Abstracts Dealing with Parasitic Angiosperms and Mycoheterotrophs
Botany 2016
Savannah, Georgia

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Randle, Christopher [1], Bayat, Soheila [2], Hammack, David [1], Ilunga, Edouard [3], Souza, Vinicius [4], Morawetz, Jeffery [5].
Phylogeny and taxonomy of the tropical clade of Orobanchaceae.
Most of the known species diversity in the parasitic plant family Orobanchaceae occurs in temperate regions. However, diversity in the well-supported clade of tropical Orobanchaceae remains largely unexplored.  With the exception of Buchnera (with 100 species), most genera of the tropical clade consist of few poorly characterized and rarely collected species. Although tribe-level classification systems include such poorly known genera, few thorough phylogenetic analyses exist to test these interpretations. In this study, we have expanded phylogenetic sampling of tropical species and genera in over previous studies through field collection of species from the paleotropics and neotropics.  For the first time, the genera Buttonia, Cycniopis, Ghikea, Gerardiina, Magdalenaea, Micrargeriella, Nothochilus, Physocalyx, Pseudosopubia and Velosiella are included in systematic analysis of the tropical clade.  Further, species sampling of genera Buchnera, Cycnium, Melasma, Sopubia, and Striga, has been greatly expanded. DNA sequences of the following loci were obtained from circa 150 ingroup and outgroup accessions of more than 100 species: the nuclear Internal Transcribed Spacer (ITS) and nuclear phyB, and chloroplast loci matK, rbcL, rpl16, and rps2.  Phylogenetic analysis was used to explore 1) monophyly and relationships among major genera and  2) proposed tribe-level taxonomies.
1 - Sam Houston State University, Department of Biological Sciences, 1900 Avenue I, Huntsville, TX, 77340, USA
2 - Sam Houston State University, Department of Biological Sciences, 1900 Avenue I, Huntsville, TX, 77340
3 - Université de Lubumbashi, Sciences Agronomiques, Lubumbashi, Katanga, DRC
4 - Universidade de São Paulo, Escola Superior de Agricultura Luiz de Que, Ciências Biológicas, Avenida Pádua Dias, 11-Caixa Postal Agronomia, Piracicaba, SP, 13418-900, Brazil
5 - Rancho Santa Ana Botanic Garden, 1500 N. College Ave., Claremont, CA, 91711, USA

Schneider, Adam [1], Baldwin, Bruce G. [1].
Host-switching as a major driver of diversification in parasitic plants: A case study in Orobanche.
Parasitism is a highly successful life strategy that has independently evolved countless times across the tree of life, including twelve times among angiosperms. The parasitic plant genus Orobanche L. is quickly becoming a focal group understanding the evolutionary consequences of parasitism across numerous taxonomic scales and levels of biological organization. A recent phylogenetic study of the clade of all species endemic to the western hemisphere, based in large part on herbarium collections, shows extensive, hitherto unrecognized host-specific diversity. Well-supported, cryptic or semi-cryptic clades within minimum-rank taxa, especially in sect. Gymnocaulis, are diagnosable by distinct host assemblages that implicate host switching in divergence of parasitic lineages. These robust but taxonomically unrecognized clades are reinforced by multiple gene trees based on nuclear and chloroplast DNA. Moreover, this phylogeny lends support to several well-known phytogeographic patterns such as multiple amphitropical colonizations of South America and post-glacial Alaska/eastern North America disjunctions.Finally, this study provides a fine-scale phylogenetic framework for a genome-skimming project currently underway to understand plastid evolution at fine scales.
1 - University of California, Berkeley, Jepson Herbarium and Department of Integrative Biology, 1001 Valley Life Sciences Building, Berkeley, CA, 94720, USA

