We found evidence of different nization in several species of Hexalectris,butnotinothers; substitution rates in putatively nonphotosynthetic taxa 2) a strongly supported phylogenetic hypothesis based on among four genes encoding the ATP synthase complex complete plastid genomes; and 3) hypothesis tests of ( , , ,and ; here excluding which atpA atpE atpF atpI H. warnockii photosynthetic loss. We further find evidence of acceler- contains pseudogenes), three ribosomal protein genes atp ated substitution rates, inter- and intra-specific variation ( , ,and ), the ATP-dependent Clp protease pro- rps3 rps4 rps8 in IR dynamics, and correlations among substitutional teolytic subunit ( ), ,and (supplementary table S2, clpP ycf1 ycf2 parameters and genomic structural features. Supplementary Material online). All other genes showed no significant substitution rate changes in association with pho- tosynthetic loss. Only two loci displayed evidence of relaxed Phylogenetic Analyses selective constraints associated with the loss of photosynthe- We recover phylogenetic relationships similar to those in pre- sis: atpA (H. warnockii excluded from analysis) and rpl14 (sup- vious studies of Bletiinae based on Sanger sequencing (nuclear plementary table S2, Supplementary Material online). ITS and four plastid DNA) that include representatives of Analyses of genes concatenated by functional class (atp, Hexalectris (Sosa 2007; Kennedy and Watson 2010; Sosa accD-clpP-matK, rpl,andrps) yielded increased substitution et al. 2016). Our results also support the revised species cir- rates associated with photosynthetic loss for the atp complex cumscriptions within the H. spicata complex proposed by (excluding H. warnockii), accD-clpP-matK as a concatenated Kennedy and Watson (2010). The main difference in topology alignment, and all rps genes, encoding small-subunit ribo- found here is in the placement of the H. arizonica-colemanii somal proteins (supplementary table S3, Supplementary clade; we find strong evidence for the former as a member of Material online). RELAX analyses of the same concatenated acladewithH. parviflora and H. revoluta, whereas relation- matrices reveal only evidence for intensified selection in the ships recovered in the most recent study placed arizonica- atp complex, again, excluding H. warnockii. colemanii as sister to revoluta-parviflora-nitida-spicata (Sosa et al. 2016). Bayesian topology tests based on whole plastome Correlations among Genomic and Substitutional Features data provide strong evidence against the latter topology. Only dS and dN are significantly correlated with one another, and the placement of H. revoluta differs between our analyses of with total plastome length, the aggregate length of protein- the LCB and CDS matrices, with strong support in both cases, coding genes, the number of functional genes, the number of as sister of H. parviflora (CDS matrix) or sister to a clade of tandem repeats, and indels (summarized in supplementary H. parviflora-colemanii-arizonica (LCB matrix).

1891 Barrett et al. . doi:10.1093/molbev/msz111 MBE

Plastid Genome Structural Evolution ndh Genes Plastome size evolution in Hexalectris follows a similar pattern We detect pseudogenes and losses of NAD(P)H dehydroge- observed in recent studies on heterotrophic angiosperms, nase subunit genes in all members of Hexalectris (i.e., coincid- including: 1) the holoparasitic Orobanchaceae (Wicke et al. ing with the loss of leaves) but not in leafy relatives Bletia and 2013, 2016), 2) fully mycoheterotrophic members of the or- Basiphyllaea.Lossofndh genes corresponds to the first stage chid genus Corallorhiza (Barrett and Davis 2012; Barrett et al. of degradation in many recent conceptual models of plas- 2014, 2018), 3) fully mycoheterotrophic members of the or- tome degradation in heterotrophic plants (Wicke et al. 2011, chid tribe Neottieae (Feng et al. 2016), and 4) the monotro- 2016; Barrett and Davis 2012; Barrett et al. 2014; Naumann poid Ericaceae (Braukmann et al. 2017). Plastomes are more et al. 2016; Graham et al. 2017; Wicke and Naumann 2018). highly reduced in Hexalectris than in Corallorhiza,butnotto Our findings are unsurprising in that loss of the ndh complex the extent observed in Orobanchaceae, Neottieae, or mono- is well documented in other clades containing hemiparasites tropoid Ericaceae. Hexalectris occupies a somewhat interme- and partial mycoheterotrophs (e.g., Barrettetal.2014; Feng diate position on the continuum of plastome degradation et al. 2016), as well as in leafy, otherwise photosynthetic relative to other groups that have been sufficiently sampled. orchids (Lin et al. 2017). Losses of ndh in the latter may be Hexalectris is estimated to have diverged from its closest leafy associated with the tendency of many orchids to grow in low- ancestor (most likely Basiphyllaea) about 33 Ma, in the late light, canopy habitats as epiphytes, or in dark, shaded woods Oligocene (Sosa et al. 2016). Hexalectris warnockii—repre- as terrestrials (e.g., Wicke et al. 2011; Lin et al. 2017). The ndh senting the earliest divergence in the genus—is estimated complex largely functions as an electron recycling system, to have split from the ancestor of the remaining Hexalectris allowing the fine tuning of photosynthesis (Peltier et al. species about 24 Ma (Sosa et al. 2016). For comparison, the 2016). Thus, in epiphytes or terrestrial orchids growing in crown age of Corallorhiza is estimated to be 9Ma,having relatively dark, forested habitats, this complex may be non- lost 20% of its plastome in the most highly reduced species, essential; this situation may be augmented by dependence C. bentleyi and C. involuta. Hexalectris arizonica,representing upon host-derived carbon and decreasing dependence upon the smallest plastome in the genus, has been reduced by 34%, photosynthetic carbon in parasites, explaining why ndh genes compared with the leafy B. roezlii (table 1, fig. 2). In contrast, are lost in nearly every parasite examined to date. the stem age and minimum plastome sizes among other The relative degree of reliance on photosynthesis in clades containing heterotrophs are estimated