Breeding system characterization - manual pollination
In order to determine the breeding system of the 14 Calceolaria species studied here (Table 1: Fig. 1A) a hand-pollination experiment was performed in greenhouse and field conditions. Seeds recollection was carried out by the authors during the 2014-15 springer-summer campaigns, at different unprotected sites throughout the country. Likewise, the field work was realized in populations that inhabit in unprotected areas, for which no government permits are required. Species identification was carried out following the review and description of Chilean species published in the Calceolaria genus monograph [17]. In this occasion, no herbarium material was deposited in any national repository.
In the greenhouse, four species were grown from seeds collected in the field in previous seasons. A total of 1500 seeds per species (thirty seeds per capsule per species) where chosen, sown and germinated in fifty pots, and then grown under daily watering and constant temperature (18°C). After germination, plants were transplanted into individual 1L-pots and grown until flowering. In the field and during the summer seasons 2016-2018, thirty plants per species (ten species) were selected and covered with a mesh at bud state. Then, both in the greenhouse and the field, six buds per plant were chosen and one of the following treatments was assigned randomly to two of those flowers a) hand self-pollination (HS), emasculated flowers were pollinated using their own pollen; b) hand cross-pollination (HC), emasculated flowers were pollinated with pollen of a plant donor situated at least 1 meter apart (in the field) or from plants grown from different seed capsules (in the greenhouse). After performing the treatments, the flower was kept isolated from further pollination with the mesh bag, and the flowers were left to develop until fructification. For each species, we quantified pollination success as in [30], using seed-set per plant to calculate an average self-incompatibility index (ISI) where, the seed-set values are the average number of seeds per fruit per treatment (ISI=HS/HC). ISI index ranges from 0 to 1, where species that present values close to zero are considered self-incompatible, while when the index values tend to one the species is considered to harbor a greater degree of self-compatibility. In our case, species where at most 20% of the seeds could be produced by self-pollination (ISI ≤ 0.2) were considered as self-incompatible (SI), while the remainders were considered as self-compatible (SC) [31].
Floral traits measurements
Thirty to 100 flowers per species were chosen for floral trait measurements. To do this, each flower was photographed from a frontal view with a camera Sony and three floral traits were measured from pictures using ImageJ 1.46r (http://rsb.info.nih.gov/ij/, Fig. 1B). We chose floral traits based on their importance for pollinator attraction (corolla area, CA; elaiophore area, EA) and the effect on the mechanical ability of the plant to self-pollinate (herkogamy, H).
In order to reduce the dimensionality and avoid correlation among floral traits, a principal component analysis (PCA) was performed on log-modified measurements, and the scores of the two principal components were used in the posterior statistical analysis. To test for potential differences in floral morphology (i.e, PCA scores) between species with different ISIs, we adjusted GLMs using a Gaussian distribution in R [32]; analyses with the raw traits are presented in the Supplementary Information). Here, our expectation was that ISI was positively and significantly explained by trait measures.
DNA extraction and genotyping
Three leaves per plant were collected and preserved in silica gel (a total of 420 individuals across the 14 species) either from field- or greenhouse-grown plants. DNA extractions were done on ~20 mg of dry material using a modified cetyltrimethyl ammonium bromide (CTAB) protocol [33]. Fifteen microsatellites previously described by [34] were tested on three samples per species. The PCR mix (10ul) was composed of: 10x PCR buffer, 5 mM MgCl2, 2.5 mM dNTP (Invitrogen), 5 mM forward primer, 5mM reverse primer, 5mM fluorescently labeled M13 universal primer, 1U GoTaq G2 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), 1μL BSA, 20 ng/ul template DNA and H2O. PCR cycling conditions were set as follows: 5 minutes of denaturation at 95oC, followed by 30 cycles of 1 minute at 95oC, 1 minute of annealing at 58oC, 1-minute extension at 72oC and 10 minutes of final extension at 72oC. Only those primers that showed amplification (9/15; see Results) were genotyped on ten individuals per species. PCR products were genotyped on a 3130xl Genetic Analyzer (Applied Biosystems, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA) at the Pontificia Universidad Católica de Chile.
We analyzed all genotypes using GeneMapper v.5 (Applied Biosystems). We checked for null alleles using Microchecker v.2.2.3 [35]. We tested for departures from Hardy-Weinberg equilibrium (HWE) using GenAlEx 6.5 [36]. We quantified genetic variation using several genetic measures: the number of alleles per locus (Na), observed (Ho) and expected (He) heterozygosity, and the fixation index FIS. Here, our expectation is that SC species will display significantly larger genetic diversity than SI species. In order to determine statistical differences in the genetic diversity parameters between SI and SC species, we ran a Kruskal-Wallis test in R [32] between values for the two groups. Finally, to determine a relationship between the level of self-incompatibility and the genetic diversity of the species, we calculated a Pearson correlation between ISI values and each genetic parameter, in R.