Plants contain organelles called chloroplasts, which is where photosynthesis takes place. Chloroplasts also contain their own DNA, which is separate from the DNA in the nucleus. This DNA does not change much over evolutionary time, so it can be used to investigate relationships between organisms. Here we created a tool that makes it easier to analyze this chloroplast DNA as well making it easier to share complete chloroplast genomes on public databases. In addition, we also found a mobile element in the chloroplast DNA of a group of ferns, which appears to be driving structural changes in their genomes.

Organellar genomes are remnants of more complex bacterial genomes reduced until reach the simplest and most efficient content. Regularly depicted as circular, these genomes can form other structures, like linear, ramified, or entangled chromosomes, or a combination of those. Nonetheless, their gene content is nearly constant throughout flowering plants based on the multiple plastid genomes (plastomes) and the comparatively few mitochondrial genomes (mitogenomes) sequenced to date. Here, I explored the evolution of the organellar genomes in orchids from a phylogenetic perspective. For this research, plastomes and mitogenomes were assembled from short pair-ended reads obtained using Illumina sequencing technology. I developed a workflow to confidently recover plastid and mitochondrial sequences, even for regions without references in databases (chapter 1). The comparison among taxa from all orchid subfamilies identified patterns of gain, loss and rearrangement of coding and non-coding DNA. Plastid and mitochondrial protein-coding genes present in all samples were used to reconstruct the phylogenetic history of orchids that was coincident in terms of topology (chapter 1). Plastomes can suffer degradation in heterotrophic species, however that is not true for mixotrophic species, as I discovered by comparing albino and green individuals of the orchid Epipactis helleborine. I found that albino plants did not suffer loss of any genes and that the sequence was almost identical to the photosynthetic plants (chapter 2). In contrast to what it is observed in angiosperm plastomes, for which the structure, content and size is conserved, plant mitogenomes are highly variable in size, which can increase by the acquisition of external DNA via horizontal gene transfer. In some orchids, the mitogenome hosts a sixteen-gene sequence transferred from a fungal mitogenome to a clade of epidendroid orchids 12-60 My ago, and has been fragmented, conserved, or fully lost since (chapter 3). Transfer RNA genes are variable in number and origin throughout orchid evolution. I identified that they had four different sources, three novel possible replacement events of the native genes with plastid-origin genes, seven tRNA remodeling events in orchids and three more in other angiosperms (chapter 4). Our comparative studies conclude that there are three main dynamics that shape the organellar genomes: gain, loss and rearrangement of genomic content. I presented examples of them in orchids (chapter 5). Additionally, I created two sets of genomic resources: one comprises eighteen new orchid mitogenomes and plastomes, and the second consists of a well-curated set of references of tRNA genes in mitogenomes discriminated by origin. These results contribute to increasing the knowledge of angiosperm organellar genomes and highlight the importance of comprehensive studies that allow the interpretation of the genomic changes in the light of the phylogenetic evolution.

Im Rahmen des ersten Teils der vorliegenden Doktorarbeit konnten zwei nicht-essentielle (rps15, rpl36) und fünf essentielle (rps3, rps16, rpl22, rpl23, rpl32) im Plastom von Nicotiana tabacum kodierte Proteine des plastidären Ribosoms bezüglich ihrer Essentialität charakterisiert werden. Diese Gene wurden durch gezielte Knockout-Experimente inaktiviert und die resultierenden Effekte untersucht. Die Ergebnisse lassen einen Rückschluss auf die Lokalisation der Gene der insgesamt sieben untersuchten ribosomalen Proteine zu, die im Plastom mehrerer parasitischer, Plastiden-besitzender Spezies nicht mehr nachweisbar sind. Im Fall von rps15 könnte tatsächlich ein Verlust des Genes stattgefunden haben, im Fall der restlichen Gene ist eher mit einem Transfer in den Nukleus zu rechnen (rpl36 ausgenommen). Dies würde bedeuten, dass die Geschwindigkeit der erfolgreichen Etablierung eines Gentransfers in vielen parasitischen Spezies gegenüber grünen Pflanzen stark erhöht ist. Alle in E. coli nicht-essentiellen