Using a Stable Isotope to Label Seeds and Seedlings of an Invasive Shrub, Lonicera maackii

Abstract Seed dispersal is a crucial process in most plant invasions, but is notoriously difficult to study. One technique to identify the maternal source of dispersed seeds and newly established seedlings is labeling with a stable isotope. We tested whether foliar application of 15N-labeled urea would result in sufficient 15N enrichment to discriminate among seeds and seedlings grown from those seeds of the invasive shrub Lonicera maackii (Amur honeysuckle). We subjected mature L. maackii to all combinations of three concentrations of 15N-labeled urea (0.025 g L−1 [0.003 oz gal−1], 0.20 g L−1, and a 0 g L−1 control) and three temporal treatments (one application in August, one application in September, and five applications spaced every three weeks from June through August). Seeds were collected September to November; some of these were analyzed for %15N and others allowed to germinate and grow into seedlings under two treatments (in potting mix in greenhouse and in woodlot soil outdoors). Seedlings were harvested midway through the next growing season. We found that seeds from plants subjected to the three different concentrations had significantly different %15N levels, and there was a significant interaction between concentration and temporal treatment: the highest seed %15N levels were from plants sprayed five times with 15N-labeled urea, and the second highest from plants sprayed once in September. Similar patterns in %15N levels were found in seedlings, except that those from the 0.025 g L−1 spray treatment were only distinguishable from controls for seedlings grown outdoors in woodlot soil. These findings demonstrate that a single foliar application of 15N in early September is sufficient to label both seeds and seedlings of this invasive shrub, enabling one to identify the source of field-collected seeds or seedlings. This provides a tool for studying patterns and processes in seed dispersal of Amur honeysuckle and potentially other invasive plants. Nomenclature: Amur honeysuckle, Lonicera maackii (Rupr.) Herder. Management Implications: Understanding the patterns and processes of seed dispersal can inform strategies for managing and eradicating invasive plants in landscapes. For example, deciding where to invest resources to detect and treat an invasive species depends on the relative importance of long-distance dispersal vs. diffusion (expansion of existing populations). For species where diffusion is important, resources should be focused on treating the advancing front of existing populations, whereas for species where long-distance dispersal is important, patrolling outside the invaded area for newly established individuals and patches would be more effective. Seed dispersal, however, is notoriously difficult to study, in part because it is challenging to determine the source (mother plant) of dispersed seeds and new seedlings. One potential method for matching seeds/seedlings with sources is stable isotope labeling, where one or more mature plants is treated with a stable isotope that is then incorporated in the tissue of the seed. This study shows that a stable isotope of nitrogen, 15N, can be used to label both the seeds and seedlings of Amur honeysuckle, one of the most problematic invasive shrubs in the Eastern and Midwestern United States. A single foliar application of 15N-labeled urea in late summer was sufficient to label seeds that matured that fall, so that they could be distinguished, via laboratory analysis of the N isotope ratio, from seeds of unsprayed plants. Furthermore, seedlings harvested midway through the next growing season retained sufficient 15N that those from sprayed mother plants could be distinguished from those from unsprayed mothers. These findings demonstrate that it is possible to use stable isotope labeling to determine the relative importance of different seed sources in an invasion, for example distinguishing how many new seedlings are from seeds of an isolated new population/horticultural planting vs. from a more distant but larger invasive population.