Epifagus Research Articles

Functional traits of Epifagus virginiana (Orobanchaceae) tubers as adaptations to the Mexican beech microenvironment

Epifagus virginiana (Orobanchaceae), known as beechdrops, is a holoparasitic plant that acquires all its resources from a narrow range of host plants, restricted to North American Fagus species. To do so, beechdrops develop a vascular connection with the host via a terminal haustoria that develops as a tuber attached to the host root. We hypothesized that microenvironmental conditions can influence functional traits of the E. virginiana tuber despite this parasite's complete reliance on the host plant for its nutrition. Therefore, the aims of this study were i) to analyze the structure of the tubers of E. virginiana; ii) to assess the variation in tuber functional traits between two E. virginiana populations; and iii) to analyze how microenvironmental factors influence functional traits in the tuber. We detected that functional trait of beechdrops tuber along Mexican beech microenvironmental covaried with traits important for below-ground processes. Boosted regression trees provided a powerful analysis tool, giving substantially superior predictive performance to generalized additive models, despite the fitting of interaction terms in the latter.

Identifying and characterizing antibiotic producers found in and on the haustorium of Epifagus virginiana.

Epifagus virginiana, a holoparsite of Fagus grandifolia, utilizes haustoria to parasitize its host. Little is known about the microbiome of this plant's parasitic organ. To shed light on these unexplored ecological interactions we collected eight individual E. virginiana specimens from Kanawha State Forest (Charleston, West Virginia) and used culture-based methods and microscopy to assess total counts and morphotypes, revealing a diverse microbial community on the haustorium surface, including 3 bacterial phyla and fungi. The internal haustorium environment was lower in counts and diversity, indicating the haustorium's internal microbiome is likely a subset of the soil microbiome. We explored the capabilities of these microorganisms to produce bioactive compounds. Fifty-eight morphospecies were investigated for cellulose degradation and antibiotic production. Forty-six isolates were challenged in a cross-streak assay against a Micrococcus roseus (Gram-positive) and Escherichia coli K12(Gram-negative) and 35% showed antagonism for at least one type. Seven surface isolates showed a range of antagonism against M. roseus from 0.85 cm ±0.21 cm to 2.25 cm ±0.07 cm, and 2 against E. coli from 0.75 cm ±0.35 cm to 100%. Two of the internal isolates showed a range of antagonism against M. roseus and E. coli from 1.25 cm ±0.07 cm to 2.35 cm ±0.07 cm and 0.4 cm ±0.00 cm to 2.05 cm ±0.21 cm, respectively. Currently, we are testing these isolates against ESKAPE pathogens and using 16S rRNA sequencing and Biolog ® to further characterize them and their potential as a source of novel antibiotics or bioactive compounds.

Cannibal plants: intraspecific autoparasitism among host-specific holoparasites

Intraspecific autoparasitism, the phenomenon in which a parasite serves as the host for another individual of the same species, is a well-described interaction among hemiparasitic flowering plants. Here, I describe a rare case of autoparasitism in Epifagus virginiana (L.) W.P.C. Barton (Orobanchaceae), a holoparasitic plant normally found exclusively on the roots of Fagus grandifolia Ehrh. (Fagaceae). Confirmation of the autoparasitic relationship was obtained through microtomography and anatomical analyses. Results show the presence of vessel connections between the two E. virginiana plants and the unobstructed passage of solutes from one plant to another, thus suggesting the autoparasitic connection to be fully functional. While several segments of nearby host roots were entangled with the adventitious roots of the parasite, no parasite–host vascular connection was detected. Additionally, an analysis of digitized herbarium specimens showed that albeit rare, the observed autoparasitic relationship in E. virginiana is not a one-of case. A total of 3097 digitized herbarium specimens were also analyzed, among which 1.69% were observed to potentially include an autoparasitic connection. Altogether, these results provide new clues for the investigation of host-specificity and haustorium development processes in parasitic flowering plants.

Evolutionary origins of the eastern North American-Mesoamerican floristic disjunction: Current status and future prospects.

