Visiting the hepaticologist Rudolf M. Schuster (1921 – 2012) in Massachusetts 2011.
Dr. Juan Carlos Villarreal
University press release
I am interested in the evolution of early seedless plants: Liverworts, mosses, hornworts, lycophytes and ferns, the closest extant relatives to the early land dwellers. I have a broad range of experience in evolutionary biology that I use to unravel the structural and genomic adaptations that the earliest colonizers faced on land. I use electron microscopy, DNA sequences, genomic data, and an array of phylogenetic and dating analyses to resolve deep and shallow divergences in seedless plants, particularly the hornworts. Current hornwort diversity is estimated at 200 – 240 species, a small number in comparison to mosses (11000 – 13000 spp., Magill 2010, liverworts (7000 – 9000 spp., von Konrat et al. 2010, lycophytes (1285 spp., Frey & Stech 2009) and ferns (11000 spp., Smith et al. 2006). However, as the closest relatives of vascular plants, hornworts have special importance for inferring ancestral trait states of the vascular plants. I have funding from the Deutsche Forschungsgemeinschaft (DFG) for my post-doctoral research, and previous work was funded by the US National Science Foundation (NSF).
Systematics, anatomy, ultrastructure, molecular evolution, and biogeography of hornworts
(Anthocerotophyta), especially American taxa. Molecular clock dating, population genetics,
genomic evolution, and genetic implications of the loss of sex in bryophytes.
I am also interested in the evolution of carbon concentration mechanisms in the hornwort chloroplast, especially the molecular and physiological implications of the presence of an algal-like pyrenoid. Other topics I have been studying are chloroplast genomes in bryophytes, RNA editing in hornworts, and hornwort transcriptomes, the last in collaboration with onekp project [onekp.com].
Why the hornworts?
The eukaryote Tree of Life is sprinkled with lineages of Paleozoic origin that have low extant diversity, including cycads (ca. 250 spp.), the Ginkgo (1 sp.), Gnetales (95 sp.), and sphenopsids (15 spp.). With their distinct morphologies, these lineages are of paramount importance for understanding character transformations and the evolution of body form. Low extant diversity is often explained by an ancient radiation followed by multiple and massive extinctions through geological time. Hornworts are the most species-poor of seedless plant phyla.
Current phylogenetic thinking places hornworts as sister to tracheophytes (vascular plants). Thus, despite their low species numbers, hornworts represent a key group in the evolution of plant form and may hold clues on the evolutionary shift from a gametophyte- to a sporophyte-dominant life cycle. To understand character evolution of tracheophytes it is critical to assess the character state in the outgroup, namely the hornworts
A. Evolution of sex in hornworts
In land plants, separate sexes (dioicy or dioecy) have evolved many times independently
(Renner & Ricklefs; Devos et al. 2011; McDaniels
et al. 2012), but little is known about the evolutionary consequences of the implied switches
among sexual systems.
On the one hand, hermaphrodites (bisexual organisms) have the advantage of reproductive assurance. However, they may experience inbreeding depression. Plants with separate sexes (dioicous or dioecious) have the advantage of outbreeding and they have developed mechanisms to “move” and “find mates” such as small spores in mosses (Crawford et al. 2009). In haploid-dominant organisms (mosses, liverworts, hornworts), hermaphrodites are thought to be highly efficient in purging slightly deleterious mutations, and inbreeding depression may therefore not be the main selective advantage of separate sexes. The evolution of separate sexes remains a puzzle.
I am currently compiling a sampling of nearly 50% of all known hornwort species to address the question of sexual system changes within the group. This will be one of the first comprehensive phylum-wide studies of sexual systems. I will also be comparing life-history traits (such as spore size, substrate, and size of male/female plants) with sexual systems to search for possible correlations.
B. Evolution of carbon concentrating mechanisms
RuBisCO has a crucial role in carbon fixation but a slow catalytic rate, a problem
overcome in some plant lineages by physiological and anatomical traits that elevate
carbon concentrations around the enzyme. Such carbon-concentrating mechanisms (CCMs)
are hypothesized to have evolved during periods of low atmospheric CO2.
