2 Materials and methods
Figure 4. (a) Map of Finland marked with the Joensuu CG and the provenances. (b) Photographs from the Joensuu CG enclosure taken in 2012 with the gas exchange measuring equipment shown being in use.
Table 1. Plant material, experiment duration, experimental growth conditions and measurements done in each experiment in (I), (II) and (III). GE-SS = Snapshot gas exchange, GE-TC = gas exchange temperature response curves, GE-LC = gas exchange light response curves, CCI = chlorophyll content index, Fv/Fm = chlorophyll fluorescence Fv/Fm measurements. Experiment Provenances Genotypes Plants Duration Lighting Photoperiod Thermoperiod Measurements Paper I 67°KI, 66°RO, 65°PO, 62°VE, 61°PU, 60°LO
K1, K2, K8, K11, K15, R3, R8, R11, R13, R15, P1, P2, P5, P7, P13, V1, V3, V4, V5, V14, Pu17, Pu25, L1, L6, L14, L15 Total: 258 plants
CG established in 2010, measured in 2013 (GE in June-July, CCI in May-July, growth in September 2012 & 2013)
Natural NaturalNatural
GE-SS, CCI, height, RGR, nbr. of leaves, leaf area, end of growth Paper II 67°KI, 61°PU
K7, K11, K27, Pu17, Pu25, Pu30 Total: 46 plants 5 weeks (pregrowth) + 101 days (main experiment) 150-600 μmol m-2 s-120/4h light/dark 20/4h at 20/16◦C day/night
GE-SS, GE-TC, GE- LC, CCI, Fv/Fm, height, nbr.of leaves+branches, mass fractions Paper III67°KI, 61°PU
K7, K27, Pu17, Pu18, Pu25, Pu30 Total: 54 plants, 27 per treatment
4 weeks (pregrowth) + 123 days (main experiment) 150-600 μmol m-2 s-124/0h (CL) 19.5/4.5h (NCL) light/dark 19.5/4.5h at 20/16◦C day/night GE-SS, height, RGR, nbr. of leaves+ branches, mass fractions, stom.dens., leaf long., leaf mass+area
The Joensuu common garden (CG) was established in 2010 (Fig. 4b) and the field experiment (I) took place during 2012 – 2013. In total, 26
genotypes of the six provenances were planted at the CG, with 10 clonal plantlets per genotype as replicates in a random block design at 1.2 x 1.2 m intervals with 258 plants available for measuring in 2013 (I). At the time of planting, 60°LO, 61°PU and 62°VE were taller than 65°PO, 66°RO and 67°KI (this was later accounted for in calculations of relative growth rate).
The CG site is more thoroughly described in (I) and in Heimonen (2015).
For the growth chamber experiments, in 2016 an effort was undertaken to restart the clonal micropropagation of 61°PU and 67°KI, which had been maintained in vitro until then. These plants were used in 2017 for the first chamber experiment (II). Also in 2017, a second round of
micropropagation was started for the second chamber experiment (III) taking place in 2018. Three genotypes per provenance were used in (II), while two northern genotypes and four southern genotypes were used for the experiment in (III) (Table 1).
Prior to 2016, the plants had been grown on Woody Plant Medium (WPM) supplemented with extra calcium, but in 2016 were transferred to Murashige-Skoog medium (MS) supplemented with 1mg/L 6-
benzylaminopurine (BAP). The micropropagated plantlets were raised in growth chambers (Microclima MC1000; Snijders Labs, the Netherlands) at 22°C in a 16/8h light/dark photoperiod in ~100 – 200 μmol m-2 s-1 of light in sterile jars (~100% RH). For tissue culture maintenance and increase of plantlet number, plants were re-transferred onto the MS-BAP medium roughly every three weeks. Before transferring to experiments, plantlets were reared for 3 weeks on a rooting medium, ½MS (half the concentration of MS-medium macronutrients) with 0.5 mg/L indoleacetic acid (IAA) in Phytagel.
In (II) and (III), the rooted plants underwent a pregrowth stage before the experiment. For this, they were randomly planted in seed flat
propagator trays (with transparent lids to keep humidity high, Fig. 5), using sterilized and unfertilized peat:perlite 1:1 (v/v), and the trays placed in growth chambers for 4 weeks in (II) and for 5 weeks in (III). Plants were given a constant temperature of 22°C, RH of 60% and the amount of light
at the plant-level was between 150 – 200 μmol m-2 s-1 (LI-250 light meter, Li- Cor, Inc., USA). This is the same amount of photosynthetically active
radiation (PAR) the plants had received during in vitro growth. The pregrowth photoperiod was 20/4h in (II) and 16/8h in (III) (both
corresponding to the photoperiods given to the in vitro plants). Morning and evening were simulated with the illumination increasing/decreasing over a 1h period. Plants were bottom-watered with 22°C deionized water 2 – 3 times a week and their position constantly randomized across the chamber. Watering through the bottom is necessary to avoid mold and algae growth and the mechanical stress of small plants moving in the substrate, which could disturb their rooting (Junker et al. 2015). Tray lids were removed toward the end of pregrowth to acclimate the plants to chamber RH.
Figure 5. Photographs from the first growth chamber experiment (II), from top left to bottom right: Plants maintained in vitro prior to planting, plants in seed flats in pregrowth prior to selection for actual experiment, a small plantlet planted to a bigger pot and ready for the experiment, plants being measured at 66 DAP (while using empty pots for equalizing plant distance to the light source).
