DOI: 10.5061/dryad.m905qfv4f
title: On the de novo emergence of ecological interactions during evolutionary diversification: A conceptual framework and experimental test

description: Ecological interactions are crucial to the structure and function of
 biological communities, but we lack a causal understanding of the forces
 shaping their emergence during evolutionary diversification. Here we
 provide a conceptual framework linking different modes of diversification
 (e.g. ecological diversification), which depend on environmental
 characteristics, to the evolution of particular interactions (e.g.
 resource partitioning) in asexual lineages. We tested the framework by
 examining the net interactions in communities of Pseudomonas aeruginosa
 produced via experimental evolution in nutritionally simple (SIM) or
 complex (COM) environments by contrasting the productivity and competitive
 fitness of whole evolved communities relative to their component isolates.
 As expected, we found that nutritional complexity drove the evolution of
 communities with net positive interactions whereas SIM communities had
 similar performance as their component isolates. A follow-up experiment
 revealed that high fitness in two COM communities was driven by rare
 variants (frequency &lt;0.1%) that antagonized PA14, the ancestral
 strain and common competitor used in fitness assays. Our study suggests
 that the evolution of de novo ecological interactions in asexual lineages
 is predictable at a broad scale from environmental conditions. Further,
 our work demonstrates that rare variants can disproportionately impact the
 function of relatively simple microbial communities.
description: <em>Evolution experiment and generation of isolate library
 and isolate mixes</em> The evolution experiment
 that produced the evolved communities used in this study is described in
 detail in Schick and Kassen (2018). Briefly, 60 replicate lines of
 <em>P. aeruginosa</em> strain PA14 were independently
 propagated in either COM or SIM environments. COM environments consisted
 of synthetic cystic fibrosis media (SCFM), which is a defined media
 composed of the essential chemical elements of sputum from cystic fibrosis
 patients (Palmer et al. 2007). The SIM environment consisted of M9 media
 (7.78 g/L Na<sub>2</sub>HPO<sub>4</sub>, 3 g/L
 KH<sub>2</sub>PO<sub>4</sub>, 0.5 g/L NaCl, 1 g/L
 NH<sub>4</sub>Cl, 493 mg/L MgSO<sub>4</sub>, 14.7
 mg/L CaCl<sub>2</sub>) supplemented with 4 g/L of glucose as
 the sole carbon source. Half of the founding strains in each environment
 contained a chromosomally inserted genetic marker,
 <em>lacZ</em>, such that they could be distinguished from
 unmarked strains on agar plates supplemented with x-galactosidase (X-Gal).
 Populations were propagated in 1.5 mL batch cultures at 37°C with daily
 serial transfers of 1% of the previous day’s culture into fresh media in
 unshaken 24-well cell culture plates (Costar® 24-well Clear TC-treated
 Multiple Well Plates), resulting in ~6.6 generations occurring each day.
 Microcosms were left unshaken to promote spatial heterogeneity, which may
 in turn promote diversification. Endpoint communities from each line were
 stored in 20% glycerol at –80°C after 32 days of experimental evolution
 (~220 generations). Unless otherwise stated, bacterial cultures in this
 study were grown in unshaken 1.5 mL microcosms at 37°C in 24-well culture
 plates. We generated working stocks of 8 randomly
 selected lines from each environment by reviving the endpoint lines from
 Schick and Kassen (2018) in Lysogeny Broth (LB) microcosms and allowing
 them to grow for 24 hrs before storing them in in glycerol at –80°C. We
 generated a library of isolates from each line by first plating a sample
 from the frozen working stock onto an agar plate (SIM media + 1.3% agar
 (w/v); Oxoid Bacteriological Agar). We then randomly selected 22 colonies
 from each plate that we grew individually in LB before storing the
 resulting cultures separately in glycerol at –80°C. We also generated
 mixes of all 22 isolates from each of the 8 examined COM lines (henceforth
 referred to as “isolate mixes”) by growing all 22 isolates from each line
 in individual LB microcosms for 24 hrs and mixing the resultant cultures
 in equal proportions before storing the mixes in glycerol at
 –80°C. <em>Cellular productivity
 assay</em> We revived evolved communities and
 isolates by transferring a small volume of frozen stock from each of the
 stored cultures into either LB or SIM microcosms. We used LB instead of
 SCFM to represent the complex environment for this assay and the
 <em>Competitive fitness assay</em> (below) for its ease of
 preparation. Like SCFM, LB is a complex medium with many carbon and
 nitrogen sources. After 24 hrs of growth, we transferred 1% of the mixture
 into a fresh microcosm containing the same media for another 24-hr growth
 period. We then diluted the cultures in SIM media by a factor of
 10<sup>6</sup> and plated 100 μL from each on SIM agar plates
 to ensure they would produce a countable number of colonies. After 48 hrs
 of growth at 37°C, we counted the number of colonies on each plate, which
 we used as our measure of cellular productivity. We measured the
 productivity of all 22 isolates from each line in each environment without
 replication. Isolates were measured without replication because we chose
 to maximize the diversity of isolate measured instead of making repeated
 measurements on individual isolates. We measured the productivity of each
 community in each environment with 8-fold replication.