Hornworts, the sister to vascular plants, have a CCM that relies on pyrenoids
(proteinaceous bodies consisting of up to 90% RuBisCO). In algae, CO2
levels near the pyrenoid are raised up to 180 times above the concentration in the rest
of the cell, enhancing photosynthesis in aquatic environments where CO2
diffusion is limited. The extent to which pyrenoids are crucial to algal CCMs, however,
has been controversial because although all pyrenoid-containing algae have CCMs, not
all algae expressing CCMs have pyrenoids. Some algal lineages without pyrenoids have
passive or active ways of accumulating CO2, and they may rely
on carbon anhydrases to create a pool of internal dissolved carbon, suggesting alternative
adaptive advantages of pyrenoids besides RuBisCO concentration and more efficient CO2
The results of my recent study imply 5 - 6 origins and an equal number of subsequent losses of pyrenoids in hornworts, with the oldest pyrenoid gained ca. 100 mya, and most others at <35 mya. The non-synchronous appearance of pyrenoid-containing clades, the successful diversification of pyrenoid-lacking clades during periods with low CO2, and the maintenance of pyrenoids during episodes of high [CO2] all argue against the previously proposed relationship between pyrenoid origin and low [CO2]. The selective advantages, and costs, of hornwort pyrenoids thus must relate to additional factors besides atmospheric CO2.
A recent paper by
collaborators suggests that small changes in the exposed helices of the nuclear subunit of
the RuBisCO (rbcS) are responsible for pyrenoid formation in the unicellular green algae
Chlamydomonas. The research group mostly
based in Cambridge is attempting to transfer the Chlamydomonas CCMs to Arabidopsis
(and eventually crop plants). I intend to further understand the biogenesis and molecular biology
of the hornwort pyrenoids. In collaboration with colleagues from Howard Griffith’s lab in
Cambridge, I intend to use in hornworts the analysis pipeline already developed for Chlamydomonas.
Arguably hornworts are better donors than algae for genetic engineering of pyrenoids in crop plants because they are phylogenetically closer, and shared features of chloroplast organization with flowering plants may allow to implement a fully functional CCM in crops. Hornworts offer a promising opportunity to study chloroplast evolution, and in particular, the acquisition and maintenance of an alternative way to concentrate carbon in terrestrial environments. However, it is critical to ascertain if hornwort pyrenoids form in an analogous fashion to those in Chlamydomonas. Once a hornwort nuclear genome is in hand, it should become possible to perform the transcriptomic and mutagenic work essential for unveiling the components of the hornwort CCM.
C. Symbiotic associations
Past and current work
I. Temporal and spatial diversity of nitrogen-fixing symbiotic bacteria
I am interested in the symbiotic associations between plants and nitrogen-fixing
cyanobacteria. Nitrogen is one of the more abundant elements in the atmosphere (~78%),
however most of this nitrogen is not accessible to plants.
Leiosporoceros dussii. Bifurcating strands of Nostoc parallel the main axis of the thallus in a female plant with one sporophyte. Nitrogen-fixing bacteria, mostly from the genus Nostoc, can fix nitrogen and make it available to plants. The input of fixed nitrogen mediated by mosses in boreal forest is estimated to be 1.5 - 2.0 kg N ha−1 year−1 (De Luca et al. 2002). The total estimated nitrogen contribution from cycads (18.8 kg N ha−1 year−1, Holub & Lajtha 2004) and cyanolichen Lobaria (2.5 - 4.5 kg N ha−1 year−1, Vessey et al. 2005) is also substantial. Hornworts form carpets in the understory of south temperate forests of Chile and New Zealand. The hornwort-mediated nitrogen contribution in Austral temperate forest is unknown.
For my master’s degree, I studied the anatomy and development of the Neotropical hornwort Leiosporoceros dussii. Using transmission electronic microscopy I have uncovered cyanobacterial diversity within the thallus and an increase of heterocyst (nitrogen-fixing cells) in symbiotic Nostoc. I have preliminary data from the cyanobacterial genes rbcLX, trnL and 16S of cyanobionts within Leiosporoceros. I have hornwort collections from all continents and I am planning to further pursue a worldwide study of symbiotic association and compare them with cycads and cyanolichens using phylogenetic and dating techniques. Hornworts and cycads share three peculiarities not due to common ancestry:
- Less than 300 species worldwide,
- species groups (genera) of very recent origin, and
- every single species has a cyanobiont, hornworts in the cavities inside the thallus and cycads in their coralloid roots.