to be 40–50 Hexalectris and close leafy relatives Bletia (with large, plicate Ma and 68 kb in Conopholis, representing holoparasitic leaves) and Basiphyllaea (with 1–2 reduced, conduplicate Orobanchaceae (Wicke et al. 2016); 90 Ma and 27 kb in leaves) remains to be explored from a physiological perspec- Hydnora visseri representing holoparasitic Hydnoraceae tive. Of particular interest would be to assess photosynthetic (Naumann et al. 2013, 2016); 80 Ma and 11 kb capacity in all species of Hexalectris, specifically to determine in Pilostyles aethiopica representing holoparasitic thedegreetowhichH. grandiflora and H. revoluta,which Apodanthaceae (Bellot and Renner 2014, 2015); and 100 retain intact reading frames for all plastid genes except ndh, Ma and 0 kb in Rafflesia lagascae representing Rafflesiaceae rely more heavily on photosynthesis than their congeners (Molina et al. 2014). Thus, time since divergence is likely a key which have physically or functionally lost most of these genes. factor in determining the degree of plastome degradation in Evidence from the visibly green, leafless Corallorhiza trifida heterotrophic lineages, in addition to the degree of weakening suggests that despite the loss of several ndh genes, this species of negative selection on photosynthetic function and associ- retains photosynthetic capacity but is photosynthetically in- ated acceleration of mutation rates associated with a purely efficient and thus relies almost entirely on mycotrophic car- heterotrophic lifestyle (Wicke and Naumann 2018). bon for nutrition (Cameron et al. 2009). The situation is not as clear in Hexalectris as it is in Corallorhiza (Barrett et al. 2014); further study is needed to determine chlorophyll content Loss of Photosynthetic Capacity among species of Hexalectris and relatives and to quantify We provide strong evidence demonstrating that several the relative importance of photosynthetic versus mycotro- independent losses of photosynthesis have occurred in phic carbon among species. Specifically, what if any photo- parallel in Hexalectris. Bayes factor comparisons con- synthetic consequences are associated with the loss of straining all taxa with genomic evidence of photosyn- the ndh complex in species otherwise capable of thetic loss to be monophyletic versus nonmonophyletic photosynthesis? allow us to conclude that a single loss of photosynthesis is highly unlikely. Ancestral state reconstructions of each plastid gene’s functional capacity and of photosynthetic Photosynthesis-Related Genes capability as a binary character under a Dollo model (i.e., Photosynthesis-related genes (e.g., ccsA, cemA, pet, psa/psb, no reversals back to photosynthesis once it is lost) suggest rbcL, ycf3,andycf4; supplementary fig. S5, Supplementary that photosynthesis has been lost multiple times in the Material online) and genes of the plastid-encoded RNA po- genus (figs. 3 and 4). Ironically, such a scenario is least lymerase that transcribes many of the former (rpoA, B, C1, parsimonious and has the lowest likelihood among vari- and C2) show extensive degradation and loss among several ous alternative models allowing photosynthetic gain, but species of Hexalectris,inparallelacrossmultiplelineages these are not biologically plausible. (fig. 3). Each of these independent transitions represents

1892 Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus . doi:10.1093/molbev/msz111 MBE the second phase in recent models of plastome degradation, correlated retention of both the plastid-encoded ATP syn- following ndh loss (see references above). This phase is largely thase and Tat genes independently across lineages of non- hypothesized to represent photosynthetic loss. photosynthetic plants and diatoms. This may explain We find evidence for functional and/or physical gene loss retention of atp genes despite loss of photosynthetic genes below the species level in four of six species in which >1 (e.g., psa, psb,andrbcL) in genera such as Hexalectris (except- individual was sampled. This suggests a continuum of plas- ing H. warnockii)andCorallorhiza, in which many recent tome degradation and functional gene capacity that extends losses of photosynthesis have occurred. to the population level, and that such patterns of variation Only atpH remains putatively functional and is under neg- may be pervasive among heterotrophs. Our findings in ative selection in H. warnockii, the species which perhaps Hexalectris corroborate those in the Corallorhiza striata com- represents the earliest loss of photosynthesis in the genus plex, in that plastome degradation at or below the species (fig. 3; Barrett and Kennedy 2018). atpH, in part, encodes a level might be very common, arguing for more intensive sam- CF0 membrane C14 ring and central “stalk” of the ATP syn- pling of plastomes within species (Barrett et al. 2018). Among thase complex embedded within the thylakoid membrane the most potentially informative are those species which dis- (Allen et al. 2011; Hahn et al. 2018). When protonated, the play evidence of being “on the edge” of photosynthetic loss. C14 ring (encoded by atpH) rotates, and distributes protons to Certainly H. grandiflora and H. revoluta would qualify, as well the proton exit channel protein encoded by plastid atpI, as some putative partially mycoheterotrophic species of allowing protons to escape across the thylakoid membrane Corallorhiza, which display evidence of the beginnings of along their pH gradient, and driving the primary mechanism plastome degradation (e.g., wpsbI, wpsbM,andwcemA; of ATP production. The extramembranous catalytic core of Barrett et al. 2014). ATP synthase, where ATP synthesis occurs, is encoded by the What drives such changes at the population level? Relaxed plastid atpA, atpB,andatpE, and nuclear atpC (Allen et al. selection and genetic drift in small populations would seem 2011). These genes have all been lost from H. warnockii along to be an appropriate explanation in most full mycohetero- with atpF, the latter comprising part of the “peripheral stalk” trophs, which typically have low population densities, patchy that acts to stabilize the rotating mechanism of