Supporting Information Methods S1
Population samples
We sampled populations throughout much of the range of Eichhornia paniculata,
including N.E. Brazil, Jamaica and Cuba in 2005, 2007 and 2008, respectively
(Supporting Information Table S1). Mature fruit were obtained from up to 20 openpollinated maternal families in each population. We also estimated the frequencies of the
three floral morphs and the size of populations. Material from both Mexico and
Nicaragua was collected as a bulk sample from an unknown number of families and
therefore these two populations were not included in our analyses of population
differentiation. We germinated seed and grew plants for DNA extraction under
glasshouse conditions at Toronto following standard protocols.
Marker development
We developed nuclear DNA markers based on EST sequences collected from a cDNA
library from leaf tissue from single plants. We purified poly-adenylated RNA from total
RNA using the Ambion Micro Poly(A) Purist kit and reverse transcribed the mRNA
using the InVitrogen Superscript cDNA synthesis kit. We cloned cDNA into E. coli and
sequenced approx. 480 clones. From these clones, we selected sequences that aligned
well to known nuclear sequences from other plants and designed primers to amplify both
coding regions and intron sequence when possible. We initially tested the primers by
sequencing the loci in highly inbred individuals with the rationale that there should be
few heterozygous sites. Loci with heterozygous sites were excluded as likely paralogs.
From these screens we chose 10 EST-derived nuclear markers of the following lengths;
EP0001- 530bp, EP0141 - 825bp, EP0143 - 846bp, EP0144 - 822bp, EP0188 - 383bp,
EP0222 - 481bp, EP0267 - 561, EP0285 - 827bp, EP0314 - 752bp and EP0317 - 651bp.
Amplification and sequencing
We extracted DNA from each of 229 individuals which was used to PCR amplify the 10
loci in each individual. We sequenced both forward and reverse strands with an ABI
3730XL fluorescent-based capillary sequencer at the Centre for Applied Genomics
facility at Sick Kids Hospital, Toronto, Ontario, Canada. We assembled and aligned
sequences using Sequencher 4.7 and edited chromatographs and alignments manually to
ensure that all base calls and polymorphisms, including heterozygotes, were reliably
scored
Sequence analysis
We generated neighbour networks using the program SplitsTree. We estimated distance
among individuals as an uncorrected number of differences and calculated the network
using the NeighborNet method. Support for the network was estimated with 10,000
bootstrap replicates and nodes with greater than 70% support are indicated on the
network (Fig. 3). We displayed the resulting network using one individual per population
that was randomly sampled using a custom computer script. In addition, we generated the
network for all 229 individuals to ensure that the network illustrated for single
individuals per population (Fig. 3) was representative of patterns across all individuals.
Using the program SITES we calculated pairwise FST for each of the 10 loci
across all populations and groups (Caribbean, Brazilian trimorphic outcrossing, Brazilian
monomorphic selfing). We calculated mean FST by averaging pairwise FST estimates
across all loci. For each pairing the program SITES also provided the number of
polymorphism within each population or group, the number of polymorphisms
segregating in both, and the number of fixed differences between them.