Research Highlights

Daniel L. Nickrent


When one hears the word “parasite,” usually the first thing that comes to mind is something unpleasant, uncomfortable, or dangerous, such as ticks, fleas, worms, and malaria!  It may surprise one to know that parasites occur among all the major branches of the Tree of Life, including flowering plants.  From a biological perspective, parasitic plants provide a wealth of opportunity for scientific research. I have devoted much of my career to addressing questions about these unusual plants.  Highlighted below are some of my research accomplishments, with some discussion of their relevance and significance to the scientific community.

I began using nucleic acid sequences as tools to investigate questions in parasitic plants as an Assistant Professor at the University of Illinois.  Previously, he used isozymes (proteins) to look at populational and systematic relationships in dwarf mistletoes (Arceuthobium), a group of economically important parasitic plants that I researched while a Doctoral student.  The switch to using nucleic acids, specifically direct ribosomal RNA sequencing, required learning many new techniques and applying these to field-collected plants.  At that time, little molecular work had been conducted outside cultivated plants, hence innovation was required to obtain usable data.  The publication by Nickrent and Franchina (1990) was one of the first studies to use complete small-subunit (SSU) rRNA sequences to estimate phylogenetic relationships in flowering plants, here the sandalwood order (Santalales).  In the late 1980s, there were less than 20 complete SSU sequences of plants in GenBank.  By comparison, today there are more than 3000 SSU rDNA sequences for green plants, and 150 of these were determined in my lab and over 300 were obtained through collaborative research. 

During the early 1990s, most plant systematists who were investigating phylogeny (evolutionary relationships) in plants were using the chloroplast gene rbcL.  This gene codes for RUBISCO, an abundant protein that fixes carbon dioxide in the process of photosynthesis.  The availability of oligonucleotide primers and the rate of evolution of this gene resulted in a virtual explosion of sequencing activity such that today, rbcL is the single most abundant gene sequence in GenBank (currently over 21,000 for land plants).  I chose to sequence nuclear SSU rRNA for two reasons: 1) a multi-user laboratory at the U of I was developed to explore this gene, and this is where his initial training took place, and 2) many of the parasitic plants that he wanted to examine were known to lack photosynthesis, hence the rbcL gene was likely missing in these plants.  Eventually, a number of researchers who had been sequencing the chloroplast gene rbcL required an independent source of data, and SSU rDNA provided such a molecular marker.  The study by Nickrent and Soltis (1995) was the first to compare the phylogenetic utility of these two molecular markers. This paper helped dispel the widely-held view that nuclear SSU rDNA contained too few nucleotide substitutions to address phylogenetic relationships among angiosperms.  Doug and Pam Soltis, and a number of collaborators, extended this work by conducting a large-scale sequencing study (Soltis et al. 1997) that used over 200 angiosperm SSU sequences.  This work showed that the nuclear SSU rDNA and rbcL topologies were highly congruent.  The above two papers effectively changed the collective view of the systematic community in demonstrating the evolutionary scale at which SSU rDNA sequences are most effective in resolving relationships.  This gene has since gained wider use within plant phylogenetics, often used as a check of the maternally inherited chloroplast gene rbcL.

At the same time nuclear SSU rDNA sequences were being obtained for phylogenetic purposes, data collected in my lab showed that for parasitic flowering plants that had lost their photosynthetic ability, the “rules of molecular evolution” were not being followed.  We published a paper (Nickrent and Starr 1994) quantifying the degree of evolutionary change (i.e. mutational rates) exhibited by these plants, which in some cases was elevated over three times that seen in other genes.  This was most remarkable because nuclear SSU rDNA sequences were thought to be extremely conservative and should not be influenced by the loss of photosynthesis, as one expects for chloroplast genes.  This phenomenon was further investigated among representatives of other “heterotrophic” plants [heterotrophs are plants that gain nutrition without photosynthesis].  Nonparasitic heterotrophic flowering plants, such as some members of the orchid and blueberry families, use fungal intermediates  and are called mycotrophs (“fungal feeders”). Some of these mycotrophs also showed increased evolutionary rates.  Thus, there is an underlying molecular phenomenon, yet to be fully explained, that was uncovered from these investigations.

