Piecing together the puzzle of parasitic plant plastome evolution

The importance of photosynthesis as a mode of energy production has put plastid genomes of plants under a constant purifying selection. This has shaped the characteristic features of plastid genomes across the entire spectrum of photosynthetic plants and has led to a highly uniform and conserved plastid genome with respect to structure, size, gene order, intron and editing site positions and coding capacity. Parasitic species that have dropped photosynthesis as the main energy provider share striking deviations from the plastid genome norm: multiple rearrangements within the circular chromosome, pseudogenization and gene deletions, promoter losses, intron losses as well as the extensive loss of mRNA editing competence have been reported. The collective loss of larger sets of functionally related genes like those for the plastid NADH–dehydrogenase complex and concomitant losses of RNA polymerase genes together with their target promoters point to “domino effects” where an initial loss might have triggered others. An example, which will be discussed in more detail, is the concomitant loss of the intron maturase gene matK and all introns that are supposedly subject to MatK-dependent splicing in two Cuscuta species.     

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales)

Premise

Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome.

Methods

We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω).

Results

Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK.

Conclusions

We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.     

Evolution of Plant Mitochondrial Intron-Encoded Maturases: Frequent Lineage-Specific Loss and Recurrent Intracellular Transfer to the Nucleus

Among land plants, mitochondrial and plastid group II introns occasionally encode proteins called maturases that are important for splicing. Angiosperm nuclear genomes also encode maturases that are targeted to the organelles, but it is not known whether nucleus-encoded maturases exist in other land plant lineages. To examine the evolutionary diversity and history of this essential gene family, we searched for maturase homologs in recently sequenced nuclear and mitochondrial genomes from diverse land plants. We found that maturase content in mitochondrial genomes is highly lineage specific, such that orthologous maturases are rarely shared among major land plant groups. The presence of numerous mitochondrial pseudogenes in the mitochondrial genomes of several species implies that the sporadic maturase distribution is due to frequent inactivation and eventual loss over time. We also identified multiple maturase paralogs in the nuclear genomes of the lycophyte Selaginella moellendorffii, the moss Physcomitrella patens, and the representative angiosperm Vitis vinifera. Phylogenetic analyses of organelle- and nucleus-encoded maturases revealed that the nuclear maturase genes in angiosperms, lycophytes, and mosses arose by multiple shared and independent transfers of mitochondrial paralogs to the nuclear genome during land plant evolution. These findings indicate that plant mitochondrial maturases have experienced a surprisingly dynamic history due to a complex interaction of multiple evolutionary forces that affect the rates of maturase gain, retention, and loss.     

Evolution of Red Algal Plastid Genomes: Ancient Architectures,Introns, Horizontal Gene Transfer,and Taxonomic Utility of Plastid Markers

Red algae have the most gene-rich plastid genomes known, but despite their evolutionary importance these genomes remain poorly sampled. Here we characterize three complete and one partial plastid genome from a diverse range of florideophytes. By unifying annotations across all available red algal plastid genomes we show they all share a highly compact and slowly-evolving architecture and uniquely rich gene complements. Both chromosome structure and gene content have changed very little during red algal diversification, and suggest that plastid-to nucleus gene transfers have been rare. Despite their ancient character, however, the red algal plastids also contain several unprecedented features, including a group II intron in a tRNA-Met gene that encodes the first example of red algal plastid intron maturase – a feature uniquely shared among florideophytes. We also identify a rare case of a horizontally-acquired proteobacterial operon, and propose this operon may have been recruited for plastid function and potentially replaced a nucleus-encoded plastid-targeted paralogue. Plastid genome phylogenies yield a fully resolved tree and suggest that plastid DNA is a useful tool for resolving red algal relationships. Lastly, we estimate the evolutionary rates among more than 200 plastid genes, and assess their usefulness for species and subspecies taxonomy by comparison to well-established barcoding markers such as cox1 and rbcL. Overall, these data demonstrates that red algal plastid genomes are easily obtainable using high-throughput sequencing of total genomic DNA, interesting from evolutionary perspectives, and promising in resolving red algal relationships at evolutionarily-deep and species/subspecies levels.     

Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus <Emphasis Type="Italic">Cuscuta</Emphasis>

Background  

Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle.     

Evidence for Transitional Stages in the Evolution of Euglenid Group II Introns and Twintrons in the Monomorphina aenigmatica Plastid Genome

Background

Photosynthetic euglenids acquired their plastid by secondary endosymbiosis of a prasinophyte-like green alga. But unlike its prasinophyte counterparts, the plastid genome of the euglenid Euglena gracilis is riddled with introns that interrupt almost every protein-encoding gene. The atypical group II introns and twintrons (introns-within-introns) found in the E. gracilis plastid have been hypothesized to have been acquired late in the evolution of euglenids, implying that massive numbers of introns may be lacking in other taxa. This late emergence was recently corroborated by the plastid genome sequences of the two basal euglenids, Eutreptiella gymnastica and Eutreptia viridis, which were found to contain fewer introns.

Methodology/Principal Findings

To gain further insights into the proliferation of introns in euglenid plastids, we have characterized the complete plastid genome sequence of Monomorphina aenigmatica, a freshwater species occupying an intermediate phylogenetic position between early and late branching euglenids. The M. aenigmatica UTEX 1284 plastid genome (74,746 bp, 70.6% A+T, 87 genes) contains 53 intron insertion sites, of which 41 were found to be shared with other euglenids including 12 of the 15 twintron insertion sites reported in E. gracilis.

Conclusions

The pattern of insertion sites suggests an ongoing but uneven process of intron gain in the lineage, with perhaps a minimum of two bursts of rapid intron proliferation. We also identified several sites that represent intermediates in the process of twintron evolution, where the external intron is in place, but not the internal one, offering a glimpse into how these convoluted molecular contraptions originate.     

Conserved Gene Order and Expanded Inverted Repeats Characterize Plastid Genomes of Thalassiosirales

Diatoms are mostly photosynthetic eukaryotes within the heterokont lineage. Variable plastid genome sizes and extensive genome rearrangements have been observed across the diatom phylogeny, but little is known about plastid genome evolution within order- or family-level clades. The Thalassiosirales is one of the more comprehensively studied orders in terms of both genetics and morphology. Seven complete diatom plastid genomes are reported here including four Thalassiosirales: Thalassiosira weissflogii, Roundia cardiophora, Cyclotella sp. WC03_2, Cyclotella sp. L04_2, and three additional non-Thalassiosirales species Chaetoceros simplex, Cerataulina daemon, and Rhizosolenia imbricata. The sizes of the seven genomes vary from 116,459 to 129,498 bp, and their genomes are compact and lack introns. The larger size of the plastid genomes of Thalassiosirales compared to other diatoms is due primarily to expansion of the inverted repeat. Gene content within Thalassiosirales is more conserved compared to other diatom lineages. Gene order within Thalassiosirales is highly conserved except for the extensive genome rearrangement in Thalassiosira oceanica. Cyclotella nana, Thalassiosira weissflogii and Roundia cardiophora share an identical gene order, which is inferred to be the ancestral order for the Thalassiosirales, differing from that of the other two Cyclotella species by a single inversion. The genes ilvB and ilvH are missing in all six diatom plastid genomes except for Cerataulina daemon, suggesting an independent gain of these genes in this species. The acpP1 gene is missing in all Thalassiosirales, suggesting that its loss may be a synapomorphy for the order and this gene may have been functionally transferred to the nucleus. Three genes involved in photosynthesis, psaE, psaI, psaM, are missing in Rhizosolenia imbricata, which represents the first documented instance of the loss of photosynthetic genes in diatom plastid genomes.     

