American Chestnut Restoration in Eastern Hemlock-Dominated Forests of Southeast

Home , Chestnut

Ohio

A thesis presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Nathan A. Daniel

June 2012

This thesis titled

American Chestnut Restoration in Eastern Hemlock-Dominated Forests of Southeast

Ohio

NATHAN A. DANIEL

has been approved for

the Program of Environmental Studies

and the College of Arts and Sciences by

James M. Dyer

Professor of Geography

Brian C. McCarthy

Professor of Environmental and Plant Biology

Howard Dewald

Interim Dean, College of Arts and Sciences 3

ABSTRACT

DANIEL NATHAN A., M.S., June 2012, Environmental Studies

American Chestnut Restoration in Eastern Hemlock-Dominated Forests of Southeast

Ohio (51 pp.)

Directors of Thesis: James M. Dyer and Brian C. McCarthy

Restoration of American chestnut (Castanea dentata (Marsh.) Borkh.) is currently underway in eastern North American forests. American chestnut and eastern hemlock

(Tsuga canadensis (L.) Carr.) trees historically co-occurred in these forests. Today, hemlock-dominated forests are in decline due to hemlock wooly adelgid (Adelges tsugae

Annand) infestation, and as such, may serve as appropriate habitat for chestnut reestablishment. To investigate this notion, I evaluated the performance of American chestnut seedlings planted under healthy eastern hemlock-dominated canopies. Two process-oriented greenhouse experiments were also performed to study the response of

American chestnut to drought stress and to test the competitive performance of chestnut against red maple (Acer rubrum (L.)), the most abundant hardwood found in the understory of regional hemlock-dominated forests. After two growing seasons, mean chestnut seedling survival in the field experiment was 6.6%. Seedling survival was significantly higher among trees that lacked a protective tree tube, suggesting low light levels played a major role in seedling mortality. In the drought stress experiment,

American chestnut exhibited significantly higher mortality under a severe drought treatment compared to the control group. The moderate stress treatment also responded poorly to drought; however, results did not differ significantly with the control group. In 4 the competition experiment, American chestnut grew significantly taller than red maple.

The results of this study caution against the underplanting of bare-root chestnut seedlings in low light conditions or where moisture may be limiting to establishing seedlings.

Approved: ______

James M. Dyer

Professor of Geography

______

Brian C. McCarthy

Professor of Environmental & Plant Biology 5

DEDICATION

To my wife, Kimberly W. Daniel 6

ACKNOWLEDGMENTS

I would like to thank Kimberly Daniel, Keith Gilland, Joseph Moosbrugger,

Lindsay Scott, Justin Schwalenburg, and Gideon Daniel for their assistance in the field. I would also like to thank my thesis committee, Dr. Brian C. McCarthy, Dr. James M.

Dyer, and Dr. Jared L. Deforest for their invaluable suggestions and guidance on this project. I thank the Southern Appalachian Botanical Society, Ohio University Graduate

Student Senate, Voinovich School of Leadership and Public Affairs, and Department of

Environmental and Plant Biology for financial support. The American Chestnut

Foundation kindly provided seedlings for this study. I am grateful to both the Ohio

Department of Natural Resources Divisions of Forestry and State Parks for granting permits to conduct this experiment. 7

TABLE OF CONTENTS Page

ABSTRACT...... 3 DEDICATION...... 5 ACKNOWLEDGMENTS ...... 6 LIST OF TABLES...... 8 LIST OF FIGURES ...... 9 INTRODUCTION ...... 10 MATERIALS AND METHODS...... 18 American Chestnut Performance in Eastern Hemlock-Dominated Forests...... 18 American Chestnut Drought Stress Experiment...... 24 Competitive Ability of American Chestnut and Red Maple...... 26 Statistical Analysis...... 29 RESULTS ...... 31 American Chestnut Performance in Eastern Hemlock-Dominated Forests...... 31 American Chestnut Drought Stress Experiment...... 34 Competitive Ability of American Chestnut and Red Maple...... 36 DISCUSSION...... 39 CONCLUSIONS ...... 44 LITERATURE CITED ...... 46 8

LIST OF TABLES

Page

Table 1. Site characteristics and soil properties of eastern hemlock-dominated study plots in the Hocking Hills region of southeast Ohio...... 23

Table 2. Analysis of variance results for height of American chestnut and red maple grown in mixtures of varying proportions and densities under two light treatments...... 36

Table 3. Overall mean height (cm) ± standard error (SE) for American chestnut and red maple grown under two light treatments ...... 37

Table 4. Mean height ± standard error (SE) for American chestnut and red maple grown across mixtures of five proportions and three densities...... 37

LIST OF FIGURES

Page

Figure 1. Native range of American chestnut (left) and eastern hemlock (right) (USGS 1999)...... 11

Figure 2. Hocking Hills region of southeast Ohio...... 19

Figure 3. Hocking Hills field site design diagram...... 20

Figure 4. Monthly rainfall (mm) and temperature (°C) measurements for the Jackson, Ohio weather observing station...... 24

Figure 5. Size-class analysis of the five most important hardwood species found in eastern hemlock-dominated forests in the Hocking Hills...... 27

Figure 6. Multiple deWit replacement design for competition study between American chestnut and red maple...... 28

Figure 7. Monthly survival (%) of American chestnut seedlings protected and unprotected by tree tubes...... 32

Figure 8. Boxplot of annual height increase (cm yr-1) in American chestnut seedlings unprotected by tree tubes...... 32

Figure 9. After two growing seasons, no significant correlation was found between American chestnut seedling survival (%) and any of the environmental variables tested...... 33

Figure 10. Monthly survival (%) of American chestnut seedlings based on drought treatment...... 34

Figure 11. Boxplot showing growth of American chestnut seedlings subjected to three watering regimes over a 120 day experiment...... 35

Figure 12. Mean height growth of American chestnut and red maple grown at five proportions, three densities, and two light treatments...... 38

INTRODUCTION

Until its functional extirpation from the overstory during the mid 20th century,

American chestnut (Castanea dentata (Marsh.) Borkh.) was a dominant foundation species in the forests of eastern North America (Braun 1950, Ellison et al. 2005). In

1904, the introduction of chestnut blight (Cryphonectria parasitica (Murr.) Barr.), an exotic invasive fungal pathogen accidentally imported from Asia, forced a precipitous decline in both abundance and ecosystem function of American chestnut (Ellison et al.