Sedaghatpour, Maryam [1], Oldham, Karoline [2], Weeks, Andrea [3].
Patterns in the population genetic diversity of the North American hemiparasite Melampyrum lineare (Orobanchaceae).
Melampyrum lineare Desr. (narrowleaf cowwheat) is a hemiparasitic annual flowering plant native to North America that obtains water and nutrients by penetrating the roots of its host species, which include maple trees, pine trees, and members of Ericaceae among others. Due to this physiological dependency, M. lineare is limited to the geographical range of its hosts. Within the United States, its distribution encompasses the southern Appalachian Mountains to throughout the northeastern states and westward to Minnesota, with disjunct populations in Idaho and Montana. Within Canada, it is distributed at lower latitudes from Newfoundland to Vancouver Island as well as further north along the more temperate Pacific coast. A recent morphological revision of its four taxonomic varieties showed that this highly polymorphic species cannot be partitioned into sub-specific categories reliably, yet morphological diversity seems greatest in the eastern-most part of its range. Molecular phylogeographic investigation was begun to uncover patterns of this species’ genetic diversity as a complement to our morphological work and to test Francis Pennell’s 1935 hypothesis that M. lineare found historical refuge in the southern Appalachian region and subsequently recolonized North America from this location. Under Pennell’s biogeographical scenario, we would expect greater genetic diversity of M. lineare in the southern-most part of it range. Molecular data were collected from 29 eastern U.S. populations ranging from Georgia to Maine and 20 Canadian populations in Alberta and British Columbia provinces. Four nuclear microsatellite markers (MsO66P, MsO70M, MsG2, and MsB58) developed for M. sylvaticum cross-amplified and showed allelic variation. A survey of 15 chloroplast gene regions found that four showed inter- and intra-population variability (trnS-trnG, psbA-trnH, trnT-trnL, and rps16-trnQ). Analysis of trnS-trnG indicated nine haplotypes are shared among 170 individuals in 49 populations. Two haplotypes are present in Canada and all nine are present in southeastern US populations, a pattern consistent with Pennell’s origin hypothesis.  Further analysis of microsatellite and other chloroplast gene regions data will refine our ability to fingerprint populations, test patterns of M. lineare’s historical range expansion, and to better describe its biological diversity in areas where it must be managed as a legally rare or threatened species.
1 - George Mason University , Department of Biology , 4400 University Dr., MSN 3E1, Fairfax, VA, 22030, USA
2 - George Mason University , School of Systems Biology, 4400 University Dr., Fairfax, VA, 22030, USA
3 - George Mason University, 4400 University Drive MSN 5F2, Fairfax, VA, 22030, USA

Yu, Wen-Bin [1], Wang, Hong [2], Jin, Jian-Jun [2], Ree, Richard [3], Li, De-Zhu [4], dePamphilis, Claude W. [5].
Evolutionary variations of chloroplast genome structure in Pedicularis (Orobanchaceae).
Chloroplast genomes shows striking variation in gene content and structure in non-photosynthetic plants. By contrast, hemiparasitic plants have almost complete chloroplast genomes in comparison to their autotrophic relatives. Pedicularis (Orobanchaceae) is a genus of hemiparasitic herbs with over 500 species. In this study, we sequenced total genomic DNA from 15 Pedicularis species. Genomic DNA was extracted using fresh leaves, then prepared for high-throughout sequencing. We first identified plastid DNA reads using reference based mapping. The filtered reads were used as targets to identify additional overlapping reads. Then, the total filtered readsset was assembled using SPAdes. The path for chloroplast genome were view and determined using Bandage. We found that chloroplast genomes of Pedicularis spp. did not lose genes. However, the inverted repeat (IR) and the short single-copy (SSC) regions showed high variations in length and gene content with large differences between species in how far into the SSC the IR extends. Phylogenomic analyses using the common conserved regions for the 15 species were well resolved, including eight recognized clades and one unplaced species (P. moupinensis) from a previous phylogeny of Pedicularis. Comparative phylogenetic analyses showed that variations of genome structure reflected phylogenetic relationship.
1 - Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Center for Integrative Conservation, Menglun, Mengla, Yunnan, 666303, China
2 - Kunming Institute of Botany, Chinese Academy of Sciences, Key Laboratory for Plant Diversity and Biogeography of East Asia, 132 Lanhei Road, Kunming, Yunnan, 650201, China
3 - Field Museum of Natural History, Department of Botany, 1400 South Lake Shore Drive, Chicago, IL, 60605, USA
4 - Kunming Institute of Botany, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming, Yunnan, 650201, China
5 - The Pennsylvania State University, Department of Biology, University Park, State College

Convolvulaceae (Cuscuta)

