DOI: 10.5061/dryad.qz612jmdq
title: Closely related tree species support distinct communities of seed-associated fungi in a lowland tropical forest

description: Previous theoretical work has highlighted the potential for natural
 enemies to mediate the coexistence of species with similar life-histories
 via density-dependent effects on survivorship. For plant pathogens to play
 this role, they must differ in their ability to infect or induce disease
 in different host plant species. In tropical forests characterized by high
 diversity, these effects must extend to phylogenetically closely related
 species pairs. Mortality at the seed and seedling stage strongly
 influences the abundance and distribution of tropical tree species, but
 the host preferences and spatial distributions of fungi are rarely
 determined. We examined how host species identity, relatedness, and seed
 viability influence the composition of fungal communities associated with
 seeds of four co-occurring pioneer trees (Cecropia insignis, C. longipes,
 C. peltata, and Jacaranda copaia). Seeds were buried in mesh bags in five
 common gardens in the understory of a lowland tropical forest in Panama
 and retrieved at intervals from 1-30 months. A subset of the seeds in each
 bag was used to determine germination success. One half of each remaining
 seed was tested for viability; the other half was used to culture and
 identify seed-infecting fungi. Seeds were infected by fungi after burial.
 Although fungal communities differed in viable vs. dead seeds, and across
 burial locations, community composition primarily varied as a function of
 plant species identity (30.7% of variation in community composition vs.
 4.5% for viability and location together), even for congeneric Cecropia
 species. Phylogenetic reconstruction showed that relatedness of fungi
 mostly reflected differences between Jacaranda (Bignoniaceae) and Cecropia
 (Urticaceae). Although the proportion of germinable seeds decreased
 gradually over time for all species, intraspecific variation in survival
 was high at the same location (e.g., ranging from 0-100% for C. peltata)
 suggesting variable exposure or susceptibility to seed pathogens.
 Synthesis: Our study provides evidence under field conditions that
 congeneric tree species with similar life-history differ markedly in
 seed-associated fungal communities when exposed to the same soilborne
 fungi. This is a critical first step supporting pathogen mediated
 coexistence of closely related tree species.
description: Seeds of all species were collected from at least five different
 maternal sources in 2012-2014 in accordance with seasonal variation in
 fruiting phenology. Mature infructescences of
 <em>Cecropia</em> spp. were collected directly from trees or
 from freshly fallen material beneath their crowns. <em>Jacaranda
 copaia </em>trees can attain 35 m in height when mature, precluding
 collection of ripe fruits directly from the crown. For this species we
 used recently fallen seeds estimated to have been on the soil surface for
 a short time (from minutes to a few hours). Seeds of <em>J.
 copaia</em> were separated from dehiscent woody seed pods and the
 wings were cut from each seed. Seeds of <em>Cecropia</em>
 (technically fruits; Lobova et al. 2003) were removed from whole
 infructescences. Immediately after collection seeds were cleaned with tap
 water and 0.7% sodium hypochlorite for 2 min to remove small pieces of
 pulp. Clean seeds of all species were air-dried at room temperature
 (∼22<sup>o</sup>C) for several days under low red:far red
 irradiance. We established five common garden plots (9
 x 15 m each) in lowland forest at Barro Colorado Island (BCI). Common
 gardens were at least 350 m apart (average distance 800 m), and were
 located under closed-canopy forest to limit seed germination (see Table S1
 for a summary of common garden characteristics; see also Zalamea et al.
 2015, Ruzi et al. 2017, and Sarmiento et al. 2017). They were placed in
 multiple forest- and soil- types that varied slope and aspect (Baillie et
 al. 2006). No adults of the study species occurred within 20 m of the
 garden edges. Dry seeds from all maternal sources of each species were
 thoroughly mixed, pooled, and placed into seed bags. Each seed bag
 contained 45 seeds of one species mixed with 10 g of sterile forest soil
 (previously autoclaved at 121<sup>o</sup>C for 2 h). Each set
 of soil and seeds was enclosed in a nylon mesh bag (pore size = 0.2 mm)
 and covered with aluminium mesh (pore size = 2 mm) to exclude seed
 predators but not microorganisms. Twenty-eight bags per plant species were
 distributed among four replicate subplots per garden, where they were
 buried 2 cm below the soil surface and 40 cm apart. A total of 140 seed
 bags and a total of 6300 seeds per species were included in the burial
 experiment. Seeds of each species were buried less than a month after seed
 collection, matching the natural phenology of fruit production and seed
 dispersal. We evaluated initial infection rates and confirmed the
 viability status of the seed lot before burial by processing 400 fresh
 seeds (i.e., seeds that were not buried) per species (200 seeds for
 germination; 200 seeds for fungal culture).  Seed
 processing and retrieval after burial:<br> Four seed bags for each
 species in each garden were collected at each of seven time points: 1, 3,
 6, 12, 18, 24, and 30 months after burial. Seeds were retrieved by rinsing
 the contents of each bag with tap water over a sieve. Seeds were
 partitioned for tests of (i) germination (10 seeds per seed bag) and (ii)
 seed viability and identification of seed-associated fungi (10 seeds per
 seed bag). In general, seeds were recovered intact, precluding lethal
 germination as a source of seed mortality. In 2% of cases, we recovered
 fewer than 20 seeds per bag, suggesting that small invertebrate seed
 predators penetrated or chewed the nylon mesh. In these cases, the
 recovered seeds were partitioned evenly among germination and culturing
 approaches. We did not find 28 (i.e., 5%) of bags that were buried, likely
 reflecting displacement by small mammals or rain events. To record the
 time series of seed germination for each species, 10 seeds from each seed
 bag were placed in a Petri dish lined with paper towel, moistened with
 sterile distilled water, and sealed with two layers of Parafilm® (Zalamea
 et al. 2015; 2018). Seeds were incubated in a shade house on BCI under ca.
 30% sunlight, high red:far-red irradiance (ca. 1.4), and ambient
 temperature (23-30°C). Germination (i.e., radicle protrusion) was scored
 over six weeks.
description: Species Plant species used in the study
 Burial_duration Recovery
 time from burial plots. There are 7 different burial durations: 1, 3, 6,
 12, 18, 24, and 30 months. 0 corresponds to fresh seeds (i.e., never
 buried) Burial_location Initials of
 the plots where seeds were buried (A=AVA, D=DRAYTON, H=25HA, P=PEARSON,
 Z=ZETEK). NA for fresh seeds  Replicate 1 - 4 replicate
 bags in burial experiment. NA for fresh seeds. 
 Fit Seed status after burial
 - records if a seed is in good shape to go through the different analyses
 (1=fit, 0=unfit) 
 Germination Score for
 seed germination (1=germinated; 0=not germinated)