Biogeographic disjunction patterns, where multiple taxa are shared between isolated geographic areas, represent excellent systems for investigating the historical assembly of modern biotas and fundamental biological processes such as speciation, diversification, niche evolution, and evolutionary responses to climate change. Studies on plant genera disjunct across the northern hemisphere, particularly between eastern North America (ENA) and eastern Asia (EAS), have yielded tremendous insight on the geologic history and assembly of rich temperate floras. However, one of the most prevalent disjunction patterns involving ENA forests has been largely overlooked: that of taxa disjunct between ENA and cloud forests of Mesoamerica (MAM), with examples including Acer saccharum, Liquidambar styraciflua, Cercis canadensis, Fagus grandifolia, and Epifagus virginiana. Despite the remarkable nature of this disjunction pattern, which has been recognized for over 75 years, there have been few recent efforts to empirically examine its evolutionary and ecological origins. Here I synthesize previous systematic, paleobotanical, phylogenetic, and phylogeographic studies to establish what is known about this disjunction pattern to provide a roadmap for future research. I argue that this disjunction pattern, and the evolution and fossil record of the Mexican flora more broadly, represents a key missing piece in the broader puzzle of northern hemisphere biogeography. I also suggest that the ENA-MAM disjunction represents an excellent system for examining fundamental questions about how traits and life history strategies mediate plant evolutionary responses to climate change and for predicting how broadleaf temperate forests will respond to the ongoing climatic pressures of the Anthropocene.

Microbiome associated with the Haustorium of Epifagus virginiana.

Epifagus virginana is a holoparsitic plant whose only host is the American Beech tree, Fagus grandifolia. Currently, little is known about the specific mechanism used by Epifagus to colonize the tree. It is thought that lignin-like or lignin degradation by-products are needed to signal the colonization process for other members of Orobanchaceae. Knowing that microbial communities that inhabit eukaryotic hosts can influence their function and development, we aimed to describe the microbial community structure of Epifagus, in particular the communities inhabiting the haustorium, (the organ used to parasitize). Using community-level physiological profiling, selective culture media, and metabarcoding we found that microbial communities in Epifagus haustorium were active and diverse and structurally similar to those from adjacent bulk soil in Fall (MRPP A= -0.004, p=0.509), but different in Summer (MRPP A= 0.169, p=0.003). Metabarcoding shows increase in the Rhizobiales, Betaproteobacteriales and Flavobacteriales in the haustorium associated soil compare to the beech tree bulk soil. We found that a larger number of endophytic isolates showed lignolytic activity compared to the haustorium associated microorganisms (HAMs). Finally, we were able to isolate bacterial isolates that use cellulose as their only carbon source. Altogether these findings lead us to further hypothesize that microbes associated with the haustorium could be involved in the production of the lignin-like haustorium inducing factors that are thought to facilitate the parasitism of other parasitic plants. These are, to our knowledge, the first published descriptions of the microorganisms inhabiting this holoparasite. Funding from the Appalachian College Association Ledford Scholars Program.

Community-level Physiological Profile, Cellulose and Lignin Degradation of the Microbial Assemblages Associated with Epifagus virginiana

Epifagus virginana is a holoparsite whose only host is the American Beech tree, Fagus grandifolia. Currently, little is known about the specific mechanism used by Epifagus to colonize the tree. It is thought that lignin-like or lignin degradation by-products are needed to signal the colonization process for other members of Orobanchaceae. Knowing that microbial communities that inhabit eukaryotic hosts can influence their function and development, we aimed to describe the microbial community structure of Epifagus, in particular the communities inhabiting the haustorium, (the organ used to parasitize). Using community-level physiological profiling as well as selective culture media, we found that microbial communities in Epifagus haustorium were active and diverse and structurally similar to those from adjacent bulk soil in Fall (MRPP A= -0.004, p=0.509), but different in Summer (MRPP A= 0.169, p=0.003). We also examined the cellulose and lignin degrading capacity of these microbial communities for their possible role in facilitating Epifagus colonization. We found that a larger number of endophytic isolates showed lignolytic activity compared to the haustorium associated microorganisms (HAMs). Finally, we were able to isolate bacterial isolates that use cellulose as their only carbon source. Altogether these findings lead us to further hypothesize that microbes associated with the haustorium could be involved in the production of the lignin-like haustorium inducing factors that are thought to facilitate the parasitism of other parasitic plants. These are, to our knowledge, the first published descriptions of the microorganisms inhabiting this holoparasite.