 Three productivity measurements in LB were lost due to
 contamination or dilution errors. Two were isolates from different COM
 lines and one was an isolate from a SIM line.
 <em>Competitive fitness assay</em>
 As above, we revived evolved communities and isolates as well as
 the ancestral strain from frozen stocks in fresh LB or SIM microcosms and
 allowed them to grow for 24 hrs. We then generated co-cultures by mixing
 an equal volume of each evolved strain with the oppositely marked
 ancestral strain, which acted as a common competitor in this experiment
 (lacZ marked ancestral strain for unmarked evolved strains and
 vice-versa), adding 50 μL of the evolved and ancestral bacterial into 900
 μL of the same media in which they were revived. Next, we diluted these
 mixtures into additional 1 mL microcosms containing the same media at a
 5-fold dilution factor and allowed these co-cultures to grow for a 24-hr
 period, resulting in ~5.6 generations of growth in the mix. Before and
 after the co-culture period, we diluted and plated a 100 μL sample of each
 mixed culture on SIM agar plates with X-Gal. We recorded the number of
 marked and unmarked colonies at both time points
 (<em>lacZ</em>-marked colonies appear blue on agar plates
 containing X-Gal), and used these counts to calculate fitness using the
 following formula:  s = (ln(n1f/n1i)-ln(n2f/n2i))/No.
 Generations         (1) Where<em> s</em>
 represents the selection coefficient,
 <em>n1<sub>f</sub></em> and
 <em>n1<sub>i</sub> </em>represent the number of
 colonies of the focal bacteria at the final and initial timepoint,
 respectively, and<em> n2<sub>f</sub></em> and
 <em>n2<sub>i</sub></em> represent the number of
 colonies of the competitor bacteria at the final and initial timepoint,
 respectively. The selection coefficient reflects the natural logarithm of
 the geometric growth rate of the ratio between focal bacteria and the
 competitor per generation. If the selection coefficient is 1, this
 reflects an e-fold increase in the ratio between focal and competitor
 bacteria each generation. For certain COM community
 replicates, zero colonies from the common competitor were present
 following co-culture (out of hundreds of colonies), meaning we could not
 estimate competitive fitness using the above formula and had to remove
 those replicates from our analyses. This may have led us to underestimate
 the competitive fitness of these communities, as the fitness of certain
 replicates exceeded our limit of detection. This occurred three times in
 each environment across three different COM lines (6.1, 6.2, and 6.15). 39
 total replicates were also lost due to contamination or dilution errors:
 38 in the COM environment (16 from COM isolates, 9 from COM communities, 9
 from MIN isolates, and 4 from MIN communities) and one in the SIM
 environment (one COM community). We measured the
 fitness of 22 isolates in LB and 11 isolates in SIM from each of the 16
 lines without replication. We measured the fitness of communities with
 8-fold replication in LB and 4-fold replication in SIM. We used fewer
 replicates in SIM because this environment tended to yield less variable
 fitness values. To investigate whether the diversity
 captured in our 22 isolate samples were representative of the diversity
 contained in six focal COM lines (6.1, 6.2, 6.8, 6.12, 6.15, 6.17), we
 also compared the fitness of communities and isolate mixes from each line
 in LB with 4-fold replication. One COM community replicate was lost due to
 exclusion of the common competitor (line 6.15). Three replicates were lost
 due to contamination or dilution errors, two from on COM community (line
 6.1) and one from a COM isolate (6.15).