2005). Although the pandemic struck before the advent of modern ecological record keeping, it is estimated that upwards of 4 billion trees or 99.9% were killed (Griffin et al.

2004). Today, from Maine to Alabama, American chestnut persists in the forest understory through a process of resprout and dieback as the trees are repeatedly infected by chestnut blight (Paillet 1988, Russell 1987). In southeast Ohio, this trend continues in the unglaciated foothills of the Appalachian Mountains.

The ecological and economic impacts of the demise of this key species are difficult to overstate. The loss of American chestnut irrevocably altered the successional trajectory of forests within its 800,000 km2 native range (Keever 1953, Jacobs 2007)

(Figure 1). Once removed from the canopy, a wide variety of other overstory trees including oaks (Quercus spp.), hickories (Carya spp.), maples (Acer spp.), and eastern hemlock (Tsuga canadensis (L.) Carr.) likely filled the resulting gaps, thereby altering the structure and productivity of these forests (Keever 1953, McCormick and Platt 1980,

Vandermast et al. 2002).

Figure 1. Native range of American chestnut (left) and eastern hemlock (right) (USGS 1999).

As an important food source for many wildlife species, the disappearance of fecund chestnut trees greatly reduced the carrying capacity of eastern forests (Diamond et al. 2000). The loss of American chestnut had grave economic implications as well. The high-quality timber was of great value and the nuts were an important regional cash crop

(Zon 1904, Lutts 2004). In order to reverse the damage wrought by chestnut blight, the restoration of American chestnut continues to be a forest management priority in eastern

North America.

Efforts to restore American chestnut to eastern forests have followed several avenues of research including failed attempts to breed blight-resistant trees in the mid- twentieth century, the use of hypovirulent strains of chestnut blight to control the disease, 12 and development of transgenic trees (Andrade et al. 2009, Griffin et al. 2004, Jacobs

2007). Since the 1980’s, a renewed effort to cultivate blight-resistant chestnut hybrids has shown promise (Burnham et al. 1986). On the forefront of this effort is The

American Chestnut Foundation (TACF) whose backcross breeding program has resulted in a hybrid tree referred to as the “Restoration Chestnut” that is 15/16 American chestnut

(the remaining 1/16 is Chinese chestnut (Castanea mollissima (Blume)) (Hebard 2005).

This hybrid tree is morphologically similar to pure American chestnut, yet is putatively blight resistant (Diskin et al. 2006). The Restoration Chestnut is currently under intense testing for blight resistance. Today, TACF and its partners are finalizing plans to begin the state-by-state restoration of this once-dominant tree. For this reintroduction to be successful, the tree’s potential for growth in habitats within its pre-blight range must be better understood.

Reminiscent of the extirpation of American chestnut, another dominant forest tree, eastern hemlock, is currently in decline across its range (Figure 1) due to an exotic species (Ellison et al. 2005). For the past 60 years, the hemlock wooly adelgid (Adelges tsugae Annand), an invasive, aphid-like insect accidently introduced from Japan, has been slowly but methodically destroying entire forest stands in the eastern US (McClure

1987). Since its introduction, the adelgid has been steadily spreading outward across eastern hemlock’s native range carried by wind, birds, and humans (McClure 1990).

While there are biological and chemical methods used to control the spread of the adelgid, they are generally labor intensive and cost prohibitive, and currently none allows 13 for sustained autogenic functioning. In the foreseeable future, it is unlikely that forest managers will be able to put a stop to the decline of eastern hemlock.

Similar to the loss of American chestnut, the demise of eastern hemlock is resulting in substantial changes to the composition and functioning of eastern forest ecosystems (Orwig et al. 2002). Eastern hemlock is one of the most long-lived canopy trees in the eastern United States reaching well over 500 years in age (Gove and

Fairweather 1988). Because of their large size and exceptional longevity, hemlock trees often form large stands where they predominate (Brooks 2001). As this massive conifer species is removed from its position of dominance in the overstory, it is replaced primarily by opportunistic intermediate shade-tolerant hardwood species including oaks

(Quercus spp.), hickories (Carya spp.), birches (Betula spp.), and maples (Acer spp.)

(Orwig and Foster 1998, Mahan et al. 2004, Eschtruth and Battles 2008). Conspicuously absent from this mix, is American chestnut.

Ample evidence exists to confirm that American chestnut and eastern hemlock historically co-occurred in eastern forests (Woods and Shanks 1959, Russell 1980, Foster and Zebryk 1993). While chestnut often dominated upland habitats, as a generalist species, it grew prolifically in mesic conditions as well (Braun 1950). In the mountains of Tennessee, chestnut was reported to attain both its largest size and greatest abundance at mid-slope and in cove habitats (Ashe 1912). More recently, Vandermast et al. (2002) documented the historical prominence of American chestnut in riparian ravines and valleys of the southern Appalachians; a habitat well-suited for eastern hemlock (Orwig et al. 2002). Palynological pollen profiles show that American chestnut and eastern 14 hemlock have maintained a dynamic relationship replacing one another as the locally dominant species throughout the past 2,500 years (Russell 1980, Foster and Zebryk

1993). Recently, eastern hemlock replaced a large portion of the canopy following the decline of chestnut (Vandermast et al. 2002). While there is much evidence showing historical co-occurrence, little is known about the performance of American chestnut in eastern hemlock-dominated forests. If chestnut performs well under healthy hemlock canopy, this would support a shade tolerant classification for the tree and would warrant further investigation of chestnut restoration in hemlock stands threatened by, or already in decline due to infestation by the hemlock woolly adelgid.

