This project aims to resolve the liverwort Tree Of Life. There is mounting evidence that liverworts (Phylum Marchantiophyta) were the first green plants to diversify on land some 500,000 million years ago and as such they are the oldest living lineage of terrestrial organisms. This species-rich group of small green plants is remarkably diverse in structure, and includes extremely ancient relictual lineages as well as more recent radiations of closely related species. The antiquity of liverworts and the rich biodiversity at all taxonomic levels provide an unparalleled window into early land plant diversification. This collaborative effort brings together experts from around the world for a multidisciplinary, highly integrated approach that combines anatomical and developmental features with DNA sequence and genome structural characters to resolve phylogenetic relationships across the entire spectrum of liverwort diversity. Three general types of data will be compiled: 1) conservative morphological and genome characters to resolve deep "backbone" relationships, 2) anatomical/developmental data to resolve intermediate-depth lineages, and 3) morphological and DNA sequence characters to resolve relationships among a sample of 800 taxa representing all genera of liverworts. A second major goal of the project is to integrate phylogenetic inferences and bionformatic efforts between this and other on-going NSF-supported projects, including several funded ATOL programs. These integrative activities include contributions to studies of genome structure and evolution across land plants, expansions of novel informatic tools to make methods, results, and implications widely accessible, and continued development of DNA sequence utilities that will benefit a broad range of scientists working on diverse organisms. This project relies on the cooperation with and integration among research programs focused on resolving the green plant Tree Of Life. Integrative activities are substantive and consequential, and include expansions to phylogenetic databases and methodological/conceptual aspects of work toward resolving the Tree Of Life. At least six post-doctoral associates and as many graduate students will be supported by the project, with substantial involvement of undergraduates as well. An effort to involve and retain individuals from underrepresented groups will be orchestrated through established federal programs that focus on such initiatives, including the Louis Stokes Alliance for Minority Participation and the McNair Scholars Program. A solid mentoring infrastructure will be developed within each research team to provide support and professional experiences that ensure productivity and success of all participants. Outreach efforts include web presentations on the morphology and identification of liverworts for educators at all levels, development of web-based materials and a workshop targeting secondary school teachers, and increasing public awareness through weekend field excursions.
Intellectual Merit Liverworts hold an important place in the history of life on Earth because they are the oldest extant plant group to evolve on land. Originating from green algal ancestors, these small obscure plants hold many clues to the complexities and events that enabled green plants to colonize and diversify out of water. Through the Liverwort Tree of Life project (LiTL), we have contributed a significant body of work that advances knowledge of the biology of liverworts. With a robust molecular phylogeny that resolves relationships within the group produced by collaborators on LiTL, my lab team conducted comparative ultrastructural studies on strategically selected taxa representing nodes of diversification within liverworts. We focused on studies of spermatogenesis, sporogenesis, the sporophyte-gametophyte junction, and the gametophytic growing point. Much of the effort entailed obtaining materials from around the world at the right stages of development and preparing these materials for future studies. A rich collection of liverwort species and materials has been processed and is available for scanning and transmission electron microscopy for generations of scientists to study. The analyses and images collected during this project will impact text book descriptions and illustrations, and will inform future research of biodiversity, conservation biology and phylogeny of these plants and other seed free plants. Summaries of results in the four focus areas follow. Four compilation plates demonstrating the types of studies and images accumulated on each structure/process are also attached. Spermatogenesis We currently have eight mature sperm cells of liverworts reconstructed. With both deep divergences and modern radiations in this earliest group of land plants, we are able to examine the evolution of motile cell architecture as illustrated in figure 1. Most notable is the independent increase in size and coiling in gametes in the simple thalloid 1 (Pellia), simple thalloid 2 (Aneura) and one leafy liverwort clade (Bazzania). Sporogenesis We have contributed important information on the biodiversity and development of spores. Figure 2 demostrates features of sporogenesis in the complex thalloid liverwort Sphaerocarpos, a liverwort that has spores that remain in tetrads. Characterization of spore development and ornamentation has provided important new characters for comparison with early fossil sporangia and spores from the Silurian, Ordivician and Devonian. In many instances, spores are the only remnants of these first colonizers of land. Gametophyte-sporophyte junction The junction between the gametophyte and sporophyte is marked by the existence of cell wall modifications in the contact cells between generations. These transfer cells vary in location and morphology across liverworts (Figure 3). Sphaerocarpos, Blasia and Marchantia in the complex thalloid lineage have elaborate wall ingrowths on both sides of the placenta. Riccardia is unique in the possession of a wall labyrinth on gametophyte cells but not on the sporophyte side. Many leafy liverworts such as Lepidozia and Scapania and a few simple thalloid liverworts such as Pallavicinia have wall elaboration on the sporophyte but not the gametophyte side. Other simple thalloids such as Pellia and Aneura lack wall ingrowths on either side of the placenta. Arabinogalacturonic acid proteins are located at the tips of ingrowths and along the inner walls, suggesting homology of this zone regardless of wall elaboration. Apical meristem Studies of liverwort apical meristems were conducted at the TEM level taxa with each of the four apical cell shapes: tetrahedral, wedge-shaped, lenticular and hemisdiscoid (Fig. 4). The tetrahedral shape is resolved as plesiomorphic. Plasmosdesmata are the most dense in apical cell walls, regardless of apical cell shape. Wall constituents vary with age of walls and location on walls, e.g., callose is associated with plasmodesmata. Broader Impacts Increasing Diversity and Training STEM Professionals Renzaglia's lab has involved three high school students and approximately 10 undergraduates in research. Of these, half have been ethnic minorities. The same is true of graduate students, many of whom are Hispanic or Native American. Three doctoral students completed degrees and obtained post-docs of academic positions in summer 2012. Two more Hispanic women are completing the doctorate in 2014. Two master’s students completed degrees and one master's student continue research in the lab. Integrating Research and Teaching Sessions on spores and spore germination were provided to local teachers in the Robert Noyce program. We continue to work with students on spores and liverwort diversity in our classes and classrooms throughout our region. Engaging the Community Renzaglia is the Director of teacher preparation programs, undergraduate research programs and community college partnerships. Through these programs countless students and teachers have learned about liverworts and how to use them in the classroom. Reaching out Beyond the Community Over 30 peer-reviewed publications have resulted from the work. One volume of Phytotaxa was co-edited by Renzaglia and a book is underway with Drs. Brown and Lemmon on Bryophyte Spores. Thousands of images illustrating liverwort diversity are being uploaded on PhytoImages for general use and viewing around the globe.
