Chromosome organization through the cell cycle at a glance.

Genome organization and the three-dimensional folding of chromosomes are now seen as major contributors to nearly all nuclear functions including gene regulation, replication and repair. Recent studies have shown that in addition to the dramatic metamorphoses in chromosome conformation associated with entry to, and exit from mitosis, chromosomes undergo continual conformational changes throughout interphase with differential dynamics in loop structure, topological domains, compartments and lamina-associated domains. Understanding and accounting for these cell-cycle-dependent conformational changes is essential for the interpretation of data from a growing array of powerful molecular techniques to investigate genome conformation function, and to identify the molecules and mechanisms that drive chromosome conformational changes. In this Cell Science at a Glance article and the accompanying poster, we review Hi-C and microscopy studies describing cell-cycle-dependent conformational changes in chromosome structure.

Modulated illumination microscopy: Application perspectives in nuclear nanostructure analysis.

The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single-molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between ≤ 200 nm and ≥ 40 nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1 nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of ≤1 nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy.

De novo chromosome level assembly of a plant genome from long read sequence data.

Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. Chromosome level genome assemblies using sequence contigs generated from long read sequencing have involved the use of proximity analysis (Hi-C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (HiFi; PacBio) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii, a genome that has been used as a model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig (six with telomere repeats at both ends) and the other six assembled from two to four main contigs. The small number of chromosome breaks appears to be the result of highly repetitive regions including ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now appears possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed.

Comparison of assembly platforms for the assembly of the nuclear genome of Trichoderma harzianum strain PAR3.

BACKGROUND: Trichoderma is a diverse genus of fungi that includes several species that possess biotechnological and agricultural applications, including the biocontrol of pathogenic fungi and nematodes. The mitochondrial genome of a putative strain of Trichoderma harzianum called PAR3 was analyzed after isolation from the roots of Scarlet Royal grapevine scion grafted to Freedom rootstock, located in a grapevine vineyard in Parlier, CA, USA. Here, we report the sequencing, comparative assembly, and annotation of the nuclear genome of PAR3 and confirm its identification as a strain of T. harzianum. We subsequently compared the genes found in T. harzianum PAR3 to other known T. harzianum strains. Assembly of Illumina and/or Oxford Nanopore reads by the popular long-read assemblers, Flye and Canu, and the hybrid assemblers, SPAdes and MaSuRCA, was performed and the quality of the resulting assemblies were compared to ascertain which assembler generated the highest quality draft genome assembly. RESULTS: MaSuRCA produced the most complete and high-fidelity assembly yielding a nuclear genome of 40.7 Mb comprised of 112 scaffolds. Subsequent annotation of this assembly produced 12,074 gene models and 210 tRNAs. This included 221 genes that did not have equivalent genes in other T. harzainum strains. Phylogenetic analysis of ITS, rpb2, and tef1a sequences from PAR3 and established Trichoderma spp. showed that all three sequences from PAR3 possessed more than 99% identity to those of Trichoderma harzianum, confirming that PAR3 is an isolate of Trichoderma harzianum. We also found that comparison of gene models between T. harzianum PAR3 and other T. harzianum strains resulted in the identification of significant differences in gene type and number, with 221 unique genes identified in the PAR3 strain. CONCLUSIONS: This study gives insight into the efficacy of several popular assembly platforms for assembly of fungal nuclear genomes, and found that the hybrid assembler, MaSuRCA, was the most effective program for genome assembly. The annotated draft nuclear genome and the identification of genes not found in other T. harzainum strains could be used to investigate the potential applications of T. harzianum PAR3 for biocontrol of grapevine fungal canker pathogens and as source of anti-microbial compounds.

Large variance in inbreeding within the Iberian wolf population.