Proteine mit Homologen in Plastiden (rps15, rpl33, rpl36) sind auch dort, trotz ~1,5 Milliarden Jahren getrennter Evolution, nicht essentiell. Dieses Ergebnis bestätigt den schon früher festgestellten hohen Konservierungsgrad der bakteriellen und plastidären Translationsmaschinerien. Die Phänotypen der KO-Pflanzen der nicht-essentiellen Gene (rps15, rpl36) weisen auf eine interessante Rolle von S15 während der Ribosomenassemblierung hin und im Fall von L36 auf eine wichtige funktionelle Rolle im Plastiden-Ribosomen sowie auf eine Involvierung der Plastidentranslation in der Generierung eines retrograden Signals, welches die Blattform zu beeinflussen im Stande ist. Des Weiteren konnte eine Verbindung der Translationsaktivität mit der Ausbildung von Seitentrieben hergestellt werden, die vermutlich auf veränderte Auxinsynthese im Chloroplast zurückzuführen ist. Aus dem Folgeprojekt, bei dem Doppel-KO-Pflanzen nicht-essentieller ribosomaler Proteine erzeugt wurden, lässt sich auf eine relativ große Plastizität der Architektur von Plastidenribosomen schließen. Im zweiten Teil der Arbeit konnte erfolgreich ein Hochdurchsatz-Screeningsystem zur semiquantitativen Analyse von 192 verschiedenen miRNAs aus Chlamydomonas reinhardtii etabliert werden. Es gelang durch die Untersuchung von 23 verschiedenen Wachstums- und Stressbedingungen sowie Entwicklungsstadien mehrere miRNAs zu identifizieren, die eine differenzielle Expression zeigen sowie unter allen untersuchten Bedingungen konstant bleibende miRNAs nachzuweisen. Dadurch konnten mehrere vielversprechende Kandidaten-miRNAs ausgemacht werden, die nun eingehender untersucht werden können. / Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway. In the second project a qRT-PCR based plattform for the analysis of miRNAs in Chlamydomonas reinhardtii has been developed. 20 different growth conditions have been scanned.

Penstemon is one of North America's largest endemic genera with over 280 described species. These species are distributed throughout most of North America from the Arctic northern latitudes to tropics of Central America. The genus has historically been divided into six subgenera, but has recently been reorganized into two subgenera following some recent phylogenetic studies. I made a comprehensive assessment of the Penstemon genus' geographic distribution utilizing herbaria databases by ecoregion to discuss the general ecologic adaptations of each historic subgenera. I also assessed the Penstemon genus' bee pollinator diversity utilizing online databases of bee specimen collections associated with Penstemon flowers. I investigated the efficacy of utilizing the plastid genomes (plastomes) of 29 species in the Lamiales order, including five newly sequenced Penstemon plastomes, for analyzing phylogenetic relationships and resolving problematic clades. I compared whole-plastome based phylogenies to phylogenies based on individual gene sequences (matK, ndhF, psaA, psbA, rbcL, rpoC2, and rps2) and concatenated sequences. I found that my whole-plastome based phylogeny had higher nodal support than all other phylogenies, which suggests that it provides greater accuracy in describing the hierarchal relationships among taxa as compared to other methods. I found that the genus Penstemon forms a monophyletic clade sister to, but separate from, the Old World taxa of the Plantaginaceae family included in our study. My whole-plastome based phylogeny also supports the rearrangement of the Scrophulariaceae family and improves resolution of major clades and genera of the Lamiales. I evaluated 16 accessions of P. cyaneus with 14 accessions of closely related Penstemon species in common garden in two distinct environments in Aberdeen, ID and Provo, UT during 2018 and 2019. I evaluated the accessions for key commercial seed production traits including survival, plant height, number of stems, and seed production. Both common gardens received supplemental irrigation during 2018, but I withheld irrigation during 2019. Plant survival in our Aberdeen, ID site was not significantly different between years, but survival was a significantly lower in 2019 than in 2018 at our Provo, UT site. The mean survival for P. cyaneus accessions ranged from 56% to 94%, and the mean seed production ranged from 91.2 kg/ha to 397.6 kg/ha. I recommend developing a commercial seed source derived from pooling germplasm of six accessions (PECY3-367, PECY3-371, PECY3-376, PECY3-443, PECY3-457, and PECY3-458). These accessions had mean survival rates of 82-94%, and seed production of 196.2-397.6 kg/ha.

