Invited Review Beyond parasitic convergence: unravelling the evolution of the organellar genomes in holoparasites.

BACKGROUND: The molecular evolution of organellar genomes in angiosperms has been studied extensively, with some lineages, such as parasitic ones, displaying unique characteristics. Parasitism has emerged 12 times independently in angiosperm evolution. Holoparasitism is the most severe form of parasitism, and is found in ~10 % of parasitic angiosperms. Although a few holoparasitic species have been examined at the molecular level, most reports involve plastomes instead of mitogenomes. Parasitic plants establish vascular connections with their hosts through haustoria to obtain water and nutrients, which facilitates the exchange of genetic information, making them more susceptible to horizontal gene transfer (HGT). HGT is more prevalent in the mitochondria than in the chloroplast or nuclear compartments. SCOPE: This review summarizes current knowledge on the plastid and mitochondrial genomes of holoparasitic angiosperms, compares the genomic features across the different lineages, and discusses their convergent evolutionary trajectories and distinctive features. We focused on Balanophoraceae (Santalales), which exhibits extraordinary traits in both their organelles. CONCLUSIONS: Apart from morphological similarities, plastid genomes of holoparasitic plants also display other convergent features, such as rampant gene loss, biased nucleotide composition and accelerated evolutionary rates. In addition, the plastomes of Balanophoraceae have extremely low GC and gene content, and two unexpected changes in the genetic code. Limited data on the mitochondrial genomes of holoparasitic plants preclude thorough comparisons. Nonetheless, no obvious genomic features distinguish them from the mitochondria of free-living angiosperms, except for a higher incidence of HGT. HGT appears to be predominant in holoparasitic angiosperms with a long-lasting endophytic stage. Among the Balanophoraceae, mitochondrial genomes exhibit disparate evolutionary paths with notable levels of heteroplasmy in Rhopalocnemis and unprecedented levels of HGT in Lophophytum. Despite their differences, these Balanophoraceae share a multichromosomal mitogenome, a feature also found in a few free-living angiosperms.

Break-induced replication is the primary recombination pathway in plant somatic hybrid mitochondria: a model for mitochondrial horizontal gene transfer.

Somatic hybrids between distant species offer a remarkable model to study genomic recombination events after mitochondrial fusion. Recently, we described highly chimeric mitogenomes in two somatic hybrids between the Solanaceae Nicotiana tabacum and Hyoscyamus niger resulting from interparental homologous recombination. To better examine the recombination map in somatic hybrid mitochondria, we developed a more sensitive bioinformatic strategy to detect recombination activity based on high-throughput sequencing without assembling the hybrid mitogenome. We generated a new intergeneric somatic hybrid between N. tabacum and Physochlaina orientalis, and re-analyzed the somatic hybrids that we previously generated. We inferred 213 homologous recombination events across repeats of 2.1 kb on average. Most of them (~80%) were asymmetrical, consistent with the break-induced replication pathway. Only rare (2.74%) non-homologous events were detected. Interestingly, independent events frequently occurred in the same regions within and across somatic hybrids, suggesting the existence of recombination hotspots in plant mitogenomes. Break-induced replication is the main pathway of interparental recombination in somatic hybrid mitochondria. Findings of this study are relevant to mitogenome editing assays and to mechanistic aspects of DNA integration following mitochondrial DNA horizontal transfer events.

Evolution of cox2 introns in angiosperm mitochondria and efficient splicing of an elongated cox2i691 intron.