The gray wolf (Canis lupus) population on the Iberian Peninsula was the largest in western and central Europe during most of the 20th century, with its size apparently never under a few hundred individuals. After partial legal protection in the 1970s in Spain, the northwest Iberian population increased to about 300-350 packs and then stabilized. In contrast to many current European wolf populations, which have been connected through gene flow, the Iberian wolf population has been isolated for decades. Here we measured changes on genomic diversity and inbreeding through the last decades in a geographic context. We find that the level of genomic diversity in Iberian wolves is low compared to other Eurasian wolf populations. Despite population expansion in the last 50 years, some modern wolves had very high inbreeding, especially in the recently recolonized and historical edge areas. These individuals contrast with others with low inbreeding within the same population. The high variance in inbreeding despite population expansion seems associated with small-scale fragmentation of the range that is revealed by the genetic similarity between modern and historical samples from close localities despite being separated by decades, remaining differentiated from other individuals that are just over 100 km away, a small distance for a species with great dispersal capacity inhabiting a continuous range. This illustrates that, despite its demographically stable condition, the population would probably benefit from favoring connectivity within the population as well as genetic exchange with other European wolf populations to avoid excessive fragmentation and local inbreeding depression.

Nuclear pore proteins and the control of genome functions.

Nuclear pore complexes (NPCs) are composed of several copies of ∼30 different proteins called nucleoporins (Nups). NPCs penetrate the nuclear envelope (NE) and regulate the nucleocytoplasmic trafficking of macromolecules. Beyond this vital role, NPC components influence genome functions in a transport-independent manner. Nups play an evolutionarily conserved role in gene expression regulation that, in metazoans, extends into the nuclear interior. Additionally, in proliferative cells, Nups play a crucial role in genome integrity maintenance and mitotic progression. Here we discuss genome-related functions of Nups and their impact on essential DNA metabolism processes such as transcription, chromosome duplication, and segregation.

Nuclear genome of a pedinophyte pinpoints genomic innovation and streamlining in the green algae.

The genomic diversity underpinning high ecological and species diversity in the green algae (Chlorophyta) remains little known. Here, we aimed to track genome evolution in the Chlorophyta, focusing on loss and gain of homologous genes, and lineage-specific innovations of the core Chlorophyta. We generated a high-quality nuclear genome for pedinophyte YPF701, a sister lineage to others in the core Chlorophyta and incorporated this genome in a comparative analysis with 25 other genomes from diverse Viridiplantae taxa. The nuclear genome of pedinophyte YPF701 has an intermediate size and gene number between those of most prasinophytes and the remainder of the core Chlorophyta. Our results suggest positive selection for genome streamlining in the Pedinophyceae, independent from genome minimisation observed among prasinophyte lineages. Genome expansion was predicted along the branch leading to the UTC clade (classes Ulvophyceae, Trebouxiophyceae and Chlorophyceae) after divergence from their last common ancestor with pedinophytes, with genomic novelty implicated in a range of basic biological functions. Results emphasise multiple independent signals of genome minimisation within the Chlorophyta, as well as the genomic novelty arising before diversification in the UTC clade, which may underpin the success of this species-rich clade in a diversity of habitats.

Using ultraconserved elements to reconstruct the termite tree of life.

The phylogenetic history of termites has been investigated using mitochondrial genomes and transcriptomes. However, both sets of markers have specific limitations. Mitochondrial genomes represent a single genetic marker likely to yield phylogenetic trees presenting incongruences with species trees, and transcriptomes can only be obtained from well-preserved samples. In contrast, ultraconserved elements (UCEs) include a great many independent markers that can be retrieved from poorly preserved samples. Here, we designed termite-specific baits targeting 50,616 UCE loci. We tested our UCE bait set on 42 samples of termites and three samples of Cryptocercus, for which we generated low-coverage highly-fragmented genome assemblies and successfully extracted in silico between 3,426 to 42,860 non-duplicated UCEs per sample. Our maximum likelihood phylogenetic tree, reconstructed using the 5,934 UCE loci retrieved from upward of 75% of samples, was congruent with transcriptome-based phylogenies, demonstrating that our UCE bait set is reliable and phylogenetically informative. Combined with non-destructive DNA extraction protocols, our UCE bait set provides the tool needed to carry out a global taxonomic revision of termites based on poorly preserved specimens such as old museum samples. The Termite UCE database is maintained at: https://github.com/oist/TER-UCE-DB/.

Dipylidium caninum draft genome - a new resource for comparative genomic and genetic explorations of flatworms.

