A comparative study across the parasitic plants of Cuscuta subgenus Grammica (Convolvulaceae) reveals a possible loss of the plastid genome in its section Subulatae.

MAIN CONCLUSION: Most species in Cuscuta subgenus Grammica retain many photosynthesis-related plastid genes, generally under purifying selection. A group of holoparasitic species in section Subulatae may have lost their plastid genomes entirely. The c. 153 species of plants belonging to Cuscuta subgenus Grammica are all obligate stem parasites. However, some have completely lost the ability to conduct photosynthesis while others retain photosynthetic machinery and genes. The plastid genome that primarily encodes key photosynthesis genes functions as a bellwether for how reliant plants are on primary production. This research assembles and analyses 17 plastomes across Cuscuta subgenus Grammica with the aim of characterizing the state of the plastome in each of its sections. By comparing the structure and content of plastid genomes across the subgenus, as well as by quantifying the selection acting upon each gene, we reconstructed the patterns of plastome change within the phylogenetic context for this group. We found that species in 13 of the 15 sections that comprise Grammica retain the bulk of plastid photosynthesis genes and are thus hemiparasitic. The complete loss of photosynthesis can be traced to two clades: the entire section Subulatae and a complex of three species within section Ceratophorae. We were unable to recover any significant plastome sequences from section Subulatae, suggesting that plastomes in these species are either drastically reduced or lost entirely.

A Bird's Eye View of the Systematics of Convolvulaceae: Novel Insights From Nuclear Genomic Data.

Convolvulaceae is a family of c. 2,000 species, distributed across 60 currently recognized genera. It includes species of high economic importance, such as the crop sweet potato (Ipomoea batatas L.), the ornamental morning glories (Ipomoea L.), bindweeds (Convolvulus L.), and dodders, the parasitic vines (Cuscuta L.). Earlier phylogenetic studies, based predominantly on chloroplast markers or a single nuclear region, have provided a framework for systematic studies of the family, but uncertainty remains at the level of the relationships among subfamilies, tribes, and genera, hindering evolutionary inferences and taxonomic advances. One of the enduring enigmas has been the relationship of Cuscuta to the rest of Convolvulaceae. Other examples of unresolved issues include the monophyly and relationships within Merremieae, the "bifid-style" clade (Dicranostyloideae), as well as the relative positions of Erycibe Roxb. and Cardiochlamyeae. In this study, we explore a large dataset of nuclear genes generated using Angiosperms353 kit, as a contribution to resolving some of these remaining phylogenetic uncertainties within Convolvulaceae. For the first time, a strongly supported backbone of the family is provided. Cuscuta is confirmed to belong within family Convolvulaceae. "Merremieae," in their former tribal circumscription, are recovered as non-monophyletic, with the unexpected placement of Distimake Raf. as sister to the clade that contains Ipomoeeae and Decalobanthus Ooststr., and Convolvuleae nested within the remaining "Merremieae." The monophyly of Dicranostyloideae, including Jacquemontia Choisy, is strongly supported, albeit novel relationships between genera are hypothesized, challenging the current tribal delimitation. The exact placements of Erycibe and Cuscuta remain uncertain, requiring further investigation. Our study explores the benefits and limitations of increasing sequence data in resolving higher-level relationships within Convolvulaceae, and highlights the need for expanded taxonomic sampling, to facilitate a much-needed revised classification of the family.

Mitochondrial phylogenomics of Cuscuta (Convolvulaceae) reveals a potentially functional horizontal gene transfer from the host.

