This project will investigate the evolutionary relationships within the large and important rose family. This group of plants contains numerous commercially valuable fruits (apples, pears, strawberries, cherries, peaches, plums, blackberries, and raspberries) and many horticulturally significant plants (roses, cotoneaster, lady\u27s mantle, bridle-wreath, flowering quince, fire-thorn, cinqfoil, and others). Despite the large and important role that these plants play in our daily lives and scientific interest, we do not have a clear view of the evolutionary relationships of members of the family. A primary reason for this situation is that insufficient evidence has been accumulated to adequately address the problem. A primary aim of Dr. Campbell\u27s project is to gather DNA sequence data to provide a reliable chronicle of evolutionary history in this group. These data will be compared with structural, chemical, and other data that have been accumulated by Dr. Campbell and other researchers to create an overall picture of evolutionary relationships in the family. Such knowledge will establish a framework for studies of key groups within the family, provide insights into the evolution of interesting features in the family (such as the fruits), and contribute to our overall understanding or plant evolution

Campbell, Christopher S.

English

University of Maine

The University of MaineDigitalCommons@UMaineUniversity of Maine Office of Research andSponsored Programs: Grant Reports Special Collections12-20-2001Molecular Phylogenetic Studies in RosaceaeChristopher S. CampbellPrincipal Investigator; University of Maine, Orono, campbell@maine.eduFollow this and additional works at: https://digitalcommons.library.umaine.edu/orsp_reportsPart of the Plant Breeding and Genetics CommonsThis Open-Access Report is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in University ofMaine Office of Research and Sponsored Programs: Grant Reports by an authorized administrator of DigitalCommons@UMaine. For moreinformation, please contact um.library.technical.services@maine.edu.Recommended CitationCampbell, Christopher S., "Molecular Phylogenetic Studies in Rosaceae" (2001). University of Maine Office of Research and SponsoredPrograms: Grant Reports. 248.https://digitalcommons.library.umaine.edu/orsp_reports/248Final Report: 9806945Page 1 of 4Final Report for Period: 10/1998 - 09/2001 Submitted on: 12/20/2001Principal Investigator: Campbell, Christopher S. Award ID: 9806945Organization: University of Maine       Title:Molecular Phylogenetic Studies in RosaceaeProject ParticipantsSenior PersonnelName: Campbell, ChristopherWorked for more than 160 Hours: YesContribution to Project: Post-docGraduate StudentUndergraduate StudentResearch Experience for UndergraduatesOrganizational PartnersOther Collaborators or Contacts(A brief summary of the impact of this proposal on the research of the first three participants is given in the section on Contributions):1.	The designated postdoctoral associate, Dr. Rodger C. Evans, worked on this grant from June 1999  through August 2000, when he took atenure-track position at Acadia University in Wolfville, Nova Scotia.2.	Dr. Timothy A. Dickinson took a full-year sabbatical leave (August 1999 to June 2000) with us to learn molecular systematic techniques forhis work on the genus Crataegus (Rosaceae).3.	Jenny Smedmark, a Ph.D. student from Sotckholm University spent most of the month of July 2000 at the University of Maine collectingDNA4.	T. Eriksson, collaborator on Rosoideae, visited UMaine twice during the period of this grant.  We discussed work on the Rosoideae and theoverall phylogeny of the family. 5.	Three undergraduate students have been involved in this project. Shane Sims, a Biology major, worked in the lab during the summer of 1999. Margaret Cox is a junior majoring in Clinial Laboratory Sciences, but she has a strong interest in plants. She has worked on various aspects ofthis project, although most of her time has been devoted to other projects. Matthew Arsenault, a junior Biology major, is currently working inthe lab.  He has been trained in all phases of lab work, from PCR to cloning to sequence analysis (He will do DNA extractions sometime nextyear).  