In 1990 the complete plastid DNA sequence of Epifagus (beechdrops, Orobanchaceae) was published.  Epifagus is a holoparasite on the roots of beech trees.  Its plastid genome (plastome) showed a marked reduction in size compared to photosynthetic plants.  But Epifagus represents just one of the ten times that holoparasitism evolved in flowering plants.  Thus, questions remained as to 1) whether plastomes exist in other holoparasites and 2) if they exist, how are their genomes structured? The plastid SSU rDNA sequences generated in my lab were the first indications that Balanophoraceae, Hydnoraceae and Rafflesiaceae (in a broad sense) contained plastid genes (Nickrent et al. 1997a, b). Structural studies of the rRNAs from these plants showed that they are the most unusual molecules known in angiosperms, indeed even among all green plants.  Southern blot hybridization work provided additional indications that a plastome existed.  These results provided compelling evidence that the plastome cannot be lost entirely, even in lineages that have lost photosynthesis many millions of years ago.  It has been speculated that the few remaining genes (really their products) in these genomes are indispensable, and parallels can be seen in other parasitic lineages such as Plasmodium, the protistan parasite that causes malaria that indeed contains a chloroplast.  These holoparasites represent a real challenge for further work because they are extremely difficult to cultivate and the level of sequence divergence of their genomes compromises the use of standard molecular techniques.  For example, the plastid large subunit (LSU) rDNA of one holoparasite, Cynomorium was cloned and sequenced.  Instead of all the plastomes being identical within the plant (as is the case for essentially all previously studied plants), Cynomorium contained numerous different copies of the LSU ribosomal gene.  This documented a phenomenon called heteroplasmy at a degree never before realized for any plant.  To put this in context, the degree of difference between the LSU rDNA clones from Cynomorium exceeds the difference seen among all land plants taken together.  This work, published in the Journal of Molecular Evolution (García et al. 2004), continues to challenge previous concepts about the organization and function of plastomes in plants.

While attempting to PCR amplify plastid genes from holoparasites, Dr. Joel Duff [a postdoctoral researcher in my lab] accidentally amplified mitochondrial SSU rDNA.  This case of serendipity was fortuitous, for it spawned a line of research that resulted in several publications.  As with plastid SSU rDNA, no mitochondrial SSU rDNA sequences of holoparasites existed prior to this work.  In fact, only six angiosperm sequences were known and five of these were crop plants.  Work in my lab greatly expanded the number of available mitochondrial SSU rDNA sequences, confirmed the highly conserved nature of its core region, further characterized the sequence and length heterogeneity of two variable domains, and documented for the first time repetitive motifs and a transversion bias [a transversion is a change in DNA from a pyrimidine to a purine, or vice versa; normally substitutions are biased towards transitions, i.e. a purine to a purine or pyrimidine to pyrimidine].  The core mitochondrial SSU rRNA sequences of the holoparasites exhibited both the highest substitution rates as well as the most divergent structural features seen in flowering plants (Duff and Nickrent 1997).  Since these lineages have similarly accelerated substitution rates in nuclear and plastid-encoded SSU rRNA genes, it appears that heterotrophy profoundly affects the molecular evolution of all three subcellular genomes.

During the course of working with parasite mt SSU rDNA sequences, my lab also investigated the utility of this gene for phylogenetic investigations of deep clades within land plants. This work (Duff and Nickrent 1999) resulted in strong support for the majority of higher-level land plant clades, such as hornworts, liverworts, mosses, lycopsids, leptosporangiate and eusporangiate ferns, gymnosperms and angiosperms.  Support for a sister relationship between Equisetum and leptosporangiate ferns and a monophyletic gymnosperm clade that was sister to angiosperms were also demonstrated – two relationships later confirmed by workers using other genes.  This paper, published in the American Journal of Botany, was the first demonstration of the utility of this gene for assessing phylogenetic relationships among plants.  Mitochondrial SSU rDNA sequences were used in combination with other genes in a collaborative study published in the the journal Molecular Biology and Evolution (Nickrent et al. 2000).

