Habitat loss and fragmentation are expected to erode genetic variation and contribute to genetic differentiation by limiting gene flow among isolated habitat patches. Yet, isolated plant populations often retain genetic diversity and exhibit limited population genetic structure. Using an assessment of genetic diversity and pollination patterns in Asclepias viridiflora across 6400 hectares of fragmented prairie remnants in western MN (USA), we aimed to (1) characterize the spatial genetic structure of A. viridiflora in a fragmented landscape and (2) evaluate if pollen movement contributes to gene flow among isolated patches. This spatial scale of our sampling area allowed us to detect pollination events over several kilometers, if they occurred. We mapped, sampled, and genotyped 102 sexually mature plants and 179 of their progeny using 19 microsatellite loci, including several new loci discovered using genomic tools. The unusual pollination system of milkweeds aided paternity assignment since we could genotype multiple offspring with shared paternity. We assessed genetic diversity and genetic structure and used paternity analysis to characterize spatial mating patterns using high-resolution mapping. Asclepias viridiflora in this fragmented landscape retained high levels of genetic diversity (H_E, expected heterozygosity, averaged 0.69 across loci) and minimal population genetic structure, indicating genetic degradation has not occurred. Paternity assignment revealed that most (74.4%) pollination events involved pollen transfer over relatively short distances (i.e., within 70 m). However, surprisingly, long-distance pollinations (up to 9 km) also occurred, and there was evidence of pollen flow from outside our study area. We found little evidence for population structure or reduced genetic variability across scattered clusters of Asclepias viridiflora within our study area. Occasional long-distance pollination events, which we identified, may be sufficient to maintain reproductive connectivity over substantial areas despite severe habitat fragmentation. Our findings suggest that even small, spatially isolated clusters of plants may not be reproductively isolated and may contribute to the persistence of fragmented plant populations.   

We extracted DNA from N = 102 Asclepias viridiflora individuals and N = 179 progeny from the harvested pods using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The DNA concentration and purity for each extracted sample were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). We then identified and optimized nineteen microsatellite primer pairs that were found to be polymorphic and amplified consistently. Twelve of these primers were previously developed for Asclepias syriaca (O’Quinn and Fishbein, 2009; Kabat et al., 2010) and tested for viability in A. viridiflora in a previous study (Kim et al., 2015). The remaining seven primer pairs were identified by one of the authors (JDM) using the published genome of A. syriaca (Weitemier et al., 2019). Briefly, the chromosomal genome assembly of A. syriaca from MilkweedBase (https://milkweedbase.org) was surveyed for microsatellite loci using QDD v3.1.2 (Méglecz et al. 2014). Geneious Prime v2023.2.1 was utilized to visualize candidate loci for primer identification to amplify trinucleotides with perfect repeats (Kearse et al., 2012). Details for the new primers used for genotyping are shown in Table 1.

Polymerase chain reaction (PCR) was then conducted in 10µl reaction volume with the following components: 0.2mM dNTPs, 1X Promega PCR buffer, 0.2µM M13 dye-labeled primer, 0.2µM reverse primer, 0.02µM M13-tagged forward primer, 0.5U Promega Taq polymerase, and approximately 20 ng template DNA. PCR reactions were performed with an initial denaturation temperature at 95°C for 5 min, followed by 35 cycles of 94°C denaturation for 30 sec, 50-55°C annealing for 30 sec, 72°C extension for 30 sec, and a final extension of 72°C for 4 min. The annealing temperature was 55°C for all primers except ASF2, ASF9, ASH8, and AS94, which had an annealing temperature of 52°C. Fragment sizes from 1.0µL of each PCR product were analyzed using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA), using ALEXA size standard (Maddox and Feldheim, 2014). Microsatellite genotypes were scored using the Microsatellite Analysis web application on Thermo Fisher Connect (Thermo Fisher Scientific).