EVOLUTIONARY STUDIES OF AMELANCHIER (SHADBUSH), PICEA (SPRUCE), AND BETULA PAPYRIFERA (WHITE BIRCH)

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
123	0613	1030	100%

Progress 10/01/98 to 09/30/04

Outputs
Work this year, during which I was on sabbatical leave, concentrated primarily on spruces and in particular completion of analyses of our data on the first internal transcribed spacer region (ITS1) of nuclear ribosomal DNA (nrDNA). We analyzed ITS1 sequence divergence and secondary structure for multiple clones from two individuals each of black spruce (Picea mariana) and red spruce (P. rubens) plus one individual each of two Asian species (P. glehnii and P. orientalis). ITS1 of Picea is 2747 to 3271 bp, the longest known of all plants, and is also remarkable for its variability within genomes. Mean sequence divergence within our six individuals (0.018 + 0.009) is more than half that between the four species (0.031 + 0.011) and may be maintained against concerted evolution by separation of Picea 18S-26S rDNA repeats on multiple chromosomes. ITS1 of the pine family (Pinaceae) is unique in containing two to six subrepeats with a motif (5'-GGCCACCCTAGTC). Picea has three such subrepeats, two of which are tandem, remote from the third, and more closely related and significantly more similar to one another than either is to the third subrepeat. This correlation between similarity and proximity may be the result of subrepeat duplication or concerted evolution within rDNA repeats. In inferred secondary structures, subrepeats generally form long hairpins, with a portion of the Pinaceae conserved motif in the terminal loop, and tandem subrepeats pair with one another over most of their length. In this paper we included ITS1 sequences from two putative hybrids of black spruce and red spruce. These sequences suggest that these individuals are later-generation hybrids, but we need to do more work to confirm this. This research has been accepted for publication in Molecular Phylogenetics and Evolution. I continued to work on the data for a paper on the phylogeny of the whole genus Picea based on DNA sequences from the chloroplast, mitochondria, and nucleus and with special reference to hybridization between the species. I was awarded a Short-term invitation fellowship by the Japanese Society for the Promotion of Science for my proposal Microsatellites in spruces (Picea, Pinaceae). I went to the Laboratory of Molecular Population Genetics, Kyushu University, Japan, where I worked with Professor Alfred E. Szmidt from 1 June to 2 July 2004. I completed the fellowship objectives, including (1) learning how to obtain, analyze, and interpret microsatellites, especially for studies of hybridization and species definition in spruces; (2) conducting field work to study vegetative and reproductive features of native Japanese spruces; and (3) establishing scientific collaborations with scientists in Japan interested in conifer systematics and evolution. I continued to work with collaborators to assemble and analyze DNA sequences from multiple chloroplast and nuclear genes to complete phylogenetic analyses at various levels within the rose family (Rosaceae). I will use these efforts in upcoming publications and in the keynote address I was invited to give at a symposium on the Rosaceae in the International Botanical Congress in Vienna in July 2005.

Impacts
The impact of this research is that it contributes to our basic understanding of the evolutionary biology and molecular phylogeny of important plants. These plants are significant to the ecology of northern landscapes; to wildlife as sources of food and shelter; and to people as sources of fiber, wood, and horticultural specimens. Our research is also informative about regions of DNA that can be useful to other scientists working on other problems and other groups of organisms.

Publications

No publications reported this period

Progress 10/01/02 to 09/30/03

Outputs
We advanced on two project objectives during the past year. We are assembling and analyzing DNA sequences from multiple chloroplast genes and the nuclear gene granule bound starch synthase I (GBSSI) to complete a phylogenetic analysis of the subfamily of the Rosaceae to which Amelanchier (shadbush) and apple belong. For our effort to understand hybridization between black spruce and red spruce, we are completing work on a manuscript concerning sequence divergence and structure of the first internal transcribed spacer (ITS1) of nuclear ribosomal DNA (nrDNA) for multiple individuals and populations of both species plus two other species of the genus. This work will be informative about the relatedness of black spruce and red spruce and about the evolution of an important genomic element (nrDNA comprises as much as 4% of the spruce genome). We are also working on a manuscript that concerns a phylogeny of the genus based on a large sample of species for two chloroplast genes. We have smaller taxonomic samples for mitochondrial nad1 B/C and nad7 1/2 intron sequences (in collaboration with scientists from the University of New Hampshire) and the nuclear 4CL gene that, together with our chloroplast data, provide evidence of past hybridization in the genus. These data create a phylogenetic context for this important genus, and they are informative about the relatedness of and hybridization between black spruce and red spruce.

