Native Plants for Georgia Part IV: Grasses and Sedges
Gary Wade, Ph.D., Extension Horticulturist (Retired)
Elaine Nash, Naturalist
Ed McDowell, Master Gardener, Amateur Botanist and Wildflower Photographer
Brenda Beckham, Master Gardener and Plant Enthusiast
“Grasses are the hair of Mother Earth”
— Karl Foerster
Few plants on Earth are more versatile or have a greater impact on the environment than grasses. They are major contributors to the total net photosynthesis and production of biomass in the world. They tame the erosive splash of raindrops, stabilize soil and assist the infiltration of water into the ground and aquifers. They interact ecologically with a diverse number of flora and fauna, both above and below ground, including insects, fungi, birds and mammals. Many insects, for example, rely on native grasses as a substrate on which they lay their eggs or as a larval food source in order to complete their life cycles. These same insects, in turn, are eaten by birds and mammals higher up the food chain. Many species of mammals, birds and insects also rely on grasses for shelter and nesting materials.
No other plant has played a more vital role in the development of civilization than grasses. For thousands of years, wild grasses have been cultivated and domesticated for human consumption and as feedstock for livestock and herds of wild animals. Most of the cultivated grains we use today, including wheat, barley, rye, oats, corn, rice, millet and sorghum, were developed from wild native grasses. Over the years, plant breeders and agronomists have made dramatic improvements in grain yields. One of the most notable was Norman Borlaug, an Iowa-born scientist and winner of the 1970 Nobel Peace Prize. He developed a high-yielding disease-resistant wheat that helped feed the world and saved millions of people in impoverished nations from starvation.
The grass family, Poaceae, is the fourth-largest plant family on Earth, with more than 10,000 species. Globally, grasses grow in all terrestrial habitats, including forests, glades, savannas, open deserts, prairies, wetlands, stream banks and floodplains. Yet grasses are relatively young plants in terms of their evolutionary history. The earliest evidence of fossilized grass pollen was found in South America and dates to the Paleocene Epoch, 55 to 65 million years ago. To put this in perspective, dinosaurs never ate grasses because grasses evolved after dinosaurs were extinct.
The southeastern United States is home to about 1,400 native grass species. Many of these species are managed for erosion control or wildlife habitats. Others have been domesticated as pasture grasses and other types of animal forage, such as grain or baled hay. A few species, such as Muhly Grass, Switchgrass and River Oats, have become popular in the landscape trade.
This publication describes and illustrates 48 grasses and 10 sedges native to Georgia. It is not the intent of the authors to describe all native grasses and sedges, but those that are most widespread or those having practical application for wildlife habitats, erosion control, restoration projects or landscape culture. A few of the plants are noted as being weedy or invasive and may not be appropriate for use in cultivated landscapes. Nonetheless, they are included to assist the reader in identifying them because they are abundant in the wild.
This publication further separates grasses into two categories: warm-season grasses and cool-season grasses. Warm-season grasses begin growing when daily temperatures are between 60°F and 65°F. They grow in the summer, flower and fruit in the fall, and then go dormant after the first frost. Examples are Broomsedge, Bluestems and Indian Grasses. Cool-season grasses grow in the late fall, winter and early spring, flower and fruit in the late spring, then go dormant in the summer. Examples include Oatgrasses, Witchgrasses and Bluegrasses.
Grasses vs. Sedges
Grasses and sedges belong to two different plant families. They are sometimes difficult to tell apart, but they can be distinguished from one another by differences in their structures, habitats or life cycles.
- Grass stems are typically round or flat and hollow inside, while those of sedges are triangular and solid inside.
- Grasses have swollen nodes or joints along their stems, while sedges do not.
- Grasses produce both vegetative and floral stems, while sedges produce only floral stems.
- The leaves of grasses are usually two-ranked, which means they occur in two rows on opposite sides of the stems, while the leaves of sedges are three-ranked and occur in three vertical planes along the stems.
- The flowers of many grasses are showy, but those of many sedges tend to be inconspicuous.
- Grasses are most abundant in dry, open habitats, while sedges prefer moist to wet areas.
- Grasses can be either annuals or perennials, but sedges are primarily perennials.
Establishing and Managing a Native Grass Meadow
Whether one is interested in the ecological management of an existing native grassland, converting an old pasture to a more diverse mixture of forbs (broadleaf herbaceous plants) and native grasses, establishing native grasses under utility easements or incorporating native grasses into a cultivated landscape, a great deal of planning is required to do the job properly. Creating a native grass meadow is a lot different from planting a mono-culture lawn with just one type of grass. Natural grass communities are diverse ecosystems with many different grasses growing in harmony with a variety of forbs. Grasses typically occupy between 65 percent and 70 percent of the total space.
Listed below are some suggested guidelines for planning, establishing and managing a native grass meadow. Each one has many alternatives and options.
Analyze the site
- Identify and make a list of ALL of the existing plants on the site, including native plants, exotic plants and weeds, as well as cool-season and warm-season annuals or perennials.
- Determine the sunlight exposure throughout the day.
- Note the topography and drainage of the site, including slopes, elevated areas that might stay drier than surrounding areas, and low spots where water can collect after rain.
- Determine the size of the area to be planted. There are 43,560 square feet in 1 acre.
- Take a sample of the soil and have it tested through the state soil testing laboratory. There is a nominal fee for this service (for information on soil sampling and testing, see http://aesl.ces.uga.edu/soiltest123/Georgia.htm). A soil test provides recommendations for lime and fertilizer prior to planting. However, while lime may be required to achieve the proper pH in the soil for optimum plant growth, fertilizer is not recommended for native grass meadows because it will encourage weeds that compete with grasses.
- Make note of any existing plants that have to be eliminated before the meadow can be established. If the site is overgrown with invasive plants, brush or scrub trees, it may be a candidate for restoration instead of rehabilitation. On the other hand, if the site contains a significant number of native plants, then adding a few native grasses to increase the diversity of the site may be all that is needed.
- Right-of-ways under power lines will require frequent monitoring during establishment because birds roosting on the lines and mammals grazing on plants in the open field may introduce unwanted weed seeds.
- Observe plant communities adjacent to the planting site and the potential for wind, water or wildlife to transport seeds from those communities into the grass meadow.
Determine your budget and equipment needs
- Determine early in the planning process how much money the project costs and how much time and energy must be devoted to the project, not only for the initial installation, but also for follow-up management.
- Determine the types of plants or seeds needed and their costs. Some grasses can be established from seeds, while others can be established from plugs (small well-rooted plants grown from seed). Consider the cost of each of these alternatives. Seeds or plugs will need to be ordered in advance. Some native grasses simply are not available in the trade, so seeds or plants must be harvested from the wild. Often this requires the assistance of a knowledgeable botanist to identify plants and to determine the best time to harvest seeds or plants. Sometimes the Georgia Native Plant Society (www.gnps.org) or the Georgia Botanical Society (www.gabotsoc.org) sponsor field trips for persons interested in collecting native plants or seeds. It is illegal to harvest seeds or plants from private property without first obtaining permission from the landowner, and the collection of plants or seeds from land owned by federal or state agencies is prohibited. Most native grasses available from nurseries are propagated from seed.
- A mower will be required during establishment to prevent annual weeds from competing with and shading out the new plants. A mower that can be adjusted to a cutting height of 5 to 6 inches is ideal.
- A sprayer for applying herbicides will be needed to eliminate unwanted vegetation prior to planting or to target specific weeds during establishment.
- Large restoration sites may require cultivation at two-week intervals prior to planting to eliminate unwanted vegetation. A contractor may be required for these large tasks, which will add to the cost.
Plan for a diverse plant community that matches the site conditions
- The typical Southeastern grass meadow is a mixture of both cool-season grasses and warm-season grasses, a few sedges and a variety of forbs, like legumes, sunflowers, mints, goldenrods and milkweeds. Make certain the newly introduced plants require the same sunlight exposure, soil type and drainage as that of the existing native vegetation.
- Including a variety of forbs along with grasses will create a more natural balanced environment. However, make certain the mature heights of the forbs added to the mix do not exceed those of the grasses. Otherwise, shading can occur, and the grasses will struggle to get established.
