Ecophysiological differentiation between life stages in filmy ferns (Hymenophyllaceae)

Samples were collected in the field and stored in plastic bags with a small amount of water to keep them fresh during transport to the lab.

To measure DT, pre-treatment maximum photochemical yield of photosystem II was measured in fresh plants after a 10 min period of dark-adaptation using a portable mini-PAM fluorometer (Walz Gmbh, Effeltrich, Germany). Samples were then transferred to desiccation chambers containing saturated salts at three different desiccation intensities or a control treatment with moist tissues (100% RH), and water withheld for either a short (2 d) or long (15 d) interval. Conditions inside the desiccation chambers were monitored during the experiment using Track-It RH/Temp dataloggers (Monarch Instrument, Amherst, NH) logging every 10 min or Hobo ProV2 dataloggers logging every 5 min. Salts used for desiccation and their corresponding mean water potentials and approximate relative humidity and VPD are as follows: LiCl (-282 MPa, 18% RH, 2.45 kPa), Mg(NO₃)₂ (-86 MPa, 58% RH, 1.25 kPa), and NaCl (-38 MPa, 80% RH, 0.60 kPa). Following the desiccation treatment, plants were rewetted and yield of photosystem II was again measured at 0.5 h, 24 h, and 48 h following rewetting. Eight individuals per treatment were used for sporophytes. No replication by species was possible for gametophytes due to their cryptic morphology. All gametophytes were subjected to the same treatment (2 d at -86 MPa). Gametophytes were later identified to species by DNA barcoding.

Relative water content (RWC) was measured in a subset of samples by recording the mass of samples prior to the DT test (fresh mass), at each step of the DT test (turgid mass), then after drying them overnight in a drying oven at 65 °C following the DT test (dry mass). Relative water content was calculated as RWC = (TM - DM) / (FM - DM), where TM is turgid mass, DM is dry mass, and FM is fresh mass. Relative water content was not calculated for gametophytes, as these were too small to measure accurately with the balances available.

To measure light responses, rapid light response curves were constructed for each species by measuring photosynthetic yield at gradually increasing levels of photosynthetically active actinic light (400 nm to 700 nm) with the Light Curve function of the mini-PAM portable chlorophyll fluorometer (Walz Gmbh, Effeltrich, Germany) as described by the manufacturer.