Impacts
The impact of this research is that it contributes to our basic understanding of the evolutionary biology and molecular phylogeny of important plants. These plants are significant to the ecology of northeastern North American landscapes; to wildlife as sources of food and shelter; and to people as sources of fiber, wood, and horticultural specimens. Our research is also informative about regions of DNA that can be useful to other scientists working on other problems and other groups of organisms.

Publications

Liston, A., Gernandt, D.S., Vining, T.F., Campbell, C.S. and Pinero, D. 2003. Molecular phylogeny of Pinaceae and Pinus. Acta Hort. (ISHS) 615:107-114. MAFES ext. pub. 2383. http://www.actahort.org/books/615/615_7.htm

Progress 10/01/01 to 09/30/02

Outputs
Significant progress was made on two project objectives during the past year. We continued our assembly of DNA sequences from chloroplast genes and the nuclear gene granule bound starch synthase I (GBSSI) for Amelanchier (shadbush) plus other members of more inclusive groups in the Rosaceae. These data are creating robust hypotheses of evolutionary relationships of Amelanchier, the rose family (Rosaceae), and groups most closely related to the Rosaceae. We used GBSSI data to falsify a 70-year-old hypothesis about the origin of the apple subfamily (see publication). For our studies of evolutionary relationships within Amelanchier, we continued field sampling in Maine. For our effort to understand hybridization between black spruce and red spruce, we completed DNA sequencing of the first internal transcribed spacer of ribosomal DNA (ITS-1) for four clones for two individuals each of black spruce and red spruce plus two other species (Picea orientalis and P. glehnii). This data set comprises over 60,000 base pairs of sequence. We completed a chloroplast DNA sequence data set for 29 species of Picea (82 % of the species in the genus) in collaboration with scientists from the University of New Hampshire. Our chloroplast phylogeny indicate that black and red spruce are closely related, that the other two species are members of distantly related groups in the genus, and that we therefore have broad representation of ITS-1 in the spruce genus. We are working on a manuscript that will address variation in spruce ITS-1 sequences within individuals, within species, and between species; explore variation in inferred molecular secondary structure; and test the utility of ITS-1 for phylogenetic analyses. We are also planning to submit our spruce chloroplast DNA sequence data for publication. In addition, we are exploring with UNH collaborators an exciting conflict between chloroplast and mitochondrial DNA hypotheses of relationships in spruces. Chloroplast DNA suggests that Serbian spruce is closely related to black and red spruce, whereas mitochondrial DNA indicates that Serbian spruce is much more closely related to Norway spruce. We are using the nuclear gene 4CL to attempt to resolve this conflict.

Impacts
The impact of this research is that it contributes to our basic understanding of the evolutionary biology and molecular phylogeny of important plants in the rose and pine families. These plants are significant in the ecology of numerous northeastern North American landscapes; to wildlife as sources of food and shelter; and to people as sources of fiber, wood, and horticultural specimens.

Publications

Evans, R.C. and C.S. Campbell. 2002. The Origin of the apple subfamily (Rosaceae: Maloideae) is clarified by DNA sequence data from duplicated GBSSI Genes. American Journal of Botany 89:1478-1484. Maine Agricultural and Forest Experiment Station ext. pub. 2530.

Progress 10/01/00 to 09/30/01

Outputs
Significant progress was made on two project objectives during the past year. We continued our collection and analysis of DNA sequences for multiple chloroplast genes and the nuclear gene granule bound starch synthase I (GBSSI) for Amelanchier (shadbush) plus many other members of the subfamily to which this genus belongs (the Maloideae). During this past year we completed a study that shows that the Maloideae, which contains apple, pear, and other important plants, evolved from an ancestor that is very close to a group of herbaceous plants of the southeastern United States. This falsifies the long-standing hypothesis that the genesis of the apple subfamily was via hybridization between evolutionarily divergent lineages. For our effort to understand hybridization between black spruce and red spruce, we completed DNA sequencing of the first internal transcribed spacer (ITS1) of ribosomal DNA for multiple individuals and populations of both species. This spacer is exceptionally long and structurally complex in the pine family (Pinaceae). Spruce ITS1 is 2741 to about 3200 base pairs in length, longer than that of any other plant, and it contains two sets of subrepeats, each with three copies. While variation between ITS repeats has been demonstrated within individuals, we are the first to show this at the level of full ITS1 sequences. We continue to develop microsatellites for our Picea work. These data will also be potentially informative about the relatedness of black spruce and red spruce and therefore about the significance of hybridization between them. I have not found a student who is appropriate to work on the white birch project.