Parasitic Beechdrops (Epifagus virginiana): A Possible Ant-Pollinated Plant

Abstract Epifagus virginiana (Beechdrop) is an annual flowering plant that parasitizes Fagus grandifolia (American Beech) roots. The pollination biology of the chasmogamous flowers of E. virginiana is unknown. In September 2011–2012, we observed insect visitors to E. virginiana flowers. Insect visitors included Bombus impatiens, Crematogaster spp., and Prenolepis imparis, but were dominated by P. imparis (over 96% of insect visits). In September 2012, we captured ants (Crematogaster and P. imparis) on E. virginiana flowers in three different areas found in the Coastal Plain and Piedmont physiographic provinces. With the use of 2,5-diphenyl tetrazolium bromide (MTT), we tested E. virginiana pollen from flowers and ant integuments for viability. We observed no significant difference (P > 0.05) between the viability of pollen taken directly from E. virginiana flowers and pollen removed from the ants. Our data suggests that ants can be playing an important role in the pollination of E. virginiana.

Sequencing and Analysis of Plastid Genome in Mycoheterotrophic Orchid Neottia nidus-avis

Plastids are the semiautonomous organelles that possess their own genome inherited from the cyanobacterial ancestor. The primary function of plastids is photosynthesis so the structure and evolution of plastid genomes are extensively studied in photosynthetic plants. In contrast, little is known about the plastomes of nonphotosynthetic species. In higher plants, plastid genome sequences are available for only three strictly nonphotosynthetic species, the liverwort Aneura mirabilis and two flowering plants, Epifagus virginiana and Rhizanthella gardneri. We report here the complete sequence of a plastid genome of nonphotosynthetic mycoheterotrophic orchid Neottia nidus-avis, determined using 454 pyrosequencing technology. It was found to be reduced in both genome size and gene content; this reduction is however not as drastic as in the other nonphotosynthetic orchid, R. gardneri. Neottia plastome lacks all genes encoding photosynthetic proteins, RNA polymerase subunits but retains most genes of translational apparatus. Those genes that are retained have an increased rate of both synonymous and nonsynonymous substitutions but do not exhibit relaxation of purifying selection either in Neottia or in Rhizanthella.

Host density drives the postglacial migration of the tree parasite, Epifagus virginiana

To survive changes in climate, successful species shift their geographic ranges to remain in suitable habitats. For parasites and other highly specialized species, distributional changes not only are dictated by climate but can also be engineered by their hosts. The extent of host control on parasite range expansion is revealed through comparisons of host and parasite migration and demographic histories. However, understanding the codistributional history of entire forest communities is complicated by challenges in synthesizing datasets from multiple interacting species of differing datatypes. Here we integrate genetic and fossil pollen datasets from a host-parasite pair; specifically, the population structure of the parasitic plant (Epifagus virginiana) was compared with both its host (Fagus grandifolia) genetic patterns and abundance data from the paleopollen record of the last 21,000 y. Through tests of phylogeographic structure and spatial linear regression models we find, surprisingly, host range changes had little effect on the parasite's range expansion and instead host density is the main driver of parasite spread. Unlike other symbionts that have been used as proxies to track their host's movements, this parasite's migration routes are incongruent with the host and instead reflect the greater importance of host density in this community's assembly. Furthermore, these results confirm predictions of disease ecological models regarding the role of host density in the spread of pathogens. Due to host density constraints, highly specialized species may have low migration capacities and long lag times before colonization of new areas.

Parallel Loss of Plastid Introns and Their Maturase in the Genus Cuscuta

Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.