Horizontal gene transfers (HGTs) from host or other organisms have been reported in mitochondrial genomes of parasitic plants. Genes transferred in this fashion have usually been found non-functional. Several examples of HGT from the mitochondrial genome of parasitic Cuscuta (Convolvulaceae) to its hosts have been reported, but not vice versa. Here we used 31 protein-coding mitochondrial genes to infer the phylogeny of Cuscuta, and compared it with previous nuclear and plastid phylogenetic estimates. We also investigated the presence of HGTs within these lineages. Unlike in plastid genomes, we did not find extensive gene loss in their mitochondrial counterparts. Our results reveal the first example of organellar HGT from host to Cuscuta. Mitochondrial atp1 genes of South African subgenus Pachystigma were inferred to be transferred from Lamiales, with high support. Moreover, the horizontally transferred atp1 gene has functionally replaced the native, vertically transmitted copy, has an intact open reading frame, and is under strong purifying selection, all of which suggests that this xenolog remains functional. The mitochondrial phylogeny of Cuscuta is generally consistent with previous plastid and nuclear phylogenies, except for the misplacement of Pachystigma when atp1 is included. This incongruence may be caused by the HGT mentioned above. No example of HGT was found within mitochondrial genes of three other, independently evolved parasitic lineages we sampled: Cassytha/Laurales, Krameria/Zygophyllales, and Lennooideae/Boraginales.

Mitochondrial genomic data are effective at placing mycoheterotrophic lineages in plant phylogeny.

Fully mycoheterotrophic plants can be difficult to place in plant phylogeny due to elevated substitution rates associated with photosynthesis loss. This potentially limits the effectiveness of downstream analyses of mycoheterotrophy that depend on accurate phylogenetic inference. Although mitochondrial genomic data sets are rarely used in plant phylogenetics, theory predicts that they should be resilient to long-branch artefacts, thanks to their generally slow evolution, coupled with limited rate elevation in heterotrophs. We examined the utility of mitochondrial genomes for resolving contentious higher-order placements of mycoheterotrophic lineages in two test cases: monocots (focusing on Dioscoreales) and Ericaceae. We find Thismiaceae to be distantly related to Burmanniaceae in the monocot order Dioscoreales, conflicting with current classification schemes based on few gene data sets. We confirm that the unusual Afrothismia is related to Taccaceae-Thismiaceae, with a corresponding independent loss of photosynthesis. In Ericaceae we recovered the first well supported relationships among its five major lineages: mycoheterotrophic Ericaceae are not monophyletic, as pyroloids are inferred to be sister to core Ericaceae, and monotropoids to arbutoids. Genes recovered from mitochondrial genomes collectively resolved previously ambiguous mycoheterotroph higher-order relationships. We propose that mitochondrial genomic data should be considered in standardised gene panels for inferring overall plant phylogeny.

The Evolution of Cytogenetic Traits in Cuscuta (Convolvulaceae), the Genus With the Most Diverse Chromosomes in Angiosperms.

Karyotypes are characterized by traits such as chromosome number, which can change through whole-genome duplication and dysploidy. In the parasitic plant genus Cuscuta (Convolvulaceae), chromosome numbers vary more than 18-fold. In addition, species of this group show the highest diversity in terms of genome size among angiosperms, as well as a wide variation in the number and distribution of 5S and 35S ribosomal DNA (rDNA) sites. To understand its karyotypic evolution, ancestral character state reconstructions were performed for chromosome number, genome size, and position of 5S and 35S rDNA sites. Previous cytogenetic data were reviewed and complemented with original chromosome counts, genome size estimates, and rDNA distribution assessed via fluorescence in situ hybridization (FISH), for two, seven, and 10 species, respectively. Starting from an ancestral chromosome number of x = 15, duplications were inferred as the prevalent evolutionary process. However, in holocentric clade (subgenus Cuscuta), dysploidy was identified as the main evolutionary mechanism, typical of holocentric karyotypes. The ancestral genome size of Cuscuta was inferred as approximately 1C = 12 Gbp, with an average genome size of 1C = 2.8 Gbp. This indicates an expansion of the genome size relative to other Convolvulaceae, which may be linked to the parasitic lifestyle of Cuscuta. Finally, the position of rDNA sites varied mostly in species with multiple sites in the same karyotype. This feature may be related to the amplification of rDNA sites in association to other repeats present in the heterochromatin. The data suggest that different mechanisms acted in different subgenera, generating the exceptional diversity of karyotypes in Cuscuta.

Karyotype asymmetry in Cuscuta L. subgenus Pachystigma reflects its repeat DNA composition.