The Mitochondrial Genome of the Entomoparasitic Green Alga Helicosporidium

Background

Helicosporidia are achlorophyllous, non-photosynthetic protists that are obligate parasites of invertebrates. Highly specialized, these pathogens feature an unusual cyst stage that dehisces inside the infected organism and releases a filamentous cell displaying surface projections, which will penetrate the host gut wall and eventually reproduce in the hemolymph. Long classified as incertae sedis or as relatives of other parasites such as Apicomplexa or Microsporidia, the Helicosporidia were surprisingly identified through molecular phylogeny as belonging to the Chlorophyta, a phylum of green algae. Most phylogenetic analyses involving Helicosporidia have placed them within the subgroup Trebouxiophyceae and further suggested a close affiliation between the Helicosporidia and the genus Prototheca. Prototheca species are also achlorophyllous and pathogenic, but they infect vertebrate hosts, inducing protothecosis in humans. The complete plastid genome of an Helicosporidium species was recently described and is a model of compaction and reduction. Here we describe the complete mitochondrial genome sequence of the same strain, Helicosporidium sp. ATCC 50920 isolated from the black fly Simulium jonesi.

Methodology/Principal Findings

The circular mapping 49343 bp mitochondrial genome of Helicosporidium closely resembles that of the vertebrate parasite Prototheca wickerhamii. The two genomes share an almost identical gene complement and display a level of synteny that is higher than any other sequenced chlorophyte mitochondrial DNAs. Interestingly, the Helicosporidium mtDNA feature a trans-spliced group I intron, and a second group I intron that contains two open reading frames that appear to be degenerate maturase/endonuclease genes, both rare characteristics for this type of intron.

Conclusions/Significance

The architecture, genome content, and phylogeny of the Helicosporidium mitochondrial genome are all congruent with its close relationship to Prototheca within the Trebouxiophyceae. The Helicosporidium mitochondrial genome does, however, contain a number of novel features, particularly relating to its introns.     

The Complete Plastid Genome of Lagerstroemia fauriei and Loss of rpl2 Intron from Lagerstroemia (Lythraceae)

Lagerstroemia (crape myrtle) is an important plant genus used in ornamental horticulture in temperate regions worldwide. As such, numerous hybrids have been developed. However, DNA sequence resources and genome information for Lagerstroemia are limited, hindering evolutionary inferences regarding interspecific relationships. We report the complete plastid genome of Lagerstroemia fauriei. To our knowledge, this is the first reported whole plastid genome within Lythraceae. This genome is 152,440 bp in length with 38% GC content and consists of two single-copy regions separated by a pair of 25,793 bp inverted repeats. The large single copy and the small single copy regions span 83,921 bp and 16,933 bp, respectively. The genome contains 129 genes, including 17 located in each inverted repeat. Phylogenetic analysis of genera sampled from Geraniaceae, Myrtaceae, and Onagraceae corroborated the sister relationship between Lythraceae and Onagraceae. The plastid genomes of L. fauriei and several other Lythraceae species lack the rpl2 intron, which indicating an early loss of this intron within the Lythraceae lineage. The plastid genome of L. fauriei provides a much needed genetic resource for further phylogenetic research in Lagerstroemia and Lythraceae. Highly variable markers were identified for application in phylogenetic, barcoding and conservation genetic applications.     