McNeal, Joel R. [1].
Comparative transcriptomics of the genus Cuscuta.
Species within the stem parasitic plant genus Cuscuta vary widely in published chromosome number and genome size estimates without strong correlation between the two values.  Analyses of transcriptome data will be used to determine how much of the genome size variation across Cuscuta can be explained by whole genome duplication events and what role these events may have played in speciation within the genus.  Rates of evolution in nuclear genes and their correlation to those in the chloroplast genome will also be addressed, with an emphasis on rate variation in photosynthetic genes encoded by the nuclear genome of Cuscuta.
1 - Kennesaw State University, Biology, 1000 Chastain Rd., Kennesaw, GA, 30144-5591, USA

Riekert, Brandy [1], McNeal, Joel R. [1].
Development of Population Genetics Markers for the Rare Parasitic Plant Cuscuta harperi.
Harper’s Dodder (Cuscuta harperi), is a rare stem parasitic annual vine that is endemic to a low number of scattered populations in Georgia and Alabama. It is a habitat specialist, occurring only on exposed sandstone and, more rarely, granite outcrops and barrens within its range. Populations of C. harperi are widely disjunct, and most populations are comprised of only a few individuals. Current methods in ecological genetics allow detection of genetic variation within and among populations, and variation can be used as an indicator of organismal fitness. Population genetics data can also be used to determine conservation priorities for populations that may be at greater risk for inbreeding depression as compared to populations with higher levels of variation. We will develop genetic markers for high-throughput sequencing to detect levels of variation within and among populations of C. harperi to elucidate population structure and identify populations that are in greatest need of conservation efforts.
1 - Kennesaw State University, Biology, 1000 Chastain Rd., Kennesaw, GA, 30144-5591, USA


Nikolov , Lachezar Atanasov [1], Davis, Charles C. [2].
The big, the bad, and the beautiful: evolution and development of the world's largest flowers (Rafflesiaceae).
Rafflesiaceae, crowned “the greatest prodigy of the vegetable kingdom,” produce the largest flowers in the world. They are also holoparasites residing inside their vine hosts, and emerge only during flowering. The floral gigantism and obligate parasitism of Rafflesiaceae have rendered their structure unrecognizable to most plant biologists. The vegetative body is composed of highly reduced strands of cells embedded in host tissue, and does not differentiate into leaves, stems, or roots. The flowers look and smell like decaying animal flesh and exhibit numerous features unknown in the vast majority of flowering plants. This unusual combination of characters has obscured the phylogenetic affinities and development of Rafflesiaceae since their discovery two centuries ago. We provide the first broad and integrated picture of the vegetative and reproductive morphology of Rafflesiaceae. Our talk will describe their diminutive vegetative body, which lacks cell differentiation. Next, we will demonstrate that shoot apex and carpel formation are facilitated by an extraordinary pattern of cell separation resulting in a secondary morphological surface, which gives rise to floral organs and ovules, respectively. Finally, for the first time, we clarify the identity of the diverse floral organs in Rafflesia, Rhizanthes, and Sapria. These findings collectively provide key insights into how these unusual plants are constructed, and offer clues on their evolution from tiny flowered ancestors to floral giants.
1 - Max Planck Institute of Plant Breeding Research, Carl-von-Linné-Weg 10, Koeln, N/A, 50829, Germany
2 - Harvard University Herbaria, 22 Divinity Ave, Cambridge, MA, 02138


