DOI: 10.5061/dryad.zs7h44jdk
title: Data from: Multiple introductions, polyploidy and mixed reproductive strategies are linked to genetic diversity and structure in the most widespread invasive plant across Southern Ocean archipelagos

description: Biological invasions in remote areas that experience low human activity
 provide unique opportunities to elucidate processes responsible for
 invasion success. Here we study the most widespread invasive plant species
 across the isolated islands of the Southern Ocean, the annual bluegrass,
 Poa annua. To analyze geographic variation in genome size, genetic
 diversity, and reproductive strategies, we sampled all major sub-Antarctic
 archipelagos in this region and generated microsatellite data for 470
 individual plants representing 31 populations. We also estimated genome
 sizes for a subset of individuals using flow cytometry. Occasional events
 of island colonization are expected to result in high genetic structure
 among islands, overall low genetic diversity, and increased
 self-fertilization, but we show that this is not the case for Poa annua.
 Microsatellite data indicated low population genetic structure and lack of
 isolation-by-distance among the sub-Antarctic archipelagos we sampled, but
 high population structure within each archipelago. We identified high
 levels of genetic diversity, low clonality, and low selfing rates in
 sub-Antarctic P. annua populations (contrary to rates typical of
 continental populations). In turn, estimates of autogamy declined in
 populations as genetic diversity increased. Additionally, we found that
 most P. annua individuals are likely tetraploid and that only slight
 variation exists in genome size across the Southern Ocean. Our findings
 suggest multiple independent introductions of P. annua into the
 sub-Antarctic, which promoted the establishment of genetically diverse
 populations. Despite multiple introductions, the adoption of convergent
 reproductive strategies (outcrossing) happened independently in each major
 archipelago. The combination of polyploidy and a mixed reproductive
 strategy likely benefited P. annua in the Southern Ocean by increasing
 genetic diversity and its ability to cope with the novel environmental
 conditions.
description: All P. annua individuals (n =
 470) were genotyped using the set of nine microsatellite loci previously
 developed by us (Mairal et al., 2022). PCR amplification of
 microsatellites was performed in two multiplex PCR reactions (see Mairal
 et al., 2022 for details). Given the sensitivity of multiplex reactions to
 genotyping errors (e.g., allele dropout), we adopted conditions
 recommended to minimize such errors, including the use of a standardized
 DNA concentrations (~100 ng/µL), special buffers for multiplexing, and
 selection of primers with similar annealing temperatures (Guichoux et al.,
 2011). All PCR reactions were carried out in 15 μL reaction volumes
 containing 1.5 μL diluted template DNA, 7.5 μL KAPA2G Fast Multiplex Mix
 (Kapa Biosystems, Cape Town, South Africa), 1.5 μL primer mix (2μM), and
 4.5 μL distilled H2O. Samples were amplified using the following PCR
 conditions: 3 min of denaturation at 95°C, 30 cycles of 15 sec of
 denaturation at 95°C, 30 sec at multiplex-specific annealing temperature
 (see Mairal et al., 2022), and 25 sec of elongation at 72°C, and a final
 extension for 10 min at 72°C. Each 96‐well PCR plate contained 93 samples plus two
 randomly selected technical replicates and one negative control (H2O). All
 technical replicates (n=19) were used to count mismatches for scored
 genotypes and thus to estimate repeatability at each locus. Gel capillary
 electrophoretic separation of amplified fragments was carried out at the
 Central Analytical Facility, Stellenbosch University (Stellenbosch, South
 Africa). All loci were scored using GeneMarker software v 2.6.4
 (SoftGenetics LLC, State College, Pennsylvania, USA) using the LIZ 500
 size standard. We applied a semi-automatic genotype scoring for each
 allele, followed by manual correction when needed, to reduce scoring
 errors (Dewoody et al., 2006). Allele scoring was combined across
 microsatellite loci to generate a multi-locus phenotype (i.e., allele
 variation with unknown dosage) for each individual. In agreement with
 previous observations of tetraploid species (Nannfeldt, 1937; Frenot et
 al., 1999), all loci displayed up to four alleles per
 individual. Unbiased analyses of microsatellite data in
 polyploids are hindered by factors such as unknown allele dosage,
 genotyping errors, and unknown mode of inheritance (Meirmans et al.,
 2018). Although some approaches have been developed to address the first
 two factors (e.g., Bruvo et al., 2004; Dufresne et al., 2014), not knowing
 modes of inheritance remains a major challenge when calculating population
 genetic metrics from allele frequency data (Meirmans et al., 2018). These
 challenges stem from the fact that the observed allelic patterns in
 allopolyploids can vary, even among microsatellite loci within species, as
 the genetic distance between parental taxa strongly determines the level
 of chromosome pairing in the resulting polyploid (Chester et al., 2012).
 As a result, a given polyploid species may not fit any of the extremes
 traditionally defined as allo- and autopolyploids, which may bias
 population genetic inferences based on allele-frequency calculations (see
 reviews in Dufresne et al., 2014; Meirmans et al., 2018).
 Previous work suggests that P. annua originated
 from a relatively recent hybridization event between two closely related
 species, P. infirma and P. supina (Nannfeldt, 1937; Tutin, 1952). As such,
 P. annua would fit Stebbins’ (1947) definition of segmental allopolyploidy
 (i.e., some degree of homology may exist between the chromosomes of
 parental subgenomes), and cytological and phylogenetic evidence appear to
 support this view (Hovin, 1958; Soreng et al., 2010). We might therefore
 expect P. annua to follow an intermediate model of disomic inheritance at
 certain loci, typically expressed as fixed heterozygosity in allelic
 profiles (e.g., García-Verdugo et al., 2013), and tetrasomic inheritance
 for loci in chromosomes showing meiotic pairing or occasional
 recombination between the parental subgenomes (Hovin, 1958). To improve
 the accuracy of our analyses, we tested this expectation by examining how
 inbreeding coefficients (FIS) varied across loci. Under a model of
 complete disomic inheritance, all loci in an allotetraploid are expected
 to show fixed heterozygosity, leading to negative FIS values, while
 deviations from this expectation would suggest tetrasomic inheritance
 (Meirmans & Van Tienderen, 2013). To test this, we calculated
 inbreeding coefficients at the ‘locus × population’ level by running the
 approach implemented in GenoDive v.3.0 (Meirmans, 2020) for testing
 deviations of allele frequencies from those expected under Hardy-Weinberg
 equilibrium conditions. Unlike our previous study that focused on
 extensive sampling of P. annua in the Prince Edward archipelago (Mairal et
 al., 2022), we found that only three loci in the present dataset (i.e.,
 Poa3, Poa8 and Poa288) had significantly negative FIS values (Table S3,
 Supplementary Material), whereas the remaining loci tended to have
 positive values, as would be expected for a predominantly selfing species
 (Mengistu et al., 2000). To segregate the allelic composition of isoloci
 (i.e., loci Poa3, Poa8 and Poa288), we used the function
 ‘allele.correlations’ implemented in the package POLYSAT v 1.6 (Clark
 & Jasieniuk, 2011) using R 3.2.5 (R Core Team, 2019). However,
 this approach failed to confidently assign alleles to isoloci, likely
 because of population genetic structure (see Results) or because allele
 sizes between parental subgenomes were shared (Clark & Schreier,
 2017). Considering these findings, we constructed two datasets to
 accommodate the heterogeneity among our microsatellite loci in all
 subsequent analyses, one containing the allelic information generated for
 all nine loci (hereafter “full dataset") and another set containing
 only the data for the six loci not following a pattern of disomic
 inheritance (hereafter “partial dataset”).