A future area of interest would be to quantify the nitrogen contribution mediated by hornworts in Austral temperate forests
II. Fungal endophytes
I recently became interested in the symbiotic associations between plants and fungi
(Glomerophytes and Endogonales). This work is a collaboration with Alessandro Desiró
(University of Turin), Silvia Pressel
(Natural History Museum, London, Jeff G. Duckett
and Martin Bidartondo (Kew Garden).
A preliminary study shows a dynamic interaction between diverse clades of Glomerophytes
and Endogonales. The fungi even interact with Nostoc colonies, an association previously
In October 2012, we made a trip to the Indian Himalayas to further explore the associations in early land plants.
D. Plastid genomics
The previously-sequenced plastome of the hornwort Anthoceros angustus differs from
that of other bryophytes by an expanded inverted repeat (IR) and the presence of a type
I intron in the 23S ribosomal RNA (rrn23) gene, found otherwise only in the chlorophyte
algae Chlamydomonas and Chlorella. The intron is found in the same exact
position and with ~50% of sequence identity between algae and A. angustus (Kugita et al. 2003).
In collaboration with Laura Forrest,
Norman Wickett and
Bernard Goffinet we
assembled the plastome of the hornwort Nothoceros aenigmaticus, contrast its architecture
to that of other bryophytes, and assess the phylogenetic significance of genomic characters
in hornwort evolution (Villarreal, Forrest, Wickett, Goffinet,
American Journal of Botany, in press).
The genome was assembled using a combination of shotgun sequencing of genomic DNA and Sanger
sequencing. Comparison with the Anthoceros plastome revealed three structural differences.
In addition, we sequenced these regions in taxa spanning the hornwort phylogeny.
The Nothoceros plastome is colinear with other bryophyte plastomes, but differs from the Anthoceros plastome as several gene regions located within the IR in Anthoceros are in the large single copy region in Nothoceros, there is no intron in the rrn23 gene and rpl2 is a pseudogene. Comparisons across the hornwort phylogeny indicate that the first two characters are restricted to Anthocerotaceae, while the rpl2 pseudogenization diagnoses the sister lineage to Anthocerotaceae.
The Nothoceros plastome is structurally similar to that of most bryophytes. However, we identified more structural differences within hornworts than have been described within either the mosses or the liverworts. The distribution of the gene duplication involving the IR and an intron in the rrn23 gene are restricted to Anthocerotaceae. Occurrence of the intron and the conserved intron sequence between Anthoceros and distantly related chlorophyte algae may be due to horizontal gene transfer.
E. Pleistocene landscape genetics and clonality in a Southern Appalachian endemic plant
My doctoral dissertation research focused on the major evolutionary events leading to the
lack of sexual reproduction in the Southern Appalachian (SA) endemic and asexual hornwort
Nothoceros aenigmaticus. This species is the only member of the genus in North America,
and there are no reported observations of sporophyte production.
Nothoceros aenigmaticus, collected in North Carolina. Note the elongated eggs inside of the thallus, probably from a damselfly (K. Tennessen, pers. com.). More studies are needed to verify the identity of the insect. In the SA region, male and female plants live ~ 30 miles apart, and male plants produce depauperate antheridia (for unknown reasons sperm cells are unable to develop functional flagella). My research had three main objectives:
- A phylogenetic delimitation of the genera Nothoceros and Megaceros
- Reconstruction of the phylogenetic origin and timeframe of the loss of sexuality in N. aenigmaticus.
- To assess the population genetic structure of clonal populations of N. aenigmaticus using microsatellites.
