DOI: 10.5061/dryad.z34tmpgqp
title: Semiannual dormancy cycling results in two seedling cohorts of annual species in the cold desert of Central Asia

description: Our aim was to quantify the life history strategies of the cold desert
 annual species Alyssum linifolium and Tetracme quadricornis
 (Brassicaceae), with particular emphasis on the seed stage.
 Freshly-matured seeds were tested for germination over a range of
 alternating temperature regimes in light and in dark, and the effects of
 seed coat scarification, cold stratification, dry after-ripening and GA3
 on dormancy-break and germination were determined. Seeds were buried in
 the field at 0 (surface), 2 and 5 cm and germination of exhumed seeds
 tested at monthly intervals for 2 years. Seedling emergence of both
 species was monitored in the cold desert, and survival to maturity of
 plants from autumn- and spring-germinated cohorts was determined. Most
 fresh seeds were dormant, and dormancy was broken by all four treatments
 tested. In the early stages of dormancy-break, seeds germinated to low
 percentages over the range of temperatures, and with additional
 dormancy-break germination percentages increased. Thus, seeds have Type 6
 nondeep physiological dormancy (PD). In the buried seed study,
 dormancy-break occurred in summer (June to August), and germination peaked
 in late summer. By late autumn (November), all non-germinated seeds had
 re-entered dormancy. During snowmelt in late winter (February to March),
 some dormancy-break occurred, and low percentages of seeds of both species
 germinated. In the field, seeds of both species germinate in autumn and in
 spring, with more seeds of A. linifolium germinating in autumn than in
 spring and more seeds of T. quadricornis germinating in spring than in
 autumn. A portion of plants from both seedling cohorts of both species
 survived to maturity (set seeds) with more spring- than autumn-germinating
 plants doing so for both species. Thus, both species behave as facultative
 winter annuals. Synthesis: Seeds of A. linifolium and T. quadricornis have
 two dormancy cycles per year. In one cycle, dormancy is broken via warm
 temperatures in summer and in the other one via cold stratification in
 late winter. Semiannual dormancy cycling results in two germination
 cohorts in one year, and it may be a bet-hedging strategy in the
 rainfall-unpredictable cold desert environment. 
description: <strong>Seed size, mass and viability
 </strong> Length, width and thickness of 20
 randomly selected seeds of each species were measured using an Olympus
 SZX16 microscope with a micrometer in one eye piece. Five replicates of
 1000 seeds of each species were weighed with a Sartorius BP 221 S
 analytical balance (0.0001 g) and mean seed mass calculated. TTC (2, 3, 5-
 triphenyl tetrazolium chloride) tests were used to assess seed viability.
 Four replications of 100 fresh seeds were incubated in 5.5-cm-diameter
 Petri dishes containing 2 ml aqueous sterile solutions of 1% (w/v) TTC for
 24 h at 20 °C in darkness. Seeds with an embryo that stained red were
 recorded as viable. <strong>Germination of fresh
 seeds</strong> Freshly matured seeds collected in
 2021 were tested for germination. Four replicates of 50 seeds of each of
 the two study species were placed in 5.5-cm-diameter plastic Petri dishes
 on two layers of Whatman No. 1 filter paper moistened with 2 ml distilled
 water. Petri dishes were sealed with Parafilm and incubated at daily
 (12/12 h) temperature regimes of 5/20, 10/25, 15/30 and 20/35 °C in light
 (12 h daily photoperiod <em>c</em>.100 μmol
 m<sup>−2</sup> s<sup>−1</sup>, cool white
 fluorescent light) or in continuous darkness by wrapping dishes of seeds
 with aluminum foil. The four temperature regimes approximate the mean
 daily minimum and maximum air temperatures for different months during the
 growing season (including the seed dispersal period in June) in the
 natural habitat: April and October, 5/20 °C; May and September, 10/25 °C;
 June and August, 15/30 °C; and July, 20/35 °C. Germination was recorded
 daily for 30 days, and treatments in darkness were checked using a green
 LED light with a spectral output between 515 - 530 nm and peaking at 520
 nm (Vanq, Shenzhen, China); photon irradiance at seed level was &lt;10
 μmol m<sup>−2</sup> s<sup>−1</sup>. Emergence of
 the radicle through the micropylar endosperm and seed coat was the
 criterion for germination. Following a test, each nongerminated seed was
 pinched with forceps to determine if it contained a firm, white embryo
 (indicating viability) or a soft, grey embryo (indicating non-viability)
 (Baskin & Baskin, 2014).
 <strong>Dormancy-breaking
 treatments</strong> The effects of gibberellic
 acid (GA<sub>3</sub>), seed coat scarification, cold
 stratification and dry storage on dormancy-break/ germination of fresh (10
 days old) seeds collected in 2021 were tested.
