Research Drought-induced lacuna formation in the stem causes hydraulic conductance to decline before xylem embolism in Selaginella Amanda A. Cardoso1 , Dominik Visel2, Cade N. Kane1, Timothy A. Batz1, Clara Garcıa Sanchez2, Lucian Kaack2, Laurent J. Lamarque3, Yael Wagner4, Andrew King5, Jose M. Torres-Ruiz6 ,Deborah Corso3 ,Regis Burlett3, Eric Badel6, Herve Cochard6 , Sylvain Delzon3 , Steven Jansen2 and Scott A. M. McAdam1 1Purdue Center for Plant Biology, Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA; 2Institute of Systematic Botany and Ecology, Ulm University, Albert-Einstein-Allee 11, Ulm 89081, Germany; 3INRAE, BIOGECO, University of Bordeaux, Pessac 33615, France; 4Department of Plant & Environmental Sciences, Weizmann Institute of Science, Rehovot 76100, Israel; 5Synchrotron Source Optimisee de Lumiere d’Energie Intermediaire du LURE, L’Orme de Merisiers, Saint Aubin-BP48, Gif-sur-Yvette Cedex, France; 6INRAE, PIAF, Universite Clermont-Auvergne, Clermont-Ferrand 63000, France Summary Author for correspondence: Lycophytes are the earliest diverging extant lineage of vascular plants, sister to all other vas- Scott A. M. McAdam cular plants. Given that most species are adapted to ever-wet environments, it has been Tel: +1 765 494 3650 hypothesized that lycophytes, and by extension the common ancestor of all vascular plants, Email: [email protected] have few adaptations to drought. Received: 18 March 2020 We investigated the responses to drought of key fitness-related traits such as stomatal reg- Accepted: 22 April 2020 ulation, shoot hydraulic conductance (Kshoot) and stem xylem embolism resistance in Selaginella haematodes and S. pulcherrima, both native to tropical understory. New Phytologist (2020) During drought stomata in both species were found to close before declines in Kshoot, with a : 10.1111/nph.16649 doi 50% loss of Kshoot occurring at À1.7 and À2.5 MPa in S. haematodes and S. pulcherrima, respectively. Direct observational methods revealed that the xylem of both species was resis- À Key words: ABA, embolism, evolution, tant to embolism formation, with 50% of embolized xylem area occurring at 3.0 and hydraulic conductance, lycophyte, stomatal À4.6 MPa in S. haematodes and S. pulcherrima, respectively. X-ray microcomputed tomogra- conductance, stomatal evolution. phy images of stems revealed that the decline in Kshoot occurred with the formation of an air- filled lacuna, disconnecting the central vascular cylinder from the cortex. We propose that embolism-resistant xylem and large capacitance, provided by collapsing inner cortical cells, is essential for Selaginella survival during water deficit. understories, others grow as epiphytes, in alpine environments Introduction and deserts, where species are frequently desiccation-tolerant Lycophytes, the earliest diverging, extant lineage of land plants (Schneider et al., 2004; Soni et al., 2012; Pampurova & Van to possess a vascular system, occupy a key phylogenetic posi- Dijck, 2014; Vanburen et al., 2018). tion as sister to all other vascular plants (Ambrose, 2013). Species of Selaginella, like all non-Isoetalean lycophytes, lack Lycophyte ancestors, or lycophyte-like plants, are found in the secondary growth. Their mature stems are usually flattened dor- oldest fossil assemblages of land plants, and had probably siventrally, consisting of a cutinized epidermis, suberized hypo- diverged by the Late Silurian (Rowe, 1988; Klaus et al., 2017). dermis, cortex and a central vascular cylinder. The vascular During the Carboniferous, lycophytes were the first group of cylinder or stele has centrally positioned xylem, surrounded by land plants to evolve secondary growth and form the first trees phloem, which is ultimately enclosed in a pericycle, generally that dominated equatorial swamp forests, comprising approxi- composed of one layer of cells (Barclay, 1931; McLean & mately half of floristic diversity at that time (Stewart & Roth- Juniper, 1979; Schulz et al., 2010) (see Supporting Information well, 2010). Lycophyte forests declined during a period of Fig. S1). The inner cortex is often described as being separated global aridification in the early Permian, with extant lycophytes from the vascular cylinder by an air-filled lacuna (Mickel & Hell- represented by just three families: Lycopodiaceae (c. 390 spp.), wig, 1969; Buck & Lucansky, 1976). The cortex and pericycle the aquatic to semi-aquatic Isoeteaceae (c. 250 spp.), and the are connected by trabeculae, highly modified, thin-walled endo- most abundant and specious family, Selaginellaceae (c. 700 dermal cells which provide a symplastic connection through spp.). Selaginella, the sole genus in Selaginellaceae, is the most abundant plasmodesmata (McLean & Juniper, 1979; Schulz ecologically diverse genus of lycophyte. While most species et al., 2010). Trabeculae cells have a well-developed Casparian grow terrestrially in ever-wet temperate and tropical rainforest strip at the anticlinal cell walls, which may be additionally Ó 2020 The Authors New Phytologist (2020) 1 New Phytologist Ó 2020 New Phytologist Trust www.newphytologist.com New 2 Research Phytologist covered with a thin layer of cutin forming the trabecular ring observed or when photosynthesis is irreparably damaged in (McLean & Juniper, 1979). The presence of a Casparian strip both species (Brodribb & Holbrook, 2004; McAdam & Bro- has been demonstrated to efficiently restrict the transport of dribb, 2013). water-soluble compounds from the vascular tissue into the Other than the evolution of desiccation tolerance in Selaginella apoplast of cortical cells (Gola & Jernstedt, 2016). Water in (Oliver et al., 2000; Zhou et al., 2016), results from the limited the xylem must traverse the trabeculae to irrigate photosynthetic number of physiological studies, and constrained ecological pref- tissue. erences of this genus (Campany et al., 2019), suggest that The vascular system of Selaginella consists primarily of trachei- Selaginella species from wet environmental conditions do not ds, while in some species vessels have been reported (Duerden, possess adaptations to survive long periods of water deficit. We 1934; Schneider & Carlquist, 2000a,b). However, treatment hypothesize that the xylem of tropical Selaginella species is highly with alcohol and various acids during sample preparation raises vulnerable to embolism formation during drought. Furthermore, questions of whether shrunken and damaged pit membranes in we expect that species from this genus have very few adaptations the bordered pits of tracheids were misinterpreted as the perfora- to tolerate water limitation, including a limited capacity to close tion plates of vessels (Zhang et al., 2017). Pit membranes are stomata during drought. To address this, we examined the described as thin in tracheids and highly porous in the supposed hydraulic vulnerability of shoots of two tropical Selaginella vessels of Selaginella species (Schneider & Carlquist, 2000b), species, S. haematodes (Kunze) Spring and S. pulcherrima Liebm. which given the importance of these structures for hydraulic effi- using three independent methods: measurement of Kshoot by ciency and protection against embolism in angiosperms (Li et al., rehydration kinetics, and embolism resistance by X-ray computed 2016), could result in an efficient yet highly vulnerable water tomography (micro-CT) and optical vulnerability (OV). These transport system (Brodersen et al., 2014; Kaack et al., 2019). two species are native to rainforest understory and are morpho- Blockage to water flow in xylem cells by embolism at negative logically representative of the majority of species in this genus, as water potentials (Ψ) progressively reduces hydraulic conductance, well as most extinct species assigned to this genus over the past limiting the transport of water within plants (Tyree & Sperry, 400 million years (Rowe, 1988; Zhou et al., 2016; Klaus et al., 1989). Single air-seeding pressure measurements of xylem con- 2017; Schmidt et al., 2020). In addition to the hydraulic proper- duits of the desiccation-tolerant species S. pallescens suggest highly ties of the xylem, we also investigated stem anatomy and the effi- vulnerable xylem, with embolism first forming at À1.5 MPa ciency and control of stomatal closure during drought. (Brodersen et al., 2014); this value matches the mean Ψ resulting Furthermore, we compared measured xylem anatomical and in a 50% decline in shoot hydraulic conductance (Kshoot) in this hydraulic traits between these two species as a preliminary assess- species (Brodribb & Holbrook, 2004). ment of the variability in these key functional traits across closely Given that extensive embolism is a primary cause of plant related species in this genus. mortality during drought (Brodribb & Cochard, 2009; Urli et al., 2013; Cardoso et al., 2020), protecting the xylem from Materials and Methods tensions that would induce embolism is of utmost importance for plant survival during prolonged drought. In seed plants, Plant material highly efficient stomatal closure is the primary means by which plants prevent declines in water status during soil Potted individuals of S. haematodes and S. pulcherrima were water-limitation (Martin-StPaul et al., 2017; Creek et al., grown in the glasshouse of the Botanical Garden of Ulm Univer- 2019). Species adapted to the most seasonally dry environ- sity. The conditions in the glasshouse were maintained at day/ ments with xylem that embolizes before declining to night temperatures above