- Where weeds are a problem, a mixture of grasses and forbs that are taller or more aggressive than the weeds may be needed.
Options when preparing the site
- If any type of soil disturbance is done, expect weed seed germination. Regular cultivation for one or two growing seasons prior to planting may be necessary to reduce weed competition. A combination of disking and shallow cultivation can be effective. Disking places some weed seeds too deeply in the soil to germinate. Light cultivation six to eight weeks after disking will kill any newly geminated weeds on the soil surface. Repeat these steps as necessary.
- Sites dominated by weeds may require an aggressive approach using a combination of cultivation to encourage weed seed germination and herbicide applications to kill the newly germinated weeds. Repeated herbicide applications will gradually deplete the weed seed bank in the soil and reduce successive weed populations.
- In some areas, controlled burning to eliminate existing vegetation is allowed. Check with your local office of the Georgia Forestry Commission to determine whether controlled burning is allowed in your area and the requirements for doing it.
- Heat sterilization (solarization) is another option for small areas. This involves placing sheets of clear plastic over the unwanted vegetation and sealing it along the edges with soil or rocks. The elevated temperatures under the plastic will kill herbaceous weeds, but it may not eliminate established woody vegetation. This technique works best during the warm summer months.
- When purchasing seeds or plants, make certain they are adapted to your geographical region. Plants produced from seed harvested from native grasses growing in the western prairies or desert regions of the U.S. may not be well adapted to the Southeast, even if the species is recommended for the area. Botanists and horticulturists often refer to “ecotypes” that are subspecies or varieties adapted to a particular set of environmental conditions. A plant?s place of origin or “provenance” is known to influence the adaptability of its offspring to a particular set of environmental conditions.
- Warm-season grasses are best planted from April to June, while cool-season grasses do best when planted from September to October.
- On sites prone to erosion, a cover crop, such as winter wheat (an annual), may need to be planted along with the grasses and forbs to help stabilize the soil during establishment.
- Plant forbs in colonies of several plants between the grasses. Clusters of forbs will not only be more visible, but also will do a better job of attracting pollinators.
- Seeds need to be in contact with the soil for best germination. When planting plugs, place the crown (the area between the base of grass blades and the roots) slightly below ground level.
- Native grasses grow well in soils having low fertility. Avoid fertilizing after planting because it will encourage weed competition.
- Supplemental irrigation may be necessary during periods of limited rainfall for at least three to six weeks while the seeds are germinating and the plants are establishing.
Managing a grass meadow requires annual observations as to how the plants are moving around, re-seeding and spreading. Also observe the balance of grasses to forbs and make note of unwanted weeds. A grass meadow is an ever-changing panorama as the balance of grasses to forbs is influenced by changing weather patterns and new plants introduced by passing wildlife or wind. Unlike a highly manicured cultivated landscape that is carefully managed and manipulated by mowing, pruning and fertilization, Mother Nature manages a native grass meadow.
- One of the greatest challenges is to distinguish the good weeds from the bad weeds. Some weeds are tame and offer little competition for the grasses and forbs. Others are considered “thugs” that spread rapidly from rhizomes or seeds and are hard to control. Examples are Burdock, Yellow Star Thistle (annual), Canada Thistle, Bermudagrass, Nutgrass, Crab Grass (annual), Crown Vetch, Canada Goldenrod, Johnsongrass and Chinese Lespedeza.
- In natural environments, grass meadows are managed by grazing from wildlife or livestock, or they are burned back by wildfires or controlled burns. Occasional mowing to a height of 5 to 6 inches can substitute for grazing. Mowing from late May through June will scatter seeds and rejuvenate cool-season grasses, then mowing again in late winter will scatter seeds and rejuvenate warm-season grasses. To encourage forbs to seed in and multiply, rake off the residue after mowing. Raking scatters the seeds of forbs, helps seed-to-soil contact and allows light to reach the new plants. Otherwise, if the planting is well-balanced with grasses and forbs, leave the mowing residue in place to act as natural mulch.
- Controlled burning is an alternative to mowing in areas where outdoor burning is allowed. Check with your local division of the Georgia Forestry Commission for laws and regulations regarding controlled burns. Annual burning once the grass meadow is fully established (three to five years after planting) will rejuvenate the planting.
Guide to Plant Descriptions
Native grasses and sedges described in this publication are listed alphabetically according to their botanical name. Grasses are divided into two categories: warm-season grasses and cool-season grasses. The appendix contains a Guide for Selecting Native Grasses and Sedges according to their growing requirements and usages. Information on each plant is provided according to the following criteria:
Common Name(s) / Botanical Name
Time of Bloom
Georgia Hardiness Zones
Common Name(s) / Botanical Name: Many of the plants have more than one common name. Those that are most often used are listed. For this publication, Flora of Southern and Mid-Atlantic States by Alan S. Weakley, North Carolina Herbarium, was used as the definitive source for botanical names. Plants that were re-classified into a new genus also show the previous botanical name in the form of a synonym (syn.) after the current name.
Life Cycle: Native grasses may be annuals or perennials, while sedges are perennials. Annuals flower, fruit and die in one growing season. Perennials flower and fruit each year, and they live for several years.
Characteristics: This section provides a botanical description of the plant that will assist the reader in identifying it. Noteworthy characteristics such as growth habit, leaf arrangement or shape, flower type and color, and seed structure are provided.
Cultural Requirements: A description of the type of growing environment the plant needs to thrive, such as the light level, soil type and soil conditions, is provided. Other information useful in managing the plant, such as pruning to remove old foliage prior to new growth or pruning before seed set to prevent seed dispersal, is included where appropriate.
Time of Bloom: The months of the year when the plant typically flowers in Georgia.
Suggested Uses: Some grasses are used for controlling erosion or restoring disturbed sites. Others are used in wildlife habitats. Still others may have ornamental value and are used in cultivated landscapes. The growing requirements and native habitat of the plant are considered when providing suggested uses.
Georgia Hardiness Zones: The Cold Hardiness Zones in Georgia to which the plant is adapted are shown here. These zones are based on the 2012 U.S. Department of Agriculture Hardiness Zone Map for the United States. Figure 2 shows the 2012 Cold Hardiness Zones for Georgia.
Size: The expected mature height and/or spread of the plant under ideal cultural conditions.
Habitat: The environment(s) in which the plant is found in the wild.
Native To: A general description of the region within the continental U.S. where the plant is presently found in its native habitat.
Comments: Additional information about the plant that the reader may find interesting.
Botanical Terms Used to Describe Grass and Sedge Plants
Terminology used to describe the parts of grasses and sedges differs from that of herbaceous or woody plants. The illustrations in figures 1, 2 and 3 show terms commonly used to describe the parts of grasses and sedges, followed by definitions of the terms used in the figures. A glossary at the end of this publication provides the reader with additional definitions of terms used elsewhere in this publication.
Definitions of Terms Shown in the Figures
Achene: A dry single-seeded fruit.
Awn: A bristle-like appendage on a floret or seed, often the extension of veins in glumes or lemmas.
Anther: The male floral part in which pollen is produced.
Blade: The broad, flattened portion of the leaf.
Bract: A modified leaf at the base of the ovary. It is also called a scale.
Bristles: Reduced or modified leaves with numerous hairs, usually in association with the ovary.
Callus: A thickened raised area of hardened tissue.
Collar: The outside area of a grass leaf where the blade and sheath join.
Crown: The basal portion of the plant just above ground level.
Culm: A hollow or pithy stalk or stem.
Filament: The stalk of the male portion of a flower to which the anther is attached.
First glume: The lower of the two glumes and just below the first floret. It is usually the smaller of the two glumes, or it may be entirely absent.
Floret: A unit within a grass spikelet usually comprised of a lemma, palea, two to three lodicules and the grass reproductive parts.
Glumes: The lower one or two sterile bracts at the base of a spikelet.
Inflorescence: A collective term used to describe the overall floral part of the plant.
Internode: The portion of the culm between two nodes.
Lemma: The lower of the two bracts enclosing a flower (floret) above the glumes. It is the most modified of the bracts and the last to disappear.
Ligule: A membranous structure on the adaxial leaf surface adjacent to the sheath.
Lodicules: Modified (reduced) perianth parts.
Nerve: The vein of a glume.
