Morphometric Approaches to Promote the Use of Exotic Germplasm for Improved Food Security and Resilience to Climate Change: a Kura Clover T Example

Plant Science 290 (2020) 110319

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Plant Science

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Morphometric approaches to promote the use of exotic germplasm for improved food security and resilience to climate change: a kura clover T example

Brandon Schlautmana,*, Luis Diaz-Garciab, Spencer Barriballa a The Land Institute, Salina, KS, USA b Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Aguascalientes, Mexico

ARTICLE INFO ABSTRACT

Keywords: Adaptation of agriculture to climate change and its associated ecological pressures will require new crops, novel Core collection trait combinations, and previously unknown phenotypic attributes to deploy in climate resilient cropping sys- Domestication tems. Genebanks, a primary source of exotic germplasm for novel crops and breeding materials, need compre- Elliptical Fourier descriptors hensive methods to detect novel and unknown phenotypes without a priori information about the species or trait Morphometrics under consideration. We demonstrate how persistent homology (PH) and elliptical Fourier descriptors (EFD), Persistent homology two morphometric techniques easily applied to image-based data, can serve this purpose by cataloging leaf morphology in the USDA NPGS kura clover collection and demarcating a leaf morphospace for the species. Additionally, we identify a set of representative accessions spanning the leaf morphospace and propose they serve as a kura clover core collection. The core collection will be a framework for monitoring the eﬀects of climate change on kura clover in situ diversity and determining the role of ex situ accessions in modern agriculture.

1. Introduction Domestication of our staple grain crops occurred on multiple con- tinents within a relatively short period of time during the climate Crop domestication and the development of agriculture beginning change following the last glacial maximum [6,7]. Adapting agriculture approximately 10,000 years ago were key steps to achieving a new kind to the potential catastrophic eﬀects of modern climate change will of food security for our human ancestors [1]. We still depend on many likewise require dramatic changes in our crops and the way we grow of those early domesticated crops and management practices (i.e. til- them. Future generations of farmers will need not only new crop vari- lage). However, agricultural productivity and food security have in- eties but also new crop species to grow grains in conditions that are creased remarkably during the past century through a combination of much more unfavorable than those of the previous century (i.e. variable intensive plant breeding, homogenization of agricultural environments weather patterns, degraded soils, water restrictions, decreased access to through irrigation and fertilization, and expansion of mechanization fossil fuels and fertilizers, and etc.) [8]. There is need for modern, ac- and fossil fuel consumption [2]. These gains in productivity have re- celerated plant domestication to design new crops (i.e. perennial grain sulted in long-term negative consequences such as expanded agri- crops) and diversiﬁed cropping systems that provision ecosystem ser- cultural land-use, losses of genetic and functional biodiversity, and vices rather than dis-services and that have the resilience to provide disruption of the nitrogen and phosphorus cycles, all which are com- stable yields under climate change and its associated ecological pres- promising essential ecosystem services at a global scale [3]. Further- sures [9,10]. more, over reliance upon a diminishing number of staple annual grain Over the past several decades, genebanks around the world have crop species and a narrowing genetic base of elite germplasm could accumulated large ex situ collections of exotic germplasm (i.e. crop wild render those crops vulnerable to emerging biotic and abiotic stresses relatives and wild species), which represent the genetic basis for [4,5]. maintaining current food production levels and confronting future