A delimitação de espécies é um aspecto de fundamental importância dentro da Biologia Evolutiva, bem como para a conservação da biodiversidade. No entanto, delimitar espécies com base em morfologia é extremamente complicado, especialmente em grupos que tiveram uma radiação recente e que apresentam pouca descontinuidade morfológica entre os táxons, como a tribo Andropogoneae (Poaceae). A presente tese utiliza sequências de DNA, como genes nucleares de cópia-única (low-copy nuclear loci) e sequenciamento completo do plastoma, para resolver a circunscrição de complexos de espécies em Andropogoneae, particularmente dos gêneros Saccharum e Eriochrysis, bem como para investigar o posicionamento filogenético dos mesmos em relação aos demais gêneros da tribo. Aspectos filogenéticos, taxonômicos e nomenclaturais foram investigados. Do ponto de vista nomenclatural, foi possível esclarecer que Andropogoneae Dumortier é o nome correto para a tribo, e não Sacchareae Martinov, como recentemente sugerido por diversos autores. As análises filogenéticas realizadas corroboram a hipótese de uma diversificação inicial rápida em Andropogoneae e a não monofilia da subtribo Saccharinae. A origem alopoliploide do gênero Saccharum foi demonstrada a partir de evidências de genes nucleares. Saccharum s.l. é polifilético e Tripidium deve ser reconhecido como um gênero distinto. As análises filogenéticas também foram capazes de resolver a circunscrição interna de Saccharum s.l., confirmando a ocorrência de três espécies nativas na América do Sul: S. angustifolium, S. asperum e S. villosum. A ocorrência de híbridos naturais entre S. villosum e S. angustifolium foi documentada. As análises filogenéticas de Eriochrysis confirmaram a monofilia do gênero e resolveram a circunscrição de suas espécies: E. villosa é um táxon distinto de E. cayennensis, bem como E. laxa é uma espécie distinta de E. warmingiana. Híbridos naturais entre E. laxa e E. villosa também foram documentados. Eriochrysis villosa é citada pela primeira vez para o Uruguai e E. laxa para o estado do Rio Grande do Sul. A presente tese demonstrou a eficiência dos genes nucleares de cópia única na delimitação de espécies e gêneros da tribo Andropogoneae, mesmo na presença de poliploidia, evolução reticulada e radiação recente. O sequenciamento completo do plastoma também se mostrou uma ferramenta extremamente promissora para inferências filogenéticas em Andropogoneae. / Species delimitation is a vital issue concerning evolutionary biology and conservation of biodiversity. However, delimiting species based on morphology is a difficult task especially in plant groups with an evolutionary history involving rapid radiation and little morphological discontinuity between taxa, as the tribe Andropogoneae (Poaceae). The present thesis uses DNA sequences, such as low-copy nuclear genes and complete plastome sequencing, to resolve the taxonomic circumscriptions of species complexes in Andropogoneae, particularly from genera Saccharum and Eriochrysis, and to investigate their phylogenetic affinities to other genera of the tribe. Phylogenetic, taxonomic, and nomenclatural aspects were investigated. We clarified that Andropogoneae Dumortier is the correct name for the tribe, rather than Sacchareae Martinov, as recently suggested by several authors. The present phylogenetic analyses support the hypothesis of an initial rapid diversification in Andropogoneae and the non-monophyly of subtribe Saccharinae. The allopolyploid origin of Saccharum was demonstrated using evidence from nuclear genes. Saccharum s.l. is polyphyletic and Tripidium should be recognized as a distinct genus. The phylogenetic analyses were also able to define the circumscriptions of the species of Saccharum s.l., confirming the occurrence of three native species in South America: S. angustifolium, S. asperum and S. villosum. The occurrence of natural hybrids between S. villosum and S. angustifolium was documented. The phylogenetic analyses of Eriochrysis confirmed the monophyly of the genus and resolved the circumscriptions of its species: E. villosa is distinct from E. cayennensis, and E. laxa is distinct from E. warmingiana. Natural hybrids between E. laxa and E. villosa were also documented. Eriochrysis villosa is reported here for the first time for Uruguay and E. laxa for the State of Rio Grande do Sul. The present thesis has demonstrated the efficiency of low-copy nuclear genes in the delimitation of species and genera from tribe Andropogoneae, even in presence of polyploidy, reticulate evolution and recent radiation. The complete plastome sequencing is also a promising tool for phylogenetic inferences in Andropogoneae.