In angiosperms, the mitochondrial cox2 gene harbors up to two introns, commonly referred to as cox2i373 and cox2i691. We studied the cox2 from 222 fully-sequenced mitogenomes from 30 angiosperm orders and analyzed the evolution of their introns. Unlike cox2i373, cox2i691 shows a distribution among plants that is shaped by frequent intron loss events driven by localized retroprocessing. In addition, cox2i691 exhibits sporadic elongations, frequently in domain IV of introns. Such elongations are poorly related to repeat content and two of them showed the presence of LINE transposons, suggesting that increasing intron size is very likely due to nuclear intracelular DNA transfer followed by incorporation into the mitochondrial DNA. Surprisingly, we found that cox2i691 is erroneously annotated as absent in 30 mitogenomes deposited in public databases. Although each of the cox2 introns is âˆ¼1.5 kb in length, a cox2i691 of 4.2 kb has been reported in Acacia ligulata (Fabaceae). It is still unclear whether its unusual length is due to a trans-splicing arrangement or the loss of functionality of the interrupted cox2. Through analyzing short-read RNA sequencing of Acacia with a multi-step computational strategy, we found that the Acacia cox2 is functional and its long intron is spliced in cis in a very efficient manner despite its length.

Cytonuclear coevolution in a holoparasitic plant with highly disparate organellar genomes.

KEY MESSAGE: Contrasting substitution rates in the organellar genomes of Lophophytum agree with the DNA repair, replication, and recombination gene content. Plastid and nuclear genes whose products form multisubunit complexes co-evolve. The organellar genomes of the holoparasitic plant Lophophytum (Balanophoraceae) show disparate evolution. In the plastid, the genome has been severely reduced and presents a > 85% AT content, while in the mitochondria most protein-coding genes have been replaced by homologs acquired by horizontal gene transfer (HGT) from their hosts (Fabaceae). Both genomes carry genes whose products form multisubunit complexes with those of nuclear genes, creating a possible hotspot of cytonuclear coevolution. In this study, we assessed the evolutionary rates of plastid, mitochondrial and nuclear genes, and their impact on cytonuclear evolution of genes involved in multisubunit complexes related to lipid biosynthesis and proteolysis in the plastid and those in charge of the oxidative phosphorylation in the mitochondria. Genes from the plastid and the mitochondria (both native and foreign) of Lophophytum showed extremely high and ordinary substitution rates, respectively. These results agree with the biased loss of plastid-targeted proteins involved in angiosperm organellar repair, replication, and recombination machinery. Consistent with the high rate of evolution of plastid genes, nuclear-encoded subunits of plastid complexes showed disproportionate increases in non-synonymous substitution rates, while those of the mitochondrial complexes did not show different rates than the control (i.e. non-organellar nuclear genes). Moreover, the increases in the nuclear-encoded subunits of plastid complexes were positively correlated with the level of physical interaction they possess with the plastid-encoded ones. Overall, these results suggest that a structurally-mediated compensatory factor may be driving plastid-nuclear coevolution in Lophophytum, and that mito-nuclear coevolution was not altered by HGT.

The minicircular and extremely heteroplasmic mitogenome of the holoparasitic plant Rhopalocnemis phalloides.

The plastid and nuclear genomes of parasitic plants exhibit deeply altered architectures,1-13 whereas the few examined mitogenomes range from deeply altered to conventional.14-20 To provide further insight on mitogenome evolution in parasitic plants, we report the highly modified mitogenome of Rhopalocnemis phalloides, a holoparasite in Balanophoraceae. Its mitogenome is uniquely arranged in 21 minicircular chromosomes that vary in size from 4,949 to 7,861 bp, with a total length of only 130,713 bp. All chromosomes share an identical 896 bp conserved region, with a large stem-loop that acts as the origin of replication, flanked on each side by hypervariable and semi-conserved regions. Similar minicircular structures with shared and unique regions have been observed in parasitic animals and free-living protists,21-24 suggesting convergent structural evolution. Southern blots confirm both the minicircular structure and the replication origin of the mitochondrial chromosomes. PacBio reads provide evidence for chromosome recombination and rolling-circle replication for the R. phalloides mitogenome. Despite its small size, the mitogenome harbors a typical set of genes and introns within the unique regions of each chromosome, yet introns are the smallest among seed plants and ferns. The mitogenome also exhibits extreme heteroplasmy, predominantly involving short indels and more complex variants, many of which cause potential loss-of-function mutations for some gene copies. All heteroplasmic variants are transcribed, and functional and nonfunctional protein-coding variants are spliced and RNA edited. Our findings offer a unique perspective into how mitogenomes of parasitic plants can be deeply altered and shed light on plant mitogenome replication.