He plans to do a capstone project on some aspect of this project.Activities and FindingsResearch and Education Activities:The submitted proposal contained three objectives, each with three or four null hypotheses:1. Explore phylogenetic relationships within Maloideae s. l.2. Study phylogeny of Rosoideae s.s., especially Potentilleae.3. Bring all available data to bear on an overall phylogeny of Rosaceae.In response to the budget reduction of about 40%, I eliminated objective 2, which was justified because (1) abundant evidence indicates thatRosoideae s.s. are monophyletic, which means that we can still consider an overall phylogeny of Rosaceae without sampling extensively withinRosoideae; (2) the interests and experience of the P.I. and the designated postdoctoral associate R. C. Evans are in Maloideae; and (3) ourFinal Report: 9806945Page 2 of 4collaborators (L. A. Alice and T. Eriksson) have a primary interest in Rosoideae s.s. and can take the lead in studying objective 2.  Overall summaryThis project concerns evolutionary relationships within the large and important rose family.  This group contains numerous commerciallyvaluable fruits - such as apples, pears, strawberries, cherries, peaches, plums, blackberries, and raspberries - and many horticulturallysignificant plants - such as roses, cotoneaster, lady's mantle, bridle-wreath, flowering quince, fire-thorn, cinqfoil, and others.  Despite the rolethat these plants play in our daily lives and long scientific interest, we do not have a clear view of the evolutionary relationships of members ofthe family.  A primary reason for this situation is that insufficient evidence has been accumulated to address the problem adequately.  The chiefaccomplishment of this project has been to obtain DNA sequence data for a reliable chronicle of the evolutionary history of the rose family. These data are being compared with structural, chemical, and other data that have been accumulated for the family to create an overall pictureof evolutionary relationships in the family.  Such knowledge establishes a framework for studies of key groups within the family, providesinsights into the evolution of interesting features in the family (such as the fruits), and contributes to our overall understanding or plantevolution.Findings:The two major contributions of this project are better understanding of evolutionary relationships of the rose family and development of alow-copy-number nuclear gene (granule-bound starch synthase; GBSSI) for phylogenetic studies. Much of the results of this project issubmitted and in preparation for submission for publication.  Below are brief summaries of four of the most significant papers (seepublications) coming from this project.1.	The granule-bound starch synthase (GBSSI) gene in Rosaceae: multiple putative loci and phylogenetic utility - This was the first phylogeneticstudy to use the 5' end of GBSSI. We sampled 1.8-2 kb, including two partial and seven complete exons and eight introns for 108 clones from18 species in 14 genera representing all subfamilies of the Rosaceae and an outgroup (Rhamnus, Rhamnaceae).  Phylogenetic analysis andsouthern hybridizations demonstrate two loci (GBSSI-1 and GBSSI-2) in all four subfamilies, even though only one locus had heretofore beendocumented from diploid flowering plants.  Phylogenetic analysis further identifies four loci (GBSSI-1A, and GBSSI-1B, GBSSI-2A, andGBSSI-2B) in subfamily Maloideae, an allopolyploid group.  Phylogenetic relationships inferred from GBSSI sequences are largely compatiblewith those from other genes.  Large clades are marked by significant intron variation: a long first intron plus no sixth intron in MaloideaeGBSSI-1, a long fourth intron in Rosoideae GBSS-1, and a GT to GC mutation in the 5'splice site of the fourth intron of all GBSSI-2sequences.2.	The origin of the apple subfamily (Rosaceae: Maloideae) is clarified by DNA sequence data from duplicated GBSSI genes -- For 70 years theleading hypothesis for the origin of the Maloideae has involved wide hybridization between ancestors of two other subfamilies. The basis ofthis hypothesis is that Maloideae have a base chromosome number of 17, while other Rosaceae are mostly x=7, 8 or 9. To investigate thishypothesis we cloned and sequenced approximately 1.8 kb from the 5' portion of granule-bound starch synthase (GBSSI or waxy) genes for 70clones from 23 Rosaceae genera. Previous studies demonstrated the presence of two copies in all Rosaceae (GBSSI-1 and GBSSI-2) and four inthe Maloideae (GBSSI-1A, GBSSI-1B, GBSSI-2A, and GBSSI-2B). Parsimony and maximum likelihood analyses nest Porteranthus (Gillenia),a genus of the southeastern United States with a base chromosome number of 9, within either Maloideae GBSSI-1 or GBSSI-2 clades.Monophyly of Maloideae-Porteranthus clades is supported by bootstrap values approaching 100%, loss of the sixth intron in all GBSSI-1sequences, intron alignability between genera, and numerous non-molecular characters. Our results falsify the wide-hybridization hypothesisand are consistent with a polyploid origin involving only members of a lineage that contained the ancestors of Porteranthus. Under thishypothesis, the subfamily originated in North America and the high Maloideae chromosome number arose via aneuploidy from x = 18.3.	