DOI: 10.5061/dryad.7m0cfxq08
title: Does competitive asymmetry confer polyploid advantage under changing environments?

description: Competitive interactions drive critical ecological processes in plant
 communities. Yet how competitive interactions are influenced by polyploidy
 which has a widespread incidence in plants remains largely unknown. To
 evaluate the hypothesis of competitive asymmetry between polyploids and
 diploids, we set up competing tetraploid and diploid plants of perennial
 herbaceous Chrysanthemum indicum L. (Asteraceae) at different relative
 frequencies under contrasting soil water contents. We quantified the
 interaction intensity between competing plants of the same (intraploidy)
 and different (interploidy) ploidy levels, and measured functional traits
 related to gas exchange and plant water use to understand the underlying
 mechanisms. The stronger competitive effect of tetraploids on diploids
 than that of diploids on tetraploids provided evidence for the competitive
 asymmetry. As a stronger competitor, tetraploids were limited more by
 individuals of their own than by diploids. Such competitive asymmetry was
 not only maintained under reduced soil water content, but also translated
 into higher aboveground biomass of tetraploids. Tetraploids showed more
 resource-acquisitive traits than diploids under high soil water content
 and more resource-conservative traits under reduced soil water content. As
 such, the higher trait plasticity in tetraploids than diploids likely
 explained the competitive asymmetry. Synthesis. These results elucidate
 the nature and magnitude of species interactions between polyploid and
 diploid plants under changing environments and the underlying mechanisms,
 and provide important insights into the prevalence and persistence of
 polyploid plants under a changing climate.
description: <strong>Study system and sampling</strong>
 The polyploid complex of <em>Chrysanthemum
 indicum</em> comprises two ploidy levels, diploid
 (2<em>x</em> = 18 chromosomes) and tetraploid
 (4<em>x</em> = 36) which likely have allopolyploid origins (Li
 et al., 2014). The diploids and tetraploids are morphologically similar,
 but tetraploids are relatively larger in plant and floral sizes (Figure
 2). These perennial plants can reproduce both sexually by seed and
 asexually by rhizomes (Shi et al., 2011). Such asexual reproduction is
 often more pronounced in tetraploids as they form large clusters of
 individuals compared to diploids that often grow singly (based on field
 observations). The diploid and tetraploid populations grow mainly in
 parapatry in China and often occur in open and dry habitats, especially
 the tetraploids (Li et al., 2014). The tetraploids that likely experienced
 a geological period of drought during the Quaternary glaciation (Yang et
 al., 2006; Li et al., 2014) are more widespread than diploids (Figure 2).
 In their overlapping region in central China, the diploids and tetraploids
 are often spatially separated. Within this region in Shennongjia, China,
 we collected seeds from four diploid populations and six tetraploid
 populations (Figure 2 and Table S1) in November 2015. As <em>C.
 indicum </em>is a common species, we did not need permission for
 collection in this area. Specifically, 3–10 plants were chosen at random
 in each population (Table S1) considering variation in population size.
 Then, from each individual, 5–10 flower heads were collected to obtain
 seeds, and three fresh leaves were stored in moist plastic bags for
 verifying the ploidy levels using flow cytometry following a previous
 protocol by Guo et al., (2016). <strong>Seedling
 cultivation</strong> In March 2016, seeds from
 different flower heads of the same maternal plant were pooled and sowed in
 peat substrates (0–10 mm; Novarbo, Lauttakylantie, Finland) in 0.25-L
 pots. A total of 60 pots for the diploid populations and 72 pots for the
 tetraploid populations were used, with each pot containing c. 10 seeds
 from the same maternal family. Seedlings were grown under 25:15ºC
 day:night temperatures and watered every other day in a naturally lit
 glasshouse at the Germplasm Bank of Wild Species (Kunming Institute of
 Botany, China). After one month, we harvested leaves from three seedlings
 of each pot to confirm seedling ploidy level using flow cytometry. Pots
 with mixed ploidy levels (N = 8 pots) were excluded from this study, which
 was likely due to dispersal when populations of the two ploidy levels grow
 close (Figure 2). In May 2016, seedlings of similar size (c. 5 cm in
 height) were used for the competition experiment and functional traits
 experiment at the same time. <strong>Competition
 experiment</strong> To examine the effects of
 intraploidy and interploidy competitions, diploid and tetraploid seedlings
 were grown together at different relative frequencies (varying in 0, 2, 3,
 or 4 plants per ploidy level in each pot; Figure S3): 0:2, 0:3, 0:4, 2:0,
 2:2, 2:3, 2:4, 3:0, 3:2, 3:3, 3:4, 4:0, 4:2, 4:3, 4:4. In this study, due
 to the concern of seedling mortality under a substantial reduction in soil
 water content (see watering treatments below), we did not include a single
 diploid or tetraploid plant growing alone during the experimental setup.