Deepred-Mt: Deep representation learning for predicting C-to-U RNA editing in plant mitochondria.

In land plant mitochondria, C-to-U RNA editing converts cytidines into uridines at highly specific RNA positions called editing sites. This editing step is essential for the correct functioning of mitochondrial proteins. When using sequence homology information, edited positions can be computationally predicted with high precision. However, predictions based on the sequence contexts of such edited positions often result in lower precision, which is limiting further advances on novel genetic engineering techniques for RNA regulation. Here, a deep convolutional neural network called Deepred-Mt is proposed. It predicts C-to-U editing events based on the 40 nucleotides flanking a given cytidine. Unlike existing methods, Deepred-Mt was optimized by using editing extent information, novel strategies of data augmentation, and a large-scale training dataset, constructed with deep RNA sequencing data of 21 plant mitochondrial genomes. In comparison to predictive methods based on sequence homology, Deepred-Mt attains significantly better predictive performance, in terms of average precision as well as F1 score. In addition, our approach is able to recognize well-known sequence motifs linked to RNA editing, and shows that the local RNA structure surrounding editing sites may be a relevant factor regulating their editing. These results demonstrate that Deepred-Mt is an effective tool for predicting C-to-U RNA editing in plant mitochondria. Source code, datasets, and detailed use cases are freely available at https://github.com/aedera/deepredmt.

Transcriptional Landscape and Splicing Efficiency in Arabidopsis Mitochondria.

Plant mitochondrial transcription is initiated from multiple promoters without an apparent motif, which precludes their identification in other species based on sequence comparisons. Even though coding regions take up only a small fraction of plant mitochondrial genomes, deep RNAseq studies uncovered that these genomes are fully or nearly fully transcribed with significantly different RNA read depth across the genome. Transcriptomic analysis can be a powerful tool to understand the transcription process in diverse angiosperms, including the identification of potential promoters and co-transcribed genes or to study the efficiency of intron splicing. In this work, we analyzed the transcriptional landscape of the Arabidopsis mitochondrial genome (mtDNA) based on large-scale RNA sequencing data to evaluate the use of RNAseq to study those aspects of the transcription process. We found that about 98% of the Arabidopsis mtDNA is transcribed with highly different RNA read depth, which was elevated in known genes. The location of a sharp increase in RNA read depth upstream of genes matched the experimentally identified promoters. The continuously high RNA read depth across two adjacent genes agreed with the known co-transcribed units in Arabidopsis mitochondria. Most intron-containing genes showed a high splicing efficiency with no differences between cis and trans-spliced introns or between genes with distinct splicing mechanisms. Deep RNAseq analyses of diverse plant species will be valuable to recognize general and lineage-specific characteristics related to the mitochondrial transcription process.

Plastomes in the holoparasitic family Balanophoraceae: Extremely high AT content, severe gene content reduction, and two independent genetic code changes.

The transition to a heterotrophic lifestyle in angiosperms is characterized by convergent evolutionary changes. Plastid genome remodeling includes dramatic functional and physical reductions with the highest degrees observed in fully heterotrophic plants. Genes related to photosynthesis are generally absent or pseudogenized, while a few genes related to other metabolic processes that take place within the plastid are almost invariably maintained. The family Balanophoraceae consists of root holoparasites that present reduced plastid genomes with an extraordinarily elevated AT content and the single genetic code change ever documented in land plant plastomes (the stop codon TAG now codes for tryptophan). Here, we studied the plastomes of Lophophytum leandri and Ombrophytum subterraneum (Balanophoraceae) that showed the remarkable absence of the gene trnE, a highly biased nucleotide composition, and an independent genetic code change (the standard stop codon TGA codes for tryptophan). This is the second genetic code change identified in land plant plastomes. Analysis of the transcriptome of Lophophytum indicated that the entire C5 pathway typical of plants is conserved despite the lack of trnE in its plastome. A hypothetical model of plastome evolution in the Balanophoraceae is presented.