I unraveled the phylogenetic origin of N. aenigmaticus and its closest Neotropical
relative, using several plastid markers (rbcL, matK, trnL-F intron,
trnL-F spacer, rps4-trnS spacer), the mitochondrial nad4-nad5
spacer, and the nuclear ITS2. The closest sexual relative of the species are Mexican populations
where males and females live sympatrically (Villarreal et al. 2012 a,b
see publication list in website). The circumscription of Nothoceros aenigmaticus has
been broadened to include similarly dioicous and fully sexual populations found in Bolivian
Punas (above 3000 m.),
the North and West Andes Páramos and in Costa Rican Páramos (above 3000 m.). The tropical
alpine populations of N. aenigmaticus show high genetic structure perhaps as a molecular
imprint of past expansions and contractions of Páramo areas in the Pleiostocene. This portion
of the study deserves further research.
The last part of my dissertation dealt with the population genetics of N. aenigmaticus, using chloroplast, mitochondrial and nuclear microsatellites (Villarreal et al. 2012b). I found a unique case of sex allopatry in a non-flowering plant that seems to reproduce clonally and to be genetically isolated from their closest relatives in Mexico. The genetic structure of the Southern Appalachian populations appears to be influenced by the geological consequences of the Last Glaciation, specifically stream capture involving the Coosa River (draining into the Alabama River) and the Ocoee-Nontootla River (draining into the Tennessee River, then Ohio and eventually Mississippi). Genotype sharing between these two isolated watersheds suggests that stream capture played a role in the contemporary genetic diversity of these Southern Appalachian plants.
Current postdoc project
Evolution of hornworts: Building a world-wide phylogeny to resolve all major clades
Crawford M., L.K. Jesson & P.J. Garnock-Jones (2009): Correlated evolution of sexual system and life-history traits in mosses. Evolution 63: 1129 – 1142.
Devos N., M.A.M. Renner, R. Gradstein, A.J. Shaw, B. Laenen, and A. Vanderpoorten (2011): Evolution of sexual systems, dispersal strategies and habitat selection in the liverwort genus Radula. New Phytologist 192:225 – 236.
Frey W. & Stech M. (2009): Marchantiophyta, Bryophyta, Anthocerotophyta. In Frey, W. (ed.), Syllabus of Plant Families – A.Engler's Syllabus der Pflanzenfamilien, 13ed. Part 3: Bryophytes and seedless Vascular Plants: Borntraeger, Berlin, 13 – 115 pp.
Kugita M. et al. (2003): The complete nucleotide sequence of the hornwort (Anthoceros formosae) chloroplast genome: insight into the earliest land plants. Nucleic Acids Research 31: 716 – 721.
Holub S.M., Lajtha K (2004): The fate and retention of organic and inorganic 15 N-nitrogen in an old-growth forest soil in Western Oregon. Ecosystems 7:368 – 380.
McDaniel S.F., J. Atwood & J.G. Burleigh. Early view. Recurrent evolution of dioecy in bryophytes. Evolution
Renner S.S. & R.E. Ricklefs (1995): Dioecy and its correlates in the flowering plants. American Journal of Botany 82: 596 – 606.
Smith A.M., Pryer K.M., Schuettpelz E., Korall P., Schneider H. & Wolf P.G. (2006): A classification of extant ferns. Taxon 55(3): 705 – 731.
Vessey J.K., Pawlowski K., Bergman B. (2005): Root-based N2-fixing symbioses: legumes, actinorhizal plants, Parasponia sp. and cycads. Plant Soil 274:51 – 78.
Villarreal J.C., L.L. Forrest, K. McFarland & B. Goffinet (2012): Chloroplast, mitochondrial and nuclear microsatellites from the Southern Appalachian hornwort, Nothoceros aenigmaticus (Dendrocerotaceae). American Journal of Botany 99: e88 – e90.
Villarreal J.C., L.V. Campos & B. Goffinet (2012): Parallel evolution of endospory in hornworts: the case of Nothoceros renzagliensis, sp. nov. Systematic Botany 37: 31 – 37.
Villarreal J.C., L.L. Forrest, N. Wickett & B. Goffinet (2013): The plastid genome of the hornwort Nothoceros aenigmaticus: Phylogenetic signal in inverted repeat expansion, pseudogenization and intron gain. American Journal of Botany 100: 467 – 477
Last update: 2014-10-30