the central distributions, and rely on self-pollination or vegetative prop- stalk and intramembranous core (Hahn et al. 2018). Even if agation to reproduce (Merckx, Smets, et al. 2013). However, the product of atpH is somehow functional in H. warnockii, an alternative hypothesis would be that site specific or re- the proton channel encoded by atpI is not, having been func- gional environmental conditions modulate selection pres- tionally lost, and thus this species may have lost the primary sures. Or, the functioning of many pathways has degraded mechanism of proton gradient maintenance across the thy- over time such that each can be disrupted idiosyncratically lakoid membrane. among populations; that is, these pathways may be “nearly It can be assumed that the thylakoid ATP-ase converting broken,” and the pattern we observe is the result of chance. ADP to ATP in H. warnockii is nonfunctional, and it remains To our knowledge, the relative importance of these scenarios to be discovered what if any function the membrane- has not been addressed in mycoheterotrophs. embedded atpH retains, as this is the only member of the complex that is putatively functional. The ability of a chloro- atp Genes plast to translocate proteins into the thylakoid membrane is This naturally leads to the question of how plastome degra- likely to be integral to the functioning of the plastid overall dation proceeds beyond loss of the ndh complex(stage1) (Mori et al. 2001; Cline and Theg 2007; New et al. 2018), and and photosynthesis-related genes (stage 2), as this seems to thus degradation of the atp complex, given its putative im- be a “sticking point” or stable equilibrium phase for many portance in maintaining the conditions necessary for basic taxa that have recently lost photosynthesis (sensu Barrett and organellar function, likely represents a major transitory event Davis 2012; Barrett et al. 2014; Naumann et al. 2016; Wicke in the basic biology of these plants, and others that have lost et al. 2016). Hexalectris warnockii is the only species that dis- ATP-ase function independently. It remains to be explored plays evidence of advanced degradation in the atp complex whether loss of the atp complex is associated with a whole- (fig. 3 and supplementary fig. S4, Supplementary Material scale loss of chloroplast function, a decreased rate of chloro- online; Barrett and Kennedy 2018). Thus, H. warnockii may plast synthesis, or some other change in cellular-organellar have entered a new state of cellular equilibrium (sensu Wicke functional dynamics. and Naumann 2018) wherein the function of a plastid ATP The situation of atp gene loss in H. warnockii lies in con- synthase is no longer needed. It has been proposed that ATP trasttothatinallotherHexalectris species, which taken to- synthase is retained in heterotrophic plants beyond photo- gether display some evidence of intensified selection. In the synthetic gene loss to maintain a proton gradient across the RELAX alternative model, small values of dN/dS decrease in thylakoid membrane, which in turn is essential for the proper the test lineages relative to the photosynthetic reference lin- functioning of the twin-arginine translocator (Tat)complex eages, whereas those sites with values dN/dS increase relative responsible for moving folded proteins across the thylakoid to reference lineages (table 3; K ¼ 1.96, P ¼ 0.031). The inter- membrane (Kamikawa et al. 2015). In fact, the pH gradient pretation is that sites under negative selection and positive across the thylakoid is the primary mechanism of Tat activity selection have both experienced intensified selection pres- and thus folded protein transport (New et al. 2018). This “atp- sures. Given the importance of the ATP synthase complex Tat” coevolutionary hypothesis is based on observations of in maintaining the proton gradient across the thylakoid

1893 Barrett et al. . doi:10.1093/molbev/msz111 MBE

Table 3. Results of RELAX Analyses Based on Genes Concatenated By Functional Class (atp, rps, and rpl; except in the case of accD, clpP, and matK Which Do Not Share Similar Functions).

Model log LK, P-val Selection Branch x1 x2 x3 All atp Alternative 28,293.4 1.96, 0.031 Intensification Test 0.01 (57.93%) 0.06 (37.31%) 10.17 (4.76%) Reference 0.07 (57.93%) 0.25 (37.31%) 3.26 (4.76%) Null 28,295.7 Test 0.00 (58.21%) 0.02 (35.58%) 6.06 (6.21%) Reference 0.00 (58.21%) 0.02 (35.58%) 6.06 (6.21%) accD, clpP, matK Alternative 26,861.3 0.85, 0.180 Relaxation Test 0.00 (3.08%) 0.43 (96.78%) 90.82 (0.14%) Reference 0.00 (3.08%) 0.37 (96.78%) 206.53 (0.14%) Null 26,862.2 Test 0.00 (1.66%) 0.40 (98.23%) 172.60 (0.10%) Reference 0.00 (1.66%) 0.40 (98.23%) 172.60 (0.10%) All rps Alternative 28,319.9 2.54, 0.097 Intensification Test 0.00 (72.92%) 0.22 (12.65%) 2.74 (14.43%) Reference 0.01 (72.92%) 0.55 (12.65%) 1.49 (14.43%) Null 28,321.2 Test 0.00 (72.30%) 0.61 (11.21%) 1.78 (16.49%) Reference 0.00 (72.30%) 0.61 (11.21%) 1.78 (16.49%) All rpl Alternative 25,321.3 1.13, 0.340 Intensification Test 0.00 (17.27%) 0.00 (79.02%) 23.74 (3.71%) Reference 0.00 (17.27%) 0.00 (79.02%) 16.41 (3.71%) Null 25,321.8 Test 0.01 (80.86%) 0.04 (15.67%) 21.22 (3.47%) Reference 0.01 (80.86%) 0.04 (15.67%) 21.22 (3.47%)

NOTE.—“Model” refers to the null versus alternative models; “log L” is the log-likelihood for a particular model; K ¼ the value of the “K” parameter, where K > 1 indicates intensified selection and K < 1 indicates relaxed selection, and P-val ¼ the significance of K; “Selection” indicates the predominant mode of selection for the test branches; “Branch” distinguishes parameter estimates for test versus reference branches; x1–x3 are the three estimates of dN/dS for the three site classes in the model, for test and reference branches, with the proportion of sites under each value given in parentheses. Analyses of single genes can be found in supplementary table S3, Supplementary Material online. membrane, it is intuitive that selection might be intensified (Barrett et al. 2014). It should be noted that the tRNAs lost in following the loss of photosynthesis in these lineages, either Hexalectris involve some level of redundancy