There has been considerable discussion as to the shade tolerance classification of

American chestnut. The tree defies easy pigeonholing, exhibiting traits of both shade- tolerant and intolerant species. American chestnut shows great plasticity in its ability to exploit light resources. In a low light environment in the forest understory, the tree is well suited to a “sit-and-wait” approach, maintaining a positive carbon balance for long periods of time (Paillet 2002). However, unlike other species such as eastern hemlock capable of persisting for long periods in the understory, chestnut is capable of rapid growth in higher light situations (McCarthy et al. 2010).

In a two-year inventory of American chestnut distribution in southern Ontario,

Tindall et al. (2004) found most chestnut trees growing under dense canopy cover

(> 50%) supporting the shade-tolerant classification and the ability of American chestnut to persist for many years under dense canopy cover. Furthermore, Paillet & Rutter

(1989) found long-established “old seedlings” surviving in low light conditions. Wang et 15 al. (2006) and Anagnostakis (2007) have supported these observations with controlled experiments. In contrast, McCament and McCarthy (2005), Joesting et al. (2009), and

Rhoades et al. (2009) suggest a more intermediate shade-tolerant classification maintaining that while resprouts may persist in low light environments, newly established transplants do not perform as well. With the ongoing alteration of eastern hemlock- dominated woodlands, an opportunity exists to further investigate the light requirements of American chestnut while testing the tree’s performance in a novel habitat.

Although ecologically disastrous, the decline of eastern hemlock could mimic a two-stage shelterwood management treatment (Rhoades et al. 2009) towards the restoration of chestnut. This silvicultural method is designed to allow intermediate shade-tolerant species an opportunity to establish in a moderate light environment while simultaneously excluding the recruitment of shade-intolerant species (Loftis 1990).

Generally, this silvicultural method employs the mechanical removal of the midstory, thus allowing a modest increase in light reaching the understory (Rhoades et al. 2009).

Over a period of several years, the intermediate shade-tolerant species establish and grow without having to contend with shade-intolerant competitors. Once the target species is well established and has a height advantage over its potential competitors, the overstory is mechanically removed (Buckley et al. 1998). Studies have shown adelgid infestation causes increasing foliar loss in the lead up to eastern hemlock mortality (Orwig and

Foster 1998). As hemlock leaf cover decreases, the amount of light reaching the forest floor increases. This process takes several years and may well mimic a two-stage shelterwood treatment. 16

Studying the ability of American chestnut to survive and grow in the understory will help researchers better understand the species’ tolerance for low light conditions.

Hibbs (1983) found that in low light environments many species capable of regenerating by resprouts (including American chestnut) do so much more readily than by seed regeneration. As the majority of American chestnut stems found in eastern forests today are resprouts, it raises the question, how well will chestnut seedlings transplanted from the nursery perform in similar low light environments?

It is common for transplanted bare-root seedlings to undergo some amount of shock following outplanting (Sutton and Tinus 1983). Much of this stress is attributable to water deficiency due to root confinement in the planting hole and limited root-soil contact (Burdett 1990). While this is true under ideal conditions, additional stochastic environmental factors such as drought have the potential to radically reduce seedling survival. Little is known about bare-root American chestnut seedling response to low soil water content (Latham 1992, Jacobs 2007). A better understanding of how bare-root chestnuts respond to dryer than normal soil conditions is an essential prerequisite for interpreting seedling survival in field studies.

For restoration efforts to succeed, American chestnut will need to successfully compete against neighboring shade-tolerant and intermediate shade-tolerant species already in situ. Recent studies show oaks (Quercus spp.), hickories (Carya spp.), birches

(Betula spp.), and maples (Acer spp.) among other species replacing hemlock throughout its range (Mahan et al. 2004, Small et al. 2005). In hemlock-dominated forests of southeast Ohio, understory species composition is now being investigated. With a clearer 17 picture of the understory community in regional hemlock-dominated forests, questions regarding chestnut’s competitive ability can be addressed.

To investigate these unknowns, this study had three objectives: 1) to evaluate whether threatened hemlock stands can be under-planted with American chestnut as both a forest reclamation and species restoration project, 2) to examine American chestnut’s tolerance and response to simulated drought conditions, and 3) to test American chestnut’s competitive ability against its most likely hardwood competitor growing in the understory of hemlock-dominated forests of southeast Ohio. 18

MATERIALS AND METHODS

American Chestnut Performance in Eastern Hemlock-Dominated Forests

Study Site

The study was conducted during the growing seasons of 2010 and 2011 in the

Hocking Hills region of southeast Ohio, USA (39.43°N, 82.54°W) (Figure 2). The

Hocking Hills lie near the western edge of the unglaciated Appalachian Plateau within the range of both American chestnut and eastern hemlock. Today, chestnut resprouts are found growing alongside hemlock trees in the Hocking Hills. The region is characterized by its highly dissected hills and valleys, rocky cliffs, and well-drained, acidic surface soils mainly composed of weathered sandstone and shale (Lemaster and Gilmore 1989).

The Hocking Hills lie at approximately 200 m to 310 m elevation above sea level (ODGS

2003). Mean annual precipitation for the Jackson weather observing station located approximately 40 kilometers from the field sites is 103.66 cm with a mean annual temperature of 11.2 °C (NCDC 2010). 19

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[ [ [ [

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0 2.5 5 10 Kilometers ¯

Figure 2. Physiographic regions of Ohio with inset map showing the unglaciated Hocking Hills region of southeast Ohio. Green areas represent state lands. Field sites are identified with black stars. Physiographic map from Ohio Division of Geological Survey (1998). Inset map from ESRI (2011). 20

Experimental Design

Chestnut seedlings were planted in eastern hemlock-dominated (> 50% basal area) forest stands adjacent to 17 long-term monitoring plots established by Dr. James M.

Dyer. All plots are located on Ohio Department of Natural Resources, Division of

Forestry and Division of Parks and Recreation lands. Plots are located mid-slope and placed across a wide range of aspects. Slope and aspect of the plots were measured with a Brunton Pocket Transit (Brunton Outdoor Group, Riverton, WY).