Down the Slippery Slope: Plastid Genome Evolution in Convolvulaceae

Cuscuta (dodder) is the only parasitic genus found in Convolvulaceae (morning-glory family). We used long PCR approach to obtain large portions of plastid genome sequence from Cuscuta sandwichiana in order to determine the size, structure, gene content, and synteny in the plastid genome of this Cuscuta species belonging to the poorly investigated holoparasitic subgenus Grammica. These new sequences are compared with the tobacco chloroplast genome, and, where data are available, with corresponding regions from taxa in the other Cuscuta subgenera. When all known plastid genome structural rearrangements in parasitic and nonparasitic Convolvulaceae are considered in a molecular phylogenetic framework, three categories of rearrangements in Cuscuta are revealed: plesiomorphic, autapomorphic, and synapomorphic. Many of the changes in Cuscuta, previously attributed to its parasitic mode of life, are better explained either as plesiomorphic conditions within the family, i.e., conditions shared with the rest of the Convolvulaceae, or, in most cases, autapomorphies of particular Cuscuta taxa, not shared with the rest of the species in the genus. The synapomorphic rearrangements are most likely to correlate with the parasitic lifestyle, because they represent changes found in Cuscuta exclusively. However, it appears that most of the affected regions, belonging to all of these three categories, have probably no function (e.g., introns) or are of unknown function (a number of open reading frames, the function of which, if any, has yet to be discovered).[Reviewing Editor: Dr. Debashish Bhattacharya]     

Dictyochophyceae Plastid Genomes Reveal Unusual Variability in Their Organization

Dictyochophyceae (silicoflagellates) are unicellular freshwater and marine algae (Heterokontophyta, stramenopiles). Despite their abundance in global oceans and potential ecological significance, discovered in recent years, neither nuclear nor organellar genomes of representatives of this group were sequenced until now. Here, we present the first complete plastid genome sequences of Dictyochophyceae, obtained from four species: Dictyocha speculum, Rhizochromulina marina, Florenciella parvula and Pseudopedinella elastica. Despite their comparable size and genetic content, these four plastid genomes exhibit variability in their organization: plastid genomes of F. parvula and P. elastica possess conventional quadripartite structure with a pair of inverted repeats, R. marina instead possesses two direct repeats with the same orientation and D. speculum possesses no repeats at all. We also observed a number of unusual traits in the plastid genome of D. speculum, including expansion of the intergenic regions, presence of an intron in the otherwise non‐intron‐bearing psaA gene, and an additional copy of the large subunit of RuBisCO gene (rbcL), the last of which has never been observed in any plastid genome. We conclude that despite noticeable gene content similarities between the plastid genomes of Dictyochophyceae and their relatives (pelagophytes, diatoms), the number of distinctive features observed in this lineage strongly suggests that additional taxa require further investigation.     

Lateral transfer of introns in the cryptophyte plastid genome

Cryptophytes are unicellular eukaryotic algae that acquired photosynthesis secondarily through the uptake and retention of a red-algal endosymbiont. The plastid genome of the cryptophyte Rhodomonas salina CCMP1319 was recently sequenced and found to contain a genetic element similar to a group II intron. Here, we explore the distribution, structure and function of group II introns in the plastid genomes of distantly and closely related cryptophytes. The predicted secondary structures of six introns contained in three different genes were examined and found to be generally similar to group II introns but unusually large in size (including the largest known noncoding intron). Phylogenetic analysis suggests that the cryptophyte group II introns were acquired via lateral gene transfer (LGT) from a euglenid-like species. Unexpectedly, the six introns occupy five distinct genomic locations, suggesting multiple LGT events or recent transposition (or both). Combined with structural considerations, RT–PCR experiments suggest that the transferred introns are degenerate ‘twintrons’ (i.e. nested group II/group III introns) in which the internal intron has lost its splicing capability, resulting in an amalgamation with the outer intron.     

AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.     

Evidence of horizontal gene transfer between land plant plastids has surprising conservation implications