Cuscuta is a cytogenetically diverse genus, with karyotypes varying 18-fold in chromosome number and 127-fold in genome size. Each of its four subgenera also presents particular chromosomal features, such as bimodal karyotypes in Pachystigma. We used low coverage sequencing of the Cuscuta nitida genome (subgenus Pachystigma), as well as chromosome banding and molecular cytogenetics of three subgenus representatives, to understand the origin of bimodal karyotypes. All three species, C. nitida, C. africana (2n = 28) and C. angulata (2n = 30), showed heterochromatic bands mainly in the largest chromosome pairs. Eighteen satellite DNAs were identified in C. nitida genome, two showing similarity to mobile elements. The most abundant were present at the largest pairs, as well as the highly abundant ribosomal DNAs. The most abundant Ty1/Copia and Ty3/Gypsy elements were also highly enriched in the largest pairs, except for the Ty3/Gypsy CRM, which also labelled the pericentromeric regions of the smallest chromosomes. This accumulation of repetitive DNA in the larger pairs indicates that these sequences are largely responsible for the formation of bimodal karyotypes in the subgenus Pachystigma. The repetitive DNA fraction is directly linked to karyotype evolution in Cuscuta.

Notes on the systematics of Cuscutasect.Subulatae (subg. Grammica) with the description of Cuscutamantiqueirana, a new species from Brazil.

Cuscutamantiqueirana Costea, S.S. Silva & Sim.-Bianch. a new species from montane cloud forests of the Serra da Mantiqueira, Brazil, is described and illustrated. The morphological and phylogenetic analyses revealed that the new species belongs to sect. Subulataeofsubg.Grammica. The new species is related to C.odoratavar.botryoides, C.rotundiflora and C.globiflora from which it differs in narrower calyx lobes and the presence of four stomatiferous lobes or projections at the distal part of the ovary. A detailed morphological comparison with C.odoratavar.botryoides, morphologically the most similar taxon, is provided along with the geographical distribution, ecology and host range of the species. The morphological and phylogenetic relationships of the new species, as well as the diversity of stomatiferous projections, are discussed in the broader context of sect. Subulatae and subg. Grammica. Cuscutabolivianavar.paranensis is considered a synonym of C.odoratavar.botryoides.

Intense proliferation of rDNA sites and heterochromatic bands in two distantly related Cuscuta species (Convolvulaceae) with very large genomes and symmetric karyotypes.

The genome size varies widely among angiosperms but only a few clades present huge variation at a low phylogenetic level. Among diploid species of the genus Cuscuta the genome size increased enormously in at least two independent lineages: in species of subgenus Monogynella and in at least one species (C. indecora) of the subgenus Grammica. Curiously, the independent events lead to similar karyotypes, with 2n = 30 mostly metacentric chromosomes. In this paper we compared the patterns of heterochromatic bands and rDNA sites of C. indecora and C. monogyna, aiming to evaluate the role of these repetitive fractions in these karyotypes. We found out that the large genomes of these species were incremented by a huge number of small heterochromatic CMA+ and DAPI+ bands and 5S and 35 rDNA sites, most of them clearly colocalized with CMA+ bands. Silver nitrate impregnation revealed that the maximum number of nucleoli per nucleus was low in both species, suggesting that some of these sites may be inactive. Noteworthy, the tandem repeats did not generate large bands or sites but rather dozens of small blocks dispersed throughout the chromosomes, apparently contributing to conserve the original karyotype symmetry.

Correction to: Caught in action: fine-scale plastome evolution in the parasitic plants of Cuscuta section Ceratophorae (Convolvulaceae).

Regrettably, an in-text citation wasn't listed in the reference section of the above mentioned publication. The citation reads Schneider et al. (2018) and the correct reference is published here.

Allopolyploid origin and genome differentiation of the parasitic species Cuscuta veatchii (Convolvulaceae) revealed by genomic in situ hybridization.