Two 20 m transects were established perpendicular to the contour, 5 m beyond the long-term plots (Figure 3). Along these transects, one chestnut seedling was planted every meter for a total of 20 seedlings per transect and 40 seedlings per plot (n = 680).

Every other seedling was protected from deer herbivory using 1.5 m, “O-style” ventilated plastic grow tubes (Plantra Inc., Mendota Heights, MN).

Figure 3. Hocking Hills field site design diagram. Black circles represent American chestnut seedlings protected with plastic tree tubes and white circles represent unprotected seedlings. 21

American chestnut planting stock consisted of 1-0 bare-root seedlings propagated at the West Virginia Division of Forestry, Clements State Tree Nursery (West Columbia,

WV) located approximately 150 miles from the study site. Seedlings were lifted in the previous fall, and placed in a cold room at 5 °C for 100 days, prior to field transplantation.

Field Methods and Site Characterization

American chestnut seedling survival was monitored monthly during both the 2010 and 2011 growing seasons from May to October. Seedling height was recorded to the nearest cm during the first and last month of both growing seasons using a meter stick.

Extent of deer herbivory was recorded at the end of each growing season based on evidence of browsing (i.e., absence of terminal bud). Insect herbivory was visually estimated as percent defoliation at the end of each growing season.

The light environment of each plot was characterized using two hemispherical photographs taken from opposite ends of the two transects. The photographs were taken one meter from the ground, approximating the height of American chestnut seedlings.

Mean canopy openness for each site was calculated from these photographs using

WinSCANOPY 2006 Pro software (Regent Instruments Inc., Nepean, Ontario, Canada).

At each site, six soil samples were collected at random from the top 15 cm of mineral soil using a 5 cm diameter corer (n = 102). Three of these samples per site were combined to create a composite of the local soil and sent to Brookside Laboratories in

New Knoxville, Ohio for pH, Carbon (%), Nitrogen (%), and C:N analysis (Table 1).

The remaining three samples were combined and passed through a 2 mm brass sieve and 22 dried at 105 °C for 24 hours. Soil texture was determined using the Bouyoucos hydrometer method to calculate percent sand, silt, and clay (Bouyoucos 1962). 23

Table 1. Site characteristics and soil properties of eastern hemlock-dominated study plots in the Hocking Hills region of southeast Ohio. Three samples per plot were homogenized prior to analysis (n = 1 per plot). Canopy Soil Texture Plot Slope (%) Aspect pH C (%) N (%) C:N Sand (%) Clay (%) Openness (%) Classification 1 24 284 12.0 4.2 3.25 0.12 27.08 22.5 15.0 Silt loam 2 13 142 9.2 4.8 2.04 0.12 17.00 5.0 17.5 Silt loam 3 24 304 12.3 4.2 2.57 0.10 25.70 22.5 17.7 Silt loam 4 26 342 5.5 4.3 2.02 0.12 16.83 65.0 12.5 Sandy loam 5 26 19 7.5 5.3 2.58 0.25 10.32 52.5 15.0 Loam 6 16 320 11.8 4.5 2.98 0.21 14.19 30.0 15.0 Silt loam 7 26 160 9.1 4.0 1.73 0.06 28.83 5.0 20.0 Silt loam 8 21 278 7.3 4.2 3.36 0.14 24.00 47.5 15.0 Loam 9 17 332 12.9 4.7 4.07 0.26 15.65 5.0 22.5 Silt loam 10 17 245 8.1 4.1 2.26 0.09 25.11 45.0 17.5 Loam 11 18 304 12.4 4.0 4.00 0.13 30.77 22.5 20.0 Silt loam 12 12 260 9.7 5.1 3.36 0.19 17.68 5.0 20.0 Silt loam 13 7 290 8.5 5.6 2.93 0.12 24.42 5.0 20.0 Silt loam 14 30 251 11.0 4.2 2.36 0.10 23.60 5.0 15.0 Silt loam 15 16 280 8.5 4.6 2.92 0.13 22.46 57.5 17.5 Sandy loam 16 31 30 11.9 4.8 2.95 0.23 12.83 67.5 7.5 Sandy loam 17 20 183 6.4 4.5 2.36 0.13 18.15 70.0 7.5 Sandy loam 24

American Chestnut Drought Stress Experiment

Experimental Design

During the first summer and fall of the field study, dry and hot conditions affected much of the Ohio Valley (NCDC 2010). Unfortunately, an Ohio weather observing station located in Logan, Ohio only 15 kilometers from the Hocking Hills failed to record precipitation data during this period of time. Records from the Jackson, Ohio weather observing station located approximately 40 kilometers from the Hocking Hills show precipitation levels during this time to be 20% below the 30 year normal (NCDC 2010)

(Figure 4). These drier than normal conditions followed an unusually wet spring in the region (NCDC 2010) and this rather abrupt change in precipitation may have affected seedling survival.

Normal Temperature Normal Precipitation 25

350 2010 Temperature 2010 Precipitation

300 20 250

15 200

Rainfall (mm) 10 Temperature (°C) Temperature 150 100 5 50

0 0 May June July August September October

Figure 4. 2010 and 1971-2000 monthly rainfall (mm) and temperature (°C) for the Jackson, Ohio weather observing station located approximately 40 kilometers from the Hocking Hills (NCDC 2010). Rainfall amounts from July to October were 20% lower than the 30 year normal. 25

Preliminary Palmer Drought Severity Index (PDSI) calculations by the National

Climate Data Center in February 2011 suggested mild drought conditions may have occurred in southeast Ohio (Division 10) during the summer and fall of 2010. According to revised calculations, southeast Ohio did not endure drought in 2010, experiencing a

Palmer Drought Severity Index (PDSI) average from July to October of -0.29 and a

Palmer Z Index value of -0.65 for the same period of time. However, the climate division is geographically large and monthly precipitation levels throughout the region were highly variable.

