Sierra Nevada Aquatic Research Laboratory

12 Jul, 06 AM - 20 Jul, 06 AM

My thesis is a proposed evolutionary history of the entire plant genus Phacelia (Boraginaceae), using nuclear and chloroplast gene sequence variation to infer lineages. Phacelia occurs in North and South America and contains three federally endangered species. A large group of two hundred species, only a fraction of the species in Phacelia have been collected for any type of molecular study, leaving the genus and sections in positions of evolutionary uncertainty. My field research will collect previously unsampled species from Valentine Eastern Sierra Reserve, for both morphological description and molecular phylogenetic analysis. Using chloroplast genes to look at the deep branch relationships and the nuclear genes to look at the more recently evolving species to species relationships in the entire genus, my data will give systematists and conservation biologists a clear and accurate picture of the entire Phacelia tree of life for the first time. Valentine Eastern Sierra Reserve contains five documented native species of Phacelia important for this study. Valentine Camp has Phacelia hastata Lehm. ssp compacta (Brand) Heckard, Phacelia hydrophylloides A. Gray, Phacelia mutabilis E. Greene, and Phacelia ramosissima Lehm. var. eremophila (E. Greene) Macbr. SNARL has Phacelia bicolor S. Watson var. bicolor (Howald and Orr 2000). One collection of P. hydrophylloides was made from Sierra County for molecular analysis but remains unassigned to section in subgenus Phacelia (Garrison 2007), and no other taxa from VESR have been collected and analyzed for phylogenetics. The protection of the reserve as a research site makes it a place that supports populations of these taxa, and ideal for systematic research for a master's thesis. The Valentine Student Grant proposal is in support of a master?s thesis to sample the entire genus Phacelia (Boraginaceae) and infer phylogenetic relationships based on chloroplast ndhF and nuclear ITS gene sequences. Phacelia is a large genus of two hundred species distributed in North and South America, including three federally endangered and three federal candidate species. Systematics of Phacelia historically divide the large genus into sections. Species included in sections have been at times contradictory or unresolved. Only a fraction of the species in Phacelia have been collected for any type of molecular study, leaving the genus and sections in a positions of evolutionary uncertainty. Complete sampling of the entire Phacelia genus is now necessary for phylogenetic analysis, to provide data on evolutionary relationships and conservation biology. This research uses chloroplast ndhF genes to determine molecular support for subgeneric sections and nuclear rITS genes to determine species level relationships within those sections. Comparing these inferred molecular phylogenetic trees to traditional morphological characters and palynology will help reconstruct evolutionary events. My research will be the first complete phylogeny of Phacelia, and it is necessary to include species from the Valentine Eastern Sierra Reserve. The history of Phacelia?s ?state of taxonomic flux? (Gillett 1960) began with A.L. Jussieu including Phacelia in the ?Ordo Boragineae? (Jussieu 1879). Name changes and revisions resulted in Asa Gray recognizing Phacelia within the family Hydrophyllaceae, and describing the ?fifty species known? from North America (Gray 1875). Today there are approximately two hundred species of Phacelia described, although ?there are many species of Phacelia not considered in recent revisions? (Ferguson 1998). Presently, molecular evidence has shown firm support to include Hydrophyllaceae within Boraginaceae, and results show Phacelia remaining as a ?core hydrophyll? monophyletic clade within Boraginaceae, warranting further molecular systematic investigations (Ferguson 1998, Olmstead et al 2000). Phacelia is a difficult group for correct field identification and a challenge for systematics, containing extremely rare species and ?morphological and geographical odds-and-ends? (Constance 1963). The genus consists of perennials, biannuals, and annual herbs united by fused corollas and glandular or hispid stems and foliage (Garrison 2007). Collection and description of new Phacelia species from Mexico in 1986 resulted in the published comment ?[i]n spite of revisional treatments for both groups, I am unable to identify the plants concerned and have little doubt that they represent undescribed species? (Turner 1986). No comprehensive taxonomic revisions of Phacelia sections have been completed recently. Morphological sections in partial taxonomic treatments have been based upon geographic distribution, cytology, and chromosome count and seed characteristics. This has resulted in sections that have been contradictory, have included species in multiple sections, or have left species as unassigned. A synthesis of classification systems contain subgenera Cosmanthus, Howellanthus and Phacelia, which contains sections Euglypta, Gymnobythus, Miltitzia, Whitlavia and Phacelia, itself containing subsections Crenulatae, Franklinii, Hispida, Humiles, Magellanicae, Pulchellae and unassigned species (Ferguson 1998). Previous molecular studies have confirmed some support for these various sections, but are restricted in utility due to limited analysis within the large genus. Gilbert et al examined fifty-three species using nrITS (2005); Garrison examined forty-two species using nrITS (2007) and Ferguson examined nineteen species using ndhF (1998). Many species remain untested using molecular techniques, and include the five Phacelia taxa in the Valentine Reserve that have not been collected for molecular analysis, and four of the species have never been included in any phylogenetic study (Howald and Orr 2000). The majority of the approximately two hundred Phacelia species occur in North America and into Mexico. The subsections Crenulatae and P. magellanica complex have an amphitropical distribution between North American and South America (Raven 1963). The prominent gap in the species range may be due to a migratory path south along the Cordilleran System, but these disjunct species pairs have not been studied in Phacelia using molecular systematic techniques (Heckard 1960). Collecting from VESR taxa from this complex such as P. hastata ssp. hastata and P. mutabilis will greatly add to systematics data. Three species of Phacelia are federally endangered in North America, Phacelia