Node: The joint of a grass stem (culm) where the leaves and branches originate.
Ovary: Part of the flower that encloses the ovules containing seeds.
Palea: The inner of the two bracts, enclosed by the edges of the lemma.
Pedicel: The stalk of a single flower.
Pistil: The female floral part.
Rachilla: The secondary axis of a compound leaf or inflorescence.
Scale: Leaflike structure found at the base or outside of the flower. It is also called a bract.
Second glume: The glume opposite to the first, usually larger glume. When the first is lacking, the second glume is on the opposite side of the first floret.
Sheath: The lower part of a grass leaf that encloses the stem.
Shoot: The above-ground portion of a plant.
Spikelet: An inflorescence with one or two glumes at the base and containing one or more florets.
Stamen: The male part of a flower.
Stigma: The distal end of the style, which is receptive to pollen.
Style: Pollen tube connecting the stigma to the ovary.
Stolon: A horizontal above-ground stem that roots along its nodes. It is also called a runner.
Rhizome: A horizontal underground stem.
Upland Bentgrass, Autumn Bentgrass / Agrostis perennans
Life Cycle: Perennial
Characteristics: A tufted grass having unbranched, leafy light-green culms. Leaf blades are medium green, 1/8 to 1/4 inch wide and 2 to 10 inches long. At the junction of each blade and sheath there is a white membranous ligule. The nodes along each culm are green and swollen. Each fertile culm terminates in an open branched panicle, 3 to 12 inches long and 1 1/2 to 6 inches wide, having a zigzag rachis. Each spikelet has two prominent glumes that resemble a pair of tiny claws. The green inflorescence is more open and airy in shady locations, and it turns tan in fall. The plant self-seeds and forms small colonies. The root system is fibrous.
Cultural Requirements: This grass adapts to a wide variety of cultural conditions, from moist to dry soils in full sun to light shade.
Time of Bloom: September, with spikelets persisting through October.
Suggested Uses: Use Upland Bentgrass in open areas, such as right of ways and meadows.
Georgia Hardiness Zones: All of Georgia
Size: 1 to 3 feet tall
Habitat: Dry or moist thickets, rocky open woodlands, thinly wooded bluffs, wooded openings, prairie swales. In woodlands, it is often found growing at the base of deciduous trees.
Native To: Maine, south to Florida, west to Texas and north to North Dakota.
Comments: A number of caterpillars feed on the foliage, and the seeds are eaten by a variety of birds and mammals. The foliage is grazed by livestock.
Big Bluestem, Turkeyfoot / Andropogon gerardii
Life Cycle: Perennial
Characteristics: Big Bluestem is a tall bunch prairie grass often used for grassland restoration in the central and southern plains. It makes premium hay. Leaves are up to 2 feet long and ½ inch wide. Lower leaves are hairy near their bases. Flower clusters are spike-like racemes with purple, brownish purple, yellow or brownish yellow coloration. The flowers rise above the foliage in late summer and branch from one central point into three 4-inch-long segments that some say resemble a turkey?s foot. The internodes of flowering stems have a barberpole appearance with rosy or creamy coloration alternating with green. Leaves take on a purple hue in fall and are russet in winter.
Cultural Requirements: Big Bluestem prefers full sun to partial shade and moist, well-drained soils. Once established, it is drought-tolerant. It also can tolerate periodic flooding. When given too much shade, too much water or too much fertilizer it will flop over and look unsightly. It spreads by seed and can be aggressive under good cultural conditions. Mow or cut back the plant in late winter to make way for new spring growth. It also comes back well after controlled burning.
Time of Bloom: August through September, with showy fruit from late September through November.
Suggested Uses: Use Big Bluestem in sunny meadows, open woodlands, wildlife habitats or sunny perennial borders. Its large stature, blue-green foliage, branched seed-heads and russet winter color add visual interest to the landscape. The plant also provides winter protection and food for birds and small mammals. It also is useful for erosion control on slopes.
Georgia Hardiness Zones: All of Georgia
Size: 4 to 8 feet tall
Habitat: Low meadows, dry barrens and woodlands, cliffs, rock outcrops and prairies.
Native To: Most of eastern North America, as far west as Montana and Arizona.
Comments: The flowering stems and leaf sheaths of most of the Andropogon species have a bluish cast on their emerging culms in the spring, so the plants are commonly called Bluestems. Big Bluestem is a larval host for the Delaware Skipper and Dusted Skipper butterflies. It also provides cover and nesting sites for a variety of songbirds. Big Bluestem was once the dominant prairie grass covering a large portion of the Midwest. It was the predominant food source for the millions of bison that once roamed the Great Plains. When settlers plowed under Big Bluestem on their western migration, there was nothing left to keep the dirt from blowing away, so the loss of this plant is said to have contributed to the historic Dust Bowl of the 1930s. Big Bluestem is also a Southeastern grass, especially in the Coastal Plain. It is less common in the Piedmont. It is quick to establish from plugs or seeds planted in late winter or early spring.
Species identification and morphological trait diversity assessment in ryegrass ( Lolium spp.) populations from the Texas Blackland Prairies
Ryegrass ( Lolium spp.) is a troublesome weed in major wheat ( Triticum aestivum L.) production regions in the United States. High diversity and adaptive potential are known to contribute to its success as a weed species and also create difficulties in correct species identification in fields. The objective of this research was to characterize diversity for 16 different morphological traits among 56 Lolium populations collected from wheat production fields across the Texas Blackland Prairies region and identify Lolium species based on taxonomic characteristics. Populations were highly diverse (both at inter- and intrapopulation levels) for the traits studied, and a taxonomic comparison with USDA-GRIN reference samples revealed that all the populations were variants of Italian ryegrass [ Lolium perenne L. ssp. multiflorum (Lam.) Husnot] with a few offtypes of perennial ryegrass ( Lolium perenne L.) or probable hybrids between the two species. Hierarchical clustering grouped the populations into six clusters based on their similarities for the morphological traits investigated. Principal component analysis showed that the variability for yield traits greatly contributed to the total diversity. Pre-flowering plant height (stage 10 on Feekes scale) was positively correlated with tiller count, shoot biomass, and spike count, but not with total seed count per plant, whereas plant height at maturity (stage 11.3 to 11.4 on Feekes scale) was highly correlated with total seeds per plant. Further, basal node color was positively correlated with plant growth habit, regrowth rate, and leaf color. Leaf blade width was positively correlated with survival to pinoxaden and multiple herbicides, whereas, spike count was negatively correlated with survival to mesosulfuron. The high levels of intra- as well as interpopulation variability documented in this study indicate the potential of this species to rapidly adapt to herbicides and emphasize the need for implementing diverse management tactics, including the integration of harvest weed seed control.
Ryegrass (Lolium spp.) is a troublesome weed in wheat (Triticum aestivum L.) production worldwide, and it is also a valuable cultivated forage, turf, and cover crop species. Nine species of Lolium are known to occur globally, and of these, perennial ryegrass (Lolium perenne L.) and Italian or annual ryegrass [Lolium perenne L. ssp. multiflorum (Lam.) Husnot] are the two most commonly occurring species globally as well as in the United States (U.S.) (Rosell Reference Rosell 1967; Scholz et al. Reference Scholz, Stierstorfer and Gaisberg 2000). They were introduced to the United States as a forage crop from Europe and used in Virginia as early as 1782 (Sullivan Reference Sullivan 1992). They can be found growing as a natural vegetation along roadsides and in crop fields, abandoned lands, orchards, pastures, and vineyards (Panozzo et al. Reference Panozzo, Collavo and Sattin 2020; Wu et al. Reference Wu, Zhang, Zhang, Lang and Sun 2012).
Lolium grows in a broad range of soil textures, adapts well to poorly drained soils (Evers Reference Evers 1995; Venuto et al. Reference Venuto, Redfearn, Pitman and Alison 2003), and shows drought and cold tolerance (Franca et al. Reference Franca, Loi and Davies 1998). It shows high adaptive potential and is a persistent species. Although these traits are favorable in permanent pasture and grazing lands (Nair Reference Nair 2004), the weedy Lolium biotypes exhibit rapid growth (Bararpour et al. Reference Bararpour, Norsworthy, Burgos, Korres and Gbur 2017), high fecundity (Beckie Reference Beckie 2006), profuse seed shattering (Salas Reference Salas 2012), and high seed dormancy (Steadman et al. Reference Steadman, Ellery, Chapman, Moore and Turner 2004), which facilitate their persistence and menace in crop fields (Peeper and Wiese Reference Peeper, Wiese and Donald 1990). Lolium emerges in the fall and continues to grow through summer and has currently become a major weed in summer forage production in many areas (Funderburg Reference Funderburg 2018).