Abbreviations: PGR, plant genetic resources; PH, persistent homology; EFD, elliptical Fourier descriptor; BLUP, best linear unbiased predictor; REML, restricted maximum likelihood ⁎ Corresponding author at: 2440 E. Water Well Rd., Salina, KS, 67401, USA. E-mail address: [email protected] (B. Schlautman). https://doi.org/10.1016/j.plantsci.2019.110319 Received 30 June 2019; Received in revised form 11 October 2019; Accepted 15 October 2019 Available online 21 October 2019 0168-9452/ © 2019 Elsevier B.V. All rights reserved. B. Schlautman, et al. Plant Science 290 (2020) 110319 challenges [11]. Genebanks play a crucial role in the conservation of EFD for this purpose. plant genetic resources (PGRs); but more importantly, they have a key Kura clover is a leguminous proto-crop from the Caucasus region in responsibility to facilitate the use of these PGRs to build a durable fu- western Asia undergoing domestication at The Land Institute (Salina, ture for humanity, its crops, and its practice of agriculture [12,13]. KS) and elsewhere. Reports indicate kura clover is well adapted to While a huge number of accessions are accessible worldwide, many – multiple climates, able to grow in low-fertility soils, is drought tolerant, especially germplasm from wild, uncultivated species – have been in- and has general resistance to multiple viruses [33,34]. Efforts have adequately characterized, making it is nearly impossible for breeders been made to introgress these desirable traits into existing clover crops and/or domesticators to predict and assess the potential role that an including white clover (T. repens), alsike clover (T. hybridum), and T. uncharacterized species and/or accession may have in climate change occidentale through wide-hybridization with limited success [34,35]. adaptation [13,14]. However, a potential new use for kura clover is as a perennial living Recent advances in high-throughput sequencing technologies have mulch in annual (i.e. corn-soybean), agroforestry, and perennial grain allowed for genome-wide genetic characterization of many accessions (i.e. intermediate wheatgrass, Kernza®) cropping systems where it has or even entire ex situ collections in a genebank [15,16]. This type of demonstrated effectiveness for suppressing weeds, reducing soil erosion genetic characterization has been used to identify specific allelic var- and nitrate leaching, and providing nitrogen credits for companion cash iants in genes of interest [16,17] and to develop “core collections”, crops [33,36–41]. The Kura clover living mulch system exemplifies the which are expected to reduce the number of accession entries required potential role for perennial herbaceous crops and the ecosystem-or- to sample the majority of a collections genetic diversity [18]. However, iented approach to cropping system design necessary for achieving comprehensive phenotypic characterization, which is expected to pro- long-term sustainability in global agriculture. mote the use of exotic germplasm and identify relevant species and The USDA NPGS kura clover (Trifolium ambiguum) collection, com- accessions as climate resilient crops, is costly and rarely performed posed of 141 unique accessions, includes accessions originating from [13]. More importantly, such characterization is essential for defining multiple geographic origins, encompasses multiple ploidy levels, and morphospaces for important plant traits (i.e. leaf shape) and meaningful contains both wild collected accessions and those that have undergone crytotypes when testing “core collections” across a summation of the some level of genetic improvement or artificial selection. The collection agronomic and environmental contexts under climate change condi- is too large and phenotypically diverse in its current state for any single tions [19]. breeder or biologist to screen and determine whether a specific kura Standard morphological ratings and descriptors exist to help crop clover accession or leaf morphotype is particularly well-adapted to a breeders and genebank curators characterize their germplasm [20,21]. certain geographic region or as a living mulch in a certain cropping Similar standards have not been developed for wild species, although system. We demonstrate how EFD and PH morphometrics, combined criteria for selecting promising domestication candidates have been with basic shape, size and color descriptors, can be used to catalog and proposed [10,22]. Even so, standardized morphological descriptors and quantify this diversity by performing the first comprehensive analysis of ratings, while useful, are likely too inflexible to observe and catalogue leaf morphology in the USDA NPGS collection and the species. the extreme phenotypes and novel, adaptive traits like perenniality or Furthermore, we use the analysis to define a kura clover leaf morpho- tolerance to heat stress that might allow new crops to thrive and space, and we propose a trait morphospace strategy for defining gen- flourish in adverse environmental conditions. ebank “core collections” composed of a subset of accessions that spans Digital images of seeds, leaves, or whole shoots, often already the leaf morphospace. Such collections could be used more practically maintained for genebank accessions, are useful and accessible data to explore and compare the fitness of particular morphotypes across the types that can be stored and reanalyzed indefinitely to extract mean- adaptive landscape of current and future crop plants, and ultimately, to ingful phenotypic variation from public repositories. Image-based identify promising candidates or morphotypes for pre-breeding and morphometric approaches, such as persistent homology (PH) and el- domestication. Finally, we show how digital representations of germ- liptical Fourier descriptors (EFD), are methods that can be used to plasm collections - through a combination of imaging, PH, EFD, linear comprehensively measure morphological variation pertaining to leaf, descriptors, and color estimates - could be a new and effective strategy fruit, and seed shapes [23–25], as well as highly branched structures for visualizing phenotypic variation among accessions in genebanks like roots and shoots [26, 27]. EFD and PH have been used to explore and, therefore, increase their utilization. This work represents an im- the genetic basis of traits [23,26,28], for phylogenetic reconstruction portant advance in using imaging and computational approaches for the and studies of trait evolution [29,30], to study seasonal heteroblasty phenotypic characterization of kura clover and PGRs of other under- [30,31], and to define leaf morphospaces for whole families of plants utilized and under characterized exotic germplasm held in genebanks. [32]. We suggest that the two image-based morphometric approaches be adopted by genebank curators as simple and standardized approaches to catalog phenotypic variation and define trait morphospaces and morphotypes among the species and accessions in their collections, and we use kura clover as a case study to validate the utility of PH and

Table 1 Traits measured in the USDA NPGS kura clover Collection, using basic descriptors, persistent homology and elliptical Fourier descriptors.