Interspecific hybridization and genome duplication to form allopolyploids are major evolutionary events in angiosperms. In the parasitic genus Cuscuta (Convolvulaceae), molecular data suggested the existence of species of hybrid origin. One of them, C. veatchii, has been proposed as a hybrid between C. denticulata and C. nevadensis, both included in sect. Denticulatae. To test this hypothesis, a cytogenetic analysis was performed with CMA/DAPI staining and fluorescent in situ hybridization using 5S and 35S rDNA and genomic probes. Chromosomes of C. denticulata were small with a well-defined centromeric region, whereas C. nevadensis had larger, densely stained chromosomes, and less CMA+ heterochromatic bands. Cuscuta veatchii had 2n = 60 chromosomes, about 30 of them similar to those of C. denticulata and the remaining to C. nevadensis. GISH analysis confirmed the presence of both subgenomes in the allotetraploid C. veatchii. However, the number of rDNA sites and the haploid karyotype length in C. veatchii were not additive. The diploid parentals had already diverged in their chromosomes structure, whereas the reduction in the number of rDNA sites more probably occurred after hybridization. As phylogenetic data suggested a recent divergence of the progenitors, these species should have a high rate of karyotype evolution.

Caught in action: fine-scale plastome evolution in the parasitic plants of Cuscuta section Ceratophorae (Convolvulaceae).

KEY MESSAGE: An exhaustive analysis of a group of closely related parasitic plants shows a predominantly gradual reduction in plastid genome composition and provides the most reduced plastomes in the genus Cuscuta. Parasitic plants have a diminished to completely absent reliance on photosynthesis, and are characterized by sweeping morphological, physiological, and genomic changes. The plastid genome (plastome) is highly conserved in autotrophic plants but is often reduced in parasites, and provides a useful system for documenting the genomic effects of a loss of photosynthesis. Previous studies have shown a substantial degree of heterogeneity in plastome length and composition across the species of the genus Cuscuta. Specifically, species in Cuscuta sect. Ceratophorae were suspected to exhibit even more dynamic plastome evolution than the rest of the genus. This complex of eight closely related species was exhaustively sampled here, and one accession per species was sequenced via a high-throughput approach. Complete plastid genomes were assembled and annotated for each of these species and were found to be 61-87 kbp in length, representing a 45-60% reduction relative to autotrophic Convolvulaceae. The most reduced plastomes on this spectrum have lost the bulk of their photosynthetic genes and are the first fully holoparasitic plastomes described for Cuscuta. The fine-scale nature of the system introduced here allowed us to phylogenetically triangulate the locations of gene loss and pseudogenization events precisely, and to construct a step-by-step model of plastome evolution in these plants. This model reveals an intense burst of gene loss along the branch leading to the most reduced plastomes, and a few idiosyncratic changes elsewhere, allowing us to conclude that the tempo of plastid evolution in sect. Ceratophorae is a blend of gradual and punctuated mode.

Punctuated plastome reduction and host-parasite horizontal gene transfer in the holoparasitic plant genus Aphyllon.

Foundational studies of chloroplast genome (plastome) evolution in parasitic plants have focused on broad trends across large clades, particularly among the Orobanchaceae, a species-rich and ecologically diverse family of root parasites. However, the extent to which such patterns and processes of plastome evolution, such as stepwise gene loss following the complete loss of photosynthesis (shift to holoparasitism), are detectable at shallow evolutionary time scale is largely unknown. We used genome skimming to assemble eight chloroplast genomes representing complete taxonomic sampling of Aphyllon sect. Aphyllon, a small clade within the Orobanchaceae that evolved approximately 6 Ma, long after the origin of holoparasitism. We show substantial plastome reduction occurred in the stem lineage, but subsequent change in plastome size, gene content, and structure has been relatively minimal, albeit detectable. This lends additional fine-grained support to existing models of stepwise plastome reduction in holoparasitic plants. Additionally, we report phylogenetic evidence based on an rbcL gene tree and assembled 60+ kb fragments of the Aphyllon epigalium mitochondrial genome indicating host-to-parasite horizontal gene transfers (hpHGT) of several genes originating from the plastome of an ancient Galium host into the mitochondrial genome of a recent common ancestor of A. epigalium Ecologically, this evidence of hpHGT suggests that the host-parasite associations between Galium and A. epigalium have been stable at least since its subspecies diverged hundreds of thousands of years ago.

Convergent Plastome Evolution and Gene Loss in Holoparasitic Lennoaceae.