Computational Detection of Plant RNA Editing Events.

Computers are able to systematically exploit RNA-seq data allowing us to efficiently detect RNA editing sites in a genome-wide scale. This chapter introduces a very flexible computational framework for detecting RNA editing sites in plant organelles. This framework comprises three major steps: RNA-seq data processing, RNA read alignment, and RNA editing site detection. Each step is discussed in sufficient detail to be implemented by the reader. As a study case, the framework will be used with publicly available sequencing data to detect C-to-U RNA editing sites in the coding sequences of the mitochondrial genome of Nicotiana tabacum.

Horizontal gene transfers dominate the functional mitochondrial gene space of a holoparasitic plant.

Although horizontal gene transfer (HGT) is common in angiosperm mitochondrial DNAs (mtDNAs), few cases of functional foreign genes have been identified. The one outstanding candidate for large-scale functional HGT is the holoparasite Lophophytum mirabile, whose mtDNA has lost most native genes but contains intact foreign homologs acquired from legume host plants. To investigate the extent to which this situation results from functional replacement of native by foreign genes, functional mitochondrial gene transfer to the nucleus, and/or loss of mitochondrial biochemical function in the context of extreme parasitism, we examined the Lophophytum mitochondrial and nuclear transcriptomes by deep paired-end RNA sequencing. Most foreign mitochondrial genes in Lophophytum are highly transcribed, accurately spliced, and efficiently RNA edited. By contrast, we found no evidence for functional gene transfer to the nucleus or loss of mitochondrial functions in Lophophytum. Many functional replacements occurred via the physical replacement of native genes by foreign genes. Some of these events probably occurred as the final act of HGT itself. Lophophytum mtDNA has experienced an unprecedented level of functional replacement of native genes by foreign copies. This raises important questions concerning population-genetic and molecular regimes that underlie such a high level of foreign gene takeover.

Multichromosomal structure and foreign tracts in the Ombrophytum subterraneum (Balanophoraceae) mitochondrial genome.

Horizontal gene transfer (HGT) is frequent in parasitic plant mitochondria as a result of vascular connections established in host-parasite relationships. Recent studies of the holoparasitic plant Lophophytum mirabile (Balanophoraceae) revealed the unprecedented acquisition of a large amount of mitochondrial sequences from its legume host. We focused on a close relative, the generalist holoparasite Ombrophytum subterraneum, to examine the incidence of HGT events in the mitochondrial genome (mtDNA). The mtDNA of O. subterraneum assembles into 54 circular chromosomes, only 34 of which contain the 51 full-length coding regions. Numerous foreign tracts (totaling almost 100 kb, ~ 14% of the mtDNA), including 12 intact genes, were acquired by HGT from the Asteraceae hosts. Nine chromosomes concentrate most of those regions and eight are almost entirely foreign. Native homologs of each foreign gene coexist in the mtDNA and are potentially functional. A large proportion of shorter regions were related to the Fabaceae (a total of ~ 110 kb, 15.4%), some of which were shared with L. mirabile. We also found evidence of foreign sequences donated by angiosperm lineages not reported as hosts (Apocynaceae, Euphorbiaceae, Lamiaceae, and Malvales). We propose an evolutionary hypothesis that involves ancient transfers from legume hosts in the common ancestor of Ombrophytum and Lophophytum followed by more recent transfer events in L. mirabile. Besides, the O. subterraneum mtDNA was also subjected to additional HGT events from diverse angiosperm lineages, including large and recent transfers from the Asteraceae, and also from Lamiaceae.

Horizontal Gene Transfer has Impacted cox1 Gene Evolution in Cassytha filiformis.