Background and AimsHorizontal gene transfer (HGT) is an important evolutionary mechanism because it transfers genetic material that may code for traits or functions between species or genomes. It is frequent in mitochondrial and nuclear genomes but has not been demonstrated between plastid genomes of different green land plant species.MethodsWe Sanger-sequenced the nuclear internal transcribed spacers (ITS1 and 2) and the plastid rpl16 G2 intron (rpl16). In five individuals with foreign rpl16 we also sequenced atpB-rbcL and trnL_UAA-trnF_GAA.Key ResultsWe discovered 14 individuals of a moss species with typical nuclear ITSs but foreign plastid rpl16 from a species of a distant lineage. None of the individuals with three plastid markers sequenced contained all foreign markers, demonstrating the transfer of plastid fragments rather than the entire plastid genome, i.e. entire plastids were not transferred. The two lineages diverged 165–185 Myr BP. The extended time interval since lineage divergence suggests that the foreign rpl16 is more likely explained by HGT than by hybridization or incomplete lineage sorting.ConclusionsWe provide the first conclusive evidence of interspecific plastid-to-plastid HGT among land plants. Two aspects are critical: it occurred at several localities during the massive colonization of recently disturbed open habitats that were created by large-scale liming as a freshwater biodiversity conservation measure; and it involved mosses whose unique life cycle includes spores that first develop a filamentous protonema phase. We hypothesize that gene transfer is facilitated when protonema filaments of different species intermix intimately when colonizing disturbed early succession habitats.     

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.     

Caryophyllales: Evaluating phylogenetic signal in trnK intron versus matK

Abstract We assess the phylogenetic information in trnK intron at the ordinal level using the Caryophyllales and compare it with that derived from matK. The trnK gene is split into two exons by an intron that includes the matK gene. The plastid trnK is a tRNA gene encoding Lysine^(UUU), whereas the matK gene is a putative group II intron maturase. The two regions are usually coamplified, and trnK intron is partially sequenced but its sequences are often excluded from phylogenetic reconstruction at deep historic levels. This study shows that the two regions are comparable in proportion of variable sites, possess a comparable pattern of substitution rates per site, and display similar phylogenetic informativeness profiles and per‐site informativeness. Phylogenetic analyses show strong congruence between phylogenetic trees based on matK and trnK intron partitioned datasets from 45 genera representing 30 of the 34 recognized Caryophyllales families. The trnK intron alone provides a relatively well‐resolved topology for the order. Combining the trnK intron with matK sequence data resulted in six most parsimonious trees, differing only in the placement of Claytonia (Portulacaceae) within the noncore group. A well‐supported major basal split in the order into core and noncore Caryophyllales with Rhabdodendraceae, Simmondsiaceae, and Asteropeiaceae as sister to remaining core lineages is evident in partitioned and combined analyses. The placement of these three families has been disputable, impacting the overall backbone topology of the Caryophyllales. This study demonstrates the cost effectiveness of using the trnK intron along with matK (both substitutions and insertions/deletions) at deeper phylogenetic level.     

A phylogenetic study of Laeliinae (Orchidaceae) based on combined nuclear and plastid DNA sequences

Background and Aims

Laeliinae are a neotropical orchid subtribe with approx. 1500 species in 50 genera. In this study, an attempt is made to assess generic alliances based on molecular phylogenetic analysis of DNA sequence data.

Methods

Six DNA datasets were gathered: plastid trnL intron, trnL-F spacer, matK gene and trnK introns upstream and dowstream from matK and nuclear ITS rDNA. Data were analysed with maximum parsimony (MP) and Bayesian analysis with mixed models (BA).

Key Results

Although relationships between Laeliinae and outgroups are well supported, within the subtribe sequence variation is low considering the broad taxonomic range covered. Localized incongruence between the ITS and plastid trees was found. A combined tree followed the ITS trees more closely, but the levels of support obtained with MP were low. The Bayesian analysis recovered more well-supported nodes. The trees from combined MP and BA allowed eight generic alliances to be recognized within Laeliinae, all of which show trends in morphological characters but lack unambiguous synapomorphies.

Conclusions

By using combined plastid and nuclear DNA data in conjunction with mixed-models Bayesian inference, it is possible to delimit smaller groups within Laeliinae and discuss general patterns of pollination and hybridization compatibility. Furthermore, these small groups can now be used for further detailed studies to explain morphological evolution and diversification patterns within the subtribe.Key words: Laeliinae, Orchidaceae, ITS, trnL intron, trnL-F spacer, matK