Among the Lolium species, L. perenne ssp. multiflorum in particular is a troublesome weed in major wheat-producing regions in the United States (Appleby et al. Reference Appleby, Olson and Colbert 1976; Appleby and Brewster Reference Appleby and Brewster 1992; Trusler et al. Reference Trusler, Peeper and Stone 2007; Tucker et al. Reference Tucker, Morgan, Senseman, Miller and Baumann 2006; Webster and Nichols Reference Webster and Nichols 2012), including parts of Texas (Neely et al. Reference Neely, Bauman and McGinty 2016). Reports indicate a 61% wheat yield reduction by 93 L. perenne ssp. multiflorum plants m −2 in Oregon (Appleby et al. Reference Appleby, Olson and Colbert 1976; Appleby and Brewster Reference Appleby and Brewster 1992) and 40% reduction by 40 plants m −2 in Texas (Stone et al. Reference Stone, Cralle, Chandler, Miller and Bovey 1999). Stone et al. ( Reference Stone, Cralle, Chandler, Bovey and Carson 1998) found that belowground root interaction between L. perenne ssp. multiflorum and wheat reduced wheat height, leaf number, tillering, leaf area, and dry weights of leaves, stems, and roots. With heavy reliance on herbicides for controlling Lolium, L. perenne ssp. multiflorum is currently the most resistance-prone weed in U.S. wheat, with resistance to eight different herbicide mechanisms of action (Heap Reference Heap 2020).
High diversity in weeds is known to favor their invasiveness and potential to adapt to various growing conditions and management regimes, including herbicide applications (reviewed in Dekker Reference Dekker 2011; Akey et al. Reference Akey, Jurik and Dekker 1990; Baker Reference Baker 1974; Fried et al. Reference Fried, Chauvel, Munoz and Reboud 2019; Jha et al. Reference Jha, Norsworthy, Bridges and Riley 2008). For example, diversity for temporal and spatial seed dispersal and staggered emergence from the soil seedbank are mechanisms that help weeds adapt to local management practices and prevailing environments (Mahaut et al. Reference Mahaut, Fried and Gaba 2018). Adaptive traits such as rapid vegetative growth, high levels of biomass production, fecundity, seed shattering, and dormancy, among others, have been reported to be associated with invasiveness and herbicide resistance (Beckie Reference Beckie 2006; Gressel and Segel Reference Gressel, Segel, LeBaron and Gressel 1982; Salas Reference Salas 2012; Watkinson and White Reference Watkinson and White 1985). Vila-Aiub et al. ( Reference Vila-Aiub, Gundel and Preston 2015) noted that accumulation of functional and adaptive traits that enhance plant fitness to a specific or a wide range of environments may favor survival of weeds to herbicides. In some cases, in addition to interpopulation diversity, intrapopulation diversity can be very high (e.g., Bangert et al. Reference Bangert, Lonsdorf, Wimp, Shuster, Fischer, Schweitzer, Allan, Bailey and Whitham 2006; Shuster et al. Reference Shuster, Lonsdorf, Wimp, Bailey and Whitham 2006), which allows for local adaptation (Dekker Reference Dekker 2011).
A robust evaluation of morphological trait diversity (both intra- and interspecific) that contributes to weed adaptation in a particular geographic region has great ecological and evolutionary implications, as high diversity leads to invasion, succession, acclimatization, and may eventually lead to new speciation (Bennett et al. Reference Bennett, Riibak, Kook, Reier, Tamme, Bueno and Pärtel 2016; Heslop-Harrison Reference Heslop-Harrison 2010). In Texas, the Blackland Prairies (Figure 1) are an important winter wheat production region, with a planted area of 1.8 million ha and a production of 1.9 billion kg in 2019 (USDA-NASS 2019). A recent survey conducted by Singh et al. ( Reference Singh, Maity, Abugho, Swart and Bagavathiannan 2020) confirmed the widespread occurrence of L. perenne ssp. multiflorum with resistance to commonly used herbicides in the Texas Blackland Prairies. However, little is known about the diversity for adaptive traits in Lolium in the Texas Blackland Prairies. The objective of this study was to determine the extent of diversity for different morphological traits and identify the correct species based on taxonomic characteristics in Lolium populations collected in the Texas Blackland Prairies.
Figure 1. Geocoordinates of Lolium collected from wheat fields in 2017 indicating (A) location within Texas, (B) population IDs, and (C) names of counties.
Materials and Methods
Field Survey and Plant Material
Field surveys were conducted during June 2017 (coinciding with wheat crop maturity window and before crop harvest) to collect Lolium seed samples from wheat production fields and field edges across the Texas Blackland Prairies (Figure 1). At each survey site, spikes from 20 to 25 plants were harvested randomly and pooled into a single sample. In total, 68 Lolium populations were collected during the survey, of which 56 were used in this study based on seed availability. The samples were dried, threshed, cleaned, and stored at room temperature (25 ± 2 C) under white fluorescent light before characterization. More specifics of the field survey methodology and sample collection can be found in Singh et al. ( Reference Singh, Maity, Abugho, Swart and Bagavathiannan 2020).
Characterization of Morphological Traits
Plant morphological traits were evaluated under controlled greenhouse conditions in a randomized complete block design, with two independent experimental runs. During each run, 50 seeds from each of the 56 Lolium populations were placed on top of two layers of filter paper (Whatman No. 1, Sigma-Aldrich, Inc., St. Louis, MO, USA) in a 9-cm-diameter petri dish, supplied with sufficient moisture throughout the germination period (21 d). The petri dishes were incubated at 25 C constant temperature, 60% relative humidity, and 12-h photoperiod. When the emerged seedlings were 21-d old (8- to 10-cm height), 15 randomly selected seedlings from each population were transplanted into individual pots (14-cm diameter and 12-cm height) filled with a potting soil mixture (LC1 Potting Mix, Sun Gro ® Horticulture, Agawam, MA, USA). The pots were transferred to the Norman Borlaug Center for Southern Crop Improvement Greenhouse Research Facility at Texas A&M University and were maintained at 26/22 C day/night temperature regime and a 10-h photoperiod.
On each of the 15 randomly selected plants within each population, observations were carried out for the following 16 morphological traits: plant height (before flowering: stage 10 on Feekes scale; and at plant maturity: stage 11.3 to 11.4 on Feekes scale), regrowth rate, growth habit, basal node color, leaf color and width, plant texture, shoot dry biomass, tiller count per plant, spike count, spike length, seed count per spikelet, seed count per spike, total seed count per plant, and seed shattering. For measuring the regrowth, 5 plants in each population were trimmed at weekly intervals (at 15-cm height from the base) starting at about 6 wk after planting for 4 consecutive weeks (stages 4 to 7 on Feekes scale). As trimming might have a significant effect on other morphological traits, these 5 plants were excluded from analysis of the other traits.
Lolium shows significant genotypic variation for shoot elongation between jointing and heading (Ullmann et al. Reference Ullmann, Herrmann, Hasler, Cai and Taube 2016) and also shows significant axillary growth during and after flowering in apical meristem (Jensen et al. Reference Jensen, Salchert and Nielsen 2001). To capture potential differences for this response, plant height was measured both before flowering (before or at booting stage; stage 10 on Feekes scale) and again at plant maturity (stages 11.3 to 11.4 on Feekes scale). For measuring growth habit, the angle of the stem/main tillers with horizontal axis was measured and scored on a scale of 1 to 5. Methodology followed for the assessment of different traits is described in Table 1, which was adopted with slight modifications from the UPOV directory Guidelines for the Conduct of Tests for Distinctness, Homogeneity and Stability, Ryegrass (Lolium spp.) (UPOV 1990), and the National Plant Germplasm System of plant expression of the U.S. Department of Agriculture–Agricultural Research Service (USDA-ARS 2005), as described by Özköse and Tamkoç ( Reference Özköse and Tamkoç 2014).
