Extensive natural hybridization between butternut and the introduced tree Japanese walnut Sean Hoban, Tim McCleary, Jeanne Romero-Severson Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, USA

Background

Results

Butternut (Juglans cinerea), a hardwood tree native to eastern North America, has experienced severe decline (75-100% mortality in many populations) in the twentieth century. Mortality is largely attributable to the disease butternut canker, caused by the fungus Sirococcus clavigignenti-juglandacearum, but other causes include changing forest management practices and seed predation1. Conservation management and reintroduction efforts are complicated by suspected (but unverified) natural hybridization with an introduced tree, Japanese walnut (Juglans ailantifolia)2. This closely related tree was introduced to America in approximately 1870, was widely planted, and soon naturalized in woodlots, abandoned fields, and roadsides3. Putative hybrids are commonly reported near old farms and other highly human impacted landscapes. Intriguingly, Japanese walnut and hybrids show significantly greater tolerance to butternut canker than butternuts4.

1 Gilbert Island, NB 2 Keswick Ridge, NB

(1) We identified 939 J. cinerea, 46 F1, 11 F2, 8 BC1 to Jc, 0 BC1 to Ja, and 9 J. ailantifolia. 98 individuals were unassigned. Of the unassigned, all but 10 had ~0.00 probability of assignment to J. cinerea, and are likely some form of hybrid.

3 Blackville, NB 4 Dave Gallagher, ON 5 Barb Boysen, ON 6 Nottawasga Lake, ON 7 Barre, VT

Naturally Regenerated Forests

8 Jerricho, VT

(2) Hybridization does not follow an apparent regional pattern or wave of hybrid expansion, but seems correlated with populated or agricultural areas (Figure 2). (3) Within populations, hybrids are locally clustered, but these clusters may be scattered over large distances. Hybrids are found in all DBH (diameter at breast height) classes. Most hybrids are healthy but some show signs of butternut canker. (4) We found a substantial bias in maternal ancestry of hybrid individuals. Approximately 92% of hybrids had the J. ailantifolia chloroplast (Table 1).

9 Green Mtn NF, VT 10 Putney, VT 11 Nicolet NF, WI 12 Waupaca, WI 13 Whitewater, WI 14 Allegheny NF, PA 15 Mammoth Cave NF, KY 16 Healing Stones, TN 17 Ozark Riverways, MO 18 St Francis NF, AR 19 Connely, MA 20 Anagnostakis, CT

Severely Human Impacted Landscapes

21 Montgomery, CT 22 Pennington, IA

Our goal was to use DNA genotyping to perform the first range wide evaluation of hybridization in this species. This project is a basis for future studies of spatial and temporal hybrid dynamics in this system.

23 Deeter, IN 24 Brosi, NC 0

20

40

60

80

100

120

140

160

180

Table 1: The chloroplast found in hybrid individuals. J cinerea

Study Questions (1) What is the extent of hybridization in naturally occurring populations and in human impacted landscapes? (2) Are there regional patterns of hybridization (i.e. a wave front spreading out from a point origin)?

Chloroplast

F1

F2

BC1 to Jc

Unassigned

Total*

J. ailantifolia

44

10

8

86

148

J. cinerea

1

1

0

10

12

(4) Is one species predominantly the maternal parent of hybrids?

F1

F2

BC1 to Jc

BC1 to Ja

not assigned

Figure 2: Extent of hybridization in sampling locations (y axis). Hybrid categories are shown as different colors. Note that no samples were found that were probable backcrosses to J. ailantifolia.

Conclusion We found extensive hybridization (>80% hybrids) in severely human impacted landscapes, and generally low or absent hybridization in naturally regenerated forests. The presence of hybrids beyond the F1, as both saplings and mature trees, suggests that hybrids will continue to persist, especially if butternut continues to decline.

*We were unable to obtain chloroplast haplotypes for three individuals. These three are not in the table.

(3) What is the spatial occurrence of hybrids (i.e. are hybrids clustered or spread out)?

J ailantifolia

3 12 13

2

11 4 6

Sean Hoban with a putative butternut in Wisconsin.

1 5 8 7 9

Methods Our samples consist of 1,111 trees from 18 naturally regenerated forests and five collections composed of backyards, abandoned fields or roadsides. We also obtained 82 J. ailantifolia (all distinct genotypes) from arboreta and nurseries as a reference group. Individuals were genotyped for 12 microsatellite loci5. We used the program NEWHYBRIDS6 to probabilistically assign individuals to either J. cinerea, J. ailantifolia, or one of four hybrid classes: F1, F2, BC1 to J. cinerea (hereafter, BC1 to Jc) and BC1 to J. ailantifolia (hereafter, BC1 to Ja). We report assignments made with ≥ 0.90 probability. Individuals that did not have at least 0.90 probability for assignment were called unassigned (see Results). Individuals were also genotyped with species specific chloroplast markers to determine recent maternal ancestry7.

A very high incidence of J. ailantifolia maternal ancestry of hybrids suggests a possible mating bias. This may be due to demographic makeup or partial reproductive incompatibility.

10

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19 14

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20, 21 References

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Putatively disease resistant butternuts have been identified in several locations, including Wisconsin and Missouri. Our finding that these trees are not hybrids suggests that some butternuts exhibit tolerance of the disease. Our study will help enable further investigation of the genetic or environmental basis of this tolerance.

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Figure 1: Sampling sites. Sites in red represent naturally regenerated forest sites. Sites in yellow represent human impacted landscapes. The shaded region is the native range of butternut.

(1) Schultz, J. (2003) Conservation assessment for butternut or white walnut ( Juglans cinerea L.). USDA Forest Service, Milwaukee, WI. (2) Ostry, M. & Woeste, K. (2004) Spread of butternut canker in North America, host range, evidence of resistance within butternut populations and conservation genetics. In Black walnut in a new century, 6th Walnut Council Research Symp. USDA Forest Service technical report NC-243. (3) Neilson J. A. (1930) Some notes on the Japanese walnut in North America. In Northern Nut Growers Report of the Proc. of the 21st Annual Meeting, pp. 39–46. Cedar Rapids, IA, USA. (4) Orchard, L. P. (1984) Butternut canker: host range, disease resistance, seedling-disease reactions and seed-borne transmission. PhD dissertation, University of Wisconsin, Madison, WI, USA. (5) Hoban, S., Anderson, R., Mccleary, T., Schlarbaum, S. & Romero-Severson, J. (2008) Thirteen nuclear microsatellite loci for butternut (Juglans cinerea L.). Mol Ecol Res 8, 643-646. (6) Anderson, E. C. & Thompson, E. A. (2002) A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160, 1217-1229. (7) McCleary, T., Robichaud, R., Nuanes, S., Anagnostakis, S., Schlarbaum, S. & Romero-Severson, J. (2009) Four Cleaved Amplified Polymorphic Sequence (CAPS) markers for the detection of the Juglans ailantifolia chloroplast in putatively native J. cinerea populations. Mol Ecol Res 9, 525-527.

For more information Contact Sean Hoban at [email protected], or visit: http://www.nd.edu/~shoban. Our lab webpage is: http://www.nd.edu/~treedna1/. Preliminary findings of this study were published in: Hoban, S., McCleary, T., Schlarbaum, S., & Romero-Severson, J. (2009) Geographically extensive hybridization between the forest trees American butternut and Japanese walnut. Biol Lett 5:3, 324-327.