A delimitação de espécies é um aspecto de fundamental importância dentro da Biologia Evolutiva, bem como para a conservação da biodiversidade. No entanto, delimitar espécies com base em morfologia é extremamente complicado, especialmente em grupos que tiveram uma radiação recente e que apresentam pouca descontinuidade morfológica entre os táxons, como a tribo Andropogoneae (Poaceae). A presente tese utiliza sequências de DNA, como genes nucleares de cópia-única (low-copy nuclear loci) e sequenciamento completo do plastoma, para resolver a circunscrição de complexos de espécies em Andropogoneae, particularmente dos gêneros Saccharum e Eriochrysis, bem como para investigar o posicionamento filogenético dos mesmos em relação aos demais gêneros da tribo. Aspectos filogenéticos, taxonômicos e nomenclaturais foram investigados. Do ponto de vista nomenclatural, foi possível esclarecer que Andropogoneae Dumortier é o nome correto para a tribo, e não Sacchareae Martinov, como recentemente sugerido por diversos autores. As análises filogenéticas realizadas corroboram a hipótese de uma diversificação inicial rápida em Andropogoneae e a não monofilia da subtribo Saccharinae. A origem alopoliploide do gênero Saccharum foi demonstrada a partir de evidências de genes nucleares. Saccharum s.l. é polifilético e Tripidium deve ser reconhecido como um gênero distinto. As análises filogenéticas também foram capazes de resolver a circunscrição interna de Saccharum s.l., confirmando a ocorrência de três espécies nativas na América do Sul: S. angustifolium, S. asperum e S. villosum. A ocorrência de híbridos naturais entre S. villosum e S. angustifolium foi documentada. As análises filogenéticas de Eriochrysis confirmaram a monofilia do gênero e resolveram a circunscrição de suas espécies: E. villosa é um táxon distinto de E. cayennensis, bem como E. laxa é uma espécie distinta de E. warmingiana. Híbridos naturais entre E. laxa e E. villosa também foram documentados. Eriochrysis villosa é citada pela primeira vez para o Uruguai e E. laxa para o estado do Rio Grande do Sul. A presente tese demonstrou a eficiência dos genes nucleares de cópia única na delimitação de espécies e gêneros da tribo Andropogoneae, mesmo na presença de poliploidia, evolução reticulada e radiação recente. O sequenciamento completo do plastoma também se mostrou uma ferramenta extremamente promissora para inferências filogenéticas em Andropogoneae. / Species delimitation is a vital issue concerning evolutionary biology and conservation of biodiversity. However, delimiting species based on morphology is a difficult task especially in plant groups with an evolutionary history involving rapid radiation and little morphological discontinuity between taxa, as the tribe Andropogoneae (Poaceae). The present thesis uses DNA sequences, such as low-copy nuclear genes and complete plastome sequencing, to resolve the taxonomic circumscriptions of species complexes in Andropogoneae, particularly from genera Saccharum and Eriochrysis, and to investigate their phylogenetic affinities to other genera of the tribe. Phylogenetic, taxonomic, and nomenclatural aspects were investigated. We clarified that Andropogoneae Dumortier is the correct name for the tribe, rather than Sacchareae Martinov, as recently suggested by several authors. The present phylogenetic analyses support the hypothesis of an initial rapid diversification in Andropogoneae and the non-monophyly of subtribe Saccharinae. The allopolyploid origin of Saccharum was demonstrated using evidence from nuclear genes. Saccharum s.l. is polyphyletic and Tripidium should be recognized as a distinct genus. The phylogenetic analyses were also able to define the circumscriptions of the species of Saccharum s.l., confirming the occurrence of three native species in South America: S. angustifolium, S. asperum and S. villosum. The occurrence of natural hybrids between S. villosum and S. angustifolium was documented. The phylogenetic analyses of Eriochrysis confirmed the monophyly of the genus and resolved the circumscriptions of its species: E. villosa is distinct from E. cayennensis, and E. laxa is distinct from E. warmingiana. Natural hybrids between E. laxa and E. villosa were also documented. Eriochrysis villosa is reported here for the first time for Uruguay and E. laxa for the State of Rio Grande do Sul. The present thesis has demonstrated the efficiency of low-copy nuclear genes in the delimitation of species and genera from tribe Andropogoneae, even in presence of polyploidy, reticulate evolution and recent radiation. The complete plastome sequencing is also a promising tool for phylogenetic inferences in Andropogoneae.