The gene cox1 is one of the most reported mitochondrial genes involved in horizontal gene transfer among angiosperms. However, whether different cox1 copies exist in different populations of a species and whether any other novel way except intron homing exists for cox1 intron acquisition is less understood. In this study, we chose Cassytha filiformis, a parasitic plant from the angiosperm family Lauraceae, as an example to study cox1 variation and evolution. We identified the stable and inheritable co-occurrence of two copies of cox1 genes, which were different in base composition and insertion/deletion among samples of a single species, C. filiformis. The bioinformatic analyses revealed that Type I copy had intact open reading frames, but type II copy had premature stop codons and was a pseudogene. Further INDEL characterization, phylogenetic analyses, and CCT comparisons consistently support two different origins for the two types of C. filiformis cox1 genes. Type I cox1 was likely vertically inherited within the magnoliids but it has captured an intron from another species, whereas the entire type II intron-containing cox1 has most likely been transferred integrally from Cuscuta or other Convolvulaceae species. The finding of the two independent horizontal gene transfer events associated with C. filiformis cox1 genes not only promotes our understanding of the evolutionary history of C. filiformis, but also leaves intriguing evolutionary questions that merits further efforts.

The complete organelle genomes of Physochlaina orientalis: Insights into short sequence repeats across seed plant mitochondrial genomes.

Short repeats (SR) play an important role in shaping seed plant mitochondrial genomes (mtDNAs). However, their origin, distribution, and relationships across the different plant lineages remain unresolved. We focus on the angiosperm family Solanaceae that shows great variation in repeat content and extend the study to a wide diversity of seed plants. We determined the complete nucleotide sequences of the organellar genomes of the medicinal plant Physochlaina orientalis (Solanaceae), member of the tribe Hyoscyameae. To understand the evolution of the P. orientalis mtDNA we made comparisons with those of five other Solanaceae. P. orientalis mtDNA presents the largest mitogenome (∼685 kb in size) among the Solanaceae and has an unprecedented 8-copy repeat family of ∼8.2 kb in length and a great number of SR arranged in tandem-like structures. We found that the SR in the Solanaceae share a common origin, but these only expanded in members of the tribe Hyoscyameae. We discuss a mechanism that could explain SR formation and expansion in P. orientalis and Hyoscyamus niger. Finally, the great increase in plant mitochondrial data allowed us to systematically extend our repeat analysis to a total of 136 seed plants to characterize and analyze for the first time families of SR among seed plant mtDNAs.

Elucidating genomic patterns and recombination events in plant cybrid mitochondria.

KEY MESSAGE: Cybrid plant mitochondria undergo homologous recombination, mainly BIR, keep a single allele for each gene, and maintain exclusive sequences of each parent and a single copy of the homologous regions. The maintenance of a dynamic equilibrium between the mitochondrial and nuclear genomes requires continuous communication and a high level of compatibility between them, so that alterations in one genetic compartment need adjustments in the other. The co-evolution of nuclear and mitochondrial genomes has been poorly studied, even though the consequences and effects of this interaction are highly relevant for human health, as well as for crop improvement programs and for genetic engineering. The mitochondria of plants represent an excellent system to understand the mechanisms of genomic rearrangements, chimeric gene formation, incompatibility between nucleus and cytoplasm, and horizontal gene transfer. We carried out detailed analyses of the mtDNA of a repeated cybrid between the solanaceae Nicotiana tabacum and Hyoscyamus niger. The mtDNA of the cybrid was intermediate between the size of the parental mtDNAs and the sum of them. Noticeably, most of the homologous sequences inherited from both parents were lost. In contrast, the majority of the sequences exclusive of a single parent were maintained. The mitochondrial gene content included a majority of N. tabacum derived genes, but also chimeric, two-parent derived, and H. niger-derived genes in a tobacco nuclear background. Any of these alterations in the gene content could be the cause of CMS in the cybrid. The parental mtDNAs interacted through 28 homologous recombination events and a single case of illegitimate recombination. Three main homologous recombination mechanisms were recognized in the cybrid mitochondria. Break induced replication (BIR) pathway was the most frequent. We propose that BIR could be one of the mechanisms responsible for the loss of the majority of the repeated regions derived from H. niger.

