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Genetic Variability in Magnolia Acuminata (L.) Populations in the Eastern United States 1,2,3

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Genetic Variability in Magnolia Acuminata (L.) Populations in the Eastern United States 1,2,3 Wollaeger 1 Genetic Variability in Magnolia acuminata (L.) Populations in the Eastern United States 1,2,3 1Heidi Wollaeger, Wittenberg University, NHRE Intern with The National Museum of Natural History at Smithsonian Institution, Washington D.C. 2Under the direction of Dr. Elizabeth Zimmer, Botany Curator, National Museum of Natural History, Washington D.C. 3Under guidance of Dr. Matthew Collier, Dr. Michelle McWhorter, and Dr. John Ritter, Wittenberg University, Springfield, OH ______________________________________________________________________________ Abstract The goal of this study was to use nuclear (ITS1, microsatellites) and chloroplast (psbK-psbI; atpF-atpH; and ndhf-rpl32) regions to determine the population genetics of Magnolia acuminata, commonly known as the cucumber tree, in the eastern United States and to correlate morphological variation with these genetic markers. DNA was extracted with Qiagen DNeasy® kits from samples of M. acuminata provided by the Magnolia Society from populations in the eastern United States. Polymerase chain reactions were performed according to the optimal temperature as determined by the touchdown PCR. The PCR product was electrophoresed in a 1.5% agarose gel stained with ethidium bromide in order to assess DNA sequence size and quality after the amplification. The PCR products for the chloroplast spacer regions (psbK-psbI; atpF-atpH; and ndhf-F-rpl32-R) were Exo-Sapped and cycle sequenced. This product was run through hydrated sephadex plates to purify the sample and was sequenced on a 3730xl DNA Analyzer. The sequences were aligned with Geneious® software. For microsatellite regions stm200 and stm49, the PCR product was diluted 1:10 with Dnase free water and was added to Rox diluted 1:10 with formamide. These products were submitted for fragment analysis and assessed on GeneMapper® software. No sequences were derived from the nuclear region, ITS1. No significant variation was detected in the chloroplast spacers but some preliminary geographical variation was shown through microsatellites stm200 and stm49. Interbreeding between close geographical populations was observed, however distinctive alleles were present across large distances. In order for conclusive evidence for the population genetics to be determined, more microsatellite markers would have to be assessed. ______________________________________________________________________________ Introduction Magnoliaceae, a family of shrubs and ecosystems. Magnolias are commercially trees, contains approximately 230 species important for the production of greenery for (Kim et al. 2001). Charles Plumier coined the the floral industry as well as for cut flowers, name "Magnolia" as a patronym in honor of a using its wood for a small percentage of fellow botanist, Pierre Magnol, in 1703. American timber production, and also to Linnaeus, famous for the binomial extract compounds to be used medicinally to nomenclature system, examined the Magnolia relieve headaches, feminine ailments, family and re-established its common name of allergies, and fever (Callaway 1994). Magnolia which is still used today (Treseder Ecologically, Magnolias are thought to have 1978). Currently, Magnolias have many co-evolved with beetles from the time of their practical and aesthetic uses in addition to first existence, but are pollinated and provide playing important ecological roles in various nectar for many other species of insects today. Wollaeger 2 This plant group is also an important nutrient of the 'Slope Magnolia' were the most similar source for migratory birds and some small to Magnolia acuminata distinguishable by the and large mammals (Treseder 1978). venation of the leaf. Magnolia acuminata The Magnolias are thought to be one of the usually has 10 to 14 secondary veins and an first angiosperms to evolve and therefore are angle from the midrib to the edge of the leaf classified as "primitive plants" (Thien 1974). of 45 to 65 degrees. The fossil leaf was most Scientists have disputed the family's specific closely identified as a direct relative of age. According to Kim et al. (2001), Magnolia acuminata due to its similarities in Magnolia broke off from its evolutionary measurements (Figlar 1993). While ancestors around 100 million years ago, but morphology of these genera seems to be Nie et al. (2008) argues that Magnolias relatively unaltered over millions of years, the specified around 93.5 million years ago taxonomy within the Magnolias is particularly (Treseder 1978). It is generally agreed upon complex. Finding a genetic basis for the Magnolias were abundant during the Neogene complexity and variability in these