Chasing Chloroplasts

Professor Fenny Dane of Auburn University presented her Castanea Migration Theory at last year's CT Chapter Annual Meeting. Each Castanea species has several distinct chloroplast types. More differences can be detected among Asian species than within European or North American species using chloroplast DNA analysis. However from analysis of samples from the Appalachian range we can hypothesize that Castanea found refuge in the southern region of the Appalachian mountain range during glacial periods and that following the retreat of the ice sheet, one lineage migrated into north easterly direction.

Dr. Dane had suggested that it would be helpful to her work if the Connecticut Chapter could provide her with samples of genetic material from known validated American chestnut examples we'd found in Connecticut. We keep accurate track of the samples and validation process, and by summer's end had twenty seven validated American chestnut that we had either successfully pollinated or felt had a high probability of flowering and providing an opportunity to pollinate.

Validation is a time and resource consuming process whereby several scientists use dichotomous keys to analyze a sample for traits indicating American lineage. We use the same group of scientists – though it evolves over time – to ensure a consistent review. It should be noted that only a small percentage of the samples found in Connecticut are actually validated using this approach. The majority (about seventy-five percent) of samples submitted are rejected as being hybrids with Asian chestnut or European chestnut traits. And also, since it time consuming and resource consuming – we do not to bother validating samples of trees with known low probability to flower. So for instance, I'll see dozens of trees during a trail hike or canoe ride or even driving – but rarely bother to report the trees unless they look large enough to flower, with enough light, and accessible by bucket truck.

Coordination and Planning

With autumn fast approaching I worked with several Chapter Members to coordinate the collection of samples Dr. Dane could use for her research. We had the good fortune to be harvesting advanced breeding nuts from some of the validated trees on the same day I had chosen for collection from the orchards. While this somewhat complicated the coordination of the three hundred mile circuit to collect the samples – it also ensured efficiency and getting the freshest and largest sample set.

I prepared by reviewing the instructions provided by Dr. Dane, and by assembling and reviewing maps of each orchard as well as planning the most efficient route. Each orchard has from one to four back-cross lines – each line representing the progeny of a single mother tree. They also have a variety of control trees planted to assist with understanding the results of resistance testing. The basic concept is that pure Chinese will show the most resistance while pure American the least. This can be used to calibrate the resistance testing on the back-cross trees which are about 15/16ths American. The trees are planted in a random pattern in the orchard to ensure proximity plays no role in apparent resistance. Subsequently, a map, along with the local orchard manager is critical to understand which trees are the targeted back-cross trees.

Guilford Conservation Commission – Nut Plains Park Orchard

First stop of the day was our youngest back-cross orchard located in Guilford. The Guilford Conservation Commission conceived of the orchard back in 2005 and planted test trees in spring of 2006. The first back-cross trees were planted in 2007. At present the orchard contains nine different lines of trees including three lines of back-cross trees, four open pollinated crosses with known American chestnuts, an F1 hybrid (half American chestnut half Chinese chestnut) and a pure Chinese chestnut.

My goal was getting samples of the three back-cross lines of which one had a mother tree that was not particularly accessible. A struggle with understanding the maps made the stop only partially successful. This was the one stop where the Orchard Manager couldn’t join me and her presence was sorely missed.

The photo to right captures the beautiful wildflowers that appear when deer are excluded from a large area such as the two acre orchard exclosure. The diversity of wildflowers attracts diverse insects which in turn support a large and diverse avian population.

Salem Orchard of Dr. David Bingham

Next stop was Dr. David Bingham’s orchard in Salem, CT. David made the stop easy by having collected samples of three local mother trees before my arrival. We walked out to the orchard to collect the samples of back-cross leaves David has growing. David’s orchard was the first TACF® back-cross research orchard planted in Connecticut. I had helped with that first memorable planting (we also erected 1100 feet of fence the same day – just the two of us). I collected three samples of back-cross trees – all of which I also had samples of the mother trees. Analysis of both samples should provide some interesting results. Between controls of pure Chinese chestnut, pure American chestnut and various F1 hybrids, David’s orchard has fifteen different chestnut tree lineages. It is a great orchard to visit to see the diversity of the various chestnuts and also the biodiversity he has allowed to develop in the orchard location.

Third stop was an orchard managed by the Northern CT Land Trust on their Swann Farm location. Approaching the orchard from the south I found a large number of large American chestnut along the road, many with burs. They had been released by a small amount of clearing in the forest – perhaps even the removal of a single large tree. It would be great to hope some survive for pollination next year.

The NCLT orchard was test planted in 2006 and following that successful test, several local back-cross lines the following year. Today the orchard has no less than fifteen different lineages including three significant back-cross trees. Orchard Managers Jim Gage and Richard bailey shown in the photo to right – met me at the orchard. In the photo you can see that those shown of the many hundreds of trees at this orchard are significantly over the heads of the two six-foot plus gentlemen shown. The trees shown were planted in 2007 and the growth shown is over just three seasons.

Jim had attended to the morning’s harvest in Willington and had also generously collected samples of several of the mother trees found in the area. I was especially glad to have them meet me since they saved me from collecting an incorrect sample from a specific known American x American cross that looked like the back-cross I sought. At least it is reassuring to know that our back-crosses are indistinguishable from known American x American crosses!