insularis var. insularis, Phacelia argillacea and Phacelia formulosa. Federal candidate species include Phacelia stellaris, Phacelia submutica, and Phacelia argentea. California is the center of diversity with ninety-three species, of which thirty-nine are endemics (Raven and Axelrod 1998) and thirty-six species are listed in the California Native Plant Society?s Inventory of Rare and Endangered Plants (CNPS 2007). There are some conservation publications with goals to conserve these rare and endangered plants, but there is little phylogenetic evidence to support these efforts. A small pilot study conducted during Fall 2007 has shown good amplification for the ndhF gene from Phacelia species DNA extractions. PCR optimization and nucleotide sequencing is currently in progress. The ndh group of chloroplast genes is highly conserved across major plant taxa, and is used to assess and define sectional evolutionary relationships (Bohs and Olmstead, 1997). This gene has been analyzed and sequenced successfully with good resolution within Phacelia; nineteen species of Phacelia were included in a study of evolutionary relationships within Boraginaceae (Ferguson 1998). Ferguson confirmed the usefulness of the chloroplast ndhF gene sequence for work that spans "a wide taxonomic range, i.e., within Boraginales and within Phacelia." (Ferguson 1998). The informative characteristics of the gene are possibly due to different functional constraints on each region (Ferguson 1998). My proposal builds upon previous work completed by Gilbert et al. (2005), Hansen (2005), and Garrison (2007). Collectively these researchers have sampled and analyzed approximately eighty species within Phacelia using 5.8S and flanking internal transcribed spacer (ITS) regions (1 and 2) of nuclear ribosomal DNA. nrITS has a high degree of variation between closely related species rendering it useful for species level analysis in phylogenetics. I plan to expand the phylogeny using Gilbert, Hansen and Garrison?s tested markers and methods and to complete the Phacelia nuclear ITS phylogeny. Phacelia species will be collected in the field during 2008 and 2009 seasons. VESR taxa will be collected during the flowering period from early June to late August 2008. Field collecting is appropriate and possible for this project due to the high species diversity centered in California and across North America. Collections target an additional one hundred and twenty remaining Phacelia species to be sampled from the genus throughout the population ranges. Mainland and Baja Mexico contain rarely collected species of importance for this study, such as three species newly described in 1986 (Turner 1986). The Crenulatae and Phacelia magellanica complex of eight taxa in Chile and Argentina represent important species pair samples to be collected (Raven 1963, Flora of Chile, Heckard 1963). A collecting trip to South America is planned for January 2009; additional research funds are currently being sought for that trip. The herbaria and DNA collections held from Hansen?s and Garrison?s research would augment field collections for ndhF analysis, and the new collections will be analyzed with nrITS. Additional sequences and herbaria specimens will be sought from GenBank, other universities, and museums. Working with botanists at institutions in the local area, field access to specimens will be arranged with required permits and permission prior to collection. Voucher specimens will be deposited in the Harry Thiers Herbarium at SFSU. Collections for molecular work will be preserved in silica gel. Fresh pollen collected from voucher specimens will be analyzed using scanning electron microscopy. Phacelia pollen has three pseudocolpi alternating with three true colpi for a group of species within the genus, and is very useful as an informative characteristic for understanding relationships but has not been surveyed for the entire genus (Ferguson 1998). My research benefits from Phacelia collection methods and nrITS protocols developed by previous researchers at SFSU (Garrison, pers. comm.), and published ndhF primers (Ferguson 1998). Molecular techniques for this project have been developed and tested at the SFSU CG Laboratory (Cipriano, pers. comm.). Analysis will result in phylogenetic relationships inferred from ndhF and ITS gene sequence data from SEQUENCER and PAUP, to be compared with morphologically based relationships, pollen surface morphology and geographic distribution. All nucleotide sequences will be deposited in GenBank. It is important to reevaluate species level taxonomy within Phacelia by complete sampling and analysis using the nuclear rITS gene. Important too are the broad relationships between sections in the Phacelia phylogeny, especially where the nuclear ITS gene has only provided varied degrees of statistical support for several of the Phacelia groups. Of interest is subgenus Cosmanthus relationship to the large subgenus Phacelia, and the problem that species in the Humiles group have been shown to not be closely related to each other and need to be reconsidered. Subsections Franklinii and Pulchellae have been hypothesized to be the origin of the Phacelia clade. The P. magellanica complex analysis will greatly benefit from VESR taxa collections. The low copy chloroplast gene ndhF has demonstrated utility for lower level phylogenetic studies with a good number of informative characters and will help provide much needed support for these subgeneric sections. My research will be the first complete investigation of the Phacelia genus using both nuclear and chloroplast sequence data. This research proposal unites both conservation biology and modern molecular techniques in an attempt to resolve relationships within Phacelia. My systematics research will offer important information to the conservation biology community by providing new ecological data and specimen collections of rare and unique species, powerful molecular analyses to infer relationships within the genus, and providing a solid foundation for further research within and beyond Phacelia

Visit #14994 @Sierra Nevada Aquatic Research Laboratory

Approved

Under Project # 9872 | Research

Phylogenetic analysis of sectional and species level relationships within Phacelia (Boraginaceae) inferred from chloroplast ndhF and nuclear ribosomal internal transcribed spacer (nrITS) sequence data.

professional - California Department of Food and Agriculture

Reservation Members(s)

Genevieve Walden Jul 12 - 20, 2008 (9 days)

Reserve Resources(s) | Create Invoice