Caraballo-Ortiz, Marcos A. [1], Carlo, Tomas [2], dePamphilis, Claude W. [3].
Elucidating evolutionary relationships and biogeography on the endemic Caribbean mistletoes Dendropemon (Loranthaceae) using multiple transcriptomes and traditional molecular markers.
Dendropemon (Loranthaceae in Santalales) is a strictly insular genus of mistletoes endemic to the Caribbean archipelago. The genus represents a unique opportunity for the study of evolution of plant species on islands because it is the only endemic genera present on most major Caribbean islands. In addition, Dendropemon presents an opportunity to exemplify how evolutionary radiations on archipelagos occur for hemiparasitic plants and to test hypotheses about how their unique lifestyle shapes processes of diversification. The objective of this study was to reconstruct phylogenetic relationships within the genus to test monophyly of Dendropemon and validate currently recognized species. The utility of traditional nuclear and chloroplast molecular markers (ITS, matK, and trnL-F) to reconstruct phylogenetic relationships for Dendropemon and outgroups was tested, but we obtained low levels of sequence divergence (3.1%, 2.0%, and 1.4%, respectively). An alternative approach was conducted by generating multiple transcriptomes for Dendropemon and outgroups using Illumina® sequencing to discover single-copy genes and build phylogenetic trees with high resolution at species level and below. Preliminary phylogenies suggest that islands contain a mixture of closely and distantly related Dendropemon species, suggesting multiple colonization events and in-situ speciation. Results obtained from molecular and morphological data support nomenclatural and taxonomic changes within the genus such as species revalidations and description of new taxa, and allow biogeographic reconstructions, estimation of divergence times, and to examine the role of hosts in the radiation of the genus.
1 - Penn State University, 208 Mueller Lab, University Park, PA, 16802, USA
2 - Penn State University, Biology, 208 Mueller Lab, University Park, PA, 16802, USA
3 - The Pennsylvania State University, Department of Biology, University Park, State College

Madden, Julie [1], Keefover-Ring, Ken [2], Duran, Kristy [3].
Correlation between dwarf mistletoe infection and monoterpene concentrations in Pinus Ponderosa.
The rapid decline of conifers throughout the world has resulted from various parasitic species such as the bark beetles and dwarf mistletoe. In an effort to combat the attacks of these species, conifers have developed various host defenses including the production of allelochemicals. There has been an increasing amount of interest surrounding one class of these chemicals known as monoterpenes, because of their potential to affect the behavior and reproduction of parasitic species. One monoterpene, limonene has been shown to deter female bark beetles.  On the other hand beta-pinene has been shown to be attractive to a number of bark beetle speices. Conifers are also susceptible to infection by dwarf mistletoe. It is possible that a correlation between mistletoe infection and monoterpene production may affect subsequent bark beetle attack. This study examines whether such correlation exists. Southwest dwarf mistletoe (Arceuthobium vaginatum) is a hemiparasitic plant that primarily infects ponderosa pines (Pinus ponderosa) and has played a major role in reshaping the forest landscape of Colorado. In an effort to determine if the levels of monoterpenes varied in ponderosa pines infected with southwest dwarf mistletoe, gas chromatography coupled with a flame ionization detector (GC-FID) was employed. Samples were collected from the Zapata Subdivision in Alamosa, Colorado within a one hectare area. The samples consisted of pine needles, in which ten samples were taken from trees that were not infected with mistletoe and ten from trees infected with mistletoe. Various monoterpenes were extracted from the needle tissue with hexane and a quantitative analysis conducted using GC-FID.  Total monoterpene concentrations were significantly higher in uninfected trees compared to infected trees (p = 0.017). Both limonene and (-) beta-pinene concentrations were significantly higher in uninfected than in infected trees (p = 0.002 and 0.021 respectively).  Bark beetles respond differently to these two monoterpenes suggesting that infected trees may be more susceptible to bark beetle infection because of lower limonene concentrations.  On the other hand, bark beetles may be more attracted to uninfected trees because of higher (-) beta-pinene concentrations.  Therefore, trees infected with dwarf mistletoe and those not infected may be equally susceptible to infection by bark beetles.
1 - Adams State University, Chemistry
2 - University of Wisconsin-Madison, Department of Entomology
3 - Adams State University, Biology and Earth Science, 208 Edgemont Blvd, Alamosa, Colorado, 81101, USA