After good rooting was achieved in pregrowth, the experimental stage began (0 DAP, days after potting) with re-potting of the plants to 1.5L pots on to sterilized and unfertilized peat:vermiculite 3:1 (v/v). The peat in this mixing ratio keeps in moisture while the vermiculite prevents excessive packing of substrate. As peat has a very low pH, it was adjusted to ~ pH 6 by adding 2 g/L of lime. The total weight of the growth substrate was 200g in (II), but adjusted to 300g in (III) for easier regulation of watering. In both experiments, two chambers were used. In (II), the chamber acted as an experimental block factor, with both chambers running the same program.
In (III), the chamber acted as a treatment for photoperiod. A total of 46 and 54 plants were randomized to the chambers in (II) and (III), respectively, meaning that in (III) there were initially 27 plants per treatment (six plants were later discarded). Plant location randomization during the
experiments was done constantly. In (II), randomization was done within chambers, but in (III) it was done within and between chambers (by alternating the chamber programs between chambers).
In (II), both chambers had a 20/4h photoperiod. In (III), there was a photoperiod treatment with two levels: continuous light (CL, 24/0h) and non-continuous light (NCL, 19.5/4.5h) (Fig.6). These photoperiods were not changed as the experiment progressed, but they correspond to the ones found at the latitudes of the provenances during midsummer (June to July) and in both articles the photoperiodic rhythms tracked real-world time of the day. Common to both experiments, the light intensity inside the chambers was equal, varying between 150 – 600 μmol m-2 s-1 from the plant bottom-level to nearest the lamps (and less horizontally from ceiling to ceiling). Chamber temperature was maintained at 20/16°C day/night with realistic thermoperiods (4h of night-time temperatures in (II), 4.5h in (III) irrespective of photoperiod). A realistic thermoperiod was desired because it can entrain a diurnal rhythm even in the absence of light-cues in CL (Velez-Ramirez et al. 2011) and 16°C is a good midway night-
temperature for silver birch, as lower temperatures can induce growth cessation and higher temperatures can increase height, stem dry weight (DW), shoot mass fraction (SMF), stem diameter and shoot:root ratio and decrease LMA (Way and Oren 2010, Mäenpää et al. 2013). Both
experiments also included simulated 1h morning and evening
increases/decreases in light and/or temperature. RH was a constant 60%, night and day. Plants were surface-watered with chamber-temperature DI- water once or twice per week. The watering volume was always equal among plants, but the volume itself varied each time depending on
substrate moisture (with approximately 60% field capacity being targeted).
The water included a fertilizer (Pot Plant Superex, Kekkilä Finland). The very first watering of both experiments was with 160, 40 and 240ppm of N, P and K, respectively (0.1% solution), while subsequent waterings had double these concentrations (0.2%). In (II), every second watering was without fertilizer. As growth rates differed by provenance and genotype, some plants eventually reached higher than others, after which the distance to the light source was equalized periodically to provide an equal amount of light for each plant. This was done in (II) from 42 DAP and in (III) from 28 DAP onwards during each watering (or when deemed necessary) by
positioning the plants on platforms so that the apices of the smaller plants were on level with the higher ones.
Figure 6. Chamber programs used during the experiments in (a) (II) and (b) (III) showing the diurnal patterns of PPFD, RH and T. The NCL treatment (19.5/4.5h) in (III) was very similar to the growth chamber conditions in (II), because the NCL photoperiod was only ½h shorter, and the day/night temperatures and RH were the same.
All leaf-level measurements were generally taken from the same spot from the right-hand side of the lamina if possible (I, II, III), also in cases where the same leaf was measured several times. Consequently, e.g., the gas exchange measurement spot included the area where stomatal density was calculated from. In the chamber experiments, the plants were mostly not removed from the chambers for measuring or were moved to another chamber with the same conditions (illumination, temperature, humidity).
As the conditions inside the chambers can be very different from the growth chamber room, the instruments and plants were allowed to acclimate to current conditions (e.g., to avoid condensation). Only fully expanded, healthy, green leaves were measured, avoiding discolored (red, yellow), damaged and diseased leaves. In (II), three youngest fully
developed (YFD) leaves on the main stem were marked on each plant during the experiment for measurements (Fig. 7), and most types of
measurements were restricted to a single leaf to avoid stressed leaves. YFD leaves are leaves of the same age, usually around the 4th to 5th leaf from the plant tip. In (III), leaves on the main stem were marked in groups of three at 4 different time points. Leaves were marked so that each
individual leaf could be identified from each group of three, named as, e.g., leaf 1-1, where the first ‘1’ identifies the first group of three (counted from the base of the plant) and the second ‘1’ indicates that this is the first (lowest and oldest) leaf of the three. Leaves 1-2 and 1-3 indicate the leaves just above the first one (constituting leaf group 1 together with leaf 1-1), and leaf 2-1 was the first leaf of the second group of three (younger and marked later), and so on (Fig. 7, Fig. 2 in III). Only the first leaf of each group was a real YFD leaf. This was done to identify vertically neighboring leaves in plants of different growth rates and to ensure that the marked leaves were of same age among all plants. As measurements were done mainly on the first leaf of the triplet, the other leaves’ traits such as leaf longevity were unaffected by measuring.
In the field, leaves on two types of shoots (LS and SS) were studied (Fig.
7, Supplementary Fig. S3 of I). However, the chamber studies utilized first year plants and all measurements can be considered to have been done on LS leaves (Fig. 1 in II and Fig. 2 in III).
Figure 7. Schematic view of the leaf types measured both in the common garden (CG) and chamber experiments. Plants are not to scale and it was not uncommon for more unmarked leaves to be located between leaf triplets than shown. LS = long shoot, SS = short shoot, YFD = youngest fully developed leaf. Figure credit: Veera Varpa. Modified by the author.