 <em>Dilution-diversity experiment</em>
 We designed this method based on similar experiments with natural
 microbial communities (Wertz et al. 2006, 2007; van Elsas et al. 2012) to
 probe the relationship between diversity and fitness in two COM
 communities (6.1 and 6.2). After reviving these communities in LB
 microcosms as above, we ran separate 10-fold dilution series on these
 cultures using LB and generating a dilution gradient that varied in
 density from ~3×10<sup>9</sup> to
 ~3×10<sup>2</sup> CFU/mL. We then took 150 μL samples from
 each of the final six wells of each dilution series, which varied in
 density from ~3×10<sup>7</sup> to
 ~3×10<sup>2</sup> CFU/mL. As such, the densest sample
 contained ~4.5x10<sup>6</sup> cells and the least dense sample
 contained ~45 cells. These density estimates are based on the cellular
 density of COM communities following 24 hrs of growth in LB (see
 Supplementary Figure 1A). We then allowed the community samples to grow
 for 24 hrs in LB before storing them in glycerol at –80°C. Although the
 density of community samples varied across the dilution gradient, we
 expect that this growth period allowed them to reach similar densities
 before storage. For each of the two focal lines, we
 produced community samples varying in diversity from three replicate
 dilution series initiated independently from frozen stocks. Each replicate
 dilution series produced six community samples varying over a wide range
 of diversity, since each was produced by a progressively smaller
 bottleneck of the original community. We measured the competitive fitness
 of each community sample in LB as described above
 (see <em>Competitive fitness assay</em>) in duplicate,
 producing six total fitness measurements for each community at each
 dilution level. This allowed us to determine at what bottleneck size the
 competitive fitness of community samples changed from community-like
 (which is to say relatively high) to isolate mix-like (which is to say
 relatively low). Five replicates were lost from line
 6.1 because the common competitor was completely excluded after the
 co-culture period. One replicate was lost from line 6.2 due to a dilution
 error. <em>Sterile spent media
 assay</em> We performed a sterile spent media
 assay to investigate whether RVs conferred high fitness in two focal COM
 lines (6.1 and 6.2) by secreting material into the media that facilitated
 other evolved genotypes and/or inhibited the ancestral strain. This assay
 was performed on community samples of both focal lines from the
 Dilution-diversity experiment. Specifically, we focused on two community
 samples from each line that were taken from adjacent dilution levels in
 which the less dilute sample contained the RV (RV<sup>+</sup>)
 and the more dilute sample was RV<sup>-</sup>, as inferred by
 their fitness values in the <em>Dilution-diversity
 experiment</em>. We grew the RV<sup>+</sup> and
 RV<sup>-</sup> samples from frozen stocks in LB for 6 hrs
 before centrifuging them in Eppendorf tubes at 4000 rpm for 10 min. A 6-hr
 growth period was used to ensure the community samples would be in the
 exponential phase of growth when their spent media was collected.
 Following centrifugation, we transferred supernatant into fresh Eppendorf
 tubes using sterile 3 mL syringes (BD) and 0.22 μm disposable syringe
 filters (Millex-GP), removing any remaining cells. As
 we prepared the sterile spent media, we grew two evolved isolates from
 each focal line as well as PA14 for 24 hrs in LB from frozen stocks. We
 then adjusted the optical density (OD<sub>600</sub>) of these
 cultures to 0.1 by diluting them in LB at dilution factors proportional to
 their density readings following the 24-hr growth period. Next, we added
 the density-adjusted cultures into one of three growth treatments with
 5-fold replication: RV<sup>+</sup> supernatant,
 RV<sup>-</sup> supernatant, or a control treatment with no
 supernatant (2 focal lines × 3 growth treatments × 3 focal lines × 5
 replicates = 90 total growth curve replicates). The
 RV<sup>+</sup> and RV<sup>-</sup> supernatant
 treatments consisted of 120 μL of 1.5× LB, 60 μL supernatant (either RV+
 or RV-), and 20 μL of the density-adjusted bacterial cultures. The no
 supernatant control consisted of 180 μL of 1× LB and 20 μL of the
 density-adjusted bacterial cultures. 1.5× LB was used in the former two
 treatments to ensure an equal density of nutrients was supplied from the
 growth media across all treatments. Once these ingredients were mixed in
 separate wells of a 96-well plate (Corning 96-well Polypropylene
 Microplate), the plate was placed in a spectrophotometer (BioTek PowerWave
 XS2 Microplate Spectrophotometer) which measured the optical density
 (OD<sub>600</sub>) of each culture every 10 min over a 12-hr
 period. The plate reader kept the cultures at 37°C and shook the plate
 prior to each density reading to minimize the development of biofilms that
 may have interfered with density readings. Four total
 growth curve replicates were lost due to contamination, one from line 6.2
 isolate 1, two from 6.2 isolate 2, and one from PA14, all grown in
 RV<sup>+</sup> spent media from line 6.2.
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