Trait Method Abbreviation Description

Length Basic size descriptors len Major axis length Width Basic size descriptors wid Minor axis length Length-to-width ratio Basic shape descriptors lw Aspect ratio Area Basic size descriptors area Leaflet projected area Perimeter Basic size descriptors perim Leaflet perimeter RGB color Basic color descriptors red/green/blue Mean color per channel Circularity Basic shape descriptors circ Compactness or shape factor Convexity Basic shape descriptors cxty Convexity of the leaflet EDF principal components Elliptical Fourier Descriptors EDF PCs 1-5 Principal components of EFD PH principal components Persistent Homology PH PCs 1-5 Principal components of PH matrix

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2. Materials and methods and kept the first five principal components. Additionally, all shape contours were loaded into an ‘Out’ object using the package Momocs 2.1. Germplasm and sample collection [45]. We calculated elliptical Fourier descriptors (EFD) using the function efourier and evaluated the contribution of the EFD harmonics 1 Seeds of 141 unique kura clover accessions were obtained from the to 6; to eliminate asymmetrical variation in leaflets, we removed har- USDA NPGS system and an additional nine experimental varieties from monics B and C as in [23]. Finally, using several functions of the Mo- the University of Minnesota (Supplementary Table 1). Included mate- mocs package, we calculated basic descriptors related to the size and the rials are accessions which are wild collected populations from six dif- shape of the leaflet. Basic size descriptors (length, width, perimeter and ferent countries: Turkey, Georgia, Iran, Russia, Ukraine, and Armenia. area) were normalized using the mean measurements of the 10 refer- Collection sites for these accessions have an elevation ranging from ences included in each picture. A summary of the traits evaluated in this 200 m to 3048 m, with most of the accessions coming from the study is provided in Table 1. Leaflets that had folds, tears, insect da- 1500–2000 m range. Other accessions available from the USDA NPGS mage, or other abnormalities were removed from the dataset, because collection were genetically improved varieties or experimental popu- they would not allow for accurate quantification of shape. Scripts for lations developed by institutions in the United States, Australia, New image processing (MATLAB) and morphometric analysis (R) are found Zealand, and the United Kingdom. A 2n = 2x through 2n = 6x ploidy in Supplementary File 1. series exist for kura clover [33], and the accessions in this study included ∼ 50% hexploid, ∼ 10% diploid, and ∼ 10% tetraploid ma- 2.3. Statistical analysis terials; the remaining ∼ 30% were of unknown ploidy. Seeds were scarified with sandpaper and planted in Jiffy-7 pellets Linear mixed-effects models were fit for each trait via Restricted (www.jiffygroup.com) in the greenhouse in February 2017. The pellets Maximum Likelihood (REML) using the function lmer from the lme4 R were surface inoculated with the appropriate rhizobium (Plant package [46]. We used models of the form: Probiotics, Lafayette, IN, USA) after planting. Depending upon germi- phenotype∼+++ column row leaflet type(1 | date ) + (1 | accession ) nation and greenhouse survival, one to eight seedlings from each accession were planted in a completely randomized design in April 2017 The terms column and row were fixed effects corresponding to the at The Land Institute in Salina, KS. Plants were spaced in a square grid field location of each plant within the experiment; the term leaflet type 0.91 m apart to minimize competition between adjacent plants. Weeds was a fixed effect referring to either central or lateral leaflet; accession were controlled with cultivation and hand weeding. The soil was a and date (i.e. the day the image was taken) were set as random effects. Cozad silt loam (coarse-silty, mixed, superactive, mesic typic haplus- Best Linear Unbiased Predictors (BLUPs) were estimated for each ac- toll) with pH, weak Bray P1, strong Bray P2, K, and organic matter cession for all leaflet basic descriptors and EFD and PH principal levels of 6.8, 13 ppm, 47 ppm, 301 ppm, and 1.8%, respectively. components. Repeatability estimates (r2) were obtained using the ac- Ten fully-expanded leaves, one leaf per shoot, were collected from cession variance-to-accession variance plus the residual variance ratio each kura clover plant in late September 2017. Three of the ten leaves Vaccession were randomly selected, dissected into three leaflets per leaf and placed VVaccession+ error on a light table. Leaves were arranged by leaf (one leaf per column from left to right) and leaflet type (top to bottom rows = left lateral, central, Correlations among leaf phenotypes were calculated using Pearson’s and right lateral leaflet) (Fig. 1, bottom right panel). A piece of clear correlations. All visualization, unless otherwise noted, was performed acrylic was placed over the leaves to flatten them on the light table. using ggplot2 in R [47]. Images were taken using a DSLR camera under controlled light conditions using a copy stand. On both sides of the image, five 2.54 cm 3. Results diameter black circular stickers were included as size references (Fig. 1). All leaf images will be uploaded into the USDA GRIN Global Leaf shape is a commonly studied morphological feature across the database. plant sciences that has been useful for delineating taxa and studying patterns of evolution [29,30] as well as for determining genetic varia- 2.2. Shape analysis tion and relationships within wild and crop species [48,49]. The effects of leaf shape in light interception, the nutrient economy of leaves, and Images were processed with a custom code based on the software thermoregulation have also been explored [50–52]. As such, leaf mor- GiNA implemented in MATLAB [42], which is described below. Raw phology, including size, shape, and color variation, remains an im- images were processed for background extraction and object identifi- portant theme in studies designed to reveal the impacts of global cli- cation using regular thresholding. For every object (i.e leaflet and size mate change on plant biodiversity, crop productivity, and the resilience reference) in the image, we extracted the mean color for all three RGB of modern cropping systems and rotations. Herein we broadly char- channels as well as the shape contours (to calculate basic descriptors acterize leaflet shape diversity in the USDA NPGS Kura Clover Collec- and elliptical Fourier descriptors, see below). Then, we converted each tion using PH and EFD, and we demonstrate how leaflet morphological object to a binary image and PH analysis was performed according to characterization in Kura Clover was useful for creating a core collec- [32,43]. Briefly, PH computes shape objects by assigning high and low tion. This phenotypically diverse kura clover core collection will be an values to pixels with many or few pixel neighbors, respectively. Then, a important resource to monitor the effects of climate change on kura Guassian density estimator is multiplied by expanding annuli (or rings), clover in situ genetic and morphological diversity, and more im- which emphasizes the shape. Given the simplicity of the leaflet shapes portantly, will assist breeders in making use of the ex situ collection for examined here, our PH pipeline computed only seven curves, corre- genetic improvement of kura clover and the development of modern, sponding to seven expanding annuli, each of those composed by 80 climate resilient cropping systems. values. For each leaflet, the numerical components of each annuli (7 × 80 = 560) were added to a n x 560 PH matrix, with n equal to the 3.1. Size and shape variation in leaflets of the USDA NPGS Kura Clover total number of leaflets in the study. Both lateral and central leaflets Collection were flagged for further analysis. Leaflet contours, RGB color descriptors, and the PH matrix were A computer vision approach was used to measure 20 size and shape loaded into R [44] for further analysis. We performed principal com- morphometric descriptors for over 7000 leaflets across 875 individual ponent analysis on the PH matrix using the base R function prcomp(), plants and 141 accessions from the USDA NPGS kura clover Collection

