Distinguishing Impatiens capensis from Impatiens pallida (Balsaminaceae) using leaf traits

Impatiens capensis (orange jewelweed) and Impatiens pallida (yellow jewelweed) are annual species with similar phenotypes that grow in similar environments throughout the eastern United States. This makes them extremely difficult to distinguish when (chasmogamous) flowers are absent. We use morphometric analyses to identify leaf characters that distinguish these species. After collecting and scanning 342 leaves from plants of each species growing in co-occurring populations in Madison, WI, we quantified: leaf size, shape (using elliptical Fourier analysis), serratedness, and color. Using leaf size and shape traits, a linear discriminate analysis assigned up to 100% of leaves to the correct species. The uppermost fully expanded leaf yielded the most accurate species assignments based on size and shape traits. This leaf was on average, smaller, less deeply serrated, with a more acute base, apex, and elliptical shape in I. capensis as compared to I. pallida. Impatiens pallida leaves had more color contrast (lighter veins and margins) than I. capensis, which were solid green throughout. Morphometric analysis is a promising technique to identify species-distinguishing characters in the absence of binary traits or molecular genetic analyses. Leaves from across these species’ ranges should be analyzed to test the robustness of the species-distinguishing characters we present.

Methods
Leaf collections

We identified three nature reserves (sites) on the University of Wisconsin – Madison campus in Madison, Wisconsin, USA: Muir Woods, Bill’s Woods, and Picnic Point Marsh (Fig. 1 in published paper) that contained co-occurring populations of I. capensis and I. pallida. These sites varied in light, soil moisture, and likely genetic composition. Within each site, we collected 3 leaves from each of 5-10 randomly selected plants of each species within each of 3 sub-locations (areas). We sampled until we reached 25 plants of each species in each site. We collected from areas where both species were growing in intermixed stands to standardize the range of environmental variability sampled across species. We could only find two areas of intermixed Impatiens in Bill’s Woods so we sampled two additional areas where only one of the species was present. We could only find two areas of I. capensis to sample in Muir Woods.

We collected leaves from three standardized locations within each plant (Supp. Fig. 1 in published paper) to test for differences in leaf shape or size depending on where they were growing on the plant (essentially leaf height, age, and susceptibility to herbivory and disease). We defined leaf position 1 as the lowest leaf on the plant borne on the main stem. The second leaf came from the middle of a branch that diverged from the main stem in the leafiest region of the plant. The third leaf was the fully expanded leaf closest to the top of the plant on the main stem. We collected leaves into envelopes labeled with the leaf position, species, site, and area and pressed them in a plant press. All leaves were collected between August and September of 2017.

Leaf scanning and image processing

We scanned pressed leaves one at a time using a CanoScan 8800F desktop scanner at a resolution of 300dpi in color photo mode with auto exposure settings and saved the scans in TIFF format. All leaves were scanned with the leaf tip positioned at twelve o’clock on the scanner bed.

Using the program FIJI (Schindelin et al. 2012), we converted leaf scans into binary black and white images. We removed any leaves that did not have entire margins and filled in any interior holes using the black paint tool. We removed leaf petioles in the scans by painting over them with the white paint tool (petioles were torn at variable lengths when collected). We retained a total of 342 leaf blade silhouettes for morphometric analysis.

The exposure settings on our original scans were not standardized to allow for meaningful comparisons of color, so we re-scanned “leaf 2” from 2 randomly selected individuals from each area and species (N = 34) to investigate differences in leaf color. We used leaf 2 for the color analysis because it was most representative of leaf color on the plant as a whole based on our field observations. Often, leaf 1 was partially senesced and leaf 3 was too young to have developed full color. We used a color card to ensure the color parameters of all leaves were standardized across scans. Using FIJI, we adjusted color threshold values to exclude the ink markings on each leaf (denoting leaf number) from the analysis. We then generated a distribution of the RGB values present in each leaf (FIJI: Analyze, Color Histogram) and calculated the mean, mode, and variance of this leaf color distribution. We chose the mode (as opposed to the mean) because it is not influenced by minor blemishes and imperfections on the leaf surface and is likely closer to the color we perceive than the mean value.