Impacts
The impact of this research is that it contributes to our basic understanding of the evolutionary biology and molecular phylogeny of important plants. These plants are significant to the ecology of northeastern North American landscapes; to wildlife as sources of food and shelter; and to people as sources of fiber, wood, and horticultural specimens.

Publications

Evans, R.C., L.A. Alice, C. S. Campbell, E. A. Kellogg, and T.A. Dickinson. 2000. The granule-bound starch synthase (GBSSI) gene in Rosaceae: multiple putative loci and phylogenetic utility. Mol. Phyl. Evol. 17:388-400.

Progress 10/01/99 to 09/30/00

Outputs
Significant progress was made on two project objectives during the past year. We were successful in obtaining DNA sequences for multiple chloroplast genes and the nuclear gene granule bound starch synthase I (GBSSI) for Amelanchier (shadbush) plus many other members of more inclusive groups in the Rosaceae and outside this large family. These data are creating robust hypotheses of evolutionary relationships of Amelanchier, the rose family (Rosaceae), and groups most closely related to the Rosaceae. We are the first lab to use GBSSI for phylogenetic information, and we have also uncovered some interesting molecular evolutionary features, such as multiple gene duplications. For our studies of evolutionary relationships within Amelanchier, we continued field sampling in Maine. We are considering use of microsatellites in this genus, with the help of a graduate student who is doing a study in our lab using microsatellites in pines. For our effort to understand hybridization between black spruce and red spruce, we are close to completing DNA sequencing of the first internal transcribed spacer of ribosomal DNA. This spacer is exceptionally long in the pine family (Pinaceae) and, as former graduate student Tom Vining demonstrated, contains multiple subrepeats within which there is a core sequence of 13-17 nucleotides conserved across the Pinaceae. Full sequence information will provide potentially useful insight into hybridization between these two species. These data will also be potentially informative about the relatedness of black spruce and red spruce and therefore about the significance of gene flow between them.

Impacts
The impact of this research is that it contributes to our basic understanding of the evolutionary biology and molecular phylogeny of important plants. These plants are significant in the ecology of numerous northeastern North American landscapes; to wildlife as sources of food and shelter; and to people as sources of fiber, wood, and horticultural specimens.

Publications

Campbell, C.S. 1999. The evolutionary role of hybridization in agamic complexes, with special emphasis on Amelanchier (Rosaceae). In L.W.D. van Raamsdonk & J.C.M. den Nijs (eds.). Plant evolution in man-made habitats. Proceedings of the VIIth International Symposium of the International Organization of Plant Biosystematists. Maine Agricutural and Forestry Experiment Station ext. publ. 2332.

Progress 10/01/98 to 09/30/99

Outputs
During this past year, we continued our studies of the evolutionary relationships among northeastern North American Amelanchier (shadbush). We have nearly completed our study that combines evidence of biased gene flow between a sexual and an asexual member of the genus; direct determination of reproductive mode (sexual or asexual) by estimates of nuclear DNA content in megagametophyte nuclei, estimates of the frequency of asexuality with progeny studies based on Random Amplified Polymorphic DNA (RAPD) markers, and testing the hypothesis that gene flow does not progress beyond the formation of F1 individuals. We also continued field sampling in Maine from Mount Desert Island eastward to the United States-Canadian border and found (1) many more samples of a taxon that we had previously known from only about three localities; (2) populations of Amelanchier bartramiana at the southern limit of distribution on the Maine coast; and (3) potential gene flow involving an apparent derivative of A. bartramiana. We decided to abandon inter-simple sequence repeat (ISSR) DNA polymorphisms because they are not confidently repeatable and to explore instead microsatellites. We collected additional DNA sequences from the granule-bound starch synthase gene (GBSSI). For our effort to understand hybridization between black spruce and red spruce, we had some success avoiding contamination of DNA extractions by endophytic fungal DNA by using only a portion of the embryo. We obtained sequences from the internal transcribed spacers of nuclear ribosomal DNA that we used to that show where spruces are located on the phylogenetic tree of conifers.