In order to investigate the effect lower than normal precipitation may have had on seedling survival in the field study, a controlled experiment was conducted over a four- month (120 day) period from May to September 2011 at the Ohio University greenhouse in Athens, OH. Planting stock was propagated, lifted, and stored using the same methods as in the field study. Three treatments, composed of 30 seedlings each, were planted for a combined total of 90 seedlings. Each seedling was planted in its own 10 × 10 × 35 cm pots using Fafard 52 (Conrad Fafard Inc., Agawam, MA) soil mixture composed of pine bark, sphagnum peat, perlite and vermiculite inoculated with 5% field soil to introduce native fungal symbionts. A control treatment and two drought stress treatments called

“moderate” and “severe” were placed in a rainout shelter under 72% shade cloth designed to mimic photosynthetic radiation levels under hemlock canopy in decline due to hemlock woolly adelgid infestation (Jenkins et al. 1999).

To establish the frequency and amount of irrigation necessary to induce the desired drought stress, the permanent wilting point (PWP) of American chestnut was 26 determined by bringing the soil of a subset of individually potted trees to field capacity and then weighing the pots repeatedly until wilting. As the trees approached the PWP, two potted trees per day were weighed and then well watered. PWP was reached when the trees failed to recover after watering (Veihmeyer and Hendrickson 1928). The mean difference in weight between pots at field capacity and PWP was determined (750 mg), and the irrigation amount for the control treatment was set at 2/3 this difference (500 ml).

Volumetric water content (VWC) was measured using an EC-5 soil moisture sensor and a

ProCheck handheld reader (Decagon Devices Inc., Pullman, WA). A watering schedule of once every five days was determined in order to keep the VWC in the control group at approximately 25%. The moderate drought treatment received half this amount of water

(250 ml) every five days to maintain a VWC of approximately 15% and the severe drought treatment received half of this amount (125 ml) to maintain a VWC of approximately 10%.

Competitive Ability of American Chestnut and Red Maple

Experimental Design

In the fall of 2009, I performed a size-class analysis of hardwood tree species found on twenty plots located in eastern hemlock-dominated forests of the Hocking Hills region of southeast Ohio. The diameter at breast height of every tree greater than 8 cm was recorded. A relative importance value (RIV) was derived from the relative frequency (RFRQ), the relative density (RDEN), and the relative basal area (RBA) of each species by the formula: 27

RFRQ + RDEN + RBA RIV = 3

All hardwood tree species with a RIV greater than 5 were then compared by size- class (Figure 3). These species included red maple (RIV 8.2), tuliptree (RIV 7.9), chestnut oak (RIV 7.3), white oak (RIV 6.4), and sweet birch (RIV 6.2). Red maple was the most abundant hardwood species found in the smaller size-classes (8-16 cm and 16-

24 cm) (Figure 5). In the Hocking Hills of southeast Ohio, chestnut will therefore likely face competition from red maple.

8 ACRU BELE 6 Stems/ha LITU 4 QUAL QUMO 2

0 8-16 16-24 24-32 32-40 40-48 48-56 56-64 >64 Size-Class (cm)

Figure 5. Size-class summary of the five most important hardwood species found in eastern hemlock-dominated woodlands of the Hocking Hills of southeast Ohio. The species are red maple (Acer rubrum) (ACRU), sweet birch (Betula lenta) (BELE), tuliptree (Liriodendron tulipifera) (LITU), white oak (Quercus alba) (QUAL), and chestnut oak (Quercus montana) (QUMO). In the smallest size-classes, red maple is most abundant. 28

In order to test the competitive ability of American chestnut against red maple, a controlled experiment was conducted at the Ohio University greenhouse during the growing season of 2010. Seedlings of both species were germinated simultaneously in three-gallon pots. Planting medium and inoculation protocol was identical to the drought experiment. Seeds were planted at varying proportions and densities using a multiple deWit replacement design (cf. Meekins and McCarthy 1999) (Figure 6). Two light treatments were used, one in full sun and a second under a 72% shade cloth designed to mimic photosynthetic radiation levels found under eastern hemlock-dominated forests in decline due to hemlock woolly adelgid infestation (Jenkins et al. 1999). All pots were kept moist throughout the duration of the experiment.

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Figure 6. Multiple deWit replacement design for competition study between American chestnut and red maple. Open circles represent chestnut and black circles represent red maple.

Statistical Analysis

Assumptions of normality and homogeneity of variance were examined prior to all analyses via the D’Agostino normality test and Levene’s equal-variance test, respectively. All analyses were done using R software (R Development Core Team

2011, Therneau 2010, Hothorn et al. 2011). The field study was analyzed as random and balanced with each seedling representing an observation. The impact of tree tubes on seedling survival was analyzed using the Cox proportional-hazards model (Cox 1972,

Fox 2002).

To account for non-normal distribution of data, Spearman’s rank correlation test was used to investigate the relationship between survival and environmental variables including: canopy openness, slope aspect, as well as soil variables including percent sand, percent carbon, percent nitrogen, and pH (Whitlock & Schluter 2009). In order to analyze slope aspect as a continuous variable, data were transformed following Beers et al. (1966) to range from 0 to 2 where Transformed Aspect = cos(45-Aspect) + 1. In cases where a correlation test failed to reject the null hypothesis, a power test was conducted to analyze the strength of the test (Onwuegbuzie & Leech 2004). For the purpose of this thesis, all results were reported regardless of power. However, only statistically non- significant results with a high power (around 0.8) are scientifically valuable as they rule out weakness of design as a possible cause of lack of correlation (otherwise known as a type II error).

In the drought stress experiment, differences in the total growth (cm) of seedlings in each treatment were analyzed using general linear model analysis of variance. 30

Subsequent multiple comparison procedures using Tukey’s Honestly Significant

Difference (HSD) test were used to examine differences among treatments. Survival analysis was performed using Cox proportional-hazards model (Cox 1972, Fox 2002).