among amino for sequence conservation (negative selection), putatively acids: each loss or modification involves a plastid-encoded novel or modified function (positive selection), or both. A tRNA that specifies at least two anticodons for a particular similar situation has been observed among lineages of holo- amino acid (i.e., trnT [n ¼ 2], trnS [n ¼ 3], and trnL [n ¼ 3] if parasitic Orobanchaceae, in which many “housekeeping” we assume the function of the latter is somehow compro- genes experience intensified selection following periods of mised; fig. 3 and supplementary fig. S6, Supplementary relaxed selection of functional/physical gene loss in other Material online). It is tempting to suggest, based on these complexes (i.e., photosynthesis-related genes; Wicke et al. observations, that tRNA genes with some level of amino 2016; Wicke and Naumann 2018). acid redundancy may be lost more easily. trnTUGU and trnTGGU are lost at nearly equal frequency among holopara- sites and full mycoheterotrophs, but trnT is by no means the Housekeeping Genes most frequently lost among all tRNAs (see supplementary Housekeeping genes are largely intact in Hexalectris,with information in Graham et al. [2017]), and trnSUGA is the least three exceptions. The transfer RNA gene trnTGGU is missing UGA frequently lost among the three tRNAs with serine from H. arizonica,andtrnS is missing from H. warnockii. anticodons. Further, indel variation in the “variable loop” region of trnLCAA in one accession of H. parviflora andbothaccessionsofH. warnockii (supplementary fig. S6, Supplementary Material on- Photosynthetic Loss line) suggests a modification or possibly loss-of-function in Loss of photosynthesis has been an ongoing process in this tRNA gene, as length of the variable loop is important in Hexalectris over the past 33 My (range ¼ 38.6–14.6 Ma recognition of aminoacyl tRNA synthetase and helps in tRNA for the stem lineage of Hexalectris based on divergence molecular stability (Sun and Caetano-Anolles 2009). These time analysis of Sosa et al. [2016]), beginning with the shared, findings, taken with the evidence of relaxed selection in a leafless condition and loss-of-function in ndh genes. Loss of single housekeeping gene (rpl14; supplementary table S3, photosynthesis genes and the atp complex in H. warnockii Supplementary Material online), open the possibility that may have begun as early as 30 Ma (Sosa et al. 2016; Barrett some species of Hexalectris have begun to shift beyond “phase and Kennedy 2018), but it is impossible to discern the order in 2” of recent models of plastome evolution in parasites (see which members of these gene functional classes were lost in citations above), defined by the loss of housekeeping genes. this lineage. Based on the phylogenetic position of the puta- We find similarities between Hexalectris and Corallorhiza, tively photosynthetic H. grandiflora, four of the five putative in which some members of the Corallorhiza striata losses of photosynthesis must have occurred within the last complexhavelosttrnTGGU just as is the case in Hexalectris 20 My in Hexalectris excluding H. warnockii. Further, due to

1894 Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus . doi:10.1093/molbev/msz111 MBE the nested placement of H. revoluta, photosynthetic losses (e.g., H. spicata) and a photosynthetic pollen recipient (e.g., among H. arizonica, colemanii,andparviflora must have oc- H. grandiflora) where they are known to co-occur locally (e.g., curred within the last 10 My. Thus, many of the indepen- Texas, USA). dent losses of photosynthesis in Hexalectris have occurred relatively recently. Evolutionary Rate Variation The possibility remains that missing or pseudogenized Functional and physical losses of plastid genes are just one copies of plastid genes have been duplicated or transferred dimension of the overall process of plastome degradation. to the nuclear genome, and that these genes may remain Although protein and RNA coding capacity are obviously of functional. Indeed, several plastid genes and gene fragments utmost importance to photosynthetic function, these were found in the nuclear and mitochondrial genomes of changes often occur in parallel with other important genomic holoparasitic Orobanchaceae, but it is doubtful that any in- changes, including rearrangements, modifications of IR volved in photosynthesis remain functional (Cusimano and boundaries, changes in base composition (e.g., GC content), Wicke 2016). We see no reason why retention of functional proliferation of tandem and dispersed repeats, increased over- copies of plastid-photosynthetic genes in the nuclear genome all substitution rates, and relaxed selective pressure on house- might be adaptive, given the heterotrophic habit of taxa such keeping genes with basic organellar functions (Wicke et al. as Hexalectris, and indeed there exist no compelling examples 2013, 2016; Barrett et al. 2018). We find evidence for increased from other studies that this is the case. For example, ndh overall substitution rates associated with photosynthetic loss genes lost from the plastome were not detected as functional for ten genes in Hexalectris (here not considering those that copies in the nuclear genomes of several orchid taxa (Lin et al. have already become pseudogenes), five of which are atp 2017). The only plausible alternative hypothesis for the pat- genes (excluding H. warnockii, which has several atp pseudo- terns observed here may be photosynthetic “rescue” via hy- genes), and five of which are housekeeping genes (supple- brid introgression. Such a scenario would require pollen to be mentary table S2, Supplementary Material online). Only transferred from a nonphotosynthetic species of Hexalectris two genes showed evidence of significantly relaxed selective to a putatively photosynthetic (and possibly now extinct) pressure (i.e., elevated dN/dS ratios), as indicated by our member of this clade, such that the offspring would inherit RELAX analysis (atpA and rpl14; supplementary table S2, a plastid genome with photosynthetic functionality. The only Supplementary Material online). Increased overall rates of known