The Lennoaceae, a small monophyletic plant family of root parasites endemic to the Americas, are one of the last remaining independently evolved lineages of parasitic angiosperms lacking a published plastome. In this study, we present the assembled and annotated plastomes of two species spanning the crown node of Lennoaceae, Lennoa madreporoides and Pholisma arenarium, as well as their close autotrophic relative from the sister family Ehretiaceae, Tiquilia plicata. We find that the plastomes of L. madreporoides and P. arenarium are similar in size and gene content, and substantially reduced compared to T. plicata, consistent with trends seen in other holoparasitic lineages. In particular, most plastid genes involved in photosynthesis function have been lost, whereas housekeeping genes (ribosomal protein-coding genes, rRNAs, and tRNAs) are retained. One notable exception is the persistence of a rbcL open reading frame in P. arenarium but not L. madreporoides suggesting a nonphotosynthetic function for this gene. Of the retained coding genes, dN/dS ratios indicate that some remain under purifying selection, whereas others show relaxed selection. Overall, this study supports the mounting evidence for convergent plastome evolution in flowering plants following the shift to heterotrophy.

Cladogenesis and reticulation in Cuscuta sect. Denticulatae (Convolvulaceae).

As traditionally circumscribed, Cuscuta sect. Denticulatae is a group of three parasitic plant species native to the deserts of Western USA (Cuscuta denticulata, Cuscuta nevadensis) and the central region of Baja California, Mexico (Cuscuta veatchii). Molecular phylogenetic studies confirmed the monophyly of this group and suggested that the disjunct C. veatchii is a hybrid between the other two species. However, the limited sampling left the possibility of alternative biological and methodological explanations. We expanded our sampling to multiple individuals of all the species collected from across their entire geographical ranges. Sequence data from the nuclear and plastid regions were used to reconstruct the phylogeny and find out if the topological conflict was maintained. We obtained karyotype information from multiple individuals, investigated the morphological variation of the group thorough morphometric analyses, and compiled data on ecology, host range, and geographical distribution. Our results confirmed that C. veatchii is an allotetraploid. Furthermore, we found previously unknown autotetraploid population of C. denticulata, and we describe a new hybrid species, Cuscuta psorothamnensis. We suggest that this newly discovered natural hybrid is resulting from an independent (and probably more recent) hybridization event between the same diploid parental species as those of C. veatchii. All the polyploids showed host shift associated with hybridization and/or polyploidy and are found growing on hosts that are rarely or never frequented by their diploid progenitors. The great potential of this group as a model to study host shift in parasitic plants associated with recurrent allopolyploidy is discussed.

On the brink: the highly reduced plastomes of nonphotosynthetic Ericaceae.

Ericaceae (the heather family) is a large and diverse group of plants that forms elaborate symbiotic relationships with mycorrhizal fungi, and includes several nonphotosynthetic lineages. Using an extensive sample of fully mycoheterotrophic (MH) species, we explored inter- and intraspecific variation as well as selective constraints acting on the plastomes of these unusual plants. The plastomes of seven MH genera were analysed in a phylogenetic context with two geographically disparate individuals sequenced for Allotropa, Monotropa, and Pityopus. The plastomes of nonphotosynthetic Ericaceae are highly reduced in size (c. 33-41 kbp) and content, having lost all photosynthesis-related genes, and are reduced to encoding housekeeping genes as well as a protease subunit (clpP)-like and acetyl-CoA carboxylase subunit D (accD)-like open reading frames. Despite an increase in the rate of their nucleotide substitutions, the remaining protein-coding genes are typically under purifying selection in full MHs. We also identified ribosomal proteins under relaxed or neutral selection. These plastomes also exhibit striking structural rearrangements. Intraspecific variation within MH Ericaceae ranges from a few differences (Allotropa) to extensive population divergences (Monotropa, Hypopitys), which indicates that cryptic speciation may be occurring in several lineages. The pattern of gene loss within fully MH Ericaceae plastomes suggests an advanced state of degradation.

Waterfowl endozoochory: An overlooked long-distance dispersal mode for Cuscuta (dodder).