In the competition experiment, data were square root transformed to meet assumptions of normality. Data were examined using general linear model analysis of variance and Tukey’s HSD was used in post-hoc analysis. The effects of species, proportion, density, and light treatment were analyzed using seedling height as the response variable. 31

RESULTS

American Chestnut Performance in Eastern Hemlock-Dominated Forests

After two growing seasons, mean chestnut seedling survival was 6.6% across all

17 plots, with survival by plot varying from 0% to 30%. Seedling survival was significantly (R2 = 0.17, P < 0.001) greater among trees that lacked a protective tree tube with 13% of unprotected trees surviving and < 1% of protected trees surviving (Figure 7).

At the end of the second growing season, mean relative annual height growth of unprotected seedlings was 4.9 cm (± 0.52 SE, n = 44) (Figure 8). No significant correlation was found between survival and any of the tested environmental variables including canopy openness (r = 0.05, P = 0.83), percent sand (r = 0.43, P = 0.08), percent carbon (r = 0.11, P = 0.68), percent nitrogen (r =0.20, P = 0.43), pH (r = 0.009, P =

0.97), and aspect (r = 0.12, P = 0.64) (Figure 9). Power analysis results for these correlations were as follows: canopy openness (Power = 0.83), percent sand (Power =

0.49), percent carbon (Power = 0.70), percent nitrogen (Power = 0.55), pH (Power =

0.97), and aspect (Power = 0.67). Approximately 35% of surviving seedlings experienced severe insect herbivory losing the majority of their leaf surface. Signs of deer browse on leaves and terminal buds were found on 7% of surviving seedlings.

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Figure 7. Monthly survival (%) of American chestnut seedlings protected and unprotected by tree tubes. Cox proportional-hazards model results indicate seedling survival based on the presence or absence of tree tube varied significantly (P < 0.001). Protected seedlings experienced a much higher level of mortality than did unprotected seedlings.

12 10 ) -1 8 6 4 Annual Growth Rate (cm yr 2 0

Unprotected Treatment

Figure 8. Boxplot of annual height increase (cm yr-1) in American chestnut seedlings unprotected by tree tubes. Whiskers represent 5th and 95th percentiles and the horizontal centerline represents the median. At the end of the second growing season, mean growth of unprotected seedlings was 4.9 cm yr1 (± 0.52 SE, n = 44). 33

35 !"<#=>=?" 35 !"<#=>BA" #"<#=>@A# #"<#=>CD# 30 30 25 25 20 20 15 15 79+:*:3;#/01# 79+:*:3;#/01# 10 10 5 5 0 0

4 6 8 10 12 14 0 10 20 30 40 50 60 70 23.,!5#6!&..&%%#/01# 73.8#/01#

35 !"<#=>EE" 35 !"<#=>G=" #"<#=>F@# #"<#=>BA# 30 30 25 25 20 20 15 15 79+:*:3;#/01# 79+:*:3;#/01# 10 10 5 5 0 0

1.5 2.0 2.5 3.0 3.5 4.0 4.5 0.05 0.10 0.15 0.20 0.25 23+4,.#/01# )*(+,-&.#/01#

35 !"<#=>==C" 35 !"<#=>EG" #"<#=>CD# #"<#=>FB# 30 30 25 25 20 20 15 15 79+:*:3;#/01# 79+:*:3;#/01# 10 10 5 5 0 0

4.0 4.5 5.0 5.5 0.0 0.5 1.0 1.5 2.0 !"# $%!&'(# Figure 9. After two growing seasons, no significant correlation was found between American chestnut seedling survival (%) and any of the environmental variables tested. Data were analyzed using Spearman’s rank correlation test. 34

American Chestnut Drought Stress Experiment

Seedling survival in the control group was 97% with 29 of 30 seedlings surviving.

The moderate drought stress group saw 77% survival with 23 of 30 seedlings surviving.

The severe drought stress treatment resulted in 66% survival with 20 of 30 seedlings surviving. A small difference in survival (Z = 1.918, P = 0.055) was found between the moderate drought stress treatment and the control treatment while the severe drought stress group exhibited significantly (Z = 2.345, P = 0.019) higher mortality than the control group (Figure 10).

100 90

<-=0#-'(>#-"?(

80 ,-./#&0/(!0#/11( !(2(34536( !"#$%$&'()*+( "(2(74788(

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May June July Aug Sept

Figure 10. Monthly survival (%) of American chestnut seedlings based on drought treatment. Seedlings under severe drought stress had significantly higher mortality than those in the control group. A small difference in survival was found between the moderate drought stress treatment and the control treatment. 35

American chestnut seedlings height growth differed significantly among treatments (F = 3.618, P = 0.032, df = 2) with mean growth of 20.4 cm yr1 in the control group, 15.8 cm yr1 in the moderate drought treatment and 14.3 cm yr1 in the severe drought treatment. In post-hoc analysis, significant differences were observed between the severe drought stress treatment and the control group (P = 0.039), but not between the moderate drought stress treatment and the control group (P = 0.131) nor between the moderate and severe treatments (P = 0.824) (Figure 11).

40 A )

-1 A B 30 B 20 10 Annual Growth Rate (cm yr 0

Control Moderate Severe Field

Figure 11. Boxplot showing annual growth rate of American chestnut seedlings subjected to three watering regimes. Tukey’s Honestly Significant Difference test indicated a significant difference between the severe drought stress treatment and the control group, but not between the moderate treatment and the control group nor between the moderate and severe treatments. Letters “A” and “B” indicate significant differences (i.e., P < 0.05). Whiskers represent 5th and 95th percentiles and the horizontal centerline represents the median. Circles above the control and moderate boxplots are outliers. Annual growth rate results from the field study are presented for comparison (and not included in statistical analyses). 36

Competitive Ability of American Chestnut and Red Maple

In the competition study, American chestnut grew significantly taller than red maple in both treatments (F = 18.04, P < 0.001) (Table 2). Under shade, chestnut grew

16% taller than did red maple, while in full sun chestnut grew 20% taller (Table 3). A significant (F = 7.99, P < 0.001) species by proportion interaction was found (Table 2).

As the proportion of either species increased per pot, the height of that respective species tended to increase as well (Table 4; Figure 12).