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Fig. 1. An example of the leaf images. References of diameter 2.54 cm are located on each side of the picture. QR code is included for automatic plant identification. Leaves are arranged by columns, leaflets within a column are arranged left lateral (top), central (C), and right lateral (bottom). and nine accessions from the former UMN breeding program. Of the 20 PC1-5 explained 98.58% of the variation, with the first two components descriptors computed here, 10 correspond to basic leaflet shape (length- explaining 88.36% and 4.96%. Given that previous studies reported to-width-ratio, circularity, convexity), size (length, width, area, peri- that higher-order PCs captured mostly complex and cryptic variation, meter) and color (RGB channels) attributes (Table 1). We observed we explored only PC1-5 [23]. EFD PC1 seems to explain the elongation values for leaflet area ranging from 0.23 cm2 to 14.47 cm2, with a mean variation of the leaflet, whereas EFD PC2 (Fig. 2E) and EFD PC3 of 3.74 cm2 (Fig. 2A); leaflet lengths ranging from 0.68 cm2 to 6.97 cm, (Fig. 2F) are more related to the orientation and flatness of the basal with a mean of 2.90 cm2 (Fig. 2B); and leaflet widths ranging from point (where the petiole is located). The contribution of EFD harmonics 0.44 cm2 and 3.79 cm2, with a mean of 1.68 cm2 (Fig. 2C). For the basic 1–6 to the mean leaflet shape is shown in Fig. 2G. Raw measurements shape descriptor length-to-width ratio, we observed leaflets with values and REML-adjusted values for all traits and all accessions are provided between 1 (similar length and width) and 3.86, with a mean of 1.75 in Supplementary File 2. (Fig. 1D). The large variation observed within accessions (among re- plicated plots) and low observed repeatability estimates (see below) 3.2. Correlation among shape and size phenotypes resulted in REML-generated Best Linear Unbiased Predictors (BLUPs) with observed dispersion considerably smaller than what was observed Basic descriptors related to leaflet size (length, width, area and in raw measurements. For example, based on BLUP estimates for leaflet perimeter) were highly correlated (Supplementary File 2, Pearson’s length and area, the accession NSL86671 had the largest leaflet with correlation coefficient R2 > 0.76, p = 0). Similarly, basic shape de- values of 3.94 cm and 5.22cm2, respectively. Repeatability values scriptors length-to-width ratio and circularity were highly correlated (Supplementary File 2) ranged from 0.03 (PH PC5) to 0.53 (length-to- with each other (R2 = 0.86, p = 0) and with both PH PC1 and EFD PC1 width ratio). In general, basic size descriptors showed low-to-medium (R2 > 0.73 (p = 0). Basic size and shape descriptors showed a sig- repeatability values (0.39-0.45), whereas shape descriptors (including nificant correlation (p < 0.05) with each other, with R2 coefficients EFD PC1 and PH PC1) showed medium values (0.42-0.53). PC3-5 of ranging between -0.25 and 0.45. Pearson’s correlation between PC1 of both EFD and PH showed low or very low repeatability estimates both PH and EFD techniques was very high (Fig. 3A, R2 = 0.93, p = 0), (< 0.25). implying that these are explaining the same shape characteristics (i.e. In addition, both PH and EFD yielded 5 attributes (principal com- leaflet elongation). However, the remaining PH and EFD principal ponents 1–5) each, all of them describing object shape variation. PH components (PC2-PC5) seem to explain other aspects of shape variation and EFD captured a significant amount of the shape variation from (Fig. 3B and C). An interesting relationship was observed between PH leaflets. In particular, the PH first principal components (1–5) captured PC1 and PH PC3 (Fig. 3D), where although R2 was non-significant (at 90.35% of the total phenotypic variance observed; PH PC1 and PC2 p > 0.05), a quadratic regression resulted in an r2 = 0.66, with PH captured 70.46% and 12.74%, respectively. On the other hand, EFD PC3 values rapidly decreasing when PH PC1 differ from 0 (on both

