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).