Impacts
The impact of this research is that it contributes to our basic understanding of the evolutionary biology and molecular phylogeny of important plants. These plants are significant in the ecology of numerous northeastern North American landscapes; to wildlife as sources of food and shelter; and to people as sources of fiber, wood, and horticultural specimens.

Publications

Campbell, C.S., L.A. Alice, and W.A. Wright. 1999. Comparisons of within-population genetic variation in sexual and agamospermous Amelanchier (Rosaceae) using RAPD markers. Plant Systematics and Evolution 215:157-167. MAFES ext. pub. 2166.

Progress 10/01/97 to 09/30/98

Outputs
Progress was made on all three objectives during the first year of this project. To uncover evolutionary relationships among northeastern North American Amelanchier (shadbush) we carried out field sampling in Maine, extracted DNA, and have been surveying samples for useful inter-simple sequence repeat (ISSR) polymorphisms. These data plus quantitative morphology will be used to test the hypothesis that some units in this group are clones perpetuated asexually. We completed data collection for a study involving our research site in western Maine, which we have used for almost 15 years. This study brings together related aspects of the reproductive biology of shadbushes at this site, including (1) biased gene flow between a sexual and an asexual member of the genus; (2) direct determination of reproductive mode (sexual or asexual) by estimates of nuclear DNA content in megagametophyte nuclei; (3) estimates of the frequency of asexuality with progeny studies based on Random Amplified Polymorphic DNA (RAPD) markers; and (4) testing the hypothesis that gene flow does not progress beyond the formation of F1 individuals. For our effort to understand hybridization between black spruce and red spruce, we have been working on methods to obtain DNA without contamination by endophytic fungal DNA. This problem, which has plagued researchers here and elsewhere, must be solved before we can collect molecular data. We are exploring surface-sterilization techniques as well as use of emrbyos and megagametophytes. We collected material to begin our study of reproductive mode of Betula papyrifera (white birch).

Impacts
(N/A)

Publications

Dibble, A.C., W.A. Wright, C.W. Greene, and C.S. Campbell. 1998. Quantitative morphological analyses at a taxonomically complex site in the Amelanchier agamic complex (Rosaceae). Systematic Botany 23:31-40. MAFES ext. publ. 2165.

Progress 10/01/96 to 09/30/97

Outputs
We completed significant aspects of all 3 components of this project. We published a nuclear ribosomal DNA (nrDNA) phylogeny of subfamily Maloideae of the rose family and 4 Amelanchier (shadbush) studies: nrDNA polymorphisms; quantitative morphology of a hybrid swarm; morphological variation in Maine populations; and status of Amelanchier nantucketensis. We concluded Amelanchier studies of genetic variation based on Random Amplified Polymorphic DNA (RAPD) and quantitative morphology. Two papers from A. C. Dibble's Ph.D. thesis (in 1995) that relate to this project are in press with CANADIAN JOURNAL OF BOTANY. In our study of spruce hybridization, we are attempting to avoid problem of leaf fungal endophyte DNA contamination by using megagametophytes, which have the added advantage for RAPD analysis of genetic haploidy. We are also collaborating with conifer biologists at Oregon State University on a molecular phylogeny of spruces and all other genera of the pine family. We collected demographic data in four Maine populations of the Federally Threatened Isotria medeoloides for the twelfth straight year. We are collaborating with P. Vitt on a publication based on these demographic data. We are in the process of analyzing data for the fifth growing season following an experimental habitat manipulation designed to enhance this species growth. We have a paper on Isotria reproductive biology in press with RHODORA.

Impacts
(N/A)

Publications

Campbell, C.S. and Wright. 1996. Agamospermy, hybridization, and taxonomic complexity in northeastern North American Amelanchier (Rosaceae). Folia Geobotancia & Phytotaxonomica 31:345-354.
Campbell, C.S., M.F. Wojciechowski, B.G. Baldwin, L.A. Alice, and M.J. Donoghue. 1997. Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier (Rosaceae) agamic complex. Molecular Biology and Evolution 14:81- 90. MAFES ext. publ. 2039.
Campbell, C.S., W.A. Wright, T.F. Vining, and W.A. Halteman. 1997. Morphological variation in sexual and agamospermous Amelanchier (Rosaceae). Canadian Journal of Botany 75:1166-1173. MAFES ext. publ.
Dibble, A. C. and C. S. Campbell. 1997. Distribution and conservation of nantucket shadbush (Rosaceae). Rhodora 97:339-349. MAFES ext.