Genome-scale transfer of mitochondrial DNA from legume hosts to the holoparasite Lophophytum mirabile (Balanophoraceae).

Angiosperm mitochondrial horizontal gene transfer (HGT) has been widely reported during the past decades. With a few exceptions, foreign sequences are mitochondrial genes or intronic regions from other plants, indicating that HGT has played a major role in shaping mitochondrial genome evolution. Host-parasite relationships are a valuable system to study this phenomenon due to the high frequency of HGT. In particular, the interaction between mimosoid legumes and holoparasites of the genus Lophophytum represents an outstanding opportunity to discern HGT events. The mitochondrial genome of the holoparasite L. mirabile has remarkable properties, the most extraordinary of which is the presence of 34 out of 43 mitochondrial protein genes acquired from its legume host, with the stunning replacement of up to 26 native homologs. However, the origin of the intergenic sequences that represent the majority (>90%) of the L. mirabile mtDNA remains largely unknown. The lack of mitochondrial sequences available from the donor angiosperm lineage (mimosoid legumes) precluded a large-scale evolutionary study. We sequenced and assembled the mitochondrial genome of the mimosoid Acacia ligulata and performed genome wide comparisons with L. mirabile. The A. ligulata mitochondrial genome is almost 700 kb in size, encoding 60 genes. About 60% of the L. mirabile mtDNA had greatest affinity to members of the family Fabaceae (∼49% to mimosoids in particular) with an average sequence identity of ∼96%, including genes but mostly intergenic regions. These findings strengthen the mitochondrial fusion compatibility model for angiosperm mitochondrion-to-mitochondrion HGT.

Towards a comprehensive picture of C-to-U RNA editing sites in angiosperm mitochondria.

KEY MESSAGE: Our understanding of the dynamic and evolution of RNA editing in angiosperms is in part limited by the few editing sites identified to date. This study identified 10,217 editing sites from 17 diverse angiosperms. Our analyses confirmed the universality of certain features of RNA editing, and offer new evidence behind the loss of editing sites in angiosperms. RNA editing is a post-transcriptional process that substitutes cytidines (C) for uridines (U) in organellar transcripts of angiosperms. These substitutions mostly take place in mitochondrial messenger RNAs at specific positions called editing sites. By means of publicly available RNA-seq data, this study identified 10,217 editing sites in mitochondrial protein-coding genes of 17 diverse angiosperms. Even though other types of mismatches were also identified, we did not find evidence of non-canonical editing processes. The results showed an uneven distribution of editing sites among species, genes, and codon positions. The analyses revealed that editing sites were conserved across angiosperms but there were some species-specific sites. Non-synonymous editing sites were particularly highly conserved (~ 80%) across the plant species and were efficiently edited (80% editing extent). In contrast, editing sites at third codon positions were poorly conserved (~ 30%) and only partially edited (~ 40% editing extent). We found that the loss of editing sites along angiosperm evolution is mainly occurring by replacing editing sites with thymidines, instead of a degradation of the editing recognition motif around editing sites. Consecutive and highly conserved editing sites had been replaced by thymidines as result of retroprocessing, by which edited transcripts are reverse transcribed to cDNA and then integrated into the genome by homologous recombination. This phenomenon was more pronounced in eudicots, and in the gene cox1. These results suggest that retroprocessing is a widespread driving force underlying the loss of editing sites in angiosperm mitochondria.

Metazoan Ribosome Inactivating Protein encoding genes acquired by Horizontal Gene Transfer.

Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes are widely distributed among plants and their presence has also been confirmed in several bacterial species. Recently, we reported for the first time in silico evidence of RIP encoding genes in metazoans, in two closely related species of insects: Aedes aegypti and Culex quinquefasciatus. Here, we have experimentally confirmed the presence of these genes in mosquitoes and attempted to unveil their evolutionary history. A detailed study was conducted, including evaluation of taxonomic distribution, phylogenetic inferences and microsynteny analyses, indicating that mosquito RIP genes derived from a single Horizontal Gene Transfer (HGT) event, probably from a cyanobacterial donor species. Moreover, evolutionary analyses show that, after the HGT event, these genes evolved under purifying selection, strongly suggesting they play functional roles in these organisms.