Here, we present a draft genome of the tapeworm Dipylidium caninum (family Dipylidiidae) and compare it with other cestode genomes. This draft genome of D. caninum is 110 Mb in size, has a repeat content of ~13.4% and is predicted to encode ~10,000 protein-coding genes. We inferred excretory/secretory molecules (representing the secretome), other key groups of proteins (including peptidases, kinases, phosphatases, GTPases, receptors, transporters and ion-channels) and predicted potential intervention targets for future evaluation. Using 144 shared single-copy orthologous sequences, we investigated the genetic relationships of cestodes for which nuclear genomes are available. This study provides first insights into the molecular biology of D. caninum and a new resource for comparative genomic and genetic explorations of this and other flatworms.

Comparative genomics of the coconut crab and other decapod crustaceans: exploring the molecular basis of terrestrial adaptation.

BACKGROUND: The complex life cycle of the coconut crab, Birgus latro, begins when an obligate terrestrial adult female visits the intertidal to hatch zoea larvae into the surf. After drifting for several weeks in the ocean, the post-larval glaucothoes settle in the shallow subtidal zone, undergo metamorphosis, and the early juveniles then subsequently make their way to land where they undergo further physiological changes that prevent them from ever entering the sea again. Here, we sequenced, assembled and analyzed the coconut crab genome to shed light on its adaptation to terrestrial life. For comparison, we also assembled the genomes of the long-tailed marine-living ornate spiny lobster, Panulirus ornatus, and the short-tailed marine-living red king crab, Paralithodes camtschaticus. Our selection of the latter two organisms furthermore allowed us to explore parallel evolution of the crab-like form in anomurans. RESULTS: All three assembled genomes are large, repeat-rich and AT-rich. Functional analysis reveals that the coconut crab has undergone proliferation of genes involved in the visual, respiratory, olfactory and cytoskeletal systems. Given that the coconut crab has atypical mitochondrial DNA compared to other anomurans, we argue that an abundance of kif22 and other significantly proliferated genes annotated with mitochondrial and microtubule functions, point to unique mechanisms involved in providing cellular energy via nuclear protein-coding genes supplementing mitochondrial and microtubule function. We furthermore detected in the coconut crab a significantly proliferated HOX gene, caudal, that has been associated with posterior development in Drosophila, but we could not definitively associate this gene with carcinization in the Anomura since it is also significantly proliferated in the ornate spiny lobster. However, a cuticle-associated coatomer gene, gammacop, that is significantly proliferated in the coconut crab, may play a role in hardening of the adult coconut crab abdomen in order to mitigate desiccation in terrestrial environments. CONCLUSION: The abundance of genomic features in the three assembled genomes serve as a source of hypotheses for future studies of anomuran environmental adaptations such as shell-utilization, perception of visual and olfactory cues in terrestrial environments, and cuticle sclerotization. We hypothesize that the coconut crab exhibits gene proliferation in lieu of alternative splicing as a terrestrial adaptation mechanism and propose life-stage transcriptomic assays to test this hypothesis.

The genome of a thorny species: comparative genomic analysis among South and North American Cactaceae.

MAIN CONCLUSION: The first South American cactus nuclear genome assembly associated with comparative genomic analyses provides insights into nuclear and plastid genomic features, such as size, transposable elements, and metabolic processes related to cactus development. Here, we assembled the partial genome, plastome, and transcriptome of Cereus fernambucensis (Cereeae, Cactaceae), a representative species of the South American core Cactoideae. We accessed other genomes and transcriptomes available for cactus species to compare the heterozygosity level, genome size, transposable elements, orthologous genes, and plastome structure. These estimates were obtained from the literature or using the same pipeline adopted for C. fermabucensis. In addition to the C. fernambucensis plastome, we also performed de novo plastome assembly of Pachycereus pringlei, Stenocereus thurberi, and Pereskia humboldtii based on the sequences available in public databases. We estimated a genome size of ~ 1.58 Gb for C. fernambucensis, the largest genome among the compared species. The genome heterozygosity was 0.88% in C. fernambucensis but ranged from 0.36 (Carnegiea gigantea) to 17.4% (Lophocereus schottii) in the other taxa. The genome lengths of the studied cacti are constituted by a high amount of transposable elements, ranging from ~ 57 to ~ 67%. Putative satellite DNAs are present in all species, excepting C. gigantea. The plastome of C. fernambucensis was ~ 104 kb, showing events of translocation, inversion, and gene loss. We observed a low number of shared unique orthologs, which may suggest gene duplication events and the simultaneous expression of paralogous genes. We recovered 37 genes that have undergone positive selection along the Cereus branch that are associated with different metabolic processes, such as improving photosynthesis during drought stress and nutrient absorption, which may be related to the adaptation to xeric areas of the Neotropics.