Table 1. Methodology for assessing morphological traits across the Lolium perenne ssp. multiflorum populations collected in the Texas Blackland Prairies.
a The methodologies used to assess the morphological traits are adopted from Rosell ( Reference Rosell 1967), UPOV (1990), and Özköse and Tamkoç ( Reference Özköse and Tamkoç 2014), with slight modifications aligned with the location, growth habits of the populations, and available resources for the current study.
Comparison with Reference Samples for Species Identification
The Lolium populations used in the study exhibited high levels of diversity, and it was unclear whether the populations represented different Lolium species. To facilitate accurate species identification and differentiation, known reference samples of L. perenne ssp. multiflorum, L. perenne L., rigid ryegrass (Lolium rigidum Gaudin), and Darnel ryegrass (Lolium temulentum L.) were obtained from the Germplasm Resources Information Network (GRIN) of the USDA-ARS (USDA-ARS Plant Germplasm Introduction and Testing Research, Pullman, WA, https://npgsweb.ars-grin.gov). These reference samples were grown alongside the Texas populations in the greenhouse during the second experimental run and were taxonomically compared (Figures 2 and 3).
Figure 2. Seed morphological comparison among Lolium species obtained from the USDA-GRIN collections: (A) Lolium rigidum, (B) Lolium temulentum, (C) Lolium perenne ssp. multiflorum, and (D) Lolium perenne.
Figure 3. Positive identification of Lolium perenne ssp. multiflorum (A) in populations surveyed from the Texas Blackland Prairies, based on morphological comparison with different known Lolium species obtained from the USDA-GRIN collections (B–E): (B) Lolium rigidum, (C) Lolium perenne, (D) L. perenne ssp. multiflorum, and (E) Lolium temulentum.
Plant morphological trait data were analyzed using JMP PRO v. 14 (SAS Institute, Cary, NC, USA). Interpopulation diversity for each trait is presented as range, mean, and standard error of the means of the populations. This illustrates the variability in population mean values for the different traits evaluated. For detecting intrapopulation variation for a given trait within a specific population, the difference between the highest and lowest value for the trait among the 20 individual plants (10 plants in each of the two runs) investigated within the population was calculated and presented as a frequency histogram. For determining regrowth rate, a total of 10 plants (5 plants per run) were used.
Populations were subjected to cluster analysis based on the 16 morphological traits measured. Hierarchical cluster analysis was performed based on Ward’s minimum variance method in JMP PRO v. 14, and cluster summaries are presented to show the grouping of populations. This method calculates Euclidean distance and creates cluster groupings by minimizing within-group ANOVA sum of squares. A principal component analysis was conducted using JMP to determine the association among the populations and the contribution of the different traits toward the total variability. Further, a Pearson correlation analysis was conducted to ascertain association between the morphological traits and herbicide resistance status. For this purpose, secondary data (from Singh et al. Reference Singh, Maity, Abugho, Swart and Bagavathiannan 2020) on herbicide-resistance profiles of the test populations for two acetyl coenzyme-A carboxylase (ACCase)-inhibiting herbicides (diclofop-methyl, Hoelon ® ; Bayer Crop Science, Research Triangle Park, NC, USA and pinoxaden, Axial XL ® ; Syngenta Crop Protection, Greensboro, NC, USA) and one acetolactate synthase–inhibiting herbicide (mesosulfuron-methyl, Osprey ® ; Bayer Crop Science) were used.
Results and Discussion
Inter- and Intrapopulation Diversity
There were high levels of inter- (Table 2) as well as intrapopulation diversity (Figure 4) for all the morphological traits investigated in the study. The differences for these traits were highly significant (P < 0.001) among the Lolium populations evaluated in this study (Table 2). Mean plant height at maturity varied 2.2-fold between the shortest (51 cm) and the tallest (110.5 cm) populations (Table 2, T2), with an intrapopulation plant height difference ranging as high as 101 to 110 cm in 2% of the populations and about 51 to 60 cm in 45% of the populations (Figures 4B and 5B–E). Regrowth rate among the populations ranged from 2.2 to 7.0 on a scale of 1 to 7 (Table 2, T3), with 80% of the populations showing slow regrowth potential (<4). However, 87% of the populations showed high intrapopulation variability for this trait (Figure 4C).
Table 2. Diversity for morphological traits across the wild populations of Lolium perenne ssp. multiflorum from the Texas Blackland Prairies.
a The ratio of higher and lower extremes of the range. Values have been rounded to the nearest decimal point.
b Indicates significant difference among the populations for a particular trait.
c Regrowth rate was scored as follows: (1) <2 cm d −1 , (3) 2–3.5 cm d −1 , (5) 3.5–5 cm d −1 , (7) >5 cm d −1 .
d Growth habit was scored as follows: (1) >60° from the horizontal axis, (3) 30°–60°, (5) <30°; a greater angle indicates erectness and a smaller angle indicates prostrate growth habit.
e Leaf color was scored as follows: (1) light green, (3) green, (5) dark green.
f Basal node color was scored as follows: (1) light green, (3) green, (5) reddish.
g Plant texture was scored as follows: (1) very smooth, (3) smooth, (5) rough, (7) very rough.
Figure 4. Frequency distribution of the difference between maximum and minimum values for a given trait, illustrating intrapopulation variation. (A) Plant height before flowering (cm); (B) plant height at maturity (cm); (C) regrowth rate measured on a scale of 1 to 7, with 1 being very slow and 7 representing very fast growth; (D) growth habit measured on a scale of 1 to 5, with 1 being erect and 5 being prostrate; (E) leaf color measured on a scale of 1 to 5, with 1 being light green and 5 being dark green; (F) basal node color measured on a scale of 1 to 5, with 1 being light green and 5 being reddish; (G) leaf blade width (mm); (H) plant texture measured on a scale of 1 to 7, with 1 being very smooth and 7 being very rough; (I) tiller count per plant; (J) shoot dry biomass per plant (g); (K) spike count per plant; (L) spike length (cm); (M) seed count per spikelet; (N) seed count per spike; (O) seed count per plant; and (P) seed shattering (%). For example, in A, 4% of the populations had the greatest intrapopulation plant height difference of 61 to 70 cm between the shortest and tallest plants, whereas 5% of the populations had the lowest difference of 0 to 10 cm.
Figure 5. Image showing intrapopulation diversity for plant height (B vs. C; D vs. E), maturity (A–C), and growth habit (prostrate vs. erect) (B vs. C; D vs. E) in Lolium perenne ssp. multiflorum populations collected in the Texas Blacklands. Plants in A–C belong to a single population, as plants in D and E.
Table 3. Clustering of the Lolium perenne ssp. multiflorum populations from the Texas Blackland Prairies based on their morphological traits. a
a Abbreviations: PF, plant height before flowering (cm); PM, plant height at maturity (cm); RG, regrowth rate scored as follows: (1) <2 cm d −1 , (3) 2–3.5 cm d −1 , (5) 3.5–5 cm d −1 , (7) >5 cm d −1 ; PG, plant growth habit scored as follows: (1) >60° from the horizontal axis, (3) 30°–60°, and 5 for <30°; LC, leaf color scored as follows: (1) light green, (3) green, (5) dark green; NC, basal node color scored as follows: (1) light green, (3) green, (5) reddish; LW, leaf blade width (mm); PT, plant texture scored as follows: (1) very smooth, (3) smooth, (5) rough, (7) very rough; TC, tiller count per plant; SD, shoot dry biomass per plant (g); SP, spike count per plant; SL, spike length (cm); SS, seed count per spikelet; SC, seed count per spike; TS, total seed count per plant; SH, seed shattering (%).
b Indicates the number of populations falling within a given cluster. Among the 56 populations used in the experiment, 51 had sufficient data for conducting cluster analysis.
