Spore liberation in mosses revisited

Abstract

The ability to perform hygroscopic movements has evolved in many plant lineages and relates to a multitude of different functions such as seed burial, flower protection or regulation of diaspore release. In most mosses, spore release is controlled by hygroscopic movements of the peristome teeth and also of the spore capsule. Our study presents, for the first time, temporally and spatially well-resolved kinematic analyses of these complex shape changes in response to humidity conditions and provides insights into the sophisticated functional morphology and anatomy of the peristome teeth. In Brachythecium populeum the outer teeth of the peristome perform particularly complex hygroscopic movements during hydration and desiccation. Hydration induces fast inward dipping followed by partial re-straightening of the teeth. In their final shape, wet teeth close the capsule. During desiccation, the teeth perform an outward flicking followed by a re-straightening which opens the capsule. We present a kinematic analysis of these shape changes and of the underlying functional anatomy of the teeth. These teeth are shown to be composed of two layers which show longitudinal gradients in their material composition, structure and geometry. We hypothesize that these gradients result in (i) differences in swelling/shrinking capacity and velocity between the two layers composing the teeth, and in (ii) a gradient of velocity of swelling and shrinking from the tip to the base of the teeth. We propose these processes explain the observed movements regulating capsule opening or closing. This hypothesis is corroborated by experiments with isolated layers of peristome teeth. During hydration and desiccation, changes to the shape and mass of the whole spore capsule accompany the opening and closing. Results are discussed in relation to their significance for humidity-based regulation of spore release.

Keywords: Brachythecium populeum; Bryophyta; hygroscopic; moss; plant movement; spore dispersal.

Figure 1.

Mature spore capsule of Brachythecium populeum without operculum. (A) Whole capsule, (B) longitudinal section (thickness: 3 µm; staining: toluidine blue; embedding: Technovit). ip: inner peristome teeth (endostome); op: outer peristome teeth (exostome); cw: capsule wall; co: columella.

Figure 2.

Movement of an initially dry, single outer peristome tooth of Brachythecium populeum during immersion in water. (A) Hydration phase I = phase H-I, inward movement (starting from a ‘hook-like’ shape), (B) hydration phase II = phase H-II, partial re-straightening with a remaining curvature below the middle region of the tooth. The interior of the spore capsule is located on the right side of the tooth, the exterior on its left side [see Supporting Information—Video 1].

Figure 3.

Movement of a single outer peristome tooth of Brachythecium populeum during desiccation. (A) Desiccation phase I = phase D-I, opening movement with form change into a ‘S-shaped’ form, (B) desiccation phase II = phase D-II, restoration of a more upright position and change into a ‘hook-like’ shape typical for the dry state (see Fig. 2). The interior of the spore capsule is located on the right side of the tooth, the exterior on its left side [see Supporting Information—Video 2A, 2B].

Figure 4.

Illustration summarizing the hygroscopic movement of a single outer peristome tooth of Brachythecium populeum observed during hydration (phases H-I and H-II) and desiccation (phases D-I and D-II). ‘in’ designates the inside of the spore capsule, ‘out’ the outside.

Figure 5.

Capsule with a single outer peristome tooth exposed to different levels of relative air humidity. The other outer peristome teeth were removed in order to provide a better visibility of the tooth shape and orientation. Shapes of two other outer peristome teeth submitted to the same conditions were very similar (data not shown).

Figure 6.

Hygroscopic shape change of a spore capsule of Brachythecium populeum. (A) Hydration by immersion in water (min 1) with volume increase (outward bulging of the capsule walls) and erection of the capsule, total duration of the swelling process 10 min, (B) desiccation at T = 25 ± 1 °C and RH = 39.5 ± 2 % with volume reduction and inward buckling of the capsule accompanied by emersion of a water droplet squeezed through the peristome (min 8 and min 9); then evaporation of the water droplet, and typical outward flicking movement of the outer peristome teeth during desiccation (temporary outwards bent shape visible at min 17) [see Supporting Information—Video 3A, 3B].

Figure 7.

Mass differences (mean values and standard deviations) of dry and wet spore capsules and comparison of closed capsules (with the operculum still attached) and open capsules (without operculum). Wet capsules were measured after immersion in water, n = 10 in each set. Wet capsules have a significantly higher mass than dry capsules (paired t-test, t(9) = −5.33, P = 4.73×10⁻⁴ for capsules with operculum; paired t-test, t(9) = −9.98, P = 3.64×10⁻⁶ for open capsules). Dry capsules without operculum have significantly lower masses than dry capsules with operculum (unpaired t-test, t(11.47) = 3.89, P = 2.33×10⁻³), whereas—due to the better penetration of water into the capsules—wet capsules without operculum have significantly higher masses than wet capsules with operculum (unpaired t-test, t(18) = −2.55, P = 1.99×10⁻²).

Figure 8.

Variation of capsule diameter measured at the thickest middle region over time during (A) hydration and (B) desiccation, respectively.

Figure 9.

Spores (arrows) sifting through the peristome and clinging at the peristome teeth of a spore capsule of Brachythecium populeum (images selected from a high-speed video taken at a time distance of 4 ms, when peristome teeth start to open during desiccation). The capsule itself was not hydrated [see Supporting Information—Video 4].

Figure 10.

Longitudinal sections of Brachythecium populeum peristome teeth (thickness: 3 µm; staining: (A) and (B) FCA, (C) and (D) toluidine blue; embedding: Technovit). The different staining of the outer layer l_o (blue by FCA/dark green by toluidine blue) and the inner layer l_i (pink by FCA/light blue or green by toluidine blue) visualizes the two-ply structure of outer peristome teeth (op). The articulation-like structure at the insertion of the tooth at the capsule rim is also composed of two layers but they stain in a different colour than the outer and inner layer of the peristome tooth itself (red in FCA staining: A and B). cw: capsule wall. In (D) spores (s) are clinging to an inner peristome tooth (ip).

Figure 11.

(A) LT-SEM image of outer and inner peristome teeth of Brachythecium populeum; (B) close up of the middle region of the outer peristome tooth shown in (A). op: outer peristome tooth; ip: inner peristome tooth; l_o: outer layer of the outer peristome tooth; l_i: inner layer with partly visible ridges; ri: ridges; pa: papillae. Broad arrows: smooth substance coating the papillae and segment borders, with an increasing gradient of coated area from the base to the tip of the outer peristome teeth.

Figure 12.

Isolated inner layer (A) and outer layer (B) of an outer peristome tooth of Brachythecium populeum, immersed in water. Due to the unavoidable destruction of one layer during preparation the depicted layers originate from two different teeth. The outer layer bears remnants of the inner layer at the apical part (arrow). The inner layer has adopted a straight shape, whereas the outer layer is bent inward (bending direction given in relation to the original arrangement of the tooth on the capsule) [see Supporting Information—Video 5A, 5B].

Figure 13.

Variation in length over time during hydration (A) and desiccation (B) of an isolated outer and an isolated inner layer of an outer peristome tooth of Brachythecium populeum. Due to the unavoidable destruction of one layer during preparation the two layers originate from two different peristome teeth. t = 0 corresponds to the contact with water in (A) and the disappearance of the water film in (B), as visible in the light microscope.