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Fig. 2. Variation in leaflet phenotypes computed through traditional methods (basic descriptors) and both persistent homology and Elliptical Fourier Descriptor approaches. Leaflet variation (n = 7050) for the basic descriptors (A) area, (B) length, (C) width, and (D) length-to-width ratio. Black and red vertical lines correspond to the mean and median of the data points, respectively. Principal components (E) 1 and 2, and (F) 1 and 3, representing shape variance in Elliptical Fourier Descriptor analysis. (G) Harmonic contribution to leaflet shape derived from Elliptical Fourier Descriptor analysis (using amplification factors of 0, 1, 2, 5 and 10). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) sides). Leaflet area and EFD/PH PC1 showed no correlation (Fig. 3E and sets of leaves (two lateral and one central leaflet per leaf), we divided F). the area of the central leaflet by the mean area of both lateral leaflets To better characterize the clover collection, we recorded the posi- (i.e. values less than one represent leaves with larger lateral than cen- tion/type (central or lateral) of each leaflet, which allowed us to tral leaflets). Central-to-lateral leaflet ratios varied from 0.44 (PI compute the lateral-to-central leaflet size-ratio. Using only complete 631895) to 1.64 (NSL 448196), and we observed greater among then

Fig. 3. Persistent Homology and Elliptical Fourier Descriptors explain only basic shape descriptors. Linear regression models and R2 are shown for (A) EDF PC1 and PH PC1, (B) EDF PC2 and PH PC2, (C) EDF PC1 and EDF PC3, (D) PH PC1 and PH PC3, (E) area and EDF PC1, (F) area and PH PC1.

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Fig. 4. Variation in central-to-external leaflet ratio and relationship between size and shape. (A) Distribution of central-to-external leaflet ratio across clover accessions; horizontal error bars correspond to the standard error; values greater than 1 indicate leaves with central leaflets larger than external leaves, whereas values lower than 1 indicate the opposite. Extreme phenotypes are labeled. (B) Leaflets with extreme PH PC1 values (stratified in intervals of 10) have smaller area.

within accession variation for the trait (Fig. 4A). accessions (47 accessions, 1897 leaflets analyzed here), and contributed In general, leaflet size variation does not seem to be shape depen- accessions from all three ploidy levels: 6x (85%), 4x (13%) and 2x (2%). dent (Fig. 3E and F); however, extreme shapes (i.e. very elongated For certain countries, only one ploidy level is represented in the col- leaflets or leaflets with a length-to-width ratio close to 1) seem to occur lection. For example, Armenia and Iran contributed only 4x materials, only in smaller leaflets (Fig. 4B). Therefore, we expect that it is possible whereas Turkey contributed only 2x accessions. Similarly, countries to select and/or generate new leaf morphotypes (i.e. combinations of like Ukraine and Georgia are missing certain ploidy level, in this case 2x leaflet shape, size, and color) that are highly adapted to target agri- and 4x, respectively. It is unclear whether these patterns represent true cultural environments (i.e. specific climates or altitudes, living mulch geographic distributions of the ploidy levels across the Caucasus region, systems, etc.) through breeding. or if the observed variation is due to current gaps in the collection.

