Parasitism in plants is often accompanied by a suite of morphological and physiological changes resulting in a condition known as the ‘parasitic reduction syndrome’. With changes including extreme vegetative reduction, frequently beyond any resemblance to its photosynthetic relatives, accompanied by significant losses of genes linked to photosynthesis, the study of parasitic plants can be challenging. Conopholis (Orobanchaceae) is a small holoparasitic genus distributed across eastern and southwestern North America and Central America. This genus has never been the subject of a molecular phylogenetic or morphometric analyses. In addition, very little is known of the relationships among populations and of their post-glacial history. To investigate the species limits and phylogenetic relationships in Conopholis, we conducted a comprehensive molecular phylogenetic study of the genus as well as a fine-scale morphometric study. Based on plastid and nuclear sequences, Conopholis was found to contain three distinct and well-supported lineages which have varying degrees of overlap with previously proposed taxa. The clustering and ordination analyses of the morphometric study corroborated the molecular data, demonstrating the morphological differentiation between the three lineages detected within Conopholis. A taxonomic re-alignment is proposed for the genus that recognizes three species, C. americana, C. panamensis, and C. alpina. To address genetic diversity and phylogeographic structure of C. americana in eastern North America, microsatellite markers were developed and characterized for the first time in this species. Using these newly generated markers along with sequences from the plastid genome, the persistence of a minimum of two glacial refugia at the last glacial maximum were inferred, one in Florida and southern Alabama and another in the Appalachian Mountains near the southern tip of Blue Ridge Mountains. The diversity seen across the southern Appalachian Mountains supports the hypothesis that populations derived from the southern and northern refugia come together in this area.

Parasitism in plants is often accompanied by a suite of morphological and physiological changes resulting in a condition known as the ‘parasitic reduction syndrome’. With changes including extreme vegetative reduction, frequently beyond any resemblance to its photosynthetic relatives, accompanied by significant losses of genes linked to photosynthesis, the study of parasitic plants can be challenging. Conopholis (Orobanchaceae) is a small holoparasitic genus distributed across eastern and southwestern North America and Central America. This genus has never been the subject of a molecular phylogenetic or morphometric analyses. In addition, very little is known of the relationships among populations and of their post-glacial history. To investigate the species limits and phylogenetic relationships in Conopholis, we conducted a comprehensive molecular phylogenetic study of the genus as well as a fine-scale morphometric study. Based on plastid and nuclear sequences, Conopholis was found to contain three distinct and well-supported lineages which have varying degrees of overlap with previously proposed taxa. The clustering and ordination analyses of the morphometric study corroborated the molecular data, demonstrating the morphological differentiation between the three lineages detected within Conopholis. A taxonomic re-alignment is proposed for the genus that recognizes three species, C. americana, C. panamensis, and C. alpina. To address genetic diversity and phylogeographic structure of C. americana in eastern North America, microsatellite markers were developed and characterized for the first time in this species. Using these newly generated markers along with sequences from the plastid genome, the persistence of a minimum of two glacial refugia at the last glacial maximum were inferred, one in Florida and southern Alabama and another in the Appalachian Mountains near the southern tip of Blue Ridge Mountains. The diversity seen across the southern Appalachian Mountains supports the hypothesis that populations derived from the southern and northern refugia come together in this area.

Plastome condensation during adaptation to a heterotrophic lifestyle is generally well understood and lineage-independent models have been derived. However, understanding the evolutionary trajectories of comparatively old heterotrophic lineages that are on the cusp of a minimal plastome, is essential to complement and expand current knowledge. We study Hydnoraceae, one of the oldest and least investigated parasitic angiosperm lineages. Plastome comparative genomics, using seven out of eight known species of the genus Hydnora and three species of Prosopanche, reveal a high degree of structural similarity and shared gene content; contrasted by striking dissimilarities with respect to repeat content [inverted and direct repeats (DRs)]. We identified varying inverted repeat contents and positions, likely resulting from multiple, independent evolutionary events, and a DR gain in Prosopanche. Considering different evolutionary trajectories and based on a fully resolved and supported species-level phylogenetic hypothesis, we describe three possible, distinct models to explain the Hydnoraceae plastome states. For comparative purposes, we also report the first plastid genomes for the closely related autotrophic genera Lactoris (Lactoridaceae) and Thottea (Aristolochiaceae).

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.

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.