Functional Gene Losses Occur with Minimal Size Reduction in the Plastid Genome of the Parasitic Liverwort Aneura mirabilis

Aneura mirabilis is a parasitic liverwort that exploits an existing mycorrhizal association between a basidiomycete and a host tree. This unusual liverwort is the only known parasitic seedless land plant with a completely nonphotosynthetic life history. The complete plastid genome of A. mirabilis was sequenced to examine the effect of its nonphotosynthetic life history on plastid genome content. Using a partial genomic fosmid library approach, the genome was sequenced and shown to be 108,007 bp with a structure typical of green plant plastids. Comparisons were made with the plastid genome of Marchantia polymorpha, the only other liverwort plastid sequence available. All ndh genes are either absent or pseudogenes. Five of 15 psb genes are pseudogenes, as are 2 of 6 psa genes and 2 of 6 pet genes. Pseudogenes of cysA, cysT, ccsA, and ycf3 were also detected. The remaining complement of genes present in M. polymorpha is present in the plastid of A. mirabilis with intact open reading frames. All pseudogenes and gene losses co-occur with losses detected in the plastid of the parasitic angiosperm Epifagus virginiana, though the latter has functional gene losses not found in A. mirabilis. The plastid genome sequence of A. mirabilis represents only the second liverwort, and first mycoheterotroph, to have its plastid genome sequenced. We observed a pattern of genome evolution congruent with functional gene losses in parasitic angiosperms but suggest that its plastid genome represents a genome in the early stages of decay following the relaxation of selection pressures.

Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii

BackgroundThe holoparasitic plant genus Cuscuta comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different Cuscuta species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species Cuscuta reflexa and Cuscuta gronovii. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of Epifagus virginiana, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in C. reflexa to a restricted photosynthetic activity and degenerated chloroplasts in C. gronovii to an achlorophyllous state in E. virginiana.ResultsThe newly sequenced plastid genomes of C. reflexa and C. gronovii reveal that the chromosome structures are generally very similar to that of non-parasitic plants, although a number of species-specific insertions, deletions (indels) and sequence inversions were identified. However, we observed a gradual adaptation of the plastid genome to the different degrees of parasitism. The changes are particularly evident in C. gronovii and include (a) the parallel losses of genes for the subunits of the plastid-encoded RNA polymerase and the corresponding promoters from the plastid genome, (b) the first documented loss of the gene for a putative splicing factor, MatK, from the plastid genome and (c) a significant reduction of RNA editing.ConclusionOverall, the comparative genomic analysis of plastid DNA from parasitic plants indicates a bias towards a simplification of the plastid gene expression machinery as a consequence of an increasing dependency on the host plant. A tentative assignment of the successive events in the adaptation of the plastid genomes to parasitism can be inferred from the current data set. This includes (1) a loss of non-coding regions in photosynthetic Cuscuta species that has resulted in a condensation of the plastid genome, (2) the simplification of plastid gene expression in species with largely impaired photosynthetic capacity and (3) the deletion of a significant part of the genetic information, including the information for the photosynthetic apparatus, in non-photosynthetic parasitic plants.

Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus Cuscuta

BackgroundPlastid 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.ResultsAside from loss of all ndh genes, Cuscuta exaltata retains photosynthetic and photorespiratory genes that evolve under strong selective constraint. Cuscuta obtusiflora has incurred substantially more change to its plastid genome, including loss of all genes for the plastid-encoded RNA polymerase. Despite extensive change in gene content and greatly increased rate of overall nucleotide substitution, C. obtusiflora also retains all photosynthetic and photorespiratory genes with only one minor exception.ConclusionAlthough Epifagus virginiana, the only other parasitic plant with its plastid genome sequenced to date, has lost a largely overlapping set of transfer-RNA and ribosomal genes as Cuscuta, it has lost all genes related to photosynthesis and maintains a set of genes which are among the most divergent in Cuscuta. Analyses demonstrate photosynthetic genes are under the highest constraint of any genes within the plastid genomes of Cuscuta, indicating a function involving RuBisCo and electron transport through photosystems is still the primary reason for retention of the plastid genome in these species.

A subset of conserved tRNA genes in plastid DNA of nongreen plants.

The plastid genome of the nonphotosynthetic parasitic plant Epifagus virginiana contains only 17 of the 30 tRNA genes normally found in angiosperm plastid DNA. Although this is insufficient for translation, the genome is functional, so import of cytosolic tRNAs into plastids has been suggested. This raises the question of whether the tRNA genes that remain in E. virginiana plastid DNA are active or have just fortuitously escaped deletion. We report the sequences of 20 plastid tRNA loci from Orobanche minor, which shares a nonphotosynthetic ancestor with E. virginiana. The two species have 9 intact tRNA genes in common, the others being defunct in one or both species. The intron-containing trnLUAA gene is absent from E. virginiana, but it is intact, transcribed, and spliced in O. minor. The shared intact genes are better conserved than intergenic sequences, which indicates that these genes are being maintained by natural selection and, therefore, must be functional. For the most part, the tRNA species conserved in nonphotosynthetic plastids are also those that have never been found to be imported in plant mitochondria, which suggests that the same rules may govern tRNA import in the two organelles. A small photosynthesis gene, psbI, is still intact in O. minor, and computer simulations show that some small nonessential genes have an appreciable chance of escaping deletion.