Average plant growth habit was erect but varied widely from prostrate to erect (2.7-fold variation) (Table 2, T4; Figure 5, B vs. C and D vs. E), with 39% of the populations showing an intrapopulation difference of 3.1 to 4.0 on a scale of 1 to 5 (Figure 4D). Lolium is a highly tillering species and, on average, produced about 10 tillers per plant (Table 2, T9); mean tiller count varied from 5 to 14 across populations, with 27% of the populations showing an intrapopulation difference of 9.1 to 18 tillers per plant (Figures 4I and 6D and E). In general, most populations had green leaf and basal node color but varied greatly at the intrapopulation level (Figure 4E and F). In particular, leaf color varied from light green to dark green (Figure 6A–C). Mean leaf blade width, which is considered a key taxonomic trait in Lolium (Bararpour et al. Reference Bararpour, Norsworthy, Burgos, Korres and Gbur 2017), varied between 2 and 12 mm at an individual leaf level across all plants (Figure 7), with 30% of the populations having the highest intrapopulation difference of 4.1 to 6 mm (Figure 4G). Though the average plant texture was smooth, there was a high intrapopulation variability for this trait (Figure 4H). Average plant size varied tremendously among the populations, and there was an 11.3-fold interpopulation difference for shoot biomass (Table 2, T10); about 9% of the populations exhibited the highest intrapopulation shoot biomass difference of 12.1 to 16.5 g (Figure 4J).
Table 4. Principal components analysis (PCA) of wild populations of Lolium perenne ssp. multiflorum from the Texas Blackland Prairies based on 16 morphological traits.
Figure 6. An example of diversity among the Lolium perenne ssp. multiflorum populations evaluated in this experiment for leaf color (A–C) and tillering habit (D and E).
Figure 7. Diversity among the Lolium perenne ssp. multiflorum populations evaluated in this experiment for leaf blade width.
Plant yield traits were also highly variable among the Lolium populations (P < 0.001; Table 2). Spike count per plant, which determines fecundity, had a mean interpopulation variability of 2 to 11, but some populations had an intrapopulation difference as high as 40 spikes per plant (Figure 4K). Spike length, however, was relatively consistent across populations and also less variable at the intrapopulation level (Figure 4L). Mean seed count per spikelet varied from 1.4 to 5.7 at the interpopulation level (Table 2, T13), with 18% of the populations exhibiting an intrapopulation difference of 6.1 to 8 seeds per spikelet (Figure 4M). Across all plants, seed production per spikelet varied from 1 to 20 (data not shown). The mean seed number was 432 seeds per plant, and there was 11.3-fold variation for this trait among the different populations (Table 2, T15). It is important to note that the spikes showed a greater taxonomic variation, ranging from branched rachis (i.e., panicle type) to spike with multiple spikelets originating at the same point (Figure 8). The seeds also highly varied for the presence and length of awns (Figure 3). Seed shattering, an important weedy trait, varied from 5% to 54% among the populations (Table 2, T16). With respect to intrapopulation variability, seed shattering showed a continuous frequency distribution across all the populations investigated (Figure 4P).
Figure 8. Diversity among Lolium perenne ssp. multiflorum populations collected in the Texas Blackland Prairies for spike morphology and maturity differences: (A) short or no awn, rare panicle type with 12–15 seeds per spikelet on main rachis but 5–7 seeds per spikelet on branches; (B) short or no awn with 5–7 seeds per spikelet; (C) long outer glume and awn, but early maturity with 5–7 seeds per spikelet; (D) long awn, spikelet arrangement on rachis is alternate spiral with 5–7 seeds per spikelet; (E) long awn, spikelet arrangement on rachis is alternate distichous with 8–10 seeds per spikelet; (F) long outer glume and awn, but late maturity with 5–7 seeds per spikelet; (G) short awn with 14–16 seeds per spikelet; (H) very long awn with 14–16 seeds per spikelet; and (I) spike with multiple spikelets at the same point.
Overall, a high degree of intra- as well as interpopulation diversity was observed for the different morphological and yield traits investigated here. High intrapopulation diversity favors field survival of a weed population under varying environmental and management conditions, which ultimately enhances the persistence of the population (Dekker Reference Dekker 2011). The specific adaptive trait(s) favorable to a population can be governed by various factors, including the nature of the selection agent and the environment, but the existence of diversity for such traits allows for selection to act on them. Our findings show that Lolium is well equipped for adaptive evolution due to the presence of high levels of diversity.
Bararpour et al. ( Reference Bararpour, Norsworthy, Burgos, Korres and Gbur 2017) also found high diversity for different traits in a large collection of L. perenne ssp. multiflorum in Arkansas. Similar findings of high diversity in L. perenne ssp. multiflorum were also reported by Rosell ( Reference Rosell 1967) and Terrell ( Reference Terrell 1968). Lolium perenne ssp. multiflorum is a highly outcrossing species (Lopes et al. Reference Lopes, Reis, Barata and Nunes 2009; Tobina et al. Reference Tobina, Yamashita, Koizumi, Fujimori, Takamizo, Hirata, Yamada and Sawada 2008), and continuous gene flow and allele exchange could be attributed to the high degrees of intra- and interspecific diversity observed in this species. Cross-pollination between diverse biotypes of L. perenne ssp. multiflorum and other compatible species can lead to numerous intermediate types with wide variation for morphological and yield traits (Cresswell et al. Reference Cresswell, Sackville, Roy and Viegas 2001; Schoth and Weihing Reference Schoth, Weihing, Hughes, Heath and Metcalfe 1962; Wang et al. Reference Wang, Cogan, Pembleton and Forster 2015). Further, the lack of intensive domestication in this species may also have contributed to the wide diversity for adaptive traits (Lopes et al. Reference Lopes, Reis, Barata and Nunes 2009). From an adaptive evolution standpoint, the occurrence of high intra- and interspecific diversity can greatly favor invasion and persistence of Lolium across changing management regimes and climatic conditions, as has been reported with a large number of other weeds (Dukes and Mooney Reference Dukes and Mooney 1999; Nicotra et al. Reference Nicotra, Atkin, Bonser, Davidson, Finnegan, Mathesius, Poot, Purugganan, Richards, Valladares and van Kleunen 2010). This also allows for rapid invasion and colonization of new habitats by the species without the need for local selection (Williams et al. Reference Williams, Mack and Black 1995).
Diversity coupled with selection by crop management practices favors the accumulation of beneficial traits and promotes weed species persistence (Dekker Reference Dekker 2011; Owen et al. Reference Owen, Goggin and Powles 2015). For example, populations with reduced growth rate and higher seed dormancy in L. rigidum (Vila-Aiub et al. Reference Vila-Aiub, Neve and Powles 2005), reduced growth rate in goosegrass [Eleusine indica (L.) Gaertn.] (Han et al. Reference Han, Vila-Aiub, Jalaludin, Yu and Powles 2017) and kochia [Bassia scoparia (L.) A.J. Scott] (Murphy and Tranel Reference Murphy and Tranel 2019), and reduced plant height and fecundity in L. perenne (Yanniccari et al. Reference Yanniccari, Vila-Aiub, Istilart, Acciaresi and Castro 2016) were reported to be associated with herbicide resistance. It has been reported that tall plant height and high shoot biomass generally enhance species competitiveness (Blair Reference Blair 2001; Fraga et al. Reference Fraga, Agostinetto, Vargas, Nohatto, Thurmer and Holz 2013), and the presence of diversity for these traits may facilitate species dominance in a competitive environment. Seed shattering is another important weedy trait, and diversity for this trait offers a weed population an extended window for seed dispersal (Burton et al. Reference Burton, Beckie, Willenborg, Shirtliﬀe, Schoenau and Johnson 2017; Delouche et al. Reference Delouche, Burgos, Gealy, Zorrilla de San Martin, Labrada, Larinde and Rosell 2007) and facilitates adaptation to management (Ashworth et al. Reference Ashworth, Walsh, Flower, Vila-Aiub and Powles 2016). For instance, the success of harvest weed seed control (HWSC) strategies relies on the ability of weeds to retain seed at the time of harvest (Walsh et al. Reference Walsh, Newman and Powles 2013). Selection imposed by HWSC can lead to adaptation by favoring early-shattering phenotypes if there is sufficient diversity for seed shattering in the weed population (Ashworth et al. Reference Ashworth, Walsh, Flower, Vila-Aiub and Powles 2016).