Table 2. Analysis of variance results for height of American chestnut and red maple grown in mixtures of varying proportions and densities under two light treatments Mixture and source of variation df SS F P

Species 1 14.40 18.04 < 0.001 Proportion 4 4.07 1.28 0.281 Density 2 3.12 1.95 0.145 Treatment 1 0.21 0.26 0.608 Species × Proportion 2 12.75 8.00 < 0.001 Species × Density 2 4.20 2.63 0.075 Proportion × Density 8 4.25 0.67 0.721 Species × Treatment 1 0.08 0.10 0.749 Proportion × Treatment 4 13.19 4.13 0.003 Density × Treatment 2 0.14 0.09 0.920

Table 3. Overall mean height (cm) ± standard error (SE) for American chestnut and red maple grown under two light treatments American Chestnut Mean Height (cm) ± SE Red Maple Mean Height (cm) ± SE Shade Sun Overall Shade Sun Overall 22.2 ± 1.0 22.4 ± 1.0 22.3 ± 0.7 18.6 ± 1.1 18.0 ± 1.1 18.3 ± 0.8

Table 4. Mean height ± standard error (SE) for American chestnut and red maple grown across mixtures of five proportions and three densities Shade Mean Height ± SE Sun Mean Height ± SE Density and American American proportion Red Maple Red Maple Chestnut Chestnut

4: 0 : 100….. -- 26.5 ± 3.9 -- 19.5 ± 3.5 25 : 75….. 27.0 ± NA 17.0 ± 4.4 14.0 ± NA 25.7 ± 6.9 50 : 50….. 23.0 ± 4.0 8.5 ± 5.5 27.0 ± 9.0 28.0 ± 5.0 75 : 25….. 26.3 ± 3.4 18.0 ± NA 20.7 ± 5.2 29.0 ± NA 100 : 0 ….. 23.3 ± 3.1 -- 22.8 ± 4.3 -- 8: 0 : 100….. -- 22.1 ± 2.9 -- 18.4 ± 4.7 25 : 75….. 17.5 ± 0.5 25.0 ± 3.6 12.5 ± 0.5 16.7 ± 2.4 50 : 50….. 17.0 ± 4.8 9.5 ± 3.1 16.3 ± 2.1 24.0 ± 2.9 75 : 25….. 22.5 ± 3.1 8.5 ± 3.5 22.3 ± 3.7 20.0 ± 3.0 100 : 0 ….. 19.9 ± 2.0 -- 24.9 ± 3.6 -- 12: 0 : 100….. -- 18.9 ± 1.7 -- 17.0 ± 1.5 25 : 75….. 19.3 ± 4.8 18.8 ± 3.0 20.0 ± 2.1 17.9 ± 3.0 50 : 50….. 21.7 ± 3.4 14.7 ± 2.1 23.7 ± 3.0 17.2 ± 2.8 75 : 25….. 26.9 ± 3.0 7.0 ± 2.3 28.1 ± 3.3 8.3 ± 2.0 100 : 0 ….. 21.8 ± 2.6 -- 20.6 ± 2.0 --

Shade Treatment Sun Treatment 30 Density = 4 30 Density = 4 25 25 20 20 15 15 10 10 5 5 0 0

30 Density = 8 30 Density = 8 25 25 20 20 15 15 10 10 5 5 0 0 Height Growth (cm)

30 Density = 12 30 Density = 12 25 25 20 20 15 15 10 10 5 5 0 0 0:100 25:75 50:50 75:25 100:0 0:100 25:75 50:50 75:25 100:0 Proportion

Figure 12. Mean growth in height of American chestnut and red maple grown at five proportions, three densities, and two light treatments. American chestnut heights are represented by circles. Squares indicate red maple heights.

DISCUSSION

American chestnut seedlings established and grew poorly after transplant into eastern hemlock-dominated forests. Chestnut seedlings did well during the spring and early summer of the first growing season with survival in May and June of 99% and 97%, respectively. This occurred directly after transplant when sapling energy reserves in the roots were high and rainfall levels were much higher than 30-year normals (NCDC

2010). Seedling survival dropped precipitously during and after July. This decline was primarily due to the low light environment found under hemlock canopy. A secondary factor driving seedling mortality was likely the effect of drier than normal conditions experienced in the area during the middle and late summer of 2010 (NCDC 2010).

The use of protective tree tubes in the low-light environment found under hemlock-dominated canopy appears to have reduced seedling survival. Seedlings protected from deer herbivory by tree tubes had significantly lower survival compared to seedlings left unprotected. In fact, seedlings protected with a tube faced nearly 100% mortality as compared with 87% mortality of unprotected trees. Here, unprotected seedlings exhibited higher survival despite evidence of deer herbivory on 7% of surviving unprotected seedlings. The use of tree tubes in these circumstances apparently reduced the amount of photosynthetically active radiation enough to hinder seedling survival.

This conclusion supports the intermediate shade-tolerant classification when American chestnut is planted as bare-root seedlings.

Nevertheless, seedling survival of 13% in the unprotected treatment suggests that while American chestnut is capable of resprouting and remaining alive under dense 40 canopy cover (including eastern hemlock-dominated understory), transplanted bare-root seedlings are quite susceptible to low-light conditions. Transplanted seedlings appear to be less robust than their shrubby resprout counterparts that already possess well- developed root systems. Shirley (1945) showed that in low-light environments, understory plants tend to focus resources on leaf growth at the expense of root development. Although the current study did not investigate the potential depression of resources allocated to chestnut roots, it is conceivable that low light levels under hemlock-dominated canopy reduced seedling root development. During the first years of life after transplant into such low light environments, seedlings may succumb to the pressures of simultaneously developing a root system and above ground biomass. This is in contrast to already established resprouts, which are capable of persisting in low light situations for extended periods of time (Paillet 2002).