3.3. Genetic improvement status, collection location, and ploidy affect 4. Discussion leaflet size and shape characteristics 4.1. Utility of EFD and PH in cataloguing phenotypic diversity held in Metadata available for each accession allowed us to examine if the genebanks improvement status, geography, origin and ploidy shape kura clover leaflet morphology. First, by comparing the leaflet area and the col- A key purpose of genebanks is to facilitate access to and use of the lection site elevation of 86 wild accessions (a total of 4071 leaflets and plant germplasm resources held in their collections by providing reli- an elevation range of 250-3250MASL), we observed that samples from able evaluation data that are easily retrievable by systematists, bree- higher altitudes tend to have smaller leaves than those from lower al- ders, domesticators, and others [53]. Reliable phenotypic data gen- − titudes (Fig. 5A; p<2×10 16, t-test). Moreover, leaflet area and erally must be collected by trained staff using calibrated and a priori shape (in this case, PH PC1) are, in general, affected by the ploidy level. standardized measuring systems and previously characterized check Both 2x and 4x accessions had smaller leaflets than 6x accessions accessions [53]. For most species of exotic, wild germplasm, a priori (Fig. 5B, p < 2.1 × 10-8, t-test); likewise, all three ploidy levels dif- standardized measuring formats do not exist, and in the case of do- fered in leaflet shapes ranging from more linear (2x accessions) to or- mestication or adaptation to climate change, the actual desired mor- bicular (4x accessions) (Fig. 5C; p < 2.4 × 10-10, t-test). Unexpectedly, photypes might be completely unknown. Exotic germplasm can be ploidy levels within a particular countries of origin seem to have a characterized with sequence-based genotypic data without a priori in- differentiated impact on leaflet shape and size (Fig. 5D). Both 6x im- formation; however, sequencing remains too expensive to characterize proved and 6x collected materials from Ukraine have larger leaflet exotic germplasm without obvious and immediate utility, and sequence areas than lower ploidy levels. Conversely, the 2x materials from data does not necessarily provide relevant information about a species’s Georgia are larger than the 6x material from Georgia as well as the 2x or accession’s utility or adaptive potential. Therefore, new approaches and 4x improved materials. At least for the 6x ploidy level, improved for comprehensive germplasm characterization that can capture emer- materials are characterized by a larger leaflet areas compared to lower gent properties of complex phenotypes and that do not require the ploidy levels. If the increment of leaflet area is desired from a breeding presupposed importance of individual traits are necessary [54]. perspective, incorporating 6x wild accessions from Ukraine could be EFD are useful morphometric tools that do not require presupposed favorable. Accessions from the United States and Australia, both con- landmarks for measuring simple seed and leaf shapes if shapes are sidered improved material, include all three ploidy levels (2x, 4x and consistent across every sample and major sources of shape variance (i.e. 6x), however, accessions from New Zealand, which are also improved, varying number of lobes or leaflets) are not present in the dataset [32]. include only 4 × . Among the collected/wild materials, Russian Fed- For the seeds of most potential grain crops, this is probably a valid eration is the most represented country of origin in terms of number of assumption and EFD will work well. However, for species with variable

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Fig. 5. Leaflet area and shape depends on the origin, elevation and ploidy of the kura clover accessions. (A) Leaflet area depends on the elevation of the collection site. Accessions with elevation information correspond only to wild collected materials (n = 4071 leaflets). (B) Leaflet area and (C) PH PC1 grouped by ploidy level; leaflets per ploidy level were

n2x = 652, n4x = 589, and n6x = 3268 (D) Distribution of leaﬂet area and PH PC1 sorted by origin and ploidy level. Within the boxplots, vertical red lines correspond to the mean value. Category “Improved” includes accessions that are varieties or experimental populations developed in the United States, New Zealand and Australia. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

leaflet numbers per leaf and for more complex traits like root, in- properties that are interesting to a wide variety of users and that are florescence, and shoot architecture, PH is a much more appropriate relevant to a wide range of potential end purposes. Both PH and EFD topological method that can be used to analyze disparate leaf shapes provide standardized strategies for collecting comprehensive pheno- and branching architecture of plants [32,55]. typic data across multiple time periods (i.e. seasonal heteroblasty), Once a core collection is developed, genebanks might expect a environments (i.e. climate gradients), and research studies without substantial increase in the request and use of those accessions as the depending on well-developed ontologies or coordinated approaches. PH core collection becomes a framework for further studies. It is important has been proposed as an adaptable solution for providing a common that this increased use also leads to an increase in the knowledge about standardized approach in the plant sciences to interpret and describe the phenotypic diversity that is present in the collection. It is therefore diverse phenotypes (2D, 3D, and 4D plant forms) and to expand known necessary that, even without a priori knowledge about traits of interest, plant and trait morphospaces through observations across times, en- standard phenotyping methods are adopted by genebanks that are vironments, and any other number of dimensions [27]. Such an ap- flexible and that can comprehensively detect characteristics or proach is essential to bridge knowledge sharing between curators,