Among the total populations (68) collected from the Texas Blackland Prairies, four were found to be L. perenne based on the morphological and taxonomic comparison with GRIN reference samples (Maity et al. Reference Maity, Abugho, Singh, Subramanian, Smith and Bagavathiannan 2019). However, all 56 Lolium populations evaluated in the current study were variants of L. perenne ssp. multiflorum with a few offtypes of L. perenne or probable hybrids between the two species (Figures 2 and 3).
The high inter- as well as intrapopulation diversity observed here for various traits can easily lead to misinterpretation that they belong to different species (Balfourier et al. Reference Balfourier, Imbert and Charmet 2000). Lolium species identification has always been challenging for growers and seed certification officers (Rosell Reference Rosell 1967). Plant height, growth habit, leaf blade width, spikelet count per plant, and presence/absence of awn are considered as key taxonomically important traits for Lolium identification (Rosell Reference Rosell 1967). However, the variation observed for these traits in our populations indicate that these traits are not solely reliable for species identification. Other studies have proposed that presence/absence of awn, leaf blade width (Bararpour et al. Reference Bararpour, Norsworthy, Burgos, Korres and Gbur 2017), and spikelet count per plant (Terrell Reference Terrell 1968) can be used for this purpose, yet our results do not support these suggestions. Moreover, as argued by Jenkin ( Reference Jenkin 1933), Lolium is still relatively new in origin and is still accumulating morphological and physiological changes that may eventually lead to divergence of new ecotypes and even speciation.
In hierarchical cluster analysis, six distinct clusters were formed based on the 16 different morphological traits at a 47% similarity level (Figure 9). These six clusters (1 to 6) consisted of 16, 8, 5, 14, 5, and 3 populations, respectively. In general, the clusters did not consistently follow any specific geographic association for population grouping (Figures 1 and 9), but there were several notable commonalities for functional traits. The populations in the cluster 5 represented erect and tall plants, which in turn produced the maximum shoot dry biomass per plant (7.3 g), spikes per plant (9), and spike length (23 cm), and the second highest tiller count per plant (11), representing the most vigorous group. Plants in this cluster also had the narrowest leaves (1.2-mm wide) and the greatest seed shattering (29%) (Table 3).
Figure 9. Hierarchical clustering showing grouping of the Lolium perenne ssp. multiflorum populations based on 16 morphological traits, computed based on correlations. In the dendrogram, the relative distances between clusters are given by the horizontal distances between vertical lines that join the clusters. The blue line at the bottom represents the similarity index among the clusters which indicates 0%–100% from left to right. The change in slope of the blue line indicates that the differences in clusters that are joined up to the point are comparatively small. The vertical red line marks the similarity level at which the number of clusters was chosen. WC with a specific number indicates the population number used in the study.
Cluster 6 had only three populations, representing short plants (24.9 cm before flowering and 61.3 cm at maturity), with a rapid regrowth rate (Table 3). However, the regrowth rate did not correspond to shoot dry biomass production or seed production, probably due to the adverse effect of repeated trimming during the peak growth period, which may have partitioned more photosynthates toward foliar growth (Table 3). Populations in cluster 4 had the greatest tiller count per plant, which resulted in the maximum seed count per plant (Table 3). Cluster 1 generally produced low seeds per spikelet (3) or per plant (453), with a fair amount of seed shattering (16%) (Table 3). It was observed in cluster 1 that plants with spreading growth habit also had dark green leaves. Populations in cluster 3 generally had prostrate to semi-erect plants with wide leaves and rough plant texture.
Several studies have clustered wild biotypes and/or landraces of Lolium based on morphological and genetic diversity (Charmet et al. Reference Charmet, Balfourier and Monestiez 1994; Jafari et al. Reference Jafari, Hessamzadeh, Abdi and Saeedi 2003; Loos Reference Loos 1994; Lopes et al. Reference Lopes, Reis, Barata and Nunes 2009; Monestiez et al. Reference Monestiez, Goulard and Charmet 1994; Oliveira et al. Reference Oliveira, Lindner, Bregu and González 1997). In all these studies, Lolium showed a wide range of diversity and was grouped predominantly based on functional trait similarity. Blackmore et al. ( Reference Blackmore, Thomas, McMahon, Powell and Hegarty 2015, Reference Blackmore, Thorogood, Skøt, McMahon, Powell and Hegarty 2016) grouped European ecotypes of Lolium in relation to their geographic origin and distribution. However, Panozzo et al. ( Reference Panozzo, Collavo and Sattin 2020) did not find any grouping based on geographic origin among different Lolium ecotypes studied in Italy. In the current study, the clusters did not show any association with the geographic origin of the populations; rather, they were grouped by common functional traits. The occurrence of high levels of gene flow in Lolium (Balfourier et al. Reference Balfourier, Charmet and Ravel 1998) may have masked differences among geographically close populations (Balfourier et al. Reference Balfourier, Imbert and Charmet 2000). Further, movement of equipment such as combine harvesters among nearby farms could also be contributing to propagule dispersal and reduced population differentiation (Anderson and Hartzler Reference Anderson and Hartzler 2018). Likewise, Lopes et al. ( Reference Lopes, Reis, Barata and Nunes 2009) reported high similarity for adaptive traits among closely occurring Lolium populations in Portugal, suggesting the contribution of gene flow. However, local selection as influenced by management practices implemented within a production field, which is known to vary greatly among fields within a geography, might be contributing to random population differences for certain adaptive traits that are eventually clustered together. In addition to local selection, random propagule dispersal in Lolium through contaminated planting seed (Benvenuti Reference Benvenuti 2007), dispersal by cattle manure and soil amendments (Fischer et al. Reference Fischer, Poschlod and Beinlich 1996; Yamada et al. Reference Yamada, Matsuo and Tamura 1972), irrigation water (Tosso et al. Reference Tosso, Ferreyra and Muñoz 1986), random movement of custom farm equipment (Burke et al. Reference Burke, Kahl and Tautges 2017; Hodkinson and Thompson Reference Hodkinson and Thompson 1997), and other anthropogenic means may be contributing to the clustering pattern observed here.
Principal Component Analysis
The principal components analysis (PCA) revealed that the first four components explain 59% of the total variation among the populations. The first four components and traits with the greatest loadings are given in Table 4. The first principal component (PC1) is attributed mainly to the yield traits, whereas PC2 and PC3 pertain primarily to the vegetative traits. A biplot and a loading plot for the first two principal components are presented in Figure 10. Total seed count per plant (TS) contributes to the most variability in the PC1, followed by the seed count per spike (SC), spike length (SL), and tiller count per plant (TC), whereas the basal node color (NC) contributes the highest in PC2, followed by the leaf color (LC), regrowth rate (RG), and seed count per spike (SC) (Figure 10). PC1 and PC2 together accounted only for 39% of the variability, so the distinct grouping of Lolium populations was not clear from the biplot. However, some populations show distinct separation from the main group. For example, WC22 was placed in PC2 away from the other populations; close examination of the data reveals that this population had reddish basal nodes with dark green leaves, fast regrowth rate, and moderately high seed count per spike (SC), whereas the other populations in PC2 had green basal nodes and leaves.
Figure 10. Principal component analysis of Lolium perenne ssp. multiflorum populations with 16 morphological traits evaluated in this study. (A) Loading plot: WC with a specific number indicates the population number used in the study. (B) Biplot or scoring plot: PF, plant height before flowering (cm); PM, plant height at maturity; RG, regrowth rate scored as follows: (1) <2 cm d −1 , (3) 2–3.5 cm d −1 , (5) 3.5–5 cm d −1 , (7) >5 cm d −1 ; PG, plant growth habit scored as follows: (1) >60° from the horizontal axis, (3) 30°–60°, (5) <30°; LC, leaf color scored as follows: (1) light green, (3) green, (5) dark green; NC, basal node color scored as follows: (1) light green, (3) green, and (5) reddish; LW, leaf blade width (mm); PT, plant texture scored as follows: (1) very smooth, (3) smooth, (5) rough, (7) very rough; TC, tiller count per plant; SD, shoot dry biomass; SP, spike count per plant; SL, spike length (cm); SS, seed count per spikelet; SC, seed count per spike; TS, total seed count per plant; SH, seed shattering (%).