example even remotely approaching such a scenario is substitution and indels may be coincident with relaxed selec- in the orchid genus Cymbidium, between the leafless, partial tive constraints on plastid genes, and further may obscure mycoheterotroph Cymbidium macrorhizon and the leafy rel- detection of relaxed selection via elevated dN/dS ratios using ative Cymbidium ensifolium (Ogura-Tsujita et al. 2014). conventional methods, as both the numerator and denomi- However, in that case, the hybrid was the result of artificial nator are elevated. Further, we interpret these findings with pollination, and the plastid-photosynthetic machinery is still caution based on the often low information content in single largely intact in the leafless Cymbidium macrorhizon gene analyses, in which there are few variable positions. (Motomura et al. 2010; Kim and Chase 2017; Kim et al. What causes increases in substitution rates in heterotro- 2017; Suetsugu et al. 2018). phic plants? A number of possibilities exist, including relaxed Under a scenario of gene flow between a nonphotosyn- negative selection, positive selection, low effective population thetic pollen donor and a photosynthetic recipient, we would sizes, growth form differences (thus related to generation expect maternal inheritance of a “functionally photo- time and the inheritance of mutations), and genome-wide synthetic” plastome, and also plastid-nuclear gene tree dis- selection for increased overall substitution rates to help evade cordance. In fact, based on nuclear ITS data from a previous host defenses in coevolutionary arms races (Bromham et al. study (Kennedy and Watson 2010), putatively photosynthetic 2013). First, increased substitution rates may be a result of H. grandiflora and H. revoluta occupy very similar phyloge- relaxed selective pressures on genes being analyzed, even netic positions, nested among nonphotosynthetic species of housekeeping genes. However, although this is obvious for Hexalectris, suggesting that this scenario is unlikely. If such a photosynthesis-related genes, it does not fully explain why scenario was to have occurred in Hexalectris,itislikelythat both synonymous and nonsynonymous rates tend to in- Mu¨ller-Dobzhansky incompatibilities would arise crease overall and across all three plant genomes, nor does (Dobzhansky 1936;sensuOrr 1995). In other words, a puta- it account for increased rates in noncoding regions (e.g., tively photosynthetic plastome would not likely be able to Nickrent and Starr 1994; Bromham et al. 2013). In the present carry out photosynthetic function within a nuclear-genomic, study, dN/dS ratios among protein-coding genes are elevated cellular “environment” of photosynthetic loss-of-function, or for only a few genes, whereas a substantially greater number evenformaviablezygoteforthatmatter(seediscussionin of genes display evidence of overall rate increases associated Barrett et al. [2018]). It seems unlikely that photosynthesis with the putative loss of photosynthesis (supplementary table could be “rescued” in such a way through hybridization, as- S2, Supplementary Material online). suming the extensive degradation of the plastid-encoded One possibility is relaxed selective pressure on the func- photosynthetic machinery is paralleled in the nuclear genome tions of nuclear-encoded genes with products involved in (e.g., Hypopitys, Ravin et al. 2016; Gastrodia, Yuan et al. 2018). DNA replication, recombination, and repair (Day and This hypothesis could potentially be tested by experimental Madesis 2007; Barnard-Kubow et al. 2014; Zhang et al. 2015, crosses in vivo between a nonphotosynthetic pollen donor 2016). These genes include, for example, “proofreading

1895 Barrett et al. . doi:10.1093/molbev/msz111 MBE enzymes” that repair DNA breaks, misincorporated bases, or ecological niche parameters should also be investigated to illegitimate recombinants (e.g., WHIRLY1 functions in plas- provide much needed biological context for evolutionary tome stability; Marechal et al. 2009). Relaxed selection in mechanisms that drive radical genomic, morphological, and some of these genes, and presumed decreases in their overall physiological changes observed in Hexalectris and relatives. functional performance, shows a positive correlation with the Heterotrophic plants such as Hexalectris afford the ability to degree of plastome rearrangement (e.g., in Geraniaceae; examine systems-level alterations to entire pathways as a re- Zhang et al. 2016). However, the reasons as to why these sult of loss- or gain-of-function in others, and provide genes with genome-stabilizing functions would experience forward-genetic opportunities beyond mutational studies in relaxed selection in heterotrophs are unclear, though theo- model systems. retical models predict that increased mutation rates overall may be beneficial to parasites in situations of coevolutionary Materials and Methods arms races, despite the “cost” associated with high mutational Plastid Genomes loads (e.g., Haraguchi and Sasaki 1996). An alternative explanation is that increased substitution Material, DNA Extraction, and Sequencing rates are directly linked to nutritional mode, that is, the loss of We collected 21 accessions across sites in North America, photosynthesis leading to a completely heterotrophic lifestyle including 19 accessions of Hexalectris (9 species), and 1 rep- itself causes the observed increase. A plausible, but as-of-yet resentative each for the green, leaf-bearing Ba. corallicola and untested hypothesis is that in order to compensate for carbon B. roezlii (table 1). Six of the nine species of Hexalectris are deficits resulting from the loss of photosynthesis, fully myco- represented by more than one individual. We dried floral and heterotrophic plants need to “ramp up” their metabolic rates. bract material in silica gel and deposited voucher specimens in This may cause an increase in reactive free radical species, and the Willard Sherman Turrell Herbarium at Miami University associated oxidative stress, which is well known to be linked (Oxford, OH) or the West Virginia University Herbarium to increased DNA damage. For