 <strong>GA<sub>3</sub>.</strong> Four
 replicates of 50 freshly matured seeds of each species were tested for
 germination in 0 (CK), 0.01, 0.1, 1 and 10 mmol L<sup>-1
 </sup>GA<sub>3</sub> solutions at 5/20, 10/25, 15/30 and
 20/35 °C (12/12 h) in light/ darkness (12/12 h, hereafter light) or in
 continuous darkness. GA<sub>3</sub> was dissolved in 2 ml of
 75% ethanol (including the control), which was then diluted with distilled
 water to prepare a total solution volume of 100 ml at the final
 concentrations. The ethanol content of the solution was very low and thus
 it is unlikely to have had an effect on seed germination. Germination was
 recorded daily for 30 days, and viability of nongerminated seeds was
 tested at the end of the experiment. <strong>Seed
 coat scarification</strong>. To test whether the (water-permeable)
 seed coat mechanically restricts embryo growth, the seed coat of four
 replicates of 50 freshly matured seeds of each species was scarified using
 fine sandpaper, being careful not to damage the embryo. Scarified and
 intact seeds (freshly matured seeds) were tested for germination in light
 at 5/20, 10/25, 15/30 and 20/35 °C. <strong>Cold
 stratification</strong>. Freshly matured (10-day-old) seeds were
 placed on two layers of filter paper in a metal box (20 cm length × 10 cm
 width × 10 cm depth) containing wet sand (12% distilled water content, wet
 weight basis). The metal box was covered with a lid and kept in darkness
 in a refrigerator at 4°C. After 0 (CK), 1, 2, 4, 10 and 12 week(s) of cold
 stratification, four replicates of 50 seeds each were arbitrarily chosen
 to test germination in light at 5/20, 10/25, 15/30 and 20/35 °C.
 <strong>Dry storage. </strong>To determine the effect
 of after-ripening on seed dormancy-break, freshly matured (10-day-old)
 seeds were dry stored in paper bags at (1) room temperature (15-25 °C,
 30-50 % relative humidity) for 0, 1, 2.5, 4 and 8 months, (2) high
 temperature (20-35 °C, 30-50 % relative humidity) for 0, 1, 2.5 and 4.5
 months and (3) a greenhouse in summer (30-45 °C, 30-55 % relative
 humidity) for 0, 0.5, 1 and 2 months. After each period and condition of
 storage, four replications of 50 seeds of each species were arbitrarily
 chosen and tested for germination in light at 5/20, 10/25, 15/30 and 20/35
 °C. <strong>Effect of seed burial depth in field
 on dormancy-break and germination</strong>
 Following collection in 2021, about 1500 fresh matured
 (10-day-old) seeds each of the two species were put into nylon-mesh bags
 (5 cm length × 3 cm width). These bags were placed at soil depths of 0
 (soil surface), 2 and 5 cm on 9 June 2021, in a stable sandy area (rather
 than on mobile sand dunes) to prevent wind accumulation of sediments,
 thereby altering soil thickness above the seeds. In total, we prepared 144
 seed bags: 24 months × 3 depths × 2 species = 144 bags. Soil temperature
 and moisture at depths of 0, 2 and 5 cm were recorded hourly by a data
 collector (Em50 METER Co. Ltd, USA). One seed bag from each depth of each
 species was exhumed at monthly intervals from July 2021 to June 2023. At
 the time of sampling each month, we inspected all the seed bags on the
 soil surface and recorded the number of seeds that had germinated in them
 during that month. In subsequent monthly samplings, we recorded the number
 of viable and non-germinated seeds and seeds that germinated in the
 current month. The sum of these data (previously recorded germinated
 seeds, viable non-germinated seeds and the current month’s germinated
 seeds) equaled the initial count of 1500 buried seeds. This accounting
 method ensured that we could confidently rule out the possibility of seed
 decay being counted as germination. From the seed bags
 excavated each month, the viable non-germinated seeds buried in the soil
 were separated from soil (sand) particles and tested in light and in dark
 at 5/20, 10/25, 15/30 and 20/35 °C to simulate the response of seeds to
 seasonal temperature conditions, thereby accurately reflecting their
 dormancy status. Following the methods of Baskin and Baskin (1983, 1986,
 2014) and Cao et al. (2012, 2014) for soil-buried seed dormancy cycling
 experiments, the seeds from each bag were divided into replicates.
 Specifically, four replicates of 25 seeds (or one-fourth of the remaining
 seeds if fewer than 200) were used in each test. The percentages of
 <em>A. linifolium</em> and <em>T.
 quadricornis</em> seeds remaining in the soil seedbank over time
 during a 2-year period were calculated.