Nickrent, D. L. (1) and K. Neubig (2), Su, Huei-Jiun (3)
Multigene phylogeny places Brachynema in Erythropalaceae (Santalales)
Past molecular phylogenetic analyses have advanced our understanding of relationships within the sandalwood order (Santalales), however, one genus has remained unplaced: Brachynema. In 1857, Bentham described the first species, B. ramiflorum from Brazil, and since then one additional species, B. axillare from northern South America, has been described. Its ordinal and familial placement has been enigmatic, including Ebenaceae, Linaceae, Icacinaceae, Lecythidaceae, and Olacaceae s.l.  Sequences of nuclear SSU and LSU ribosomal DNA, chloroplast rbcL, matK and accD and mitochondrial matR were obtained for 170 members of Santalales. The analysis included one sample of B. ramiflorum collected July 1980 (Pará, Brazil, C. A. Cid Ferreira 1482, NY). The total DNA obtained from this herbarium specimen was subjected to genome skimming using an Illumina HiSeq which provide complete sequences of the above six genes. A concatenated data set was analyzed with maximum parsimony and likelihood. All separate gene partitions and the concatenated data set yielded the same result: Brachynema is sister to Maburea trinervis (Erythropalaceae), a monospecific shrub from Guyana first described in 1992 by Maas et al.  This result is fascinating because Brachynema displays a number of morphological features that are aberrant in the order: glandular-dentate leaf margins, upper and lower pulvinar thickenings, and a 4-5 chambered ovary with axile placentation (1 ovule pendulous in each locule). Placentation in nearly all members of Santalales is free-central with apical, pendulous ovules; however, Maburea was described as being 2-3 locular, also with axile placentation (1-2 somewhat pendulous ovules per locule). Leaf anatomical characters are notably similar between the two genera. This study demonstrates how particular suites of morphological characters support affinities that are in conflict with strongly supported molecular phylogenetic results.
1. Southern Illinois University, Department of Plant Biology, 1125 Lincoln Drive, Carbondale, IL, 62901-6509, USA. nickrent@plant.siu.edu
2. Southern Illinois University Carbondale, Plant Biology, Carbondale, IL, 62901, USA. kneubig@siu.edu
3. National Taiwan University, Institute of Ecology and Evolutionary Biology, Life Science Building R1227, Roosevelt Road Section 4, Taipei, Taiwan


Barrett, Craig [1], Sass, Chodon [2].
A phylogeographic approach to assessing plastid genome evolution in a non-photosynthetic orchid using targeted sequence capture.
Studies of plastid genome (plastome) evolution have focused on single representatives of species, even in comparative investigations, yet few if any studies have addressed plastome evolution at the infraspecific level.  Parasitic and mycoheterotrophic plants display accelerated rates of plastome evolution, often with drastic reduction in size and gene content due to relaxed selective constraints of photosynthesis.  The leafless orchid genus Corallorhiza contains both partially and fully mycoheterotrophic species, and has thus become a model for understanding the relationship between plastome evolution, nutritional mode, and fungal host specificity.  The C. striata complex, with three putative species (C. bentleyi; C. involuta; C. striata, with three varieties), displays the most evidence of plastome modification in the genus.  However, plastomes in this complex have not been severely reduced as in other, well-studied parasites (e.g. holoparasitic Orobanchaceae), making it a useful system to address questions associated with the early stages of plastome degradation.  For example, what is the degree of variation in plastome size and gene content among members of this complex?  What is the tempo of plastome reduction?  Do members of this complex follow a single trajectory of degradation, and is this similar to that observed in other parasites?  Here we use genome skimming and targeted sequence capture via Agilent microarray to generate complete to nearly complete plastomes for 53 individuals across the geographic range of the C. striata complex, from Mexico to northern North America.  Briefly, genome skimming was used to generate five annotated reference plastomes representing each putative taxonomic entity of the complex.  Targeted sequence capture was then used to expand sampling of individuals, in a cost-effective manner.  Plastome sizes vary widely from 124 kb to 141 kb among members of the complex, with the highest degree of reduction in the endangered C. bentleyi (eastern North America) and C. involuta (southern Mexico).  Members of the complex show varying degrees of degradation of photosynthesis-related gene complexes.  Based on patterns of deletions and pseudogenes, two distinct degradation pathways have occurred within the complex, in addition to that observed in the C. maculata complex.  This suggests that C. bentleyi/C. involuta and C. striata sensu stricto represent two losses of photosynthesis from a putatively photosynthetic ancestor.  Divergence times, rates of degradation, and ancestral plastome reconstructions are discussed in the context of phylogeographic history of the complex.  Further, relative merits and challenges of targeted sequence capture are discussed for closely related taxa displaying rapid genome evolution.
1 - West Virginia University, Plant and Soil Sciences, G153 South Agricultural Sciences Building, Morgantown, WV, 26506, USA
2 - University of California, Berkeley, Department of Plant and Microbial Biology, Department of Integrative Biology and the University and Jepson Herbaria, Berkeley, CA, 94720, USA

