DOI: 10.5061/dryad.2z34tmpkm
title: Germination and epiphytic seed fungi on Festuca roemeri and Danthonia californica

description: Demographic studies measure drivers of plant fecundity such as seed
 production and survival, but few address environmental drivers of seed
 viability, such as germination and dormancy. Variation in climate and seed
 type may both directly and indirectly alter seed germination via altered
 fungal pathogen abundance. We examined seed germination and microbial
 communities of seeds of Danthonia californica, which are either
 chasmogamous (external, wind-pollinated) or cleistogamous (internal,
 self-fertilized) and Festuca roemeri, which are solely chasmogamous. Seed
 populations were sourced across environmental gradients. We tested
 germination and characterized seed fungal community structure, using
 high-throughput sequencing. For F. roemeri, maternal plant significantly
 influenced germination as did climate and pathogens; germination increased
 in wetter, cooler sites. For D. californica, the main drivers of
 germination were maternal plant, seed type and pathogens; on average, more
 chasmogamous seeds germinated. Fungal composition depended on seed type,
 with fewer fungi associated with cleistogamous seeds. Seed fungal
 composition varied with climate, plant density and mean proportion of
 seeds germinated. Putative pathogens that were negatively correlated with
 germination were more abundant for both Danthonia and Festuca chasmogamous
 seeds than Danthonia cleistogamous seeds. In D. californica, cleistogamous
 and chasmogamous seeds contain vastly different fungal communities.
description: Germination: Sterile Petri dishes (90 mm) were lined with sterile
 filter paper and up to 25 seeds per dish (range 12-25) were arranged to
 reduce contact, with <i>D. californica</i> seeds separated
 into different dishes according to their seed type (up to 25 seeds/seed
 type/maternal family) . Hands, forceps and countertop were cleaned with
 alcohol between seed families to reduce carryover of seed fungi. All petri
 dishes were watered to dampness with distilled water, placed immediately
 into cold storage at 4°C for four weeks to break seed dormancy, and
 watered when necessary to keep damp but not wet. After cold stratification
 the seeds were kept at room temperature. Germinated seeds were recorded
 and removed every two days and petri dishes re-watered as
 needed. Seed epiphytic
 fungi<strong>:</strong> When the number of germinating seeds
 approached zero (approximately 6 weeks after removal from 4°C), all ungerminated
 seeds
 (N=5,521), were examined with a dissecting microscope.
 Microbes on the seeds were categorized as fungi, bacteria, or
 unknown. When possible, fungi were keyed to genus. A small subset of
 seeds that germinated (N=27) were also examined
 for microbes. To identify the fungi growing on
 ungerminated seeds to species and to verify the accuracy of
 morphology-based genus-level identifications, we isolated the fungi on
 culture media before extracting and sequencing DNA. The seeds were
 cultured on diameter 90 mm plates of 2% water agar medium supplemented
 with 20 mL/L of an antibiotic solution (5 g/L of penicillin, 5 g/L
 streptomycin, and 1.5 g/L chloramphenicol) to inhibit bacterial growth.
 Fungi growing from the cultured seeds were isolated and maintained on 2%
 malt agar plates. A representative sample of each morphotype
 was subcultured in 2% malt
 liquid media and incubated at approximately 25 C for 1-2 weeks to ensure
 sufficient mycelial growth for DNA extraction. DNA extraction
 and Sanger sequencing protocols followed Thomas et al. (2016). Sequences
 were then aligned against the UNITE fungal database using the BLAST
 algorithm (Altschul<i> et al.</i>, 1990; Abarenkov<i> et
 al.</i>, 2010) to obtain species hypotheses (Koljalg<i> et
 al.</i>, 2013).
description: Please see the associated ReadMe files.