7 B. Schlautman, et al. Plant Science 290 (2020) 110319 breeders, taxonomists, and other members of the plant science com- Core collections, while they do not replace whole collections, reduce munity as we work together to meet the urgent need for new resilient the size of germplasm collections to optimum sets of dissimilar groups crops [56]. that can be assessed across multiple experiments [57]. Core collections Herein, we demonstrate how high-throughput image analysis, using of this type are critical for pre-breeding studies or studies of new can- PH and EFD morphometric methods and combined with conventional didate species for domestication that aim to evaluate the full range of shape, size, and color descriptors, quantitatively cataloged leaf and genetic and phenotypic characteristics for a species, to compare the leaflet morphological variation in the USDA NPGS Kura clover collec- responses of particular morphotypes or an entire morphospace to a tion for the first time. While PH PC1 and EFD PC1 were highly corre- range of unique environments or management practices (i.e. morpho- lated with traditional shape parameters (length-to-width ratio) de- type x environment interactions), or to identify optimal or missing scribing the orbicular (round) to linear (elongated with parallel margis) morphotypes that can serve as selection criteria in their breeding pro- gradient of kura leaflet shapes (Figs. 2E&3), other PCs were mostly grams. Perennial, herbaceous grain crops have been identified as one uncorrelated with traditional descriptors and represent complex mor- such morphotype which is missing from global agriculture, but that phometric variation that was not measurable using standard proce- could improve carbon sequestration, improve nutrient and water use dures. For example, EFD PC 2 quantitatively describes leaflet variation efficiency, prevent soil erosion and degradation, and provide various ranging from ovate (egg shaped, wide at base/petiole attachment) to other ecosystem services to agricultural lands [8,58,59]. A recent in- obovate (egg, shaped, narrow at base/petiole attachment) (Fig. 2E), ventory of the Fabaceae found that 6644 perennial, herbaceous species and EFD PC 3 quantitatively describes variation in leaflet cordateness were present in the family [60]; however, no strategies were presented (degree of heart-shaped lobing) (Fig. 2F). Collectively, EFD PCs 1–3 for identifying the most promising species for domestication as per- quantitatively describe 96.5% of leaflet shape variation spanning ennial grain legumes or living mulches. We expect if such core collec- standard leaf shape terms such as: lanceolate, ovate, cordate, obcor- tions existed for a range of wild, undomesticated species, coordinated date, acicular, truncate, elliptic, and etc (Fig. 2E & F). and effective strategies could be developed to screen and detect can- Instead of assigning each kura clover leaflet shape to one or more didate species with novel or agronomically useful traits that merit their traditional leaf shape categories, we use PH and EFD PCs to demarcate a inclusion in domestication pipelines to develop climate resilient crops. kura leaflet shape morphospace spanning all standard categories, and To facilitate future breeding and domestication efforts in kura we project the position of each accession in the USDA NPGS collection clover, we propose a core collection for this species based on the leaf onto that morphospace. Using the PCs shifts the treatment of leaf shape morphospace developed here (Fig. 2B). The original leaf morphospace as a traditional qualitative trait description to one that is instead spanned 56 (37%) of the total accessions. We further reduced the quantitative and usable in downstream statistical analyses. Ultimately, number of accessions for the core collection by grouping highly cor- this strategy should allow clover breeders to manipulate leaf shape related traits and removing the leaflet traits with low repeatability es- using quantitative genetics and recurrent selection; allow evolutionary timates. In particular, the green color channel was used as an estimate and ecological biologists to measure or monitor changes in clover leaf of leaf color; length, width, area, and perimeter, were used as estimates shape variation across geographic ranges, over evolutionary time of leaflet size; length-to-width ratio, circularity, PH PC1 and EFD PC1 frames, and during climate change; and allow plant physiologists to were used as estimates of leaflet aspect ratio; and PH PC2-3 and EFD establish links between quantitative variation in clover leaf shape and PC2-3 were considered individually as unique leaflet shape measures. light interception. Our analysis of the available metadata suggested that The blue and red color channels, leaflet convexity, PH PC4-5, and EFD geography, genetic improvement, and ploidy number affect kura clover PC4-5, were not taken into consideration. Based on this approach, we leaf morphology and demonstrated the effectiveness of PH and EFD to identified 30 unique accessions (20% of the total accessions) that re- quantify this variation and enable such studies (Fig. 5). However, we present most of the leaflet color, shape and size variation, of which half hesitate to provide inferences about our observations because metadata corresponded to improved and half to wild collected materials (Fig. 5B; was only available for a subset of accessions; furthermore, while the Supplementary File 2). wild collection was large and diverse, it was made over many decades without attempting to thoroughly or equally sample all regions and 4.3. Imaging to create digital illustrations of entire germplasm collections ploidy levels. Establishing a core collection and characterizing it with quantitative 4.2. Defining a kura clover leaf morphospace and building a kura clover data is an important way to promote its utilization. Visualizing an ac- “core collection” of distinct leaf morphotypes cession prior to dedicating precious field space and resources to it, is an invaluable tool for breeders and many other potential users. Images are To define a phenotypically-diverse kura clover leaf morphospace, one of the easiest ways to make information about genebank accessions we identified accessions with mean and extreme values in both the wild accessible [12, 61]. In fact, at the 2018 USDA Alfalfa Crop Germplasm collected and genetically improved groups for the 20 leaf phenotypes Committee meeting, breeders asked curators to collect, if nothing else, evaluated here (based on accession BLUP values) (Fig. 6A). We chose to at least an image of the accessions during seed multiplication (personal separate and visualize the two groups by projecting the mean leaf size, communication, Heathcliffe Riday). These images are useful, but we shape, and color for each accession representing a key morphotype have found it to be difficult to simultaneously visualize multiple ac- separately onto the overall morphospace (Fig. 6A). Following this cessions for comparison purposes, especially if the images are taken at strategy, we ended up with 56 (37%) unique morphotypes (i.e. acces- different times or in different ways. sions) combined for the wild collected and genetically improved PH and EFD both begin with a step that compresses the complex groups. This current kura clover leaf morphospace is diverse in that it dimensionality of an object shape to provide a scalable approach for was defined by wild collected accessions from across the native range describing and comparing shapes within a scaled and standardized for T. ambiguum, genetically improved accessions from multiple pro- morphospace. At least for EFD, a few descriptors can be used to recreate grams in multiple countries, and accessions representing each of the and visualize, at any given time, digital illustrations of leaves or other three ploidy levels. However, the morphospace is specific to measure- plant organ types using existing functions from various software ments collected from a single location-year and is expected to expand to packages. We developed an illustration of leaf morphological variation include new or missing morphotypes when the accessions are tested in that allows users to simultaneously visualize the leaflet diversity con- new environments, when additional collection trips are completed, and tained in the entire kura clover collection (Fig. S1) and could aid po- as plant breeders develop new improved populations by selecting and tential users in making side by side visual comparisons to select ac- combining extreme morphotypes. cessions that are most likely to fit their particular goals.