Acknowledging that caution must be taken when comparing greenhouse results with those of a field study, the annual height growth of seedlings in the drought stress experiment planted under intermediate light levels were on average three times higher than the height growth attained by unprotected seedlings growing under healthy hemlock canopy. This large difference in annual growth between seedlings planted under intermediate and low light conditions supports the notion that American chestnut should be classified as intermediate shade-tolerant. There is a strong likelihood that a survival threshold exists for bare-root chestnut seedlings between 28% canopy openness as mimicked in the greenhouse experiment and 13% canopy openness found under hemlock-dominated cover. Above this threshold, bare-root chestnut seedlings appear to 41 maintain a positive carbon balance while below it the seedlings may exhaust their energy reserves.

While low light levels appear to be the primary causative factor in poor seedling survival, a probable secondary contributor was the inability of bare-root seedlings to handle the stress of lower than normal precipitation during the summer and fall of 2010.

These newly transplanted seedlings appear to have exhibited an increased susceptibility to water deficiency as a result of poorly developed root systems (Burdett 1990). Results of the greenhouse experiment support this conclusion. Under a rainout shelter and in moderate light conditions, bare-root American chestnut seedlings exposed to both severe drought and moderate drought conditions exhibited higher levels of mortality than did seedlings in the control group. However, survival across both drought treatments was considerably higher than survival in the field experiment. These results suggest that the drier than average conditions experienced in the field experiment in 2010 may have had a negative effect on seedling establishment, but the primary factor for low survival remains the low-light level found under healthy hemlock canopy.

Two other moisture-related factors potentially inhibited chestnut seedling establishment during the field study. These include reduction in moisture that reached the forest floor due to interception by overstory trees and competition between overstory and understory species for moisture that did reach the forest floor. In every precipitation event, moisture is intercepted by overstory foliage, trunks and branches (Rowe and

Hendrix 1951, Anderson et al. 1969). The amount of precipitation that eventually reaches the soil is directly related to storm intensity where weaker storms allow less 42 penetration (Coffay 1962). In mixed hemlock-hardwood forests, Mitchell (1929) found that in light rain events (≤1.5 mm) up to 40% of precipitation was intercepted by trees before reaching the forest floor. During stronger storms (≤ 13 mm), a still considerable

20% was intercepted. Overstory and understory species then compete for what moisture does reach the soil. During July 2010 when the majority of seedling mortality occurred, data recorded by the Remote Automated Weather Station located in Zaleski State Forest approximately 20 km from the field site showed 7 of the total 14 rain events were under

1.5 mm (WRCC 2011). Of the remaining 7 events, 4 were less than 13 mm. Therefore, in the field study a considerable portion of the lower than normal rainfall experienced during the summer was likely intercepted by hemlock-dominated overstory. This, coupled with competition with mature trees with well-developed root systems for soil moisture may have also impacted seedling establishment.

The light environment found across study plots showed little variability with canopy openness ranging from 5% to 13%. This relatively homogeneous light environment may account for the lack of correlation found between canopy openness and survival across study plots. The lack of correlation between seedling survival and pH helps to rule out this environmental variable as a significant factor in low seedling survival. Post-hoc power analyses on these correlations indicate these tests were strong enough to preclude the likelihood a type II error (a false negative) was committed.

However, power tests on percent sand, percent carbon, percent nitrogen, and aspect show these correlations to have been of insubstantial power to dismiss the possibility of a type

II error. 43

Results of the size-class analysis indicate that, with the exception of eastern hemlock itself, red maple is the most abundant species found in the hemlock-dominated understory. The fact that red maple is performing well in the Hocking Hills relative to other hardwood species is not surprising. The abundance of red maple throughout eastern forests has been on the rise for at least a century due to the tree’s generalist characteristics such as the ability to survive under a wide range of light environments (Abrams 1998).

However, in the greenhouse competition experiment, American chestnut seedlings outgrew red maple in both light treatments across a range of densities and proportions.

These results bode well for American chestnut restoration efforts as we continue to see the prevalence of red maple on the rise throughout the region (Widmann et al. 2009). 44

CONCLUSIONS

This study was conducted under healthy eastern hemlock canopy with the assumption that if American chestnut seedlings performed well under these low light conditions, they would perform equally well or better in hemlock stands in decline due to hemlock wooly adelgid infestation. While American chestnut did not perform well under healthy hemlock cover, its performance in eastern hemlock-dominated woodlands in decline remains unknown. Recent studies by McCarthy et al. (2008, 2010) show higher levels of survival in hybrid chestnut compared to pure American chestnut on reclaimed mine lands. What differences (genetics, physiology, etc.) that may exist between hybrid and pure seedling performance in hemlock-dominated forests remains unstudied.

Results of this study suggest that bare-root chestnut seedlings are unsuited for transplant into low-light environments. The use of protective tree tubes under these conditions may exacerbate the problem by further reducing the already limited amount of light reaching the seedlings. In this study, the benefit of reduced deer herbivory was outweighed by increased mortality brought about by the reduction of light inside of the tubes.

Another factor potentially affecting chestnut seedling establishment may be eastern hemlock allelopathy (Daubenmire 1930). Vandermast et al. (2002) suggested that the ability of American chestnut and eastern hemlock to suppress one another’s growth by allelopathy may have played an important role in a long-term, dynamic competitive relationship between these two foundation species. If this were true, it would have 45 important implications for chestnut restoration efforts in eastern hemlock-dominated forests.

Seedling survival in these forests may be also be limited due to lack of obligatory mycorrhizal associations. However, this is unlikely as chestnut mainly interacts with ectomycorrhizae (ECM) typically found in forests containing other members of the

Fagaceae family (Palmer et al. 2008). In the Hocking Hills, oak species are commonly found and presumably so are networks of these ECM.

Whether or not American chestnut will survive in eastern hemlock stands in decline due to hemlock woolly adelgid infestation, forest managers must consider what successional path is most ecologically and economically advantageous in these changing forests. While the results of this study do not support American chestnut restoration in healthy eastern hemlock-dominated forests, the potential ecological and economic utility of this restoration plan warrants further study, especially in stands already degraded by hemlock woolly adelgid infestation. 46

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