Introduction

The globally distributed duckweed family (Lemnaceae) or subfamily (Lemnoideae) is composed of 36 species (Bog et al., 2020) of very small floating or submerged aquatic plants (Landolt, 1986; Sree, Bog, & Appenroth, 2016). Duckweeds have a long history of scientific study given their highly specialized morphology, widespread distribution, high abundance and production of the world’s smallest flowers (Hillman, 1961; Jacobs, 1947; Landolt, 1986; Landolt, 1992). More recently, there has been an explosion in research interest given their potential applied uses including for agricultural feed (Cheng & Stomp, 2009), bioremediation (Ekperusi, Sikoki, & Nwachukwu, 2019; Gupta & Prakash, 2013) and biofuel production (Cui & Cheng, 2015). Furthermore, their use as a model system to experimentally study numerous topics in ecology and evolutionary biology is quickly expanding (Laird & Barks, 2018). This growing basic and applied interest stems from their ability to reproduce clonally very quickly with population doubling times in as little as 1.5 days (Ziegler, Adelmann, Zimmer, Schmidt, & Appenroth, 2015). In addition, they are amenable to large-scale manipulative experiments in both the lab and field mesocosms (Armitage & Jones, 2019; Hart, Turcotte, & Levine, 2019; O’Brien et al., 2022; Tan, Kerstetter, & Turcotte, 2021), and have growing genomic data and tools (, 2020; Ho, Bartkowska, Wright, & F, 2019; Wang et al., 2014; Xu et al., 2019) and characterization of their microbiome and herbivore communities (Acosta et al., 2020; Subramanian & Turcotte, 2020). Finally, duckweed express variation in numerous traits across species and among genotypes (clonal lineages) within species (Anneberg, O’neill, Ashman, & Turcotte, 2023; Chen et al., 2020; Hart et al., 2019; Hitsman & Simons, 2020; Steveninck, Steveninck, & Fernando, 1992). Therefore, being able to identify genotypes may also be beneficial in many ecological studies to assess differences in traits among genotypes and to determine how these genotypes may respond to different environmental conditions.

Genetic markers, such as microsatellite markers, are important tools to study population genetics. Microsatellites, also known as simple sequence repeats (SSR), are tandem repeats two to ten base pairs in length, that are flanked by conserved sequences and occur ubiquitously throughout eukaryotic genomes (Tautz & Renz, 1984). They are highly informative as locus-specific genetic markers due to their high abundance, high reproducibility, co-dominance, and polymorphic nature (Morgante & Olivieri, 1993; Powell, Machray, & Provan, 1996). The length of the sequence repeats can be determined through PCR amplification using primers specific to their flanking regions; variation in PCR product length is a function of the number of repeated sequences. The high levels of polymorphisms observed in SSR markers (Schlötterer & Tautz, 1992; Tautz, 1989) and the relative ease of detection of these polymorphisms by PCR amplification have led to the wide applications of microsatellites as genetic markers (Vieira, Santini, Diniz, & Munhoz, 2016). Such within-species markers have numerous applications including quantifying biogeographic distributions, population genetic structure, evolutionary history, and mating systems.

Moreover, a growing number of experimental evolution studies use SSR markers to track changes in genotypic composition of asexually reproducing populations over multiple generations (Agrawal, Johnson, Hastings, & Maron, 2013; Hart et al., 2019; Turcotte, Reznick, & Hare, 2011) in large replicated experiments for which genotyping-by-sequencing remains too costly. These cost savings are magnified when several loci can be multiplexed and genotyped in the same reaction (Markoulatos, Siafakas, & Moncany, 2002).

With the growing interest in duckweed, microsatellite markers have been developed for a few duckweed species.Wani, Shah, Reshi, Atangana, and Khasa (2014) developed nine polymorphic and 24 monomorphic haplotype chloroplast DNA-based microsatellite primers for L. minor.Xu et al. (2018) developed 60 microsatellite primers for Spirodela polyrhiza, 19 of which were polymorphic within three populations of S. polyrhiza from China. Feng et al. (2017) developed three microsatellite primers for the identification of S. polyrhiza and Landoltia punctata (G. Mey.) Les & D. J. Crawford haplotypes. More recently,Fu et al. (2020) developed 70 microsatellite primers within coding regions for L. gibba L. It is important to continue developing and reporting new microsatellite markers as populations can differ in which markers function (e.g. due to null alleles) and are polymorphic (Chapuis & Estoup, 2007).