Whole-genome sequencing reveals asymmetric introgression between two sister species of cold-resistant leaf beetles.

A large number of genetic variation studies have identified cases of mitochondrial genome introgression in animals, indicating that reproductive barriers among closely related species are often permeable. Because of its sheer size, the impact of hybridization on the evolution of the nuclear genome is more difficult to apprehend. Only a few studies have explored it recently thanks to recent progress in DNA sequencing and genome assembly. Here, we analysed whole-genome sequence variation among multiple individuals of two sister species of leaf beetles inside their hybrid zone, in which asymmetric mitochondrial genome introgression had previously been established. We used a machine learning approach based on computer simulations for training to identify regions of the nuclear genome that were introgressed. We inferred asymmetric introgression of ≈2% of the genome, in the same direction that was observed for the mitochondrial genome. Because a previous study based on a reduced-representation sequencing approach was not able to detect this introgression, we conclude that whole-genome sequencing is necessary when the fraction of the introgressed genome is small. We also analysed the whole-genome sequence of a hybrid individual, demonstrating that hybrids have the capacity to backcross with the species for which virtually no introgression was observed. Our data suggest that one species has recently invaded the range of the other and/or some alleles that where transferred from the invaded into the invading species could be under positive selection and may have favoured the adaptation of the invading species to the Alpine environment.

Some Molecular and Cellular Stress Mechanisms Associated with Neurodegenerative Diseases and Atherosclerosis.

Chronic stress is a combination of nonspecific adaptive reactions of the body to the influence of various adverse stress factors which disrupt its homeostasis, and it is also a corresponding state of the organism's nervous system (or the body in general). We hypothesized that chronic stress may be one of the causes occurence of several molecular and cellular types of stress. We analyzed literary sources and considered most of these types of stress in our review article. We examined genes and mutations of nuclear and mitochondrial genomes and also molecular variants which lead to various types of stress. The end result of chronic stress can be metabolic disturbance in humans and animals, leading to accumulation of reactive oxygen species (ROS), oxidative stress, energy deficiency in cells (due to a decrease in ATP synthesis) and mitochondrial dysfunction. These changes can last for the lifetime and lead to severe pathologies, including neurodegenerative diseases and atherosclerosis. The analysis of literature allowed us to conclude that under the influence of chronic stress, metabolism in the human body can be disrupted, mutations of the mitochondrial and nuclear genome and dysfunction of cells and their compartments can occur. As a result of these processes, oxidative, genotoxic, and cellular stress can occur. Therefore, chronic stress can be one of the causes forthe occurrence and development of neurodegenerative diseases and atherosclerosis. In particular, chronic stress can play a large role in the occurrence and development of oxidative, genotoxic, and cellular types of stress.

Genome sequence of the coffee root-knot nematode Meloidogyne exigua.

Root-knot nematodes (Meloidogyne spp.) cause serious damages on most crops. Here, we report a high-quality genome sequence of Meloidogyne exigua (population Mex1, Costa Rica), a major pathogen of coffee. Its mitogenome (20,974 bp) was first assembled and annotated. The nuclear genome was then constructed consisting of 206 contigs, with an N50 length of 1.89 Mb and a total assembly length of 42.1 Mb.

[Risk of Multiple Sclerosis: Analysis of Interactions Between Variants of Nuclear and Mitochondrial Genomes].