example, genes involved in (Morgantown, WV; table 1). We extracted total genomic oxidative stress response in albino Epipactis helleborine are DNAs using a modified CTAB protocol (Doyle and Doyle upregulated, possibly in association with increased reliance 1987) and checked nucleic acid quality with a NanoDrop on fungal carbon (Suetsugu et al. 2017). Although many eco- Spectrophotometer (Thermo Fisher Scientific, Waltham, physiological studies in partially and fully heterotrophic plants MA). We quantified double stranded DNA using nucleic have focused on photosynthetic loss per se, few have focused acid staining via Qubit fluorometry (Thermo Fisher Scientific). on relative respiratory metabolic rates among lineages of leafy, We sheared total gDNAs to 450 bp with a Covaris E220 partially heterotrophic, and fully heterotrophic plants. Such a Focused-ultrasonicator (Covaris, Inc., Woburn, MA) and pre- study would be particularly powerful in a phylocomparative pared/indexed libraries for shotgun sequencing following framework, to tease apart the association of metabolic rate, Glenn et al. (2016) with slight modifications as in Barrett substitution rate, and phylogenetic relationships. One would and Kennedy (2018). We quantified indexed libraries using expect to observe increased respiration rates and also in- nucleic acid staining and pooled at equimolar ratios, and then creased expression of respiratory genes and genes involved assessed library size distribution on an Agilent Bioanalyzer in oxidative stress responses in fully heterotrophic taxa rela- (Agilent Technologies, Santa Clara, CA) prior to sequencing tive to partial heterotrophs and especially to leafy relatives, on two lanes of an Illumina HiSeq1500 (Illumina, Inc., San under identical conditions. Diego, CA) at the West Virginia University-Marshall University Shared Sequencing Facility (100-bp paired ends), with ten libraries from another project. Conclusions We demonstrate a minimum of four losses of photosynthesis Data Processing, Plastome Assembly within a single orchid genus of only nine species, which is We conducted initial assemblies using Novoplasty v. 2.7.2, a hitherto unprecedented among heterotrophic plants. Thus, a PERL-based assembler that begins with a plastid sequence as a previous estimate of 30 transitions to full mycoheterotrophy starting seed, and generates a complete, circularized plas- in orchids is likely an underestimate. A conservative estimate tome, given sufficient coverage (Dierckxsens et al. 2017). based on the current study and a previous study in Weusedthefollowingparameters:targetgenomesize50– Corallorhiza (2 transitions; Barrett et al. 2018) increases 200 kb, kmer size ¼ 45, and insert size range ¼ 1.8.Wealso this number to a minimum of 35 independent transitions used VELVET v.1.2.1 for de novo assembly (Zerbino and within the orchids, underscoring the importance of dense Birney 2008), with the following settings: kmer length ¼ 71, sampling within clades containing partial and full hetero- insert size ¼ 450 bp, scaffolding ¼ off, minimum contig trophs. Future studies should include additional sampling of length ¼ 300, and minimum coverage ¼ 10.Wethen plastomes of several Hexalectris species at the population level aligned VELVET contigs to circularized draft plastomes from to further reveal details of the patterns by which photosyn- NOVOPlasty using the native Geneious v.10 “map to refer- thesis is lost. Data from nuclear genomes, transcriptomes, ence” function. Further, we mapped original read pools to proteomes, physiology (carbon and nitrogen isotopic data, each draft reference to check for misassemblies or coverage chlorophyll content, etc.), basic cellular data (relative popu- gaps. Accessions with very high coverage were downsampled lations of mitochondria, chloroplasts and other plastids), and to 200 coverage depth and reassembled de novo as

1896 Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus . doi:10.1093/molbev/msz111 MBE above, to avoid errors associated with misinterpretation of 10 independent searches from random starting seeds and sequencing errors as polymorphisms by the assembler. We assessed support via nonparametric “standard” bootstrap- visually verified contiguous coverage and spanning of the IR ping, with 1,000 pseudoreplicates. We generated Bayesian boundaries using paired end data, and then used the self- estimates of plastid relationships in MrBayes v.3.2.6 dotplot function in Geneious to verify the IR. We extracted (Ronquist et al. 2012), under a GTR þ C model with four and aligned the two IR copies for each accession using the rate categories, using both unpartitioned and partitioned MAFFT v.7 Geneious plugin (Katoh and Standley 2013)to models, as above. We ran four independent chains (three verify identical sequence. We completed annotations for all heated, one cold) for 107 generations, discarding the first accessions in DOGMA (Wyman et al. 2004) and submitted 20% as burn-in. We confirmed chain stationarity and conver- them to GenBank under NCBI accessions MK726066– gence by standard deviation of split frequencies <0.01, and MK726085. effective sample size >500 for each parameter in Tracer v.1.7.1 (http://tree.bio.ed.ac.uk/software/tracer/; last accessed December 10, 2018). Data Set Construction, Alignment We initially aligned the annotated plastomes, excluding one Plastid Genome Structural Evolution copy of the IR, using the ProgressiveMAUVE algorithm in Physical and Functional Gene Losses Geneious (Darling et al. 2010; parameters ¼ autocalculate We scored each gene present in the “typical” monocot an- seed weight, minimum LCB score), which identifies locally giosperm gene set as putatively functional, physically lost (i.e., collinear, syntenic blocks (LCB), and allows visualization of completely deleted), or functionally lost as a pseudogene genomic structural rearrangements. We scored the degree among the accessions. Here, we denoted pseudogenes as of rearrangement as the DCJ distance from each accession “w,” and defined these conservatively by at least 10% of the to B. roezlii, which shows no evidence of genomic rearrange- open reading frame being disrupted