PCA has been successfully utilized for understanding the multidimensional variability through linear combinations in Lolium (Bennett Reference Bennett 1997; Lopes et al. Reference Lopes, Reis, Barata and Nunes 2009). Diversity for morphological and yield traits among Lolium populations have been shaped by the environments they were exposed to, which directly influence grouping and contribution of specific traits to the total variability (Lopes et al. Reference Lopes, Reis, Barata and Nunes 2009). In a study involving various Lolium species, Bennett ( Reference Bennett 1997) showed that PC1 pertained to vegetative traits, whereas PC2 and PC3 were influenced by yield traits. In the current study, most of the populations were grouped together, suggesting natural hybridization and propagule movement leading to a lack of population differentiation (Arojju et al. Reference Arojju, Barth, Milbourne, Conaghan, Velmurugan, Hodkinson and Byrne 2016; Blackmore et al. Reference Blackmore, Thorogood, Skøt, McMahon, Powell and Hegarty 2016; Jonaviciene et al. Reference Jonaviciene, Statkeviciute, Kemesyte and Brazauskas 2014; Thorogood et al. Reference Thorogood, Yates, Manzanares, Skot, Hegarty, Blackmore, Barth and Studer 2017). However, some populations such as WC22, WC4, and WC31(2), tend to be positioned away from the others in the PCA, which likely reflects their distinctive traits evolved through continuous adaptation in a particular habitat as a result of local selection (Rosell Reference Rosell 1967).
Correlation among the Different Morphological and Yield Traits
A correlation matrix heat map of the different traits being studied provided vital information on the nature of association among the traits (Figure 11). Plant height (before flowering) showed positive correlation with tiller count (P = 0.02), spike count (P = 0.0003), shoot dry biomass (P = 0.006), and seed count (P = 0.05), but not with spike length, seed count per spike, or total seed count; however, plant height at maturity was significantly correlated with spike length and total seed count, but not with tiller count (Figure 11). Regrowth rate was positively correlated with leaf and basal node color; the darker the basal node color, the greater the regrowth rate. Plants with dark basal node color also had a prostrate growth habit. Tiller count was highly correlated with shoot dry biomass (P = 0.002), spike length (P = 0.01), spike count (P = 0.0001), and seed count per spike (P = 0.04), which further contributed to its positive association with total seed count per plant (P = 0.0007). Moreover, the different yield traits were highly correlated with one another (P < 0.0001) (Figure 11). Seed shattering was not significantly associated with any of the morphological and yield traits studied.
Figure 11. Correlation matrix showing the strength of association between different morphological traits in the Lolium perenne ssp. multiflorum populations collected in the Texas Blackland Prairies. The marker band with numbers on the right side indicates significance level, where red (0) represents the highest significance level, and blue (1) represents the lowest significance level.
Correlation between taxonomically as well as agronomically important traits offers valuable insights into how traits evolve and interact under diverse cropping intensities and production practices (Korres et al. Reference Korres, Norsworthy, Tehranchian, Gitsopoulos, Loka, Oosterhuis, Gealy, Moss, Burgos, Miller and Palhano 2016). Further, knowledge of correlation between different traits allows for combining desirable traits for breeding improved Lolium cultivars for use as forage or turf species (Jian et al. Reference Jian, ShengRong and AiXing 2000). Many studies have examined correlation among different morphological and yield traits in relation to herbicide sensitivity and have found variable degrees of association (Owen et al. Reference Owen, Goggin and Powles 2015; Vila-Aiub et al. Reference Vila-Aiub, Yu and Powles 2019; Watkinson and White Reference Watkinson and White 1985). Parallel evolution of strongly associated traits leads to better adaptation and/or elimination of a less fit biotype in a cropping environment, which has tremendous ecological and agronomic importance (Oliveira et al. Reference Oliveira, Lindner, Bregu and González 1997). Evidence suggests that cropping systems and management practices influence trait association in different weed species, improving their fitness in their environments (Fried et al. Reference Fried, Chauvel, Munoz and Reboud 2019; Vila-Aiub et al. Reference Vila-Aiub, Yu and Powles 2019).
Correlation between Morphological and Yield Traits and Herbicide Resistance
The leaf blade width showed positive correlation with survival to pinoxaden (r = 0.3, P < 0.05) and multiple herbicides (r = 0.3, P < 0.05), whereas spike count was negatively correlated with survival to mesosulfuron-methyl (r = −0.3, P < 0.05) (Figure 11). Survival to diclofop was negatively correlated with plant height at maturity (r = −0.3, P < 0.07) and positively with plant texture (r = 0.2, P < 0.07).
It has been reported that intensive herbicide use patterns have selected for increased or decreased expression of certain traits parallel to resistance evolution in weeds (Darmency et al. Reference Darmency, Colbach and Corre 2017; Jasieniuk et al. Reference Jasieniuk, Brule-Babel and Morrison 1996; Martinez-Ghersa et al. Reference Martinez-Ghersa, Ghersa, Benech-Arnold, Mac Donough and S´anchez 2000; Mortimer Reference Mortimer 1997). Different plant adaptive traits such as competitiveness, biomass, height, leaf area, regrowth rate, photosynthetic rate, or seed production potential have been investigated concurrent with herbicide-resistance evolution (reviewed in Mortimer Reference Mortimer 1997; Watkinson and White Reference Watkinson and White 1985). Ghanizadeh and Harrington ( Reference Ghanizadeh and Harrington 2019) reported that triazine-resistant common lambsquarters (Chenopodium album L.) phenotypes were shorter and produced less biomass than susceptible phenotypes. Delayed germination was recorded in diclofop-resistant L. perenne ssp. multiflorum (Ghersa et al. Reference Ghersa, Martínez-Ghersa, Brewer and Roush 1994; Gundel et al. Reference Gundel, Martinez-Ghersa and Ghersa 2008) and L. rigidum (Vila-Aiub et al. Reference Vila-Aiub, Neve and Powles 2005). Henckes et al. ( Reference Henckes, Cechin, Schmitz, Piasecki, Vargas and Agostinetto 2019) reported greater plant height, shoot dry matter, and absolute growth rate and reduced number of tillers and leaf area ratio in L. perenne ssp. multiflorum biotypes resistant to glyphosate, iodosulfuron-methyl, and pyroxsulam compared with susceptible biotypes. In our study, plant height, leaf width, plant texture, and spike count were the only traits that were correlated with herbicide resistance. Contrary to the report of Henckes et al. ( Reference Henckes, Cechin, Schmitz, Piasecki, Vargas and Agostinetto 2019), our finding of positive correlation between leaf width and herbicide resistance indicates that resistant plants may have a greater photosynthetic potential, leading to greater fitness, though this phenomenon is yet to be confirmed in our populations. Nevertheless, it appears that the specific correlations observed vary greatly across weed species, traits, and herbicides and may be influenced by various genetic, biological, and management factors.
All Lolium populations investigated in this study were L. perenne ssp. multiflorum, which confirms the predominant occurrence of this species in the Texas Blackland Prairies. Lolium perenne ssp. multiflorum boasts a high level of diversity, both intra- as well as interpopulation, which allows it to constantly adapt to changing habitat conditions and invade new geographic regions. The range of diversity found in the Texas Blacklands Lolium populations is consistent with the observation of multiple herbicide resistance in them. The high outcrossing nature of the species and long-distance propagule movement may further worsen the situation. Given the high diversity and adaptive potential of Lolium, management practices must involve diverse tactics within an integrated weed management framework. Additionally, managing Lolium populations occurring in field edges, roadsides, and other noncultivated areas may be a useful stewardship practice. Information generated from this study provides an improved understanding of adaptive trait diversity in L. perenne ssp. multiflorum and association among them, offering valuable insights into species persistence and informing management considerations. Elucidating the physiological, biochemical, and molecular mechanisms underpinning the correlation between various plant morphological traits and herbicide resistance warrants further investigation.
The Netaji Subhas-Indian Council of Agricultural Research International Fellowship (ICAR-IF) and the Tom Slick Fellowship from the College of Agriculture and Life Sciences, Texas A&M University, provided to AM are gratefully acknowledged. We thank Seth Abugho for assisting with the field survey and all graduate students, student workers, and interns of the Texas A&M Weed Science Research program for assisting with the characterization of the Lolium populations. The authors declare that no conflicts of interest exist.