Dissimilar evolutionary histories of two resistance gene families in the genus Solanum.

Genomic analyses have shown that most genes in eukaryotic lineages belong to families. Gene families vary in terms of number of members, nucleotide similarity, gene integrity, expression, and function. Often, the members of gene families are arranged in clusters, which contribute to maintaining similarity among gene copies and also to generate duplicates through replication errors. Gene families offer us an opportunity to examine the forces involved in the evolution of the genomes and to study recombination events and genomic rearrangements. In this work, we focused on the evolution of two plant resistance gene families, Sw5 and Mi-1, and analyzed the completely sequenced nuclear genomes of potato and tomato. We first noticed that the potato genome carries larger resistance gene families than tomato, but all gene copies are pseudogenes. Second, phylogenetic analyses indicated that Sw5 and Mi-1 gene families had dissimilar evolutionary histories. In contrast to Sw5, Mi-1 homologues suffered repeated gene conversion events among the gene copies, particularly in the tomato genome.

Unparalleled replacement of native mitochondrial genes by foreign homologs in a holoparasitic plant.

Horizontal gene transfer (HGT) among flowering plant mitochondria occurs frequently and, in most cases, leads to nonfunctional transgenes in the recipient genome. Parasitic plants are particularly prone to this phenomenon, but their mitochondrial genomes (mtDNA) have been largely unexplored. We undertook a large-scale mitochondrial genomic study of the holoparasitic plant Lophophytum mirabile (Balanophoraceae). Comprehensive phylogenetic analyses were performed to address the frequency, origin, and impact of HGT. The sequencing of the complete mtDNA of L. mirabile revealed the unprecedented acquisition of host-derived mitochondrial genes, representing 80% of the protein-coding gene content. All but two of these foreign genes replaced the native homologs and are probably functional in energy metabolism. The genome consists of 54 circular-mapping chromosomes, 25 of which carry no intact genes. The likely functional replacement of up to 26 genes in L. mirabile represents a stunning example of the potential effect of rampant HGT on plant mitochondria. The use of host-derived genes may have a positive effect on the host-parasite relationship, but could also be the result of nonadaptive forces.

Comparative and evolutionary analyses of Meloidogyne spp. Based on mitochondrial genome sequences.

Molecular taxonomy and evolution of nematodes have been recently the focus of several studies. Mitochondrial sequences were proposed as an alternative for precise identification of Meloidogyne species, to study intraspecific variability and to follow maternal lineages. We characterized the mitochondrial genomes (mtDNAs) of the root knot nematodes M. floridensis, M. hapla and M. incognita. These were AT rich (81-83%) and highly compact, encoding 12 proteins, 2 rRNAs, and 22 tRNAs. Comparisons with published mtDNAs of M. chitwoodi, M. incognita (another strain) and M. graminicola revealed that they share protein and rRNA gene order but differ in the order of tRNAs. The mtDNAs of M. floridensis and M. incognita were strikingly similar (97-100% identity for all coding regions). In contrast, M. floridensis, M. chitwoodi, M. hapla and M. graminicola showed 65-84% nucleotide identity for coding regions. Variable mitochondrial sequences are potentially useful for evolutionary and taxonomic studies. We developed a molecular taxonomic marker by sequencing a highly-variable ~2 kb mitochondrial region, nad5-cox1, from 36 populations of root-knot nematodes to elucidate relationships within the genus Meloidogyne. Isolates of five species formed monophyletic groups and showed little intraspecific variability. We also present a thorough analysis of the mitochondrial region cox2-rrnS. Phylogenies based on either mitochondrial region had good discrimination power but could not discriminate between M. arenaria, M. incognita and M. floridensis.