Botany Distinguishing Impatiens capensis from Impatiens pallida (Balsaminaceae) using leaf traits Journal: Botany Manuscript ID cjb-2020-0022.R1 Manuscript Type: Article Date Submitted by the 20-Mar-2020 Author: Complete List of Authors: Whitfield, Heather; University of Wisconsin Madison, Botany Toczydlowski, Rachel; Michigan State University, Integrative Biology jewelweed, leaf shape, linear discriminate analysis, morphometric Keyword: analysis, speciesDraft identification Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 29 Botany Distinguishing Impatiens capensis from Impatiens pallida (Balsaminaceae) using leaf traits Running head: Impatiens leaf traits Heather L. Whitfield Rachel H. Toczydlowski1 Authors’ Affiliation: Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison, WI 53706 Draft For: Botany 1Corresponding author: Rachel H. Toczydlowski, [email protected] 1 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 29 Abstract 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,Draft 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. 2 https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 29 Botany Keywords: jewelweed, leaf shape, linear discriminate analysis, morphometric analysis, species identification Introduction Both Impatiens capensis (Meerb.) and Impatiens pallida (Nutt.), commonly referred to as orange and pale touch-me-not, or jewelweed, respectively, are challenging to distinguish annual dicot species. These species share many characteristics in both physical appearance and growing environment (Voss and Reznicek 2012). Both species are native to North America, and the range of I. pallida is completely nested within the range of I. capensis (USDA 2007). Although I. capensis tends to grow more frequently Draftin wetter and shadier areas whereas I. pallida prefers a more mesic environment, there is considerable overlap in the environmental niches of these species (Lechowicz 1988). In fact, the two species sometimes grow in completely intermixed stands (Lechowicz 1988). It is therefore difficult to identify the species based solely on habitat requirements. The species can be reliably distinguished from one another using (chasmogamous) flower color and shape – I. pallida flowers are light yellow, usually with limited spotting, and about as long as they are wide (Fernald 1950; Gleason and Cronquist 1991). In contrast, I. capensis flowers are orange, commonly with varying degrees of red spotting, and longer than they are wide (Fernald 1950; Schoen and Latta 1989). Chasmogamous flowers are typically only present from late July through mid-September or the first frost, however (Rust 1977). Aside from genetic analysis, which is costly and time intensive, there remains no concrete way to distinguish these species in the absence of chasmogamous flowers. Although some argue that petiole color can be used as a 3 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 29 distinguishing character – red on I. capensis and green on I. pallida – petiole color is sensitive to light levels (among other environmental factors) and is thus not a reliable trait for species identification (personal observations). Experienced botanists can often distinguish morphologically similar species from one another based on “gestalt” or their “overall impression” of the plant (Harris et al. 2015). These perceptual skills require considerable time and effort to acquire, and when experts are able to detect species distinguishing patterns, they often find it difficult to convey this knowledge to others (Harris et al. 2015; Daston 2008). Recent advances in digital imaging and Draftcontemporary computing power now allow us to quantify and analyze subtle, complex, and/or continuous variation in shape and size (Rohlf and Marcus 1993; Adams et al. 2004). These modern or geometric morphometric outline analyses may allow us to identify, quantify, and describe the “gestalt” characters used by experienced botanists to distinguish highly similar species (Cope et al. 2011). Complex outlines (e.g. of a leaf) are decomposed into a set of mathematical coefficients using Elliptical Fourier analysis (EFA) that quantitatively describe the (size and) shape of the outline (McLellan and Endler 1998; see Bonhomme et al. 2014 for an accessible in-depth explanation). These mathematical coefficients from EFA can be used to visualize average shapes. They can also be summarized using principal components analysis (PCA) and fed into a discriminant analysis to test how accurately these continuous shape traits can assign individuals to species. For example, EFA has been used to distinguish agricultural crops from weeds (Neto et al. 2006), species of grapes (Klein et al. 2017), soft corals (Carlos et al. 2011), mosquitos (Rohlf and Archie 1984), and 4 https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 29 Botany hominids (Gonzalez-Jose et al. 2008). We applied modern morphometric techniques to investigate the extent to which I. capensis and I. pallida can be distinguished based on leaf shape, size, and color. 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) 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 speciesDraft 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) 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 5 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 6 of 29 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 thatDraft 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. Morphometric analysis – quantifying leaf shape, size, and color To characterize leaf shape, we performed elliptical Fourier analysis using the Momocs package V1.2.9.3 (Bonhomme et al. 2014) in R (R Core Team 2017). Elliptical Fourier analysis mathematically characterizes and quantifies complex shapes by decomposing them into a series of ellipses or harmonics that vary in shape, size, and orientation. We interpolated 500 points around each leaf margin and defined 4 landmarks at the maximum and minimum points in the horizontal and vertical directions to maintain a consistent