A phylogeny of the Maloideae - Relationships of the approximately 30 genera of Maloideae have long been a challenge. Low levels ofevolutionary divergence and intergeneric hybridization may have contributed to our poor understanding of the genera.  Alignability of GBSSIintrons, which is facile in the Maloideae, is not possible in any of the other major Rosaceae clades. Other data indicating little evolutionarydivergence among genera of the Maloideae include the ease with which many different maloid genera may be graft successfully, numerousintergeneric natural hybrids, and low sequence divergence in five chloroplast regions (matK, ndhF, rbcL, rps16 intron, and trnT-L. This lack ofdivergence is surprising in a group that extends back to at least the Middle Eocene.  Past intergeneric hybridization could well have obscuredthe phylogeny with reticulation. The limited evolutionary divergence could be the result of a combination of relatively slow rates of evolutionin woody plants or of continued gene flow between the genera.  We have completed a data set of sequences for four chloroplast DNA regions --matK, ndhF, rps16 intron, and trnT-L - for a sample of 31 maloid genera.  We have completed a data set of internal transcribed spacer (ITS)sequences, and have about 80% of a GBSSI data set for all four loci for the same sample of genera.  Our goal is to provide a robust phylogenyof this subfamily and to provide insight into potential past episodes of hybridization.4.	A phylogeny of the Rosaceae - Along with collaborators, I am assembling a multi-gene data set to attempt to establish the overall phylogenyof the family.  The largest Rosaceae phylogenetic study, the rbcL study of Morgan et al. (1994), did resolve numerous larger clades but did notresolve well the relationships among these clades.  We will use several chloroplast regions, ITS, and GBSSI toward this effort.  We plan toFinal Report: 9806945Page 3 of 4present this phylogeny at the Rosaceae colloquium during the American Society of Plant Systematists (ASPT) meetings in Madison Wisconsinin August 2002 (see below).  We are in the process of determining who among about six collaborators will be responsible for which genes andother components of this large undertaking.Training and Development:1. The postdoc, Dr. Rodger C. Evans, obtained a lot of new data, especially using granule-bound starch synthase (GBSSI) sequences andconsiderable experience in cloning and sequencing a low-copy-number nuclear gene. He coauthored one significant publication, submittedanother, and has several others in preparation.  He developed a number of collaborations and was selected for a tenure-track position at AcadiaUniversity in Wolfville, NS.2. Dr. Timothy A. Dickinson's visit to the Campbell lab at the University of Maine provided him with an opportunity to learn techniques ofmolecular systematics from C. S. Campbell, post-doc R. C. Evans, and others at the University of Maine. Funding for this visit came fromDickinson's home institution, the Royal Ontario Museum (ROM) and from his research grant from the Natural Sciences and EngineeringResearch Council of Canada (NSERCC). Supplementary funding to help offset living expenses in Orono was obtained from the University ofMaine. Using DNA extracted from samples collected prior to and during the sabbatical, substantial progress was made toward an ITSphylogeny of Crataegus, with Mespilus and other, apparently closely related Maloideae (e.g. Amelanchier) as outgroups. Amplification andsequencing of the chloroplast gene trnL was also done, in order to evaluate its utility in Crataegus and to collect data for a collaborativemulti-gene study of Maloideae phylogeny. The ITS results were presented at Botany 2000 in Portland OR in August 2000. These resultsindicate that at least two variants of ITS are present in Crataegus, of which only one (apparently restricted to Crataegus) shows phylogeneticallysignificant variation. Further work has been delayed until recently by the move of the ROM's botanical collections from offsite locations intothe main Building of the ROM.  Dickinson's work in Maine was facilitated by access not only to the resources in Campbell's lab and those ofthe Department of Biological Sciences, but also to the University of Maine DNA Sequencing Facility (www.ume.maine.edu/dnaseq/).  3. Jenny Smedmark wanted GBSSI sequences to provide perspective on the history of extensive polyploidy in Geum (there are few knowndiploids).  