In 1994, interspecific relationships in Arceuthobium (dwarf mistletoes, Viscaceae) were addressed using nuclear ribosomal internal transcribed spacer (ITS) sequences (Nickrent et al. 1994).  This was just one year after a publication by Baldwin who first used this spacer for addressing phylogenetic relationships among plant species.  Today, over 50,000 ITS sequences for land plants exist on GenBank and this spacer is commonly used to assess phylogenetic relationships among species.  Through a collaborative project with fellow scientists from Spain (García and Martín) and R. Mathiasen from Northern Arizona University, a revised molecular phylogeny of Arceuthobium, using both ITS and a chloroplast spacer (trnT-L-F), was published (Nickrent et al. 2004).  This study was notable in that sampling of all known species of Arceuthobium was achieved, something that previously had not been done with a genus of this size (46 species). This work showed how different conclusions can be reached when classifications are based upon morphological vs. molecular characters.  For diminutive parasites such as A. pusillum and A. douglasii, morphological convergence can lead to erroneous classifications that is only detected using genetic markers.  It also showed that many of the mistletoe pathogens from the western U.S. are so closely related they could be considered one biological species.  Examination of the chloroplast trnT-L-F sequences showed that structural changes (insertion/deletion events) can occur in parallel in unrelated lineages (homoplasy), thus suggesting caution when inferring phylogenetic pattern from such events.

Nickrent and Franchina (1990) published the first molecular phylogenetic investigation of the sandalwood order (Santalales) and several projects, funded by the NSF, have since been initiated on these fascinating plants. The major goal of this research is to determine the phylogenetic relationships among members of the order which traditionally includes six families and over 2200 species of mainly parasitic angiosperms.  My lab now has DNA samples from 147 of the 160 known genera in this order, thus representing an extremely valuable genetic resource.  Sequencing has expanded beyond nuclear SSU rDNA and now includes nuclear LSU rDNA as well as chloroplast rbcL, matK and accD.  A major portion of the sequencing work by Valéry Malécot (Paris, France) for his Ph.D. research was conducted at SIUC. Nickrent continues to collaborate with Dr. Malécot and the first of several papers on phylogenetic relationships in basal Santalales, specifically Olacaceae, has been published (Malécot et al. 2004).  Earlier work (Nickrent and Malécot 2001) on sandalwood phylogenetic relationships  provided an overall framework for more detailed studies within the component families. While confirming some traditionally-held ideas about relationships in the order, several novel affinities emerged, such as the relationship of Schoepfia with Loranthaceae and the presence of parasitic and nonparasitic clades in Olacaceae.  This work has been instrumental in revising the classification of Santalales by the Angiosperm Phylogeny Group (APG 1998, 2003).

The family for which the sandalwood order is named is Santalaceae, and this group has been the subject of a recent M.S. degree by Josh Der (July 2005).  The Nickrent lab was able to obtain DNA samples of all 38 genera in this family.  From these Der produced a data matrix of three genes and conducted robust molecular phylogenetic analyses using maximum parsimony, maximum likelihood and Bayesian inference.  Santalaceae have never been monographed on a worldwide  scale, thus this work will produce the first classification that includes all genera in the family.  Moreover, the classification will follow modern principles (e.g. monophyly) and will for the first time circumscribe and name natural evolutionary groups (six new families).  This work showed that the mistletoe habit (aerial parasitism) arose in Santalaceae (in a broad sense) three times independently.  We are currently preparing this work for publication.

Another group in the sandalwood order is Loranthaceae, a pantropical family comprising over 70 genera and 900 species. Work in my lab established the utility of the chloroplast gene matK as a useful phylogenetic marker in this family.  The goal of a recently expired NSF project (Phylogeny and Biogeography of the Gondwanan Mistletoe Family Loranthaceae) was to elucidate the phylogeny of this family and test several biogeographic hypotheses previously proposed based on tectonics and cytology.  Work by two graduate students, Jonathan Cabrera and Romina Vidal-Russell, has generated a molecular data set comprising nuclear SSU and LSU rDNA and chloroplast matK.  Analyses of these data provides a number of insights into the evolution of Loranthaceae.  For example, Nuytsia floribunda is the most basal member of the family followed by Atkinsonia and Gaiadendron. The first two genera are endemics to Australia and the latter from the New World tropics and all have previously been reported to be primitive based on the fact that they are root parasites (all other genera in the family are aerial parasites). From a biogeographic perspective, the arrangement of clades on the Loranthaceae tree does not follow the known pattern of continental separation in the southern hemisphere, thus suggesting that dispersal has been of primary importance in the evolution of major groups in the family. A phylogenetically-based classification of Loranthaceae will be of great interest to other biologists because loranths are important keystone species in many tropical ecosystems.  They have intricate coevolutionary relationships with birds for both flower pollination and seed dispersal. Molecular phylogenetic trees indicate that bird pollination has evolved in several different waves from insect-pollinated ancestors.  Biogeographic distributions and chromosome number have played an important role in interpreting the origin and radiation of this Gondwanan family.  A molecular phylogeny for the family will allow cytogeographic hypotheses to be tested using an independent source of data. 