Pigment composition of putatively achlorophyllous angiosperms

Chlorophyll and carotenoid pigment composition was determined for ten species of putatively achlorophyllous angiosperms using high-performance liquid chromatography. Four families were represented:Lennoaceae (Pholisma arenarium);Monotropaceae (Allotropa virgata, Monotropa uniflora, Pterospora andromedea, Sarcodes sanguinea); Orobanchaceae (Epifagus virginiana, Orobanche cooperi, O. uniflora);Orchidaceae (Cephalanthera austinae, Corallorhiza maculata). Chlorophylla was detected in all taxa, but chlorophyllb was only detected inCorallorhiza maculata. The relative amount of chlorophyll and chlorophyll-related pigments in these plants is greatly reduced compared to fully autotrophic angiosperms.

Understory Influence of the Invasive Norway Maple (Acer platanoides)

WYCKOFF, PETER H. AND SARA L. WEBB (Biology Department, Drew University, Madison NJ 07940). Understory influence of the invasive Norway maple (Acer platanoides). Bull. Torrey Bot. Club 123:197-205. 1996.Norway maple (Acer platanoides) is invasive within a Fagus grandifolia-Acer saccharum-Quercus spp. forest preserve in New Jersey. To assess the community-level consequences of this invasion, we compared understory composition, richness, and structure beneath canopies of invasive Acer platanoides and of native Acer saccharum and Fagus grandifolia. Understory species richness was significantly lower beneath Acer platanoides and highest beneath Fagus grandifolia. Stem densities were high beneath Acer platanoides but consisted primarily of its own seedlings and saplings. When these Acer platanoides stems were removed from the analysis, there was a trend toward lower stem densities beneath the exotic tree compared with the native canopies. Individual shrub and forb species were too patchy for any canopy affinities to demonstrate significance within this small forest preserve, with two exceptions: Epifagus virginiana, a beech (Fagus) root parasite, was less abundant under Acer platanoides than under either Fagus grandifolia or Acer saccharum; and the shrub Lindera benzoin was less abundant under Acer platanoides and Fagus grandifolia than under Acer saccharum. Among tree seedlings, the exotic Acer platanoides far outnumbered all others, beneath its own canopy and also beneath the two native trees. A surprising paucity of Fagus grandifolia reproduction seems unrelated to the biological invasion. Acer saccharum has some prospects for self-replacement judging from sapling distributions but has sparse reproduction under the other canopy species relative to its exotic congener.

Transcription, splicing and editing of plastid RNAs in the nonphotosynthetic plant Epifagus virginiana.

Expression of the vestigial plastid genome of the nonphotosynthetic, parasitic flowering plant Epifagus virginiana was examined by northern analysis and by characterization of cDNAs. Probes for each of 12 plastid genes tested hybridized to all lanes of northern blots containing total RNA prepared from stems and fruits of Epifagus and from leaves of tobacco. Certain transcript patterns in Epifagus plastids are highly complex and similar to those of tobacco operons. In contrast, genes such as rps2, which have become orphaned in Epifagus as a result of evolutionary loss of formerly cotranscribed genes, show simpler transcript patterns in Epifagus than in tobacco. Sizing and sequencing of cDNAs generated by reverse transcriptase-PCR for three genes, rps12, rpl2, and clpP, show that their transcripts are properly cis- and/or trans-spliced at the same five group II intron insertion sites used in photosynthetic plants. A single, conventional C-->U edit in rps12 was found among the total of 1401 nucleotides of cDNA sequence that was determined for the three genes. An octanucleotide sequence identical to a putative guide RNA of plant organelles and perfectly complementary to the rps12 edit site itself was identified just 200 bp upstream of the edit site. These data, together with previous results from the complete sequencing of the Epifagus plastid genome, provide compelling evidence that this degenerate genome is nonetheless expressed and functional. Analysis of the putative maturase MatK, encoded by the group II intron of trnK in photosynthetic land plants but by a freestanding gene in Epifagus, leads us to hypothesize that it acts 'in trans' to assist the splicing of group II introns other than the one in which it is normally encoded.