Jim Gage noticed this beautiful Grey Tree Frog nestled in the branch of one of the back-cross trees. The land for the orchard is not far from a vernal pond and the orchard has clearly been adopted by its brethren.

Manchester

Jim Gage led me to the nearby Manchester Mother Tree to collect a sample. The tree was pollinated several years ago and manages to hang on even producing a large crop of nuts. This leaf measured more than twelve inches in length and was found on new stems from the root crown of a portion of the tree which had died back from blight. The progeny of this tree are planted in the Woodbridge Orchard which I would be visiting later in the day.

Burlington

Next Stop was Burlington where we had pollinated a native American chestnut Mother Tree last year. I was thrilled to find the tree, find that it was still alive and in fact had a crop of fertile burs. I also found a large number of other possible mother trees in the area – many with burs. Hopefully they will lend their genes to another generation.

Next stop was New Hartford where I collected samples from two trees that have been validated as being pure American chestnut. All along the trip between Burlington and New Hartford I passed potential American chestnut. I kept thinking about the Prediction Model done for Litchfield County earlier this year by Christine Cadigan and Bill Moorhead. Memo to file – remember to look up and see if the trees I spotted were in a model hotspot!

Great Mountain Forest – Falls Village

Approaching the Great Mountain Forest Orchard the sky turned dark and a few spots of rain fell on the windshield. Ellery Sinclair met me at the Orchard and like the other managers had a few samples of leaves he had collected earlier in the day during harvests on Mt. Riga. The orchard was started just three years ago as a partnership with the Housatonic Valley Regional High School, the Great Mountain Forest Corporation and The American Chestnut Foundation. Today there are fourteen lines of trees represented amongst the four hundred plus trees. I collected the back-cross samples I sought and took a few photos in the light rain.

It is a beautiful site with Canaan Mountain towering in the background. One of the lines planted is the progeny of a tree on South Canaan Road which died shortly after harvest. The tree had been measured that summer and entered in the record book as the largest American chestnut in CT. It had a circumference of 43 inches, a height of 71 feet, and an average spread of 39 feet. When pollinated the tree had significant blight and while mudpacks extended its life through to the harvest season, it did not survive to the next year. Fortunately its progeny do survive in the GMF Orchard ready to donate their genes to the next generation of blight resistant American chestnut in Connecticut!

In the photo to the right Ellery Sinclair points to several distinctive features on one of the Chinese chestnut controls in the Great Mountain Forest Orchard. Note the strong alternate but regular leaf arrangement on the green stems as well as leaf shape, color, shine and prominent petiole at base of leaf. These are all notable features of Chinese chestnut.

Warren Tree Sample – Litchfield

From Falls Village I headed south toward the Milton section of Litchfield to meet up with John Baker. John had just come back from harvesting nuts from a tree in Warren that had been pollinated this summer with TACF advanced breeding pollen. John had leaf samples from that tree as well as several others he and fellow Litchfield Hills Audubon members had found throughout Litchfield.

The Litchfield Hills Audubon has developed a back-cross orchard at Wigwam Brook and completed a test planting of thirty American chestnut seedlings. The test planting recorded excellent survival at eighty percent including several trees visibly removed by animals. The test planting also recorded and stupendous growth. One of the seedlings is shown at right is pushing thirty inches which is quite respectable for a seed planted American chestnut seedling of about four months age. We look forward to planting this orchard next year.

Woodbridge Land Trust

Last stop was Woodbridge to meet with Orchard Manager Dr. Philip Arnold. Dr. Arnold and Dr. Robert Gregg – both members of the Woodbridge Land Trust and The American Chestnut Foundation – had worked together to collect the samples I needed for Dr. Dane’s research – thus making my job easy. I had been up to the Woodbridge Orchard just a few weeks earlier and had these photos on hand from that visit.

The trees in the Woodbridge Orchard have thrived under the care of Dr. Arnold with trees planted in 2006 now up to and exceeding fourteen feet in height. The tree in the photo at right is one of the Calistro (of Woodbridge origin) back-cross trees and was planted in 2006. Many of these trees are ready for inoculation to test resistance – though we may push that back a year to allow some of the other lines planted at Woodbridge to catch up. Inoculation will tell us which of the trees have best resistance and can be used for breeding BC4F2 generation of highly blight resistant trees. One of our big projects is lining up the land required to use for planting those BC4F2 trees which will be our seed orchard for producing highly blight-resistant American chestnut. It is exciting that that time has come, and that have three orchards with material that is almost ready to breed the BC4F2 generation.

Philip had recorded the ORTV segment for Crossroads Magazine of an interview with Philip, myself and Sandy Anagnostakis from CAES – which provided a great chuckle. I labeled the samples, placed them in my icebox in the back seat, and hit the road hoping to make it home in time for dinner.

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Summary

Total journey was just over three hundred miles and the collection totaled thirty five mother tree samples and twelve orchard back-cross samples. We don’t yet have a clear understanding what the cpDNA analysis will reveal for us – but we’re hoping the sample size is large enough, and of adequate quality to provide Dr. Dane with the sample she needs to make progress on her hypothesis.

Bill Adamsen
Member and Director – The American Chestnut Foundation®