Romina's husband, Guillermo Amico, has also been involved in the work on Loranthaceae.  His previous work (with M. A. Aizen) was published in Nature (2000; 408:929-930) on mistletoe seed dispersal by the marsupial Dromiciops australis (Chilean opossum, monito del monte).  Guille expressed interest in collecting molecular data to further investigate genetic differentiation in the mistletoe (Tristerix corymbosus). With funding from the National Geographic Society, Guille began a project looking at genetic variation within T. corymbosus populations using RAPDs.  These data did not indicate that the northern and southern populations had fixed genetic differences.  Because RAPDs are rapidly evolving and have limitations with respect to their application above the species level, we decided to collect DNA sequence data.  We looked at nuclear ITS and the atpB-rbcL and trnL-F spacers from the chloroplast for most  species of Tristerix.  We found that the taxonomy of the genus requires revision, i.e. that  T. verticillatus and T. penduliflorus, previously considered part of subgenus Metastachys, belonged in subgenus Tristerix.

Concomitant with phylogenetic analysis of Santalales, my lab has also been involved in determining evolutionary relationships within three holoparasite families: Hydnoraceae, Balanophoraceae, and Rafflesiaceae (in the broad sense). Classification of these nonphotosynthetic plants using traditional means has been difficult owing to morphological reductions and losses. Determining evolutionary relationships within and among these holoparasites is arguably the most difficult avenue of inquiry remaining in angiosperm phylogenetics. These plants were among the few listed in the first APG classification as “position uncertain.” In 1997 a collaborative project with Y.-L. Qiu and his student A. Blarer (Zürich, Switzerland) was initiated.  The Zürich group were interested in sequencing mitochondrial genes such as atp1 and matR and using these data to place Rafflesiaceae among photosynthetic angiosperms. My lab had DNA samples and nuclear small-subunit sequences from many of these plants, hence this was a natural collaboration. This work resulted in a publication (Nickrent et al. 2002) that placed Hydnoraceae, which contains just two genera, Prosopanche (South and central America) and Hydnora (Africa/Madagascar), within Piperales [order of the black pepper plant].  The second APG classification (2003) now reflects this relationship.

Our group then moved forward to address the molecular phylogeny of Rafflesiaceae (in the broad sense, = s. lat.).  Four groups can be defined by morphology and these groups were confirmed by DNA sequences from both the nucleus and mitochondrion: 1) Cytinus and Bdallophyton (Cytinaceae), 2) Rafflesia, Rhizanthes, and Sapria (Rafflesiaceae in the strict sense), 3) Apodanthes, Pilostyles, and Berlinianche (Apodanthaceae), and 4) Mitrastema (Mitrastemonaceae).  Although the work by Barkman et al. (2004) showed that Rafflesia was placed with the order Malphiales and Mitrastema with Ericales, they did not include phylogenetic information the remaining two clades, Cytinaceae and Apodanthaceae.  Sequencing work by our group produced data that showed that these two clades were not related to the Rafflesia or Mitrastema clades (Nickrent et al. 2004).  The former is strongly supported as a component of Malvales (the order of cotton).  The affinity of the latter family is variable depending upon the gene used (Malvales or Cucurbitales) and is complicated by rate heterogeneity and likely horizontal gene transfer (HGT) events.  This work helped to clarify a long-standing debate about the phylogenetic placement of Rafflesiaceae s. lat.  From an analytical perspective, this work is significant in that it shows that model-based methods (maximum likelihood and Bayesian analyses), when applied to a nuclear small-subunit rDNA data set, recover the same topology as the mitochondrial matR gene tree, thus providing independent confirmation of the earlier result for Rafflesia and Mitrastema.  This is important because of the difficulties in determining the phylogenetic placement of taxa that have increased evolutionary rates [i.e. they exhibit classic “long-branch attraction” artifacts].  More recently, collaborations with Charles Davis and Ken Wurdack resulted in a paper (and cover!) in the journal Science wherein we showed that Rafflesiaceae is closely related to Euphorbiaceae.  This is a surprising result because flowers in Euphorbiaceae are very small.  Calculations showed that the stem lineage of Rafflesia experienced a 79-fold size increase over ca. 46 million years.