PREMISE OF THE STUDY: Dispersal of parasitic Cuscuta species (dodders) worldwide has been assumed to be largely anthropomorphic because their seeds do not match any previously known dispersal syndrome and no natural dispersal vectors have been reliably documented. However, the genus has a subcosmopolitan distribution and recent phylogeographic results have indicated that at least18 historical cases of long-distance dispersal (LDD) have occurred during its evolution. The objective of this study is to report the first LDD biological vector for Cuscuta seeds. METHODS: Twelve northern pintails (Anas acuta) were collected from Suisun Marsh, California and the contents of their lowest part of the large intestine (rectum) were extracted and analyzed. Seed identification was done both morphologically and using a molecular approach. Extracted seeds were tested for germination and compared to seeds not subjected to gut passage to determine the extent of structural changes caused to the seed coat by passing through the digestive tract. KEY RESULTS: Four hundred and twenty dodder seeds were found in the rectum of four northern pintails. From these, 411 seeds were identified as Cuscuta campestris and nine as most likely C. pacifica. The germination rate of C. campestris seeds after gut passage was 55%. Structural changes caused by the gut passage in both species were similar to those caused by an acid scarification. CONCLUSIONS: Endozoochory by waterbirds may explain the historical LDD cases in the evolution of Cuscuta. This also suggests that current border quarantine measures may be insufficient to stopping spreading of dodder pests along migratory flyways.

DNA barcodes from four loci provide poor resolution of taxonomic groups in the genus Crataegus.

DNA barcodes can facilitate identification of organisms especially when morphological characters are limited or unobservable. To what extent this potential is realized in specific groups of plants remains to be determined. Libraries of barcode sequences from well-studied authoritatively identified plants represented by herbarium voucher specimens are needed in order for DNA barcodes to serve their intended purpose, where this is possible, and to understand the reasons behind their failure to do so, when this occurs. We evaluated four loci, widely regarded as universal DNA barcodes for plants, for their utility in hawthorn species identification. Three plastid regions, matK, rbcLa and psbA-trnH, and the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA discriminate only some of the species of Crataegus that can be recognized on the basis of their morphology etc. This is, in part, because in Rosaceae tribe Maleae most individual plastid loci yield relatively little taxonomic resolution and, in part, because the effects of allopolyploidization have not been eliminated by concerted evolution of the ITS regions. Although individual plastid markers provided generally poor resolution of taxonomic groups in Crataegus, a few species were notable exceptions. In contrast, analyses of concatenated sequences of the 3 plastid barcode loci plus 11 additional plastid loci gave a well-resolved maternal phylogeny. In the ITS2 tree, different individuals of some species formed groups with taxonomically unrelated species. This is a sign of lineage sorting due to incomplete concerted evolution in ITS2. Incongruence between the ITS2 and plastid trees is best explained by hybridization between different lineages within the genus. In aggregate, limited between-species variation in plastid loci, hybridization and a lack of concerted evolution in ITS2 all combine to limit the utility of standard barcoding markers in Crataegus. These results have implications for authentication of hawthorn materials in natural health products.

Phylogeny, character evolution, and biogeography of Cuscuta (dodders; Convolvulaceae) inferred from coding plastid and nuclear sequences.

PREMISE OF THE STUDY: The parasitic genus Cuscuta, containing some 200 species circumscribed traditionally in three subgenera, is nearly cosmopolitan, occurring in a wide range of habitats and hosts. Previous molecular studies, on subgenera Grammica and Cuscuta, delimited major clades within these groups. However, the sequences used were unalignable among subgenera, preventing the phylogenetic comparison across the genus. METHODS: We conducted a broad phylogenetic study using rbcL and nrLSU sequences covering the morphological, physiological, and geographical diversity of Cuscuta. We used parsimony methods to reconstruct ancestral states for taxonomically important characters. Biogeographical inferences were obtained using statistical and Bayesian approaches. KEY RESULTS: Four well-supported major clades are resolved. Two of them correspond to subgenera Monogynella and Grammica. Subgenus Cuscuta is paraphyletic, with section Pachystigma sister to subgenus Grammica. Previously described cases of strongly supported discordance between plastid and nuclear phylogenies, interpreted as reticulation events, are confirmed here and three new cases are detected. Dehiscent fruits and globose stigmas are inferred as ancestral character states, whereas the ancestral style number is ambiguous. Biogeographical reconstructions suggest an Old World origin for the genus and subsequent spread to the Americas as a consequence of one long-distance dispersal. CONCLUSIONS: Hybridization may play an important yet underestimated role in the evolution of Cuscuta. Our results disagree with scenarios of evolution (polarity) previously proposed for several taxonomically important morphological characters, and with their usage and significance. While several cases of long-distance dispersal are inferred, vicariance or dispersal to adjacent areas emerges as the dominant biogeographical pattern.