Progress 10/01/95 to 09/30/96

Outputs
In Amelanchier (shadbush), we completed studies of nuclear ribosomal DNA polymorphisms and of a hybrid swarm and breeding systems in a local Amelanchier population. We continued our study of hybridization using random amplified polymorphic DNA (RAPD) analysis and initiated a similar study at another site. The manuscript on morphological and genetic variation in Amelanchier that we submitted to the AMERICAN JOURNAL OF BOTANY was rejected, and so we submitted it to CANADIAN JOURNAL OF BOTANY. We worked with A. C. Dibble (completed PH.D. here in 1995) to publish papers from her thesis on Amelanchier. Two papers from her thesis have been submitted to CANADIAN JOURNAL OF BOTANY. I presented a paper on our Amelanchier research at the annual meeting of the Botanical Society of America. In our study of hybridization between black spruce and red spruce, we are still working out methods for RAPDs. We continued our effort to determine levels of genetic diversity in the Federally Endangered orchid, Isotria medeoloides, small whorled pogonia. We collected demographic data in four Maine populations of this species for the eleventh straight year (eight years by individuals in my lab). We are collaborating with P. Vitt (completed M.S. here in 1991) in analyzing 10 years of demographic data and writing a manuscript. We are in the process of analyzing data for the fourth growing season following an experimental habitat manipulation designed to enhance this species growth.

Impacts
(N/A)

Publications

Campbell, C.S., M.F. Wojciechowski, L.A. Alice, and W.A. Wright. 1996. Hybridization, agamospermy and microspecies in Amelanchier (Rosaceae). American J. Botany 83:144.
Anderson, D.S., R.B. Davis, S.C. Rooney, and C.S. Campbell. 1996. The ecology of sedges (Cyperaceae) in Maine peatlands. Torrey Bot Club Bull. 123:100-110.

Progress 10/01/94 to 09/30/95

Outputs
We performed experimental crosses for a third year in our study of the frequencyof asexual reproduction in A. laevis. We collected Amelanchier samples from Maine and Vermont as part of our study of nuclear ribosomal DNA (nrDNA) polymorphisms. We have found that the Vermont plants, which are the easternmost representatives of the species A. humilis, have the western North American type of nrDNA. This finding logically identifies one of the parents of our polymorphic plants. We completed a study of a hybrid swarm and breeding systems in a local Amelanchier population. We submitted two manuscripts involving our Amelanchier work. I presented four papers (two were invited) on our Amelanchier research at one regional and two international meetings. In our study of hybridization between black spruce and red spruce using RAPD markers, we are still working out methods for RAPD analysis and are considering switching from leaf to seed material next year. Spruce seed set was poor in this year s drought. We continue our effort to determine levels of genetic diversity in the Federally Endangered orchid, Isotria medeoloides, small whorled pogonia. We collected demographic data in four Maine populations of this species for the tenth straight year (seven years by individuals in my lab) and anticipate analyzing a large body of data and preparing one or more manuscripts next year. We collected data for the third growing season following an experimental habitat manipulation designed to enhance this species growth.

Impacts
(N/A)

Publications

Campbell, C.S., M.J. Donoghue, B.G. Baldwin, and M.F. Wojciechowski. 1995. Phylogenetic relationships in Maloideae (Rosaceae): Evidence from sequences of the internal transcribed spaces of nuclear ribosomal DNA and Amer. J. Botany 82(7): 90
Cambell, C., et al. 1995. The Checklist of Vascular Plants in Maine. Maine Agr. & For. Exp. Sta. Bulletin 844.
Baldwin, B., M. Sanderson, J. Porter, M. Wojciechowski, C. Campbell, & M. Donoghue. 1995. The ITS region of nuclear ribosomal DNA. Annals Missouri Botanical Garden 82:247-277.