taxa, time. The Paleogene and Neogene are parts of specifically of the species M. acuminata, is the Cenozoic Era, encompassing the Pliocene, the focus of this investigation. Miocene, Oligocene, Eocene, and Paleocene This study focuses on Magnolia acuminata epochs Figure 1. This time period was an (Figure 3). ideal time for the uprising of many southern species. Figure 1. A time line of the Cenozoic era, containing the Neogene and Paleogene periods, the periods of increasing prevalence of the Magnolia (Eon, Epochs, Etc. 2010). The Atlantic Coastal Plain emerged from Figure 2. A comparison of the fossil 'Slope Magnolia' below water and many of these eroded rock and a living Magnolia acuminata (Figlar 1993). layers are easily accessible today. The Appalachian Mountains were present but only a couple thousand meters high. Many of the species, particularly ones that had East Asian relatives (such as Magnolia acuminata, commonly known as the cucumber tree), spread easily through the coastal plain beginning at the lower latitudes and moving north (Braun 1955). Amazingly, the Magnolia species found today are quite similar to those living millions of years ago. Fossil fruits of a relative of the Magnolias, found in a Miocene Figure 3. Magnolia acuminata (Arboretum Freiburg- fossil bed in Idaho, appear remarkably similar Günterstal. Accessed July 23, 2010. <http://hubertus- to those of M. acuminata in Figure 2. Leaves nimsch.de/pages/arbor_magnolien-tal.htm>). Wollaeger 3 The family Magnoliaceae is comprised of the flower color and petiole anatomy, and sub-family known as Magnoliodeae that pubescence was observed, Figure 4. Leaf contains one genus, Magnolia (Figlar and shape, however, is the most variable character Nooteboom 2004). Magnolia acuminata of all the different phenotypes (Hardin 1954). belongs to that genus and subgenus Yulania, On the broader scope, in order to evaluate section Tulipastrum (Treseder 1978; Kim et the lineage of Magnoliaceae in comparison al. 2001). Plants assigned to the species M. with similar morphological families, Canright acuminata are most commonly deciduous (1960) examined floral characteristics, such trees or shrubs approximately 30 meters tall, as carpels and stamens, in a series of papers. with straight bark, often light brown to gray. Previous to molecular techniques, for Leaves are elliptical, oblong, or ovate to example, the carpels were examined obvate 4-16 centimeters long and 2-8 microscopically, by gross view, and also by centimeters wide. M. acuminata belongs to taking cross sections of the tissue for the anatomical group within the genus where distinguishing characters. The results from the the flowers close at night with papillose anatomical study for the Magnolias were very (textured) stigmata around the other female inconclusive, due to large variation in number floral parts (Thien 1974). The flowers have and style of carpels and the amount of fusion six petals and have a radius of 2-5 cm from among them (Canright 1960). the center of the flower (Thien 1974). The flower color and pubescence of the twigs help to define the varieties within the species acuminata. Yellow-green flowers and glabrous twigs characterize Magnolia acuminata var. acuminata. Magnolia acuminata var. subcordata often has yellow flowers and pubescent twigs. Additionally, four other varieties exist: ludoviciana, ozarkensis, aurea, and albamensis. Furthermore, there are dozens of cultivars and hybrids within M. acuminata due to its showy and fairly hardy characteristics (Callaway 1994). Observation of extremely variable morphological characteristics in M. acuminata spurred a series of studies on the species and its relationship to other genera in Figure 4. The range of morphological variation in the family Magnoliaceae. Early studies by leaf base (left), pubescence level (top), and the Whittaker (1933) compared the venation of respective characters by variety (bottom). Solid the leaves, the presence of vessels, and their Line: M. ozarkensis, Dashes: M. cordata, Dots: M. structure. Whittaker (1933) examined species acuminata (Hardin 1954). in the genus Magnolia and found that they had pinnate leaf venation and the presence of sclariform tracheids and vessels. Hardin Through this lineage study, Canright similarly (1954) was the first to examine extensively observed the variance in the Magnolia family. the varying characters in M. acuminata and Perhaps the complexity and variation within group them according to those characters. the family and genera arise from the Differing pubescence on the lower leaf origination of the species from the North surface, petal length and width, base structure, American and Asian continents cross- Wollaeger 4 pollinated over the land bridge many years acuminata. A small sample of individuals ago (Treseder 1978). This conclusion has among different populations throughout M. been backed up only
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