The last remaining holoparasite group that had not been placed within angiosperms was Balanophoraceae s. lat.  These plants are very strange, with morphologies so modified and reduced that botanists in the 19th century classified them among the fungi!  For over 150 year, one member of this group, Cynomorium, was considered by some botanists distinct from the remaining 17 genera.  Cynomorium [the “Maltese Mushroom”] has long been known to the Muslim world as a medicinal plant and this knowledge was passed to Europeans in the 16th century.  Molecular phylogenetic work on Balanophoraceae and Cynomorium (Nickrent et al. 2005) cleared up a number of taxonomic issues.  This work showed that Cynomorium is not related to Balanophoraceae but to Saxifragales [the order with Sedum or stonecrops].  And the most surprising result obtained was that Balanophoraceae appears related (using both nuclear and mitochondrial genes) to the sandalwood order (Santalales).  Although some previous classifications placed these two groups together, most workers assumed this was overly influenced by the presence of parasitism and that any morphological similarities were simply a result of convergence.  Further work is needed to confirm which members of the order are most closely related to Balanophoraceae.

In addition to molecular phylogenetic studies, my lab has also remained active in pursuing questions that center around plant population genetics.  These studies began years ago with isozyme analyses of mistletoes and have continued to apply this methodology to questions in other groups.  For example, genetic diversity in Mead's Milkweed was assessed using isozymes in a collaborative project between Diane Tecic (SIUC graduate student) and Marlin Bowles (Morton Arboretum).  Asclepias meadii is a federal threatened milkweed that was once more common in the prairies of the Midwest.  The few remaining small populations in Illinois, Iowa, and northern Missouri persist vegetatively but no longer produce seeds and are vulnerable to stochastic extinction processes.  Isozyme electrophoresis was used to measure the amount and distribution of genetic variation in A. meadii and to provide guidance for its recovery and restoration (Tecic et al. 1998). The data indicate that the introduction of additional genotypes into declining populations is necessary.  These data, and results of artificial crosses made by M. Bowles with geographically distant sources, show that outbreeding depression is less a concern than previously thought, at least for these plants.  Another isozyme study conducted on two rare Illinois legumes (Dalea foliosa and Astragalus tennesseenis) was begun by a graduate student (Bethany Wiltshire) in my lab in 1994.  This work was “resurrected” by Dr. Adrienne Edwards at the Illinois Natural History Survey who helped collect additional data and conduct more analyses.  These data were published in the Journal of the Torrey Botanical Society (Edwards et al. 2004). 

My lab has also applied DNA methods to plant population genetic questions.  Although many studies focus on rare and endangered plants, a former graduate student in the Nickrent lab (Danny Gustafson) was interested in characterizing the genetic diversity and genetic identity of species that are dominants in the Illinois tallgrass prairie.  RAPD (randomly amplified polymorphic DNA) as well as isozyme data were collected for Andropogon gerardii, Sorghastrum nutans, and Dalea purpurea populations from remnant and restored Illinois tallgrass prairies, Konza Prairie Research Natural Area (Kansas), and several commercially available cultivars of Andropogon gerardii and Sorghastrum nutans.  This work has shown that 1) there are genetic difference between local and non-local seed sources, 2) it is not correct to assume that small remnant populations have low genetic diversity relative to larger populations, and 3) there are differences in plant performance between local and non-local Andropogon gerardii, Sorghastrum nutans, and Dalea purpurea seed sources.  Results from Gustafson’s dissertation research have been published in several research journals (Gustafson et al. 1999, 2001, 2002, 2004a, b).


SIUC / Plant Biology / Faculty / Dan Nickrent / projects /
URL: http://www.plantbiology.siu.edu/nickrent/projects.html
Last updated: 25-Sept-07 / dln