by internal stop codons, ment relative to the highly conserved plastome configuration nontriplet frameshifts, or large deletions. Accessions display- characteristic of monocot angiosperms (as assessed by a pre- ing evidence of functional or physical loss in 10 vious alignment in MAUVE as above to Typha latifolia [NCBI photosynthesis-related genes were concluded to have lost accession GU195652; Poales, Typhaceae] and Phoenix dacty- the ability to carry out photosynthesis. We chose this conser- lifera NCBI accession GU811709 [Arecales, Arecaceae]). We vative definition to avoid assigning a loss of photosynthesis to realigned each collinear block identified by MAUVE using the accessions with only limited evidence of functional gene loss. MAFFT plugin for Geneious (default parameters). We then We used tRNAscan-SE (Lowe and Chan 2016)and concatenated the blocks, and filtered sites with missing data ARAGORN (Laslett and Canback 2004)todetectputative or gaps; hereafter, we refer to this as the “LCB” matrix, which transfer RNA pseudogenes, secondary structural rearrange- includes both coding and noncoding sequence. We also ments, or substitutions affecting anticodon sites. For each, extracted each protein-coding DNA sequence (CDS; hereafter we chose default search parameters and submitted full sets the “CDS” matrix) from the data set using a custom UNIX of tRNAs (excluding duplicates from the IR) as unaligned command and aligned each with MACSE v.2 (Ranwez et al. FASTA files. We then aligned each homologous tRNA across 2018), which preserves reading frame and allows incorpora- all accessions in MAFFT and annotated the anticodon iden- tion of sequencing errors or sequences with frameshifts (de- tified by ARAGORN in Geneious to check for substitutions or fault parameters). We kept genes for which all taxa had intact indels. reading frames, and then concatenated these into a single plastid supermatrix using TriFusion (http://odiogosilva. Evolution of Photosynthetic Capacity github.io/TriFusion/; last accessed December 10, 2018). Topology Tests for a Single Loss of Photosynthesis We constrained all accessions with evidence of degradation in Phylogenetic Analysis photosynthetic machinery (see above) to be monophyletic Estimation of Relationships (i.e., all accessions excluding Bletia, Basiphyllaea, H. grandiflora, We subjected the two data matrices above to parsimony, and H. revoluta). We set an alternative, negative constraint likelihood, and Bayesian analyses. We conducted parsimony specifying nonmonophyly of these accessions. Further, to test searches in TNT v.1.1 (Goloboff et al. 2008), using Bletia as the a recent phylogenetic hypothesis of relationships in outgroup (as for all subsequent phylogenetic analyses). We Hexalectris (Sosa et al. 2016), we specified a monophyly con- ran 1,000 random addition sequences and Tree Bisection- straint including all accessions of H. spicata, H. nitida, H. parvi- Reconnection branch swapping, keeping up to 1,000 trees. flora,andH. revoluta. We used stepping-stone sampling in We assessed parsimony branch support via Jackknife resam- MrBayes v.3.2.6, specifying a GTR þ CþI model with four rate pling, with 37% character removal probability. We chose the categories on the filtered data set of concatenated LCBs, and best-fit substitution model for each data set in Mega v.7 using 107 steps of the Markov Chain Monte Carlo sampler (Kumar et al. 2016), under the Bayesian Information with the first 20% as burn-in, and sampling every 5,000 steps. Criterion. We generated maximum likelihood trees in We checked convergence by verifying that the standard de- RAxML v.8.2 (Stamatakis 2014) under an unpartitioned and viation of split frequency for each run was <0.01. We then gene-partitioned GTR þ C model, respectively, with the de- used the average estimates of the marginal likelihoods in a fault number of rate categories across sites (25). We initiated Bayes factor comparison, given by BF ¼ the average of the

1897 Barrett et al. . doi:10.1093/molbev/msz111 MBE marginal likelihood of full mycoheterotroph monophyly di- likelihood via the “APE” package in R), multiple sequence vided by the average of the marginal likelihood of nonmono- alignment, a binary trait, and a joint maximum likelihood phyly of full mycoheterotrophs. This formula simplifies to 2 model of sequence and trait evolution to test the hypothesis the difference in log-likelihoods between the two marginal of trait/rate association versus a null model of no association likelihood averages. A Bayes factor >j100j indicates very via stochastic trait mapping. We additionally included atp strong evidence in favor of one model over another. genes, some of which are degraded in H. warnockii (Barrett and Kennedy 2018; see below), by pruning this species from Ancestral Reconstruction of Photosynthetic Status the tree in R via the “drop.tip” function (“APE”) and excluding We reconstructed photosynthetic status at the ancestral this species from the sequence alignment and trait data. nodes for the plastid tree as a discrete character with three states for each gene: functional (0; as this is the ancestral state Analysis of Relaxed Selective Constraints across Taxa among orchids and within Bletiinae), pseudogene (1), or We used the RELAX model in HyPhy v.2.4.4 (Kosakovsky physically lost (2). We specified a transition matrix allowing Pond et al. 2005; Wertheim et al. 2015) to test the hypothesis changes from 0 ! 1, 1 ! 2, and 0 ! 