Plastid genome evolution across the genus Cuscuta (Convolvulaceae): two clades within subgenus Grammica exhibit extensive gene loss.

The genus Cuscuta (Convolvulaceae, the morning glory family) is one of the most intensely studied lineages of parasitic plants. Whole plastome sequencing of four Cuscuta species has demonstrated changes to both plastid gene content and structure. The presence of photosynthetic genes under purifying selection indicates that Cuscuta is cryptically photosynthetic. However, the tempo and mode of plastid genome evolution across the diversity of this group (~200 species) remain largely unknown. A comparative investigation of plastid genome content, grounded within a phylogenetic framework, was conducted using a slot-blot Southern hybridization approach. Cuscuta was extensively sampled (~56% of species), including groups previously suggested to possess more altered plastomes compared with other members of this genus. A total of 56 probes derived from all categories of protein-coding genes, typically found within the plastomes of flowering plants, were used. The results indicate that two clades within subgenus Grammica (clades 'O' and 'K') exhibit substantially more plastid gene loss relative to other members of Cuscuta. All surveyed members of the 'O' clade show extensive losses of plastid genes from every category of genes typically found in the plastome, including otherwise highly conserved small and large ribosomal subunits. The extent of plastid gene losses within this clade is similar in magnitude to that observed previously in some non-asterid holoparasites, in which the very presence of a plastome has been questioned. The 'K' clade also exhibits considerable loss of plastid genes. Unlike in the 'O' clade, in which all species seem to be affected, the losses in clade 'K' progress phylogenetically, following a pattern consistent with the Evolutionary Transition Series hypothesis. This clade presents an ideal opportunity to study the reduction of the plastome of parasites 'in action'. The widespread plastid gene loss in these two clades is hypothesized to be a consequence of the complete loss of photosynthesis. Additionally, taxa that would be the best candidates for entire plastome sequencing are identified in order to investigate further the loss of photosynthesis and reduction of the plastome within Cuscuta.

Plastid genome evolution in mycoheterotrophic Ericaceae.

Unlike parasitic plants, which are linked to their hosts directly through haustoria, mycoheterotrophic (MHT) plants derive all or part of their water and nutrients from autothrophs via fungal mycorrhizal intermediaries. Ericaceae, the heather family, are a large and diverse group of plants known to form elaborate symbiotic relationships with mycorrhizal fungi. Using PHYA sequence data, we first investigated relationships among mycoheterotrophic Ericaceae and their close autotrophic relatives. Phylogenetic results suggest a minimum of two independent origins of MHT within this family. Additionally, a comparative investigation of plastid genomes (plastomes) grounded within this phylogenetic framework was conducted using a slot-blot Southern hybridization approach. This survey encompassed numerous lineages of Ericaceae with different life histories and trophic levels, including multiple representatives from mixotrophic Pyroleae and fully heterotrophic Monotropeae and Pterosporeae. Fifty-four probes derived from all categories of protein coding genes typically found within the plastomes of flowering plants were used. Our results indicate that the holo-mycoheterotrophic Ericaceae exhibit extensive loss of genes relating to photosynthetic function and expression of the plastome but retain genes with possible functions outside photosynthesis. Mixotrophic taxa tend to retain most genes relating to photosynthetic functions but are varied regarding the plastid ndh gene content. This investigation extends previous inferences that the loss of the NDH complex occurs prior to becoming holo-heterotrophic and it shows that the pattern of gene losses among mycoheterotrophic Ericaceae is similar to that of haustorial parasites. Additionally, we identify the most desirable candidate species for entire plastome sequencing.