 These 15 density combinations constituted the basic unit of the
 competition experiment (Figure S3) and were replicated 10 times, with 720
 total plants in 150 7-L pots filled with 1.5 kg custom potting mixture
 (1:1, peat:perlite). To reduce potential confounding effects of maternal
 family and/or population-specific responses (Rosche et al., 2018; Wei et
 al., 2020) on competitive interactions, seedlings from different maternal
 plants and populations were mixed at random and transplanted for the
 competition experiment. Soil water content was maintained at field
 capacity (‘FC’; 0.9 kg water per 1.5 kg potting mixture) by watering every
 two days for 3 weeks. On June 1, 2016, we started the watering treatments,
 with 5 replicates maintained at 80% field capacity (FC) and 5 replicates
 at 20% FC (referred to as ‘normal watering’ vs. ‘reduced watering’,
 respectively). To maintain the corresponding soil water content, pots were
 weighed every two days to record water loss and re-water accordingly,
 which was effective in maintaining the soil water content (Ma et al.,
 2010) when the weight of the matrix (soil and water) was much higher than
 plant biomass (kg vs. g). This experiment used a split plot design for the
 ease of watering, where watering treatments were assigned to whole plots
 with the replicated 15-density combinations randomized within the whole
 plots. Pots in the glasshouse were not rotated during the experiment due
 to the large size of the pots and fragile creeping stems under reduced
 watering. Thus pot positions (i.e. column and row unique to individual
 pots in the glasshouse) were considered in data analyses later to account
 for the potential influence of variation in microenvironmental conditions.
 The competition experiment was run for 2 months because (1) soil at 20% FC
 became very dry over the course of the experiment and plants already
 exhibited some leaf wilting (e.g. in diploids) and (2) we focused on
 growth rather than mortality-related responses in this
 experiment. On July 30, 2016, we harvested the
 aboveground plant materials by ploidy level in each pot, but not the
 belowground due to challenges in separating roots between diploid and
 tetraploid plants. Trials with diploid and tetraploid plants growing
 separately indicated a similar root-to-shoot ratio between the ploidy
 levels (both ~1.0 under normal watering and 1.5 under reduced watering),
 and thus aboveground dry biomass is expected to correlate closely with
 whole plant dry biomass in both ploidy levels. The harvested plant
 materials were dried at 80ºC for 72 h to obtain the aboveground dry
 biomass of diploid and tetraploid plants in each pot. Due to occasional
 plant mortality during the competition experiment, pots that did not match
 their original density combination (N = 10) were excluded from data
 analyses. <strong>Functional traits
 experiment</strong> We measured a suite of
 functional traits that are relevant to plant growth and drought tolerance
 (Table 1) in a separate experiment, to avoid the potential influence of
 plant tissue collection on the competition experiment (Methods S1). In the
 functional traits experiment, seedlings of the same ploidy level (i.e. two
 diploid or two tetraploid seedlings in a pot; Methods S1) were
 transplanted with a total of 56 pots (112 seedlings) for each ploidy
 level. Ten ‘empty’ pots filled with the same potting mixture but no
 seedlings were included for measuring evaporation. As in the competition
 experiment, on June 1, 2016, half of the experimental (N = 28 pots per
 ploidy level) and ‘empty’ pots (N = 5) were assigned at random to the
 normal watering treatment (80% FC) and half to the reduced watering
 treatment (20% FC) for 2 months. No seedling mortality occurred during
 this experiment. Functional traits were measured
 primarily at the end of the experiment during July 20–30, 2016.
 Instantaneous gas exchange (maximum photosynthetic rate,
 A<sub>max</sub>; stomatal conductance,
 g<sub>s</sub>; transpiration rate, E) was measured for a
 random subset of 13 pots per ploidy level per treatment using a LI-6400
 portable photosynthesis system (LI-COR, Lincoln, NE, USA). Measurements
 were taken on a fully expanded green leaf from one plant of each pot
 between 09:30 and 12:00 h at a saturating irradiance (1200 μmol
 m<sup>-2</sup> s<sup>-1</sup>). The cuvette CO2
 concentration was maintained at 400 μmol mol<sup>-1</sup>.
 Relative humidity and leaf temperature were maintained at 50–60% and
 20–25ºC, respectively. Instantaneous water use efficiency (WUEi) was
 calculated as A<sub>max</sub>/E. From the
 same subset of pots used for gas exchange measurements, 10 pots per ploidy
 level per treatment were selected for determining leaf mass per unit area
 (LMA) and relative water content (RWC). Specifically, the largest, fully
 expanded green leaf from one plant in each pot was excised and weighed
 immediately for fresh mass. The leaf was scanned and the leaf area was
 estimated using ImageJ v1.45 (Schneider et al., 2012). The same leaf was
 then submerged in distilled water overnight to obtain the saturated mass,
 and dried at 80ºC for 48 h for dry mass. LMA was calculated as leaf dry
 mass/leaf area, and RWC was calculated as (fresh mass – dry
 mass)/(saturated mass – dry mass) × 100. In addition, one mature leaf was
 collected at 06:00 h from each pot (8 pots per ploidy level per treatment)
 for measuring predawn leaf water potential
 (Ψ<sub>predawn</sub>) using a WP4C Dewpoint Potential Meter
 (METER Group, Inc., Pullman, WA, USA). Another mature leaf was collected
 and dried for measuring carbon isotope composition
 (δ<sup>13</sup>C, ‰) using an isotope mass spectrometer
 (Thermo Finnigan MAT GmbH, Bremen, Germany) at the Institute of Tibetan
 Plateau Research. Plant total water use (TWU) and
 long-term water use efficiency (WUE<sub>L</sub>) were measured
 in a different subset of 10 pots per ploidy level per treatment, to avoid
 the influence of destructive leaf collection. As the experimental and
 ‘empty’ pots were weighed every two days to record water loss (for
 maintaining soil water content), total water use in each pot was
 determined as the difference between accumulated water loss in each
 experimental pot (transpiration and evaporation) and the average
 accumulated water loss per empty pot (evaporation). Plant TWU was
 estimated as the average TWU in each pot. To determine
 WUE<sub>L</sub>, plants in each pot were harvested for
 estimating dry biomass (including both belowground and aboveground).
 WUE<sub>L</sub> was calculated as the ratio between plant dry
 biomass and TWU (Ma et al., 2010).