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Fig. 6. Leaﬂet size and shape variation as guidelines for building a kura clover core collection. (A) Digital representation of extreme and mean phenotypes from improved and wild collected accessions. For every trait and group (improved or collected), the three digital leaﬂets correspond with the accessions having the minimum, mean, and maximum BLUP value. Each shape was computed by averaging the replicates available for each accession. (B) Proposed kura clover core collection based on highly informative leaf traits. Accessions marked with (i) and (c) correspond to improved and wild collected materials, respectively.

In further studies, such digital illustrations could also help breeders We have demonstrated how EFD and PH, two morphometric tech- and researchers visualize variations in leaf shape that exist across niques easily applied to image-based data, are useful for characterizing “cryptotypes” (plant ontologies, unique environments, agricultural and cataloguing phenotypic variation in germplasm collections, de- practices, etc.). While the current example is limited to leaf mor- marcating trait morphospaces, producing digital illustrations for phology, we can imagine expanding these results in future to create germplasm visualization, and creating core collections. We have fo- digital illustrations of more complex structures using 3D imaging or cused here on leaf form, but the techniques could and should similarly image reconstructions. PH in particular is useful for quantifying and be used to develop morphospaces for other plant organs (flowers, fruits, analyzing highly topological 3D structures, and was recently used to tendrils) or whole plant phenotypes (shoots, roots). We believe these characterize wild grapevine inflorescence architecture [55]. Such stu- tools offer the ease and flexibility to be adopted by genebank curators dies suggest that virtual reality could soon be a tool that plant breeders, and to be used during accession multiplication. Furthermore, devel- curators, and even plant taxonomists can use to explore and compare oping morphospaces and core collections will allow for the design of materials in their programs, collections, or herbaria using 3D plant future plant science experiments exploring the ecophysiology of adap- resource databases [62]. tation to adverse conditions likely under climate change, promote the use of exotic germplasm in modern crop domestication efforts, and ultimately play an important role in enabling the research necessary to 4.4. Conclusions and future directions develop new cropping systems using an ecosystem first approach that will ensure long term global food security. Breeding kura clover to be a better companion in living mulches is a challenge because no a priori information exists about which traits di- Author contributions rectly affect the compatibility of kura clover with its companion grain crops. Similar situations exist for all crops in the early stages of do- BS conceptualized the project. BS and SB collected and curated data. mestication, because yield component studies have not been thoroughly LDG and BS performed investigations, developed methodology, and conducted to identify criteria for selection. We observed variation in performed data analysis, validation, and visualization. BS and LDG leaf area in this study corresponding to collection site elevation that wrote the original draft. BS, LDG, and SB reviewed, edited, and ap- suggests that divergent adaptation of certain leaf morphotypes (Fig. 5). proved the final manuscript. Similarly, we have observed that plants with smaller, bluer leaves ap- pear to be more drought tolerant in Kansas than other morphotypes (Schlautman, unpublished). Such environmentally dependent variation Declaration of Competing Interest suggests that certain kura clover morphotypes will be more adapted fl than others to new agricultural environments, including kura clover- The authors have no con icts of interest to declare. grain living mulch systems. The kura clover core collection developed herein, which spans the species leaf morphospace, will provide us and Acknowledgments other researchers the opportunity to test this hypothesis, to select the optimal leaf morphotypes for the living mulch system, and to explore This work was made possible through the charitable donations of adaptation of leaf morphotypes to other diverse climates and environ- multiple persons (“Friends of the Land”) and organizations given to The ments. Land Institute, Salina, KS. The funders had no role in the study design.

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