On the Identification of Group II Introns in Nucleotide Sequence Data

Four Different consensus sequences (GTI, group II identifiers) have been derived from domains V of known group II introns and are used as query input sequences for sensitive database screenings with the FASTA and LFASTA programs. The set of four GTI sequences can identify all domains V of the 96 known group II introns in the completely sequenced chloroplast genomes of Marchantia polymorpha, Epifagus virginiana, Oryza saliva, Nicotiana tabacum and the completely sequenced mitochondrial genomes of Saccharomyces cerevisiae, Podospora anserina, Schizosaccharomyces pombe and Marchantia polymorpha. Seven moderately high-scoring hits can easily be rejected as false-positives since they do not fulfil secondary structure requirements. Large FASTA outputs obtained after screening the entire nucleotide sequence database are evaluated in a second step by a program (D5SCAN) that allows the assignment of variable selection criteria for potential domain V secondary structures. Database searches with these routines yield evidence for several group II intron sequences previously unrecognized. These include novel intron structures in the cyanobacterium Synechocystis and in the mitochondrial genomes of Marchantia, soybean, pea, broad bean, sugar beet and a heterobasidiomycete. Potential intron remnants are found contributing to the secondary structure of rRNAs in several trypanosome species. At a given sensitivity of 95% positively identified true domains V, the search routine produces one false positive hit per 10,000 kb.

Structure and evolution of the largest chloroplast gene (ORF2280): internal plasticity and multiple gene loss during angiosperm evolution

We have determined the nucleotide sequence of the Pelargonium x hortorum ORF2280 homolog, the largest gene in the plastid genome of most land plants, and compared it to published homologs from Nicotiana tabacum, Epifagus virginiana, Spinacia oleracea, and Marchantia polymorpha. Multiple alignment of protein sequences requires an extraordinary number of gaps, indicating a very high frequency of insertion/deletion events during the evolution of the protein; however, the overall predicted size of the protein varies relatively little among the five species. At 2,109 codons, the Pelargonium gene is smaller than other land plant ORF2280 homologs and exhibits a rate of nucleotide substitution several times higher relative to Nicotiana, Epifagus, and Spinacia. Southern-blot and restriction-mapping studies were carried out to uncover length variation in ORF2280 homologs from 279 species (representing 111 families) of angiosperms. In many independent angiosperm lineages, this gene has sustained deletions ranging in size from 200 bp to almost 6 kb. Based on the severity of deletions, we postulate that the chloroplast homolog of ORF2280 has become nonfunctional in at least four independent lineages of angiosperms.

Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function.

Many group II introns encode reverse transcriptase-like proteins that potentially function in intron mobility and RNA splicing. We compared 34 intron-encoded open reading frames and four related open reading frames that are not encoded in introns. Many of these open reading frames have a reverse transcriptase-like domain, followed by an additional conserved domain X, and a Zn(2+)-finger-like region. Some open reading frames have lost conserved sequence blocks or key amino acids characteristic of functional reverse transcriptases, and some lack the Zn(2+)-finger-like region. The open reading frames encoded by the chloroplast tRNA(Lys) genes and the related Epifagus virginiana matK open reading frame lack a Zn(2+)-finger-like region and have only remnants of a reverse transcriptase-like domain, but retain a readily identifiable domain X. Several findings lead us to speculate that domain X may function in binding of the intron RNA during reverse transcription and RNA splicing. Overall, our findings are consistent with the hypothesis that all of the known group II intron open reading frames evolved from an ancestral open reading frame, which contained reverse transcriptase, X, and Zn(2+)-finger-like domains, and that the reverse transcriptase and Zn(2+)-finger-like domains were lost in some cases. The retention of domain X in most proteins may reflect an essential function in RNA splicing, which is independent of the reverse transcriptase activity of these proteins.