DOI: 10.5061/dryad.pzgmsbd17
title: Data from: Persistence in a fragmented habitat: Genetic diversity and pollination patterns in <em>Asclepias viridiflora</em>

description: 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 (HE, 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.
   
description: We extracted DNA from N = 102 <em>Asclepias
 viridiflora</em> 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 <em>Asclepias
 syriaca</em> (O’Quinn and Fishbein, 2009; Kabat et al., 2010) and
 tested for viability in <em>A. viridiflora</em> 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 <em>A.
 syriaca</em> (Weitemier et al., 2019). Briefly, the chromosomal
 genome assembly of <em>A. syriaca</em> 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).
description: # Data from: Persistence in a fragmented habitat: Genetic diversity and
 pollination patterns in *Asclepias viridiflora* Dataset DOI:
 [10.5061/dryad.pzgmsbd17](https://doi.org/10.5061/dryad.pzgmsbd17) ##
 Description of the data and file structure DNA was extracted from dried
 leaves collected from 109 flowering individuals and 179 progeny. DNA
 samples were genotyped at 19 microsatellite loci.  ### Files and variables
 #### File: ASVI_Genotypes_10.10.25.xlsx **Description:** Microsatellite
 genotypes: This table (for mother and progeny) is a microsatellite
 genotype matrix, listing each plant’s genotype at various microsatellite
 loci, showing allele sizes for each locus. Rows (AV103, AV104, etc):
 Individual plants (each row is one plant). Columns (Asyr_B5, B102, C4,
 etc.): Microsatellite loci — specific locations on the DNA where
 microsatellites are analyzed. Values: Allele sizes at each locus, measured
 in base pairs (bp). ##### Variables * Microsatellite locus names *
 Individual plant IDS * Allele sizes in base pairs