Progress 10/01/93 to 09/30/94

Outputs
We made significant progress in all three components of this project. In Amelanchier (shadbush), we completed a manuscript on comparing quantitative morphological and genetic variation (the latter determined using Random Amplified Polymorphic DNA or RAPD markers) of six from a sexual species and six from an asexual species in Maine. We presented a paper at a national meeting on the frequency of asexuality in one Amelanchier species using RAPD markers. We performed experimental crosses for a second year of material in anticipation of taking this study to publication. We collected material of a larger sample of possible hybrid individuals for our effort to determine the extent of backcrossing in a population of a sexual species, an asexual species, and their hybrid. I visited the University of Arizona in August 1994 to work with a collaborator, Dr. M.F. Wojciechowski, on DNA sequencing of clones of Amelanchier individuals whose nuclear ribosomal DNA (nrDNA) is polymorphic. We completed a project on a molecular phylogeny of the Maloideae, the group that includes Amelanchier and relatives (such as apple and pear) using DNA sequences of nrDNA. We extracted DNA from a large sample of individuals from our study sites on coastal Maine and Isle au Haut for our study of hybridization between black spruce and red spruce. We devoted considerable effort to determine levels of genetic diversity in the Federally Endangered orchid, Isotria medeoloides, small whorled pogonia.

Impacts
(N/A)

Publications

SANDERSON, M., B. BALDWIN, G. BHARATHAN, C. CAMPBELL, ET AL. 1993. The growth of phylogenetic information and the need for a phylogenetic database. Systematic Biology 42:562-568.
DIBBLE, A.C., W.A. WRIGHT, AND C.S. CAMPBELL. 1994. Demographic monitoring of Isotria medeoloides in Maine. 1992 Report. Prepared for Natural Areas Program, Maine Dept. Economic & Community Development.
DIBBLE, A.C., W.A. WRIGHT, AND C.S. CAMPBELL. 1994. Small whorled Pogonia (Isotria medeoloides): Habitat manipulation experiment. Prepared for Natural Areas Program, Maine Dept. Economic and Community Development.

Progress 10/01/92 to 09/30/93

Outputs
We have made significant progress this year toward all objectives of this project. For our study of evolutionary relationships and population structure among northeastern North American species of Amelanchier, we have completed one study combining Random Amplified Polymorphic DNA (RAPD) markers and quantitative morphology to contrast within- and among-population phenotypic and genetic variation in a sexual and an asexual species. We demonstrated that their estimated levels of genetic variation are similar, but genetic distances between sexuals is normally distributed while that of asexuals is significantly skewed toward high levels of identity. Using a multivariate equivalent of Leven's test, we found that the asexual species shows significantly greater among- population morphological variation than the sexual. We began efforts to document the occurrence of later-generation hybrids between these two species and to estimate the level of apomixis (asexual seed production) in the asexual species. To test hypothesis of hybridization between black and red spruce using RAPD markers, we extracted DNA from 60 individuals that we have studied morphologically and undertook preliminary RAPD analyses. For our objective of determining intra- and interpopulation genetic diversity of Isotria medeoloides, a Federally Endangered orchid, we extracted DNA from over 200 individuals from several populations and carried out preliminary RAPD analyses.

Impacts
(N/A)

Publications

CAMPBELL, C.S., L.A. ALICE, AND T.G. HELENTJARIS. 1993. Genetic diversity in and hybridization between apomictic and sexual Amelanchier (Rosaceae, Maloideae). Am. J. Botany (abstract) 80:135.
CAMPBELL, C.S., B.G. BALDWIN, M.J. DONOGHUE, AND M.F. WOJCIECHOWSKI. 1993. Toward a phylogeny of Amelanchier (Rosaceae:Maloideae). Am. J. Botany (abstract) 80:135-136.
DIBBLE, A.C., C.S. CAMPBELL, W.A. WRIGHT, A.D. HAINES, C.W. GREENE, AND C.S. MAJOR. 1993. Morphological variation in Amelanchier (Rosaceae: Maloideae) at a taxonomically complex site in Maine. Am. J. Botany (abstract) 80:144.
MAJOR, C.S., C.S. CAMPBELL, AND W.A. WRIGHT. 1993. Hybridization between Picea mariana and P. rubens (Pinaceae) in Maine. Am. J. Botany (abstract) 80:162.
WRIGHT, W.A., T.F. VINING, AND C.S. CAMPBELL. 1993. Morphological diversity within and among populations of an apomictic species and sexual species of Amelanchier (Rosaceae: Maloideae). Am. J. Botany (abstract) 80:184.