A delimitação de espécies é um aspecto de fundamental importância dentro da Biologia Evolutiva, bem como para a conservação da biodiversidade. No entanto, delimitar espécies com base em morfologia é extremamente complicado, especialmente em grupos que tiveram uma radiação recente e que apresentam pouca descontinuidade morfológica entre os táxons, como a tribo Andropogoneae (Poaceae). A presente tese utiliza sequências de DNA, como genes nucleares de cópia-única (low-copy nuclear loci) e sequenciamento completo do plastoma, para resolver a circunscrição de complexos de espécies em Andropogoneae, particularmente dos gêneros Saccharum e Eriochrysis, bem como para investigar o posicionamento filogenético dos mesmos em relação aos demais gêneros da tribo. Aspectos filogenéticos, taxonômicos e nomenclaturais foram investigados. Do ponto de vista nomenclatural, foi possível esclarecer que Andropogoneae Dumortier é o nome correto para a tribo, e não Sacchareae Martinov, como recentemente sugerido por diversos autores. As análises filogenéticas realizadas corroboram a hipótese de uma diversificação inicial rápida em Andropogoneae e a não monofilia da subtribo Saccharinae. A origem alopoliploide do gênero Saccharum foi demonstrada a partir de evidências de genes nucleares. Saccharum s.l. é polifilético e Tripidium deve ser reconhecido como um gênero distinto. As análises filogenéticas também foram capazes de resolver a circunscrição interna de Saccharum s.l., confirmando a ocorrência de três espécies nativas na América do Sul: S. angustifolium, S. asperum e S. villosum. A ocorrência de híbridos naturais entre S. villosum e S. angustifolium foi documentada. As análises filogenéticas de Eriochrysis confirmaram a monofilia do gênero e resolveram a circunscrição de suas espécies: E. villosa é um táxon distinto de E. cayennensis, bem como E. laxa é uma espécie distinta de E. warmingiana. Híbridos naturais entre E. laxa e E. villosa também foram documentados. Eriochrysis villosa é citada pela primeira vez para o Uruguai e E. laxa para o estado do Rio Grande do Sul. A presente tese demonstrou a eficiência dos genes nucleares de cópia única na delimitação de espécies e gêneros da tribo Andropogoneae, mesmo na presença de poliploidia, evolução reticulada e radiação recente. O sequenciamento completo do plastoma também se mostrou uma ferramenta extremamente promissora para inferências filogenéticas em Andropogoneae. / Species delimitation is a vital issue concerning evolutionary biology and conservation of biodiversity. However, delimiting species based on morphology is a difficult task especially in plant groups with an evolutionary history involving rapid radiation and little morphological discontinuity between taxa, as the tribe Andropogoneae (Poaceae). The present thesis uses DNA sequences, such as low-copy nuclear genes and complete plastome sequencing, to resolve the taxonomic circumscriptions of species complexes in Andropogoneae, particularly from genera Saccharum and Eriochrysis, and to investigate their phylogenetic affinities to other genera of the tribe. Phylogenetic, taxonomic, and nomenclatural aspects were investigated. We clarified that Andropogoneae Dumortier is the correct name for the tribe, rather than Sacchareae Martinov, as recently suggested by several authors. The present phylogenetic analyses support the hypothesis of an initial rapid diversification in Andropogoneae and the non-monophyly of subtribe Saccharinae. The allopolyploid origin of Saccharum was demonstrated using evidence from nuclear genes. Saccharum s.l. is polyphyletic and Tripidium should be recognized as a distinct genus. The phylogenetic analyses were also able to define the circumscriptions of the species of Saccharum s.l., confirming the occurrence of three native species in South America: S. angustifolium, S. asperum and S. villosum. The occurrence of natural hybrids between S. villosum and S. angustifolium was documented. The phylogenetic analyses of Eriochrysis confirmed the monophyly of the genus and resolved the circumscriptions of its species: E. villosa is distinct from E. cayennensis, and E. laxa is distinct from E. warmingiana. Natural hybrids between E. laxa and E. villosa were also documented. Eriochrysis villosa is reported here for the first time for Uruguay and E. laxa for the State of Rio Grande do Sul. The present thesis has demonstrated the efficiency of low-copy nuclear genes in the delimitation of species and genera from tribe Andropogoneae, even in presence of polyploidy, reticulate evolution and recent radiation. The complete plastome sequencing is also a promising tool for phylogenetic inferences in Andropogoneae.