Barrett, Craig [1], Perez, Leticia [2].
Gene expression and mycoheterotrophy: an exploratory analysis of green vs. non-green Corallorhiza (Orchidaceae).
Studies of genome evolution in parasitic and mycoheterotrophic plants have focused overwhelmingly on changes occurring in the plastid genome (plastome) as a result of relaxed selection on photosynthetic function. Only a handful of studies have focused on the effects of photosynthetic loss and patterns of gene expression.  While studies have addressed gene expression patterns between holoparasites and their host plants, almost no data exist on expression related to obligate interactions between mycoheterotrophs (viz. holomycotrophs) and their fungal hosts.  Here we examine the orchid genus Corallorhiza to compare gene expression patterns across green, partially mycoheterotrophic species vs. non-green, holomycotrophic species.  Specifically, we address hypotheses concerned with loss of photosynthesis in holomycotrophs, and changes in expression patterns between orchids parasitizing different hosts.  Green species (C. trifida, C. wisteriana) are hypothesized to express photosynthesis-related genes (nuclear and plastid) at some level in contrast to non-green species (C. striata var. striata, C. maculata var. occidentalis). A single representative of each of the aforementioned species was collected at a site where they are sympatric in Montana, USA, all at the same time of day and at the same developmental stage (pre-anthesis).  Material was chopped finely in the field and stored immediately in cold RNA-later solution to preserve mRNA.  Total RNAs were extracted from whole shoots and rhizomes after cleaning with nuclease free water using the Zymo Plant RNA kit, which has a DNA-exclusion step, followed by treatment with DNAse.  Total mRNAs were sequenced after rRNA exclusion and poly-A purification on an Illumina NextSeq500 for 150 bp paired-end reads.  Shoot-derived reads were assembled de novo into contigs using Trinity after digital normalization.  Plastid contigs were mapped to previously sequenced plastomes of Corallorhiza and annotated, while nuclear contigs were annotated based on the recently published Phalaenopsis genome.  Photosynthesis related unigenes were scored as present or absent, and compared qualitatively between green vs. non-green species, as acquisition of biological replicates within species was not yet possible.  Unigenes missing from both non-green orchid datasets but present in both green orchid datasets were considered putative gene losses, pseudogenes, or extreme instances of down-regulation.  Reads from rhizome mRNAs were mapped simultaneously to recently sequenced orchid and fungal genomes (fungal families Russulaceae or Thelephoraceae), followed by de novo assembly.  Differences in transcript presence/absence is discussed, as are future efforts to collect biological replicates and other taxa in Corallorhiza and leafy relatives, with the goal of a phylo-comparative assessment of gene expression.
1 - West Virginia University, Plant and Soil Sciences, G153 South Agricultural Sciences Building, Morgantown, WV, 26506, USA
2 - California State University, Los Angeles, Biological Sciences, 5151 State University Drive, Los Angeles, CA, 90032, USA

Gerelle, Wesley [1], Lam, Vivienne K. Y.  [1], Graham, Sean W. [1].
Comparative genomics of Parasitaxus usta, a podocarp conifer with a unique form of heterotrophy.
Parasitaxus usta (Podocarpaceae) is a parasitic and possibly mycoheterotrophic conifer endemic to New Caledonia. Its plastid genome is highly reduced with only the ribosomal-protein genes, RNA polymerase genes, infA, ycf1, ycf2, matK, clpP and accD retained under putative purifying selection. Despite the loss of most plastid-encoded photosynthetic genes, it retains both nuclear and plastid-encoded genes of the CF1 complex of ATP synthase (the so-called 'lollipop head' of the enzyme). We examine how this complex is evolving based on analysis of dN/dS ratios and document the utility of each gene in phylogenetic inference. We also explore a transcriptome for this species produced by the 1KP (One Thousand Plants) project to document genome-wide patterns of gene retention, including nuclear-encoded photosynthetic genes and other plastid-targeted genes, with the goal of characterizing patterns of Gene Ontology (GO) term enrichment relative to its photosynthetic relatives in Podocarpaceae.
1 - University of British Columbia, Botany, 3529 - 6270 University Boulevard, Vancouver, BC, V6T 1Z4, Canada