Progress 10/01/91 to 09/30/92

Outputs
During my NSF Midcareer fellowship at the Univ. of Arizona, I completed a directDNA sequencing project on 24 species of Amelanchier and 20 genera of the Maloideae, the subfamily of the Rosaceae (rose family) that includes Amelanchier and such horticulturally and agriculturally important plants as apple, pear, quince, mountain ash, and cotoneaster. As reference taxa for the Maloideae study, we used samples of other genera of the Rosaceae. Both DNA sequencing efforts have yielded informative data for reconstruction of phylogeny or evolutionary history. We have nearly completed a dataset of 14 morphological variables for almost 100 individuals from a mixed population of eight taxa of shadbushes. During this sabbatical leave I also gained experience with southern analysis (RFLP's) and the recently developed technique of Random Amplification of Polymorphic DNA (RAPD). I used RAPD's to compare intrapopulation variability of a sexual species and a species that produces seed asexually in Amelanchier and to detect interspecific hybridization. We are nearing completion of our spruce hybridization project. At the University of Arizona I confirmed with flow cytophotometry our earlier finding that black and red spruce do not differ in nuclear DNA content. We have completed the crossability component of this study and anticipate finishing the multivariate analysis of morphological differences this winter.

Impacts
(N/A)

Publications

NO PUBLICATIONS REPORTED THIS PERIOD.

Progress 10/01/90 to 09/30/91

Outputs
We made significant progress in our incipient molecular genetic studies of Amelanchier, Shadbush. These studies, in collaboration with scientists at the N.Y. Agr. Exp. Sta., were aimed at using molecular techniques to test if there is a bias in the direction of pollen flow between a sexual species and an apomict, which produces seed asexually. We reported a statistically significant directional bias from apomict to sexual based on a restriction fragment length polymorphism (RFLP) in chloroplast DNA (cpDNA) between the two species. We continued our study of hybridization between black and red spruce with a second crossability experiment and further quantitative study of morphological variation. We also tried flow cytometry to distinguish the species, which have been reported to differ in their DNA content by a factor of two. While this effort was inconclusive, perhaps due to interference by secondary compounds, we plan to continue with this technique because it allows rapid determination of DNA levels for large populations of cells. We are completing work on the reproductive and evolutionary status of Amelanchier nantucketensis, which is under consideration for Federal listing as an endangered species. We initiated a new project at a site that contains about eight taxa of shadbush, some of which may be the result of interspecific hybridization. We are using morphometric analysis and flow cytometry of permanently tagged individuals to classify them and draw inferences about their interrelationships.

Impacts
(N/A)

Publications

CAMPBELL, C.S., GREENE, C.W. and DICKINSON, T.A. 1991. Reproductive biology of the Maloideae (Rosaceae). Syst. Bot. 16:333-349.
CAMPBELL, C.S., WEEDEN, N.F. , DICKSON, E. and WRIGHT, W. 1991. Biased gene between a sexual and an apomictic species of Amelanchier (Rosaceae:Maloideae) based on a chloroplast DNA restriction site difference. Amer. J. Bot. Suppl. 169.
DIBBLE, A.C. and CAMPBELL, C.S. 1991. Reproductive biology of a rare endemic Amelanchier nantucketensis (Rosaceae:Maloideae). Amer. J. Bot. 78 Suppl. 179.
DICKINSON, T.A. and CAMPBELL, C.S. 1991. Population structure in the Maloideae (Rosaceae). Syst. Bot. 16:350-362.
DICKINSON, T.A. and CAMPBELL, C.S. 1991. Introduction to the symposium. Evolution in the Maloideae (Rosaceae). Syst. Bot. 16:299-302.

Progress 10/01/89 to 09/30/90

Outputs
We continued research on all phases of this project. We completed two studies of phenotypic viability in Amelanchier, (Shadbush) one in print, (see first publications below) and another accepted for publication. I was awarded an NSF EPSCpR grant in the Maine Science and Technology Commission's 1990 Faculty Enhancement Program. I spent two weeks collaborating on molecular genetics of shadbush with N.F. Weeden's group at the New York State Agricultural Experiment Station. We worked on isozyme of seeds and seedlings to estimate the frequency of apomixis (asexual seed production) and a restriction fragment length polymorphism of chloroplast DNA to determine the percentage of hybrids between sexual and apomictic species. We will submit an abstract to present a paper of our results at the 1991 annual meetings of the American Institute of Biological Sciences. A graduate student, A.C. Dibble, began Ph.D. research on evolution and endimism in Amelanchier. We reported results of a study of breeding systems in white birch (see second publication below). A graduate student, C.S. Major, continues research on hybridization between black and red spruce.