Here, we report on the successful development of 18 and 16 new microsatellite markers, respectively, for two commonly studied and widespread duckweed species: the common duckweed Lemna minor L. and the greater duckweed Spirodela polyrhiza (L.) Schleid. A small subset of these microsatellite primers was used to differentiate genotypes in our experimental studies on evolutionary coexistence (Hart et al., 2019). In addition, we report genotyping results using these markers on individuals sampled in Europe and the United States of America (USA). We thus provide new tools and evidence that they function, which can be utilized by the growing community of duckweed researchers (Laird & Barks, 2018).

Materials and methods

Results and discussion

We successfully developed 18 L. minor and 16 S. polyrhiza microsatellite primers (Table 1 and Table 2) which were tested on samples of duckweeds from Europe or Western Pennsylvania (USA). Some markers were more successful than others (Table 1 and Table 2). All markers amplified in some samples; of these, all 18 L. minor primers and 12 of the 16 S. polyrhiza primers amplified polymorphic loci, having more than one allele. Moreover, these polymorphic loci differed in product length and can be used in multiplex reactions to increase efficiency and lower genotyping costs. We also found that some loci were much more polymorphic than others. For L. minor, these included loci amplified by primers LmR.5.C, LmR.8.B, LmR.15.A, LmR.15.B, LmR.15.C and LmR.26.B, some of which showed high allele richness even when tested on only 28 samples (Table 1). For S. polyrhiza these included loci amplified by primers Sp.1467, Sp.7286, Sp.7814, and Sp.Pso31 (Table 2). Monomorphic loci may still be useful in different duckweed populations (Chapuis & Estoup, 2007). Many microsatellite loci also showed heterogeneity (Supplemental Tables S1 and S2), which helps make the primers more informative to distinguish genotypes. We note that some primers developed in one continent were not tested on samples from the other continent (see caption in Supplemental Tables S1 and S2); we suspect these primers will work across continents given patterns observed in the others, but this remains to be tested.

Comparing between species, we saw that S. polyrhiza has lower allelic and genotypic richness across most primers, although we also tested fewer samples of this species. This is consistent with our own recent large-scale sampling (Hobble et al. In preparation) as well as other studies using different genotyping methods, that similarly found low genetic diversity in S. polyrhiza (Bog et al., 2015; Feng et al., 2017; Xu et al., 2015). It has been hypothesized that this low genetic variation in S. polyrhiza is due to its low mutation rate (Xu et al., 2019). In addition, primers differed greatly in average observed heterozygosity, but species had similar mean heterozygosities (0.256 for L. minor and 0.283 for S. polyrhiza). Given that our sampling was designed to find unique genotypes (shallow and widespread) and not characterize populations, we limit our discussion of population genetic indices. The primers we developed can help researchers address various ecological and evolutionary questions as well as better identify and catalogue genotypes for the expanding applied uses of duckweed in bioremediation, biofuel production and as a forage crop.

Supplemental data

Supplemental Table S1: Lemna minor sample collection sites and allele lengths. Supplemental Table S2: Spirodela polyrhiza sample collection sites and allele lengths.

Acknowledgements

We thank Walter Lämmler for sharing duckweed lineages from the Landolt Duckweed Collection. We are grateful to the former Levine Plant Ecology Group at ETH Zurich and the Turcotte Lab for their assistance in maintaining collections. We thank ETH Zurich Genetic Diversity Center and Mary Janecka for help troubleshooting primer development. M.M.T. was supported by the ETH Zurich Center for Adaptation to Changing Environments and now by an NSF grant DEB-1935410.

Author contributions

All authors contributed to testing, optimizing, and evaluating marker data, and contributed to reviewing the manuscript. JEK and MMT wrote the initial draft of the manuscript.

Conflict of interest statement

The authors have no conflicts of interest to report.