This gene has worked marvelously in this regard, as Smedmark was able to get most of the expected number of GBSSI copies intetraploids and hexaploids. She spent a month in our lab cloning GBSSI for her sample. She clearly demonstrates that the polyploids areallopolyploid and has identified one parental genome in several cases.Outreach Activities:NoneJournal PublicationsEvans, R.C., L.A. Alice, C. S. Campbell, E. A. Kellogg, and T.A. Dickinson., "The granule-bound starch synthase (GBSSI) gene in Rosaceae:multiple putative loci and phylogenetic utility.", Molecular Phylogenetics and Evolution, p. 388, vol. 17, (2000). PublishedEvans, R.C. and C.S. Campbell., " The origin of the apple subfamily (Rosaceae: Maloideae) is clarified by DNA sequence data from duplicatedGBSSI genes.", American Journal of Botany, p. , vol. , (    ). SubmittedCampbell, C.S., R.C. Evans, and others., "A phylogeny of the Maloideae",  Systematic Botany, p. , vol. , (    ). in preparationCampbell, C.S., R.C. Evans, and others., "A phylogeny of the Rosaceae",  American Journal of Botany, p. , vol. , (    ). in preparationBooks or Other One-time PublicationsWeb/Internet SiteOther Specific ProductsContributionsContributions within Discipline: The major contributions within the discipline are better understanding of evolutionary relationships of the rose family and development of alow-copy-number nuclear gene (granule-bound starch synthase; GBSSI) for phylogenetic studies. We are the first to use the 5' end of GBSSI inFinal Report: 9806945Page 4 of 4phylogenetic studies. More information about the evolutionary relationships within the Rosaceae is presented in the section on activities andfindings.A major contribution of this project is the organization of a colloquium, entitled 'Rosaceae phylogeny: Current knowledge, problems, andprospects.'   This colloquium has been approved by the ASPT. The primary objective is to encourage synthesis and discussion of recentmolecular and other data relevant to Rosaceae phylogeny.  The conceptual focus is that a phylogenetic perspective will advance ourunderstanding of evolution of the Rosaceae, which has been hampered by its large size, considerable geologic age, indistinct boundariesbetween taxa, hybridization, polyploidy, agamospermy, and complex relationships among the taxa.  The colloquium organizers are ChristopherS. Campbell and Timothy A. Dickinson.Pooling large amounts of recently collected data from a large number of genes and other sources and sharing perspectives will greatly enhanceour understanding of Rosaceae phylogeny. This colloquium should stimulate and direct future research in this large, ecologically andeconomically important clade. After an introduction and overview of the major challenges of the topic, we would consider the impact ofhybridization, polyploidy, and agamospermy on Rosaceae phylogeny, demonstrate the phylogenetic utility of reproductive structures, and focuson important and challenging groups within the family.  We would conclude the colloquium with a phylogeny of the family based on allavailable data. The presenters were chosen for their work on Rosaceae phylogeny and ability to contribute to a balanced presentation. Ongoingcollaborations and communications among the presenters provide a network for integration of different perspectives.  We have a commitmentfrom the presenter for all presentations except numbers 3 and 8, and we expect commitments for these two presentations.Below is a tentative list of: titles of presentations and names of presenters1. Rosaceae phylogeny: An introduction and overview - T. A. Dickinson2. Hybridization, polyploidy, and phylogeny in the Rosaceae - J. Smedmark3. Agamospermy and evolution in the Rosaceae - B, Eriksen4. Floral development and Rosaceae phylogeny - R. C. Evans 5. Reproductive character evolution in Maloideae - D. R. Morgan and K. R. Robertson 6. Evolution and phylogeny of Rosoideae - T. Eriksson7. Phylogeny and relationships of prunoids of the Rosaceae - D. Potter 8. Phylogenetic insights into a taxonomically challenging group (Rubus) - L. A. Alice9. A phylogeny of the Rosaceae - C. S. Campbell Much of the results of this project is published, submitted for publication, or in preparation for submission for publication.  Contributions to Other Disciplines: NoneContributions to Human Resource Development: Our contributions to human resource development are in the area of training, detailed in another section of this report.Contributions to Resources for Research and Education: NoneContributions Beyond Science and Engineering: None.Categories for which nothing is reported: Organizational PartnersAny BookAny Web/Internet SiteAny Product

Molecular Phylogenetic Studies in Rosaceae

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Molecular Phylogenetic Studies in Rosaceae

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