Impacts
(N/A)

Publications

CAMPBELL, S.C. and DICKINSON, T.A. 1990. Apomixis and patterns of phenotypic variation in the Maloideae (Rosaceae). Syst. Bot. 15:124-135. MAES external publ. 1369.
CAMPBELL, C.S., DEHOND, P.E. and WRIGHT, W.A. 1990. Gametophyte development in Betula papyrifera (Betulaceae). Amer. J. Bot. 77:1220.

Progress 10/01/88 to 09/30/89

Outputs
During the past year, we carried out research on all four objectives of this project. We completed a cytophotometric study (see first publication below) and are considering a related, potentially more powerful approach to the same problem. We completed a data set of morphological variables for about 170 individuals of shadbush (Amelanchier) from nine populations and two species that we are in the process of analyzing for our comparison of variability within and between populations of sexual and apomictic species. We continue to work on electrophoretic methods of estimating the frequency of apomixis in shadbush. We progressed in our study of the cellular details of pollination and mode of reproduction in white birch (Betula papyrifera). We have started our work on interspecific hybrids of black & red spruce (Picea mariana and P. rubens) on Isle au Haut, Maine by permanently marking study individuals, establishing a data set, and carrying out preliminary electrophoretic studies.

Impacts
(N/A)

Publications

CAMPBELL, C.S., RIDING, R.T., and WRIGHT, W.A. 1989. Nuclear DNA levels in megagametophytes of a sexual and an apomictic species of Amelanchier (Rosaceae, Maloideae) and their putative hybrid. Amer. J. Bot. 76:230.
CAMPBELL, C.S. and DICKINSON, T.A. 1990. Apomixis and patterns of phenotypic variation in the Maloideae (Rosaceae). Syst. Bot. 15:124-135. MAES external publ. 1369.

Progress 10/01/87 to 09/30/88

Outputs
During this past year, we have extended work on the reproductive biology of shadbushes and white-barked birches. For shadbushes, we initiated three projects. First by cytophotometric study we hope to confirm that asexually derive megagametophytes are not chromosomally reduced and that endosperm is triploid, resulting from the fusion of one unreduced polar nucleus and a reduced sperm. We are using isozymes to estimate the frequency of asexual seed production in the second project. For the second project, we collected abundant material to compare both development and gametic interactions in asexually vs. sexually producing individuals. In birches, we are examining reproduction in white birch, specifically whether a pentaploid individual produces its seed sexually or asexually.

Impacts
(N/A)

Publications

DEHOND, P.E. and CAMPBELL, C.S. In press. Multivariate analyses of hybridization between Betula cordifolia and B. populifolia (Betulaceae). Can. J. Bot. MAES External publ. 1272.
O'CONNELL, M.M., BENTLEY, M.D., CAMPBELL, C.S. and COLE, B.J.W. 1988. Betulin in four white-barked birches (Betulaceae). Phytochem. 27:2715-2716. MAES External publ. 1260.
WEBER, J.E. and CAMPBELL, C.S. In press. Breeding systems in a hybrid of a sexual and an apomictic species of Amelanchier (Rosaceae: Maloideae). Amer. J. Bot. MAES External publ. 1264.

Progress 10/01/86 to 09/30/87

Outputs
Three studies for masters theses concerning the reproductive biology of Amelanchier and Betula were completed this year. Patricia DeHond established through multivariate statistical studies the best characters for distinguishing blue birch from its parents mountain paper birch and gray birch. She also showed that blue birch is a fertile hybrid and gathered preliminary data on the reproductive biology of white birch. Margaret O'Connell, working with Barbara Cole and Mike Bentley of Chemistry and myself, used levels of the bark compound betulin as a character to support separate species status of mountain and white paper birch and the hybrid origin of blue birch. Jill Weber showed that hybrids of a sexual and asexual parent in melanchier are largely asexual and further resemble the asexual parent in being self-compatible. All three studies have contributed significantly to our understanding of the reproductive biology of these two important genera of trees.

Impacts
(N/A)

Publications