2 with equal proba- of relaxed versus intensified selective constraints based on bilities, and restricting all reversals. We use the R packages nonsynonymous/synonymous substitution ratios (x ¼ dN/ “ape,” “geiger,” and “phytools” for all analyses (Harmon et al. dS, i.e., the proportion of nonsynonymous and substitutions 2008; Revell 2012; Paradis and Schliep 2019). per respective site) in nonphotosynthetic accessions versus photosynthetic reference accessions. We used Bletia, Testing Discrete Trait Models of Photosynthetic Loss Basiphyllaea, H. grandiflora,andH. revoluta as reference pho- We then coded photosynthetic status as a binary character, tosynthetic branches, and others as “test” nonphotosynthetic based on evidence from extensive degradation of photosyn- branches. Analyses were run for all loci used in TraitRate thesis-related genes (0 ¼ photosynthetic and analyses (above). Specifically, RELAX allows testing of a null 1 ¼ nonphotosynthetic). First, we used a parsimony criterion model of no relaxed selection against an alternative model in and cost matrices to constrain character state changes in which test branches are allowed to have differing rates of x, terms of loss and gain of photosynthesis. We used equal indicated by the selection intensity parameter, K,whereK > 1 weights for character state changes in the first analysis (all indicates intensified selection and K < 1 indicates relaxed se- costs ¼ 1 step), and two cost constraints including weights lection. These are evaluated via a log-likelihood ratio test. All against loss and gain of photosynthesis. Constraints on state analyses were run via commandline on a local server, and changes were conducted in TNT by increasing the cost to the resulting .json result files were parsed with a custom UNIX maximum allowable (cost ¼ 99 steps) for the state change of script. interest. Searches for most parsimonious trees were con- ducted as above, and character state changes under each Correlations among Genomic and Substitutional Features cost matrix were optimized on the most parsimonious tree. We used the program CoEvol v.1.5 (Lartillot and Poujol 2011) We fit four Markov (mk model, sensu Lewis 2001)models to test correlations among genomic and substitutional fea- of discrete character evolution under a maximum likelihood tures. Specifically, we analyzed dS,dN,GCcontent,totalge- framework to the LCB tree, using modifications of the char- nome length, the number of functional genes (i.e., those with acter state rate matrix, that is, the “q-matrix.” We fit the no evidence of functional or physical loss), the degree of following alternative models of trait change: 1) “ER” or equal rearrangement (DCJ distance, see below), length of the IR, rates (q01 ¼ q10), 2) “ARD” or allowing rate differences (q01 ¼6 insertions/deletions, tandem repeats, and dispersed repeats. q10), 3) a “loss-only” model in which only photosynthetic loss CoEvol uses a Bayesian model to allow GC frequencies and can occur (q01 only), and 4) a “gain-only” model in which only substitution rate parameters to vary across branches of a reversals to photosynthesis from a nonphotosynthetic state user-defined tree (here, the CDS matrix topology), and mod- are allowed (q10 only). We evaluated the fit of each model els various continuous traits (phenotype, life-history, genome using the corrected Akaike information criterion (AICc; size, etc.) with the objective of testing for correlations among Burnham and Anderson 2002) and Akaike weights in the R these traits and substitution parameters while controlling for package qpcRv.1.4-1 (Spiess 2018). others. Here, we treat genomic features (repeat content, rear- rangements, etc.), as the “phenotype” of the plastome. Evolutionary Rate Variation and Photosynthetic Loss We used REPuter (Kurtz et al. 2001) to find genomic Photosynthetic Capacity and Substitution Rate Associations repeats >20 bp in length, with a Hamming distance of 3, We used TraitRateProp (Mayrose and Otto 2011; Karin, and maximum e-value of 103.Specifically,wesearchedfor Ashkenazy, et al. 2017; Karin, Wicke et al. 2017) to test asso- forward–forward, forward–reverse, forward–compliment, ciations between photosynthetic status as a binary trait and palindromic repeats. We transformed data as follows, (0 ¼ photosynthetic and 1 ¼ nonphotosynthetic) and substi- following the recommendations of Lartillot and Poujol tution rates for protein-coding genes with intact reading (2011):totalgenomelength(ex/10,000), length of coding frames. Genes with extensive loss or evidence of pseudogeni- DNA (ex/10,000), # functional genes (ex/10), DCJ distance (ex), zation in >2 taxa were excluded (e.g., psa/psb, pet,andrpo). IR length (ex/10,000), repeats classes (ex/10), and indels (ex/1 x) TraitRateProp uses an ultrametric tree (here via penalized to avoid zeroes. We ran two independent chains of CoEvol for

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105 generations of the Markov Chain Monte Carlo, discarding implications for heterotrophic angiosperms. MolBiolEvol. the first 10% as burn-in and sampling every five steps. We 31(12):3095–3112. used minimum effective sample sizes of 500 and discrepancies Barrett CF, Freudenstein JV, Taylor DL, Koljalg U. 2010. Rangewide anal- ysis of fungal associations in the fully mycoheterotrophic between chains of <0.1 for each parameter to assess conver- Corallorhiza striata complex () reveals extreme specific- gence, using the executable “tracecomp” which is part of the ity on ectomycorrhizal Tomentella (Thelephoraceae) across North CoEvol suite. Once the chains converged, we summarized the America. Am J Bot. 97(4):628–643. results with “readcoevol.” We considered pp’s of >0.9 for Barrett CF, Kennedy AH. 2018. Plastid genome degradation in the en- positively correlated attributes and <0.1 for negatively corre- dangered, mycoheterotrophic, North American orchid Hexalectris warnockii. Genome Biol Evol. 10(7):1657–1662. lated attributes (1 – pp) to be significant. Barrett CF, Wicke S, Sass C. 2018. Dense infraspecific sampling reveals rapid and independent trajectories of plastome degradation in a heterotrophic orchid complex. New Phytol. 218(3):1192–1204. Supplementary Material Bellot S, Renner SS. 2014. Exploring new dating approaches for parasites: Supplementary data areavailableatMolecular Biology and the worldwide Apodanthaceae (Cucurbitales) as an example. Mol Evolution online. Phylogenet Evol. 80:1–10. Bellot S, Renner SS. 2015. The plastomes of two species in the endopar- asite genus Pilostyles (Apodanthaceae) each retain just five or six Acknowledgments possibly functional genes. Genome Biol Evol. 8(1):189–201. Bidartondo MI. 2005. The evolutionary ecology of myco-heterotrophy. This work was supported by a WVU-PSCoR grant to C.F.B. New Phytol. 167(2):335–352. 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