La tribu d'Oleeae (Oleaceae) est un modèle biologique très intéressant pour étudier la diversification et les patterns d'évolution moléculaire chez les plantes. Les reconstitutions de l'histoire évolutive et phylogénétique des relations entre ses espèces ont été au cœur de cette thèse. Ce travail a conduit à des progrès significatifs dans la résolution des relations phylogénétiques au sein de la tribu à différents niveaux taxonomiques. Nous avons démontré que l'utilisation d'une approche de "Genome Skimming" est très appropriée pour générer plastomes complets (ptDNA) et de l'ADN ribosomal nucléaire (nrDNA), même sur un échantillon d'herbier. Nous avons montré l'utilité du ptDNA pour reconstruire la première ossature phylogénétique robuste pour la tribu, et fourni de nouvelles connaissances sur l'histoire biogéographique et sur l'évolution de certains traits. Nous avons aussi montré que l'évolution de la composition en bases du nrDNA peut être influencée par des facteurs climatiques. / The Oleeae tribe (Oleaceae) is a very interesting biological model to investigate plant diversification and molecular evolution patterns. However, a comprehensive phylogeny is missing to accurately describe the evolutionary and biogeographic history of this tribe. Reconstructions the Oleeae evolutionary history and the phylogenetic relationships between its species were the core of this thesis. This work has led to significant progress in resolving the phylogenetic relationships within the tribe at different taxonomic levels. We demonstrated that the use of a shotgun approach is a highly suitable method to generate complete plastomes (ptDNA) and nuclear ribosomal DNA (nrDNA), even on herbarium sample. We showed the usefulness of ptDNA to reconstruct highly resolved phylogeny of Oleeae and provided new insights into the biogeographic history and the evolution of some traits. We also showed that the evolution of nrDNA base-composition seems to be influenced by environmental factors.

No description available.

Plastid genomes of photosynthetic flowering plants are usually highly conserved in both structure and gene content. However, the plastomes of parasitic and mycoheterotrophic plants may be released from selective constraint due to the reduction or loss of photosynthetic ability. Here we present the greatly reduced and highly divergent, yet functional, plastome of the nonphotosynthetic holoparasite Hydnora visseri (Hydnoraceae, Piperales). The plastome is 27 kb in length, with 24 genes encoding ribosomal proteins, ribosomal RNAs, tRNAs and a few non-bioenergetic genes, but no genes related to photosynthesis. The inverted repeat and the small single copy region are only ~1.5 kb, and intergenic regions have been drastically reduced. Despite extreme reduction, gene order and orientation are highly similar to the plastome of Piper cenocladum, a related photosynthetic plant in Piperales. Gene sequences in Hydnora are highly divergent and several complementary approaches using the highest possible sensitivity were required for identification and annotation of this plastome. Active transcription is detected for all of the protein coding genes in the plastid genome, and one of two introns is appropriately spliced out of rps12 transcripts. The whole genome shotgun read depth is 1,400X coverage for the plastome, while the mitochondrial genome is covered at 40X and the nuclear genome at 2X. Despite the extreme reduction of the genome and high sequence divergence, the presence of syntenic, long transcriptionally-active open reading frames with distant similarity to other plastid genomes and a high plastome stoichiometry relative to the mitochondrial and nuclear genomes suggests that the plastome remains functional in Hydnora visseri. A four stage model of gene reduction, including the potential for complete plastome loss, is proposed to account for the range of plastid genomes in nonphotosynthetic plants.