There is increasing evidence that the interaction of the mitochondrial and nuclear genomes substantially affects the risk of neurodegenerative diseases. The role of mitonuclear interactions in the development of multiple sclerosis, a severe chronic neurodegenerative disease of a polygenic nature, is poorly understood. In this work, we analyzed the association of multiple sclerosis with two-component mitonuclear combinations that include each of seven polymorphic variants of the nuclear genome localized in the region of the UCP2, and KIF1B genes and in the PVT1 locus (MYC, PVT1, and MIR1208 genes) and each often polymorphisms of the mitochondrial genome, as well as individual genetic variants that make up these combinations. Association of the individual components of these combinations with multiple sclerosis was also evaluated. 507 patients with multiple sclerosis and 321 healthy individuals were enrolled in the study, all participants were ethnic Russians. Two mitonuclear combinations associated with multiple sclerosis were identified: the UCP2 (rs660339)*A + MT-ATP6 (rs193303045)*G combination was characterized by p-value = 0.015 and OR= 1.39 [95% CI 1.05-1.87], and the PVT1 (rs2114358)*G + MT-ND1 (rs1599988)*С combination - by p-value = 0.012 and OR = 1.77 [95% CI 1.10-2.84]. Only one of the individual components of these combinations, allele rs660339*A of the nuclear gene UCP2 encoding uncoupling protein 2 of the mitochondrial anion carrier family, was independently associated with multiple sclerosis (p = 0.028; OR = 1.36 [95% CI 1.01-1.84]). This study expands the current understanding of the role of mitonuclear interactions and variance of nuclear genes, whose products function in mitochondria, and in risk of MS.

Genetics of Arterial-Wall-Specific Mechanisms in Atherosclerosis: Focus on Mitochondrial Mutations.

PURPOSE OF REVIEW: Mutations in both nuclear and mitochondrial genes are associated with the development of atherosclerotic lesions in arteries and may provide a partial explanation to the focal nature of lesion distribution in the arterial wall. This review is aimed to discuss the genetic aspects of atherogenesis with a special focus on possible pro-atherogenic variants (mutations) of the nuclear and mitochondrial genomes that may be implicated in atherosclerosis development and progression. RECENT FINDINGS: Mutations in the nuclear genes generally do not cause a phenotype restricted to a specific vascular wall cell and manifest themselves mostly at the organism level. Such mutations can act as important contributors to changes in lipid metabolism and modulate other risk factors of atherosclerosis. By contrast, mitochondrial DNA (mtDNA) mutations occurring locally in the arterial wall cells or in circulating immune cells may play a site-specific role in atherogenesis. The mosaic distribution of heteroplasmic mtDNA mutations in the arterial wall tissue may explain, at least to some extent, the locality and focality of atherosclerotic lesions distribution. The genetic mechanisms of atherogenesis include alterations of both nuclear and mitochondrial genomes. Altered lipid metabolism and inflammatory response of resident arterial wall and circulating immune cells may be related to mtDNA damage and defective mitophagy, which hinders clearance of dysfunctional mitochondria. Mutations of mtDNA can have mosaic distribution and locally affect functionality of endothelial and subendothelial intimal cells in the arterial wall contributing to atherosclerotic lesion development.

Subchromosome-Scale Nuclear and Complete Mitochondrial Genome Characteristics of Morchella crassipes.

Morchella crassipes (Vent.) Pers., a typical yellow morel species with high economic value, is mainly distributed in the low altitude plains of Eurasia. However, rare research has been performed on its genomics and polarity, thus limiting its research and development. Here, we reported a fine physical map of the nuclear genome at the subchromosomal-scale and the complete mitochondrial genome of M. crassipes. The complete size of the nuclear genome was 56.7 Mb, and 23 scaffolds were assembled, with eight of them being complete chromosomes. A total of 11,565 encoding proteins were predicted. The divergence time analysis showed that M. crassipes representing yellow morels differentiated with black morels at ~33.98 Mya (million years), with 150 gene families contracted and expanded in M. crassipes versus the two black morels (M. snyderi and M. importuna). Furthermore, 409 CAZYme genes were annotated in M. crassipes, containing almost all plant cell wall degrading enzymes compared with the mycorrhizal fungi (truffles). Genomic annotation of mating type loci and amplification of the mating genes in the monospore population was conducted, the results indicated that M. crassipes is a heterothallic fungus. Additionally, a complete circular mitochondrial genome of M. crassipes was assembled, the size reached as large as 531,195 bp. It can be observed that the strikingly large size was the biggest up till now, coupled with 14 core conserved mitochondrial protein-coding genes, two rRNAs, 31 tRNAs, 51 introns, and 412 ncORFs. The total length of intron sequences accounted for 53.67% of the mitochondrial genome, with 19 introns having a length over 5 kb. Particularly, 221 of 412 ncORFs were distributed within 51 introns, and the total length of the ncORFs sequence accounted for 40.83% of the mitochondrial genome, and 297 ncORFs had expression activity in the mycelium stage, suggesting their potential functions in M. crassipes. Meanwhile, there was a high degree of repetition (51.31%) in the mitochondria of M. crassipes. Thus, the large number of introns, ncORFs and internal repeat sequences may contribute jointly to the largest fungal mitochondrial genome to date. The fine physical maps of nuclear genome and mitochondrial genome obtained in this study will open a new door for better understanding of the mysterious species of M. crassipes.

'Yes' to mitochondrial replacement techniques and lesbian motherhood: a reply to Françoise Baylis.

In a recent paper - Lesbian motherhood and mitochondrial replacement techniques: reproductive freedom and genetic kinship - we argued that lesbian couples who wish to have children who are genetically related to both of them should be allowed access to mitochondrial replacement techniques (MRTs). Françoise Baylis wrote a reply to our paper -'No' to lesbian motherhood using human nuclear genome transfer- where she challenges our arguments on the use of MRTs by lesbian couples, and on MRTs more generally. In this reply we respond to her claims and further clarify our position.

Unraveling the nuclear and chloroplast genomes of an agar producing red macroalga, Gracilaria changii (Rhodophyta, Gracilariales).

Agar and agarose have wide applications in food and pharmaceutical industries. Knowledge on the genome of red seaweeds that produce them is still lacking. To fill the gap in genome analyses of these red algae, we have sequenced the nuclear and organellar genomes of an agarophyte, Gracilaria changii. The partial nuclear genome sequence of G. changii has a total length of 35.8Mb with 10,912 predicted protein coding sequences. Only 39.4% predicted proteins were found to have significant matches to protein sequences in SwissProt. The chloroplast genome of G. changii is 183,855bp with a total of 201 open reading frames (ORFs), 29 tRNAs and 3 rRNAs predicted. Five genes: ssrA, leuC and leuD CP76_p173 (orf139) and pbsA were absent in the chloroplast genome of G. changii. The genome information is valuable in accelerating functional studies of individual genes and resolving evolutionary relationship of red seaweeds.

RNA-seq highlights parallel and contrasting patterns in the evolution of the nuclear genome of fully mycoheterotrophic plants.

BACKGROUND: While photosynthesis is the most notable trait of plants, several lineages of plants (so-called full heterotrophs) have adapted to obtain organic compounds from other sources. The switch to heterotrophy leads to profound changes at the morphological, physiological and genomic levels. RESULTS: Here, we characterize the transcriptomes of three species representing two lineages of mycoheterotrophic plants: orchids (Epipogium aphyllum and Epipogium roseum) and Ericaceae (Hypopitys monotropa). Comparative analysis is used to highlight the parallelism between distantly related fully heterotrophic plants. In both lineages, we observed genome-wide elimination of nuclear genes that encode proteins related to photosynthesis, while systems associated with protein import to plastids as well as plastid transcription and translation remain active. Genes encoding components of plastid ribosomes that have been lost from the plastid genomes have not been transferred to the nuclear genomes; instead, some of the encoded proteins have been substituted by homologs. The nuclear genes of both Epipogium species accumulated nucleotide substitutions twice as rapidly as their photosynthetic relatives; in contrast, no increase in the substitution rate was observed in H. monotropa. CONCLUSIONS: Full heterotrophy leads to profound changes in nuclear gene content. The observed increase in the rate of nucleotide substitutions is lineage specific, rather than a universal phenomenon among non-photosynthetic plants.