 <strong>Field emergence and survival of plants to
 maturity</strong> In October 2021, ten 1 m × 1 m
 plots were arbitrarily established in the southern edge of the
 Gurbantunggut Desert where <em>A. linifolium
 </em>and<em> T. quadricornis</em><em>
 </em>were widely distributed. Newly emerged seedlings of
 <em>A. linifolium </em>and<em> T.
 quadricornis</em> were labelled, and the date was recorded when they
 were first found. Number of surviving seedlings/plants was recorded in
 each of the 10 plots in October 2021 and March and May 2022, which
 represent emergence of the autumn-germinated (AG) seedlings, emergence of
 the spring-germinated (SG) seedlings and completion of the life history of
 adult plants, respectively. Using number of AG and SG seedlings and number
 of adult plants that had completed their life cycle, we calculated the
 survival percentages of AG and SG plants.
 <strong>Statistical analysis</strong>
 All data analyses were carried out in R software (v3.3.2, R Core
 Team 2016). The response variables were expressed as the mean ± SE
 (non-transformed data appear in all figures and tables). We calculated the
 SE using the formula , where SD is the standard deviation of the
 germination percentages and n is the number of replicates. Germination
 percentages were calculated as
 (N<sub>G</sub>/N<sub>T</sub>) × 100, where
 N<sub>G</sub> is the number of seeds that germinated and
 N<sub>T</sub> is the total number of viable seeds tested. A
 general linear model (GLM) with binomial family (‘logit’, var (y)=log (y/
 [1- y])) model (Warton & Hui, 2011) was used to test the effects
 of different experimental treatments, including temperature, time and
 light condition, on seed germination, followed by multiple comparison with
 Tukey’s HSD tests using the ‘glht’ function of the ‘multcomp’ package.
 Level of significance was set at<em> P</em> &lt;
 0.05.
description: # Semiannual dormancy cycling results in two seedling cohorts of annual
 species in the cold desert of Central Asia
 [https://doi.org/10.5061/dryad.z34tmpgqp](https://doi.org/10.5061/dryad.z34tmpgqp) ## Description of the data and file structure This dataset contains original data related to the seed size, mass, viability, germination, dormancy-breaking treatments, and field emergence of A. linifolium and T. quadricornis. The data includes measurements of seed dimensions, viability assessments using TTC tests, germination percentages under various conditions, and the effects of different treatments on seed dormancy. ### Files and variables #### File: Guo\_et\_al.\_data.xlsx Description: This dataset includes comprehensive data on seed characteristics (seed size, mass of 1000 seeds and seed viability), germination percentage, dormancy-breaking treatments, buried seeds, soil temperature and moisture conditions, and field emergence for two species: *A. linifolium* and *T. quadricornis*. ##### Variables | variables | units | description | | :------------------------------- | :----- | :-------------------------------------------------------------------------------------------------------------------------------------------------------- | | seed\_size | mm | Length, width and thickness of 20 randomly selected seeds of each species were measured | | mass\_of\_1000\_seeds | g | Five replicates of 1000 seeds of each species were weighed | | seed\_viability | % | Percentage of viable seeds | | initial\_germination | % | Germination percentages across four temperature regimes and light conditions for the two species | | GA3 | % | Effects of varying GA3 concentrations and four temperature regimes on germination percentages | | seed\_coat\_scarification | % | Effect of seed coat scarification on germination percentages of fresh seeds at different temperature regimes in light. | | cold\_stratification | % | Effect of cold stratification on germination percentages of freshly mature seeds at different temperature regimes in light. | | dry\_storage | % | Effect of dry storage at different temperatures on germination percentages of seeds at different temperature regimes. | | buried\_seeds | % | Germination percentage of seeds at different temperature regimes in light and continuous darkness following 0–24 months of burial in the natural habitat | | depletion\_of\_soil\_seed\_banks | % | The percentages of remaining viable seeds at different depths | | emergence\_and\_survival\_of\_AG | number | AG,autumn-germinated (AG) seedlings; the emergence/survival counts of the two species across different months in designated plots | | emergence\_and\_survival\_of\_SG | number | SG, spring-germinated (SG) seedlings; the emergence/survival counts of the two species across different months in designated plots | * **soil_temperature_and_moisture.xlsx** * Records soil moisture (%), maximum and minimum temperatures (°C) at different depths (0 cm, 2 cm, and 5 cm) over time from June 2021 to June 2023. ## Code/software This dataset can be analyzed using the following free and open-source software: * **R** (version 3.3.2 or later) * Perform an analysis of variance (ANOVA) followed by Duncan testing to analyze differences between groups.