New Phytologist Research

Structure–function constraints of tracheid-based xylem: a comparison of conifers and ferns

Jarmila Pittermann1, Emily Limm2, Christopher Rico1 and Mairgareth A. Christman3 1Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA 95064, USA; 2Save the Redwoods League, 114 Sansome St Suite 1200, San Francisco, CA 94104, USA; 3Institute for Ecohydrology Research, 1111 Kennedy Place Suite 4, Davis, CA 95616, USA

Summary

Author for correspondence: • The ferns comprise one of the most ancient tracheophytic plant lineages, and Jarmila Pittermann occupy habitats ranging from tundra to deserts and the equatorial tropics. Like Tel: +1 831 459 1782 their nearest relatives the conifers, modern ferns possess tracheid-based xylem but Email: [email protected] the structure–function relationships of fern xylem are poorly understood. Received: 20 April 2011 • Here, we sampled the fronds (megaphylls) of 16 species across the fern phylo- Accepted: 6 June 2011 geny, and examined the relationships among hydraulic transport, drought-induced cavitation resistance, the xylem anatomy of the stipe, and the gas-exchange New Phytologist (2011) 192: 449–461 response of the pinnae. For comparison, the results are presented alongside a doi: 10.1111/j.1469-8137.2011.03817.x similar suite of conifer data. • Fern xylem is as resistant to cavitation as conifer xylem, but exhibits none of the hydraulic or structural trade-offs associated with resistance to cavitation. On a Key words: cavitation, gas exchange, hydraulic conductivity, primary xylem, conduit diameter basis, fern xylem can exhibit greater hydraulic efficiency than sporophytes, xylem evolution. conifer and angiosperm xylem. • In ferns, wide and long tracheids compensate in part for the lack of secondary xylem and allow ferns to exhibit transport rates on a par with those of conifers. We suspect that it is the arrangement of the primary xylem, in addition to the intrinsic traits of the conduits themselves, that may help explain the broad range of cavita- tion resistance in ferns.

peaked with the appearance of the torus-margo pit mem- Introduction brane found in conifers, Gingko and some angiosperms The evolution of tracheid-based xylem in the Lower (Niklas, 1985; Sperry, 2003; Pittermann, 2010; Pittermann Devonian led to profound shifts in plant size and structure, et al., 2005; Jansen et al., 2004). The subsequent specializa- and marked the first appearance of tracheophytes, the tion of tracheids into fibers and vessels that characterized so-called true vascular plants (Pittermann, 2010; Kenrick the evolution of angiosperm wood allowed for a division of & Crane, 1997; Niklas, 1992; Bateman et al., 1998; Sperry, labor whereby short, narrow fibers provide mechanical sup- 2003). Tracheids preceded the widespread appearance of port while multicellular vessels function solely for water vessels by an estimated 150 million yr and served as the fun- transport (Bailey & Tupper, 1918; Carlquist, 1988). damental water transport tissue for some of the earliest land Although vessels may confer water transport efficiencies that plants, including Rhynia and Psilophyton, horsetails, ferns are well over three orders of magnitude greater than those of and the extinct arborescent lineages of the Late Devonian conifers (Tyree & Zimmermann, 2002; McCulloh et al., such as the lycopod Lepidodendron and pro-gymnosperms 2010), it is remarkable that tracheid-based xylem continues such as Archaeopteris (Cichan, 1985; Stewart & Rothwell, to serve as the primary transport tissue for two abundant and 1993; Taylor et al., 2009). Generally, the evolution of trac- diverse plant lineages, the conifers and the ferns. We know heids is characterized by increasing length and diameter that, on a xylem area basis, conifers and angiosperms can exhi- (particularly during the Devonian), greater deposition of bit similar hydraulic efficiencies (Pittermann et al., 2005), secondary cell wall material and progressive specialization in but how does the performance of the tracheid-based xylem of the inter-tracheid pit membranes, the complexity of which ferns compare with the more derived xylem of conifers?

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Despite the extraordinary diversity and world-wide abun- tree ferns, produced secondary xylem from a polystelic dance of terrestrial and epiphytic ferns (Moran, 2008; arrangement of several bifacial vascular cambia (Cichan, Schuettpelz & Pryer, 2009), our understanding of the vas- 1986; Wilson et al., 2008; Taylor et al., 2009). Interestingly, cular performance of these primitive plants is just gaining a recent examination of two Botrychium species excluded momentum (Calkin et al., 1985; Veres, 1990; Brodribb the possibility that these ferns exhibit true cambial-derived et al., 2005; Watkins et al., 2010). Early work on water secondary growth, although the developmental pattern of transport in several fern species showed that the main axis their rhizomes is regarded as a departure from standard of the frond exhibits progressively lower hydraulic conduc- definitions of primary and secondary growth (Rothwell & tivities along its length as a result of a decrease in conduit Karrfalt, 2008). abundance and conduit size, especially from the start of the Tracheid-based xylem is common to both conifers and leafy rachis to the tip of the frond (Gibson et al., 1985; ferns, but key differences in xylem architecture have a pro- Schulte et al., 1987). These seemingly low rates of water found effect on the overall structure of these plants, as well transport were again reported in a broad sampling of tropi- as the physical principles that guide the shape and size of cal pteridophytes, a finding that was mirrored in the the xylem conduits. Most importantly, the evolution of a concurrently low rates of gas exchange (Brodribb & bifacial vascular cambium and the resultant secondary Holbrook, 2004, Brodribb et al., 2007, Watkins et al., xylem in conifers and woody angiosperms marked a radical 2010). Low transpiration rates are consistent with the pref- departure in the evolution of xylem function as well as over- erence of these tropical ferns for the low-light habitats all plant structure because it allowed plants to have the characteristic of forest understories and dense canopies: architectural flexibility to vary the height and horizontal dis- because understory plants may only experience brief photo- play of their foliage, an otherwise impossible endeavor in synthetic peaks during sunflecks, selection places a lower plants limited by a unifacial cambium or simple strands of premium on the evolution of high vascular and gas- primary xylem (Rowe & Speck, 2005; Spicer & Groover, exchange capacity in favor of a smaller sized, nonwoody, 2010). In conifers and pro-gymnosperms, the xylem slow-growing life form with reduced metabolic costs. There acquired the capacity not only to transport water to the leaf are, however, some exceptions to these generalizations, most canopy, but also to structurally support it (Meyer-Berthaud notably in the form of tree ferns, desert-dwelling ferns and et al., 1999; Tyree & Zimmermann, 2002; Pittermann the many temperate species that appear to thrive in a broad et al., 2006a,b; Sperry et al., 2006; Pittermann, 2010). variety of temperate high-light habitats, such as Pteridium Recent work has shown that, in north-temperate conifers, aquilinum and Blechnum chilense (Page, 2002; Saldana the combined requirements for canopy support along et al., 2007). Considering that several species of ferns, such with reinforced, implosion-resistant tracheids constrain the Lygodium microphyllum and P. aquilinum, can be highly maximum hydraulic efficiency of conifer xylem because of invasive (Robinson et al., 2010), it is not unreasonable to the necessity to build a strong, secondary cell wall coupled hypothesize that ferns are capable of high rates of water with a narrower lumen diameter (Pittermann et al., 2006a; transport and photosynthesis. Sperry et al., 2006). Both the cell size and the volume of The vascular system in both fern fronds and rhizomes the conduit wall are limited by the metabolic output of the consists of tracheids that tend to be longer and wider than developing xylem cell over the growing season, so it is those of conifers, with scalariform pitting extending along impossible for conifer tracheids to be long and wide and the entirety of at least one side of the tracheid wall (Gibson also sufficiently fortified to offer structural support et al., 1985; Veres, 1990; Carlquist & Schneider, 2001). (Pittermann et al., 2006a; Sperry et al., 2006). By contrast, Cryptic vessels have been reported in the rhizomes of several fern xylem is released from the structural support require- species on the basis of what appeared to be scalariform per- ment by virtue of the hypodermal sterome, a ring of foration plates in the terminal ends of vessel elements, but schlerenchyma fibers that surrounds the main axis of the their frequency in the ferns is now presumed to be much frond as well as the thicker secondary axes in some species lower than originally thought (Carlquist & Schneider, (Rowe & Speck, 2004; Rowe et al., 2004). This tissue 2001, 2007). Because water transport in ferns occurs exclu- supports the frond and provides it with a high degree of sively through primary vascular tissue, the xylem and flexural stiffness (Niklas, 1992; Rowe et al., 2004). phloem of ferns are encased in discrete bundles that may Consequently, without a support function, fern tracheids span the length of the frond and bifurcate at each pinna. can occupy a much broader morphospace in the form of The bundles are arranged in a variety of stelar patterns rang- longer and wider tracheids that have little need for excep- ing from the simple protostele of whisk fern (Psilotum tionally reinforced cell walls. However, fern tracheids lack nudum) to the more complex siphonostele- and dictyostele- the torus-margo pitting particular to conifer tracheids and like arrangements found in more derived fern species. instead possess the ancestral, homogenous pit membrane Secondary xylem is absent in extant ferns though extinct that confers much greater pit area resistance than the torus- plants such as Medullosa, which bear some resemblance to margo arrangement, especially in derived angiosperms

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(Sperry et al., 2005; Pittermann et al., 2005; but see Hacke et al., 2007). Without torus-margo pit membranes, secondary xylem Mesic and a canopy support function, just how does the trans- port efficiency of the tracheid-based xylem of ferns compare to that of conifers? The goal of this study was to examine the structure and function of fern xylem across a broad phylogenetic sampling scheme that includes self- supporting ferns, climbers, and perennial and seasonally deciduous ferns as well as the desert-dwelling ferns in order to capture the broadest possible variation in tracheid structure. Because water transport occurs under negative pressure and xylem conduits are vulnerable to the entry air (cavitation), we examined how the cavitation resistance of fern xylem compares to that of conifers and whether ferns exhibit any of the trade-offs associated with cavitation resistance such as reduced hydraulic efficiency and increased fortification of tracheids against implosion, as observed in north-temperate conifers (Pittermann et al.,

2006a,b; Pittermann et al., 2010; Hacke et al., 2001; bogs and swamps Sperry et al., 2006).

Materials and Methods Plant material was gathered locally from campus forests at the University of California in Santa Cruz (UCSC) or from glass- house collections at UCSC and UC Berkeley, USA. Fronds were collected from a minimum of four individuals (but only three individuals for the Lygodium species) from 16 species belonging to several families and exhibiting a variety of life history strategies, statures, and native habitats (Table 1). Species were sampled in May–August of 2009 and 2010.

Gas-exchange measurements Gas-exchange measurements were performed on the same fronds used for the hydraulics and anatomical studies described below using the Li-Cor LI-6400XT portable gas- exchange system (Li-Cor Biosciences Inc., Lincoln, NE, USA) fitted with a standard opaque 2 · 3 cm LED cham- ber. All plants were watered to saturation the day before the gas-exchange measurements were collected such that the lowest midday water potentials were only )0.75 MPa, as measured using a standard pressure chamber (PMS Instruments, Corvallis, OR, USA). Each measurement was obtained on mid-rachis pinnae, requiring between 2 and L. Pteridaceae AC Glasshouse collection Erect North American warm-temperate forests Mesic

5 min for a stable reading, with leaf temperature at 21C, (Willd) Pteridaceae CB Botanical garden Erect Central American deserts Xeric Sm. Blechnaceae WF Glasshouse collection Erect Western North American temperate forests Mesic G. Forst Aspleniaceae AB Glasshouse collection Erect Southern-hemisphere temperate forests Mesic )1 (Kaulf.) C. Presl Dryopteridaceae PM Coastal Redwood forest Erect Western North American temperate forests Mesic (L.) Sw. Lygodiaceae LF Glasshouse collection Climbing Southeast Asia tropical forests Mesic (L.) Kuhn Pteridaceae PQ UCSC Campus Erect Temperate and subtropical regions Mesic (L.) J.Sm. Polypodiaceae PA Glasshouse collection Erect North and South American tropical and subtropical forests Mesic the flow rate at 300 ml min and the CO2 mixer set to (Labill) Discksoniaceae DA Glasshouse collection Erect Southern-hemisphere temperate forests Mesic (Thunb.) C. Presl Dennstaedtiaceae MS Glasshouse collection Erect Hawaiian Island tropical and subtropical forests Mesic )2 )1 (Thunb.) Sw. Lygodiaceae LJ Glasshouse collection Climbing Southeast Asia tropical forests Mesic (Kaulf.) Watt Drypteridaceae DR Coastal Redwood forest Erect Western North American temperate forests Mesic L. Osmundaceae OR Botanical garden Erect European North African, and Eastern North American

400 lmol m s . The chamber humidity ranged from 70 (L.) Beauvois Psilotaceae PN Glasshouse collection Erect Hawaiian Island tropical and subtropical regions Mesic (Thunb.) Farwell Polypodiaceae PL Glasshouse collection Erect Asian temperate regions Mesic

to 80%, only slightly higher than ambient glasshouse (L.) Pteridaceae PC Glasshouse collection Erect Southern and Northern Hemisphere temperate regions Mesic humidity. For glasshouse-grown species, the minimum ) ) A list of species used in this study and their collection sites, growth habit and native habitat features saturating light intensity was 500 lmol m 2 s 1, but it was ) ) increased to 700 lmol m 2 s 1 to ensure that the pinnae Pteris cretica Pyrrosia lingua Pteridium aquilinum Woodwardia fimbriata Psilotum nudum Polypodium aureum Polystichum munitum Table 1 SpeciesAdiantum capillus-veneris Asplenium bulbiferum Cheilanthes bonariensis Dicksonia antarctica Dryopteris arguta Lygodium flexuosum Lygodium japonica Microlepia strigosa Family Symbol Collection site Habit Endemic habitat Climate were indeed light saturated. Outdoor-growing, sun-exposed Osmunda regalis

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species such as Cheilanthes bonariensis and P. aquilinum Functional xylem area was measured on samples perfused ) ) required the equivalent of full sun (2000 lmol m 2 s 1)to for several hours with basic fuchsin whereby hand-cut cross- achieve saturation, while the understory fern Woodwardia sections were photographed and analyzed using IMAGEPRO ) ) fimbriata saturated at 700 lmol m 2 s 1. An average of 10 software (Media Cybernetics, Carlsbad, CA, USA). Xylem measurements were made on glasshouse species (one to two specific conductivity (ks) and leaf specific conductivity (kL) measurements per frond), and that was doubled for out- represent k standardized for functional xylem area and distal door-grown ferns. leaf area, respectively. Conifer gas-exchange data were collected in May to late Several workers have previously reported the presence of June in 2009 and 2010 from 20 individuals from the mucilage that often inconvenienced hydraulic measure- Pinaceae, Cupressaceae, Podocarpaceae and Araucariaceae ments on ferns, but with the exception of P. aquilinum and in the context of another project, and supplemented by data D. antarctica we did not experience this problem. In the from the literature (Pittermann et al., 2006a,b). The gas- aforementioned species, the stipe ends were carefully shaved exchange measurements were performed by J.P. and E.L. to expose the xylem, and following a mild )0.5 MPa spin on well-hydrated branches (the average midday water in the centrifuge (see below), the mucilage was eliminated potential was )0.4 MPa) sampled from trees growing at the from the cut ends. All hydraulic measurements were made UC Santa Cruz Arboretum, San Francisco Botanical promptly to minimize artifacts caused by wounding effects. Garden at Golden Gate Park and the UC Botanical Garden We used the centrifuge method to determine species’ vul- in Berkeley, all locations in coastal, central California. The nerability to cavitation in response to a range of xylem measurements were made as described in the previous para- pressures (Pockman et al., 1995; Alder et al., 1997). Stems graph, with a leaf temperature of 21C, a flow rate of were secured in a custom rotor designed to fit a Sorvall RC- )1 )2 )1 300 ml min , 400 lmol m s ambient CO2, chamber 5C centrifuge (Thermo Fisher Scientific, Waltham, MA, humidity between 40 and 60% and a light intensity of USA) and spun for 3 min at speeds that induce a known )2 )1 2000 lmol m s . Six measurements were made on four xylem pressure (Px). The per cent loss of conductivity (PLC) to six stems collected from at least two trees, depending on caused by centrifugation at each Px was calculated from the k availability at the local arboreta. measured after spinning, relative to the maximum conduc- tivity after degassing (kmax)atPx = 0 Mpa, such that Hydraulic measurements PLC ¼ 100 ð1 k=kmaxÞ; Eqn 1 The stipes represent the leafless portion of the fern frond, and unlike the frond rachis, the total xylem area is generally where kmax was determined at Px = 0 MPa following de- invariable along this segment. Stipes were collected from the gassing. The segments were spun to progressively more ) bottom 18–20 cm of the frond for a sample size of n = 6–10. negative Px at 1 MPa increments until the PLC exceeded ) Stipe diameters ranged from 2 to 8 mm, with Lygodium sp. 90%, or alternatively until Px = 10 MPa, which is the and Adiantum capillus-veneris possessing the narrowest stipes most negative Px that can be achieved using the centrifuge. and P. aquilinum and W. fimbriata the widest. Stipe samples A Weibull function was used to fit the vulnerability curves were re-cut under water to a length of 142 mm, and the distal from each stipe (Neufeld et al., 1992), and the xylem pres- ends shaved smooth with a razor blade. Although all plants sure at which segments exhibited a 50% loss of conductivity were well hydrated before collection, any remaining embo- (P50) was computed as an average ± 1 SD per species. lism was removed by submersing the segments in distilled Ferns such P. nudum and A. capillus-veneris had fronds and filtered 20 mM KCl solution (0.22 lm; E-Pure filtra- that were either too short or too weak to endure centrifuga- tion system; Barnstead International, Dubuque, Iowa, USA) tion, so these species were subjected to the bench-dry and degassing overnight under vacuum. Stipes were degassed method of obtaining vulnerability curves on at least 15 rather than flushed in order to minimize handling damage fronds. Intact fronds were cut and dehydrated down to a and wound effects arising from compressed cortex tissue. range of desired water potentials measured on mid-rachis Hydraulic conductivity (k) was measured according to pinnae, at which point the stipes were removed and the the method of Sperry (1993) and calculated as the flow rate hydraulic conductivity and PLC calculations were made as for a given pressure gradient standardized per unit of stem described above. The data were fitted with a Weibull func- length. The stipes were mounted on a tubing apparatus tion that was used to compute a single P50.. where k was measured gravimetrically under a pressure of Similarly, we used the bench-dry method to verify the 6–8 kPa using filtered 20 mM KCl solution. The flow rate validity of the centrifuge-generated PLC data on through the segments was determined without a pressure P. aquilinum, a species known to possess vessels longer than head before and after each gravimetric flow measurement. 142 mm in both the rhizome and the stipes (Carlquist These background flows were averaged and subtracted from & Schneider, 2007). Recent work on vines and ring- the pressure-induced flow in order to improve accuracy. porous species suggests that the centrifuge method may

New Phytologist (2011) 192: 449–461 2011 The Authors www.newphytologist.com New Phytologist 2011 New Phytologist Trust New Phytologist Research 453 overestimate vulnerability to cavitation (Choat et al., 2010; et al. (2006a,b), as well as unpublished data collected in Cochard et al., 2010), but we found that, in P. aquilinum, 2008–2010 by J. P. the two methods yielded vulnerability curves that were indistinguishable from one another (data not shown). Conduit length measurements Similarly, we observed no differences in kmax on P. aquilinum stipes ranging in length from 14 to 22 cm, We used the silicone injection method of Hacke et al. 22 cm approximating the length of the longest infrequent (2007) and Christman et al. (2009) to obtain conduit vessel. We would expect kmax in the 14.2-cm segment to be length distributions in species of ferns suspected to possess much higher if the removal of conduit end walls reduced a long conduits such as Lygodium flexuosum and P. aquilinum significant proportion of end-wall resistance. (Carlquist & Schneider, 2007). Five fronds of each species were injected basipetally from the base of the stipe with a silicone-fluorescent dye-hardener mix at a pressure of Anatomical measurements 50 kPa, and left overnight (Christman et al., 2009). The In north-temperate woody plants, the conduit double-wall- silicone was hardened over 3 d, after which the stipes were 2 to-lumen-span ratio, (t ⁄ b)h , is positively correlated with sectioned at regular intervals starting at 5 mm from the base cavitation resistance and reflects the potential of the xylem of the injection site. In P. aquilinum, which is known to conduits to withstand implosion caused by negative water possess vessels, the increments were progressively increased potentials (Hacke et al., 2001). This metric has since been from 5 mm to 5 cm. The fraction of silicone-filled conduits applied to woody plants across a variety of habitats at each length increment was counted and the data analyzed (Pittermann et al., 2006a; Jacobsen et al., 2007). Conduit according to Christman et al. (2009). diameters and double-wall thickness:lumen span measure- ments were determined on xylem located in the center of Results the stipe previously used for hydraulic measurements according to Pittermann et al. (2006a,b) and Hacke et al. Conduit diameter frequency distributions show that with (2001). The sections were treated with phloroglucinol to the exception of P. nudum and P. lingua, all surveyed stipes highlight lignified tissue such as xylem, rinsed, and possess conduits that are wider than 40 lm, and average mounted in glycerin. All of the xylem was photographed conduit diameters can vastly exceed even the largest maxi- under 200–400· magnification with a digital camera mum tracheid diameters (c. 60 lm) found in riparian mounted on a Motic BA400 compound microscope (JH conifer roots (Fig. 1; Pittermann et al., 2006a,b). Tracheids Technologies, San Jose, CA, USA). Because of the limited in excess of 100 lm in diameter were frequently present in amount of xylem present in the stipes, it was possible to the viney, indeterminately growing stipes of Lygodium measure all conduits located in each cross-section. Conduit flexuosum, a finding that is consistent with the need to features were measured using IMAGEPRO analysis software. maximize water transport to distal leaves by packing large Tracheid lumen areas were converted to equivalent circle conduits in narrow, 2.5-mm-diameter stipes with low xylem diameters and the hydraulic mean diameter wasP calculatedP 5 4 from tracheid diameter distributions as Dh = D ⁄ D according to Kolb & Sperry (1999). All conduits were mea- sured, with the sample size ranging from from 70 to 300 conduits depending on species xylem area. The thick- ness:span ratio was measured on adjacent conduits where at least one cell (although usually both) was within 10% of Dh. The double-wall thickness was determined from the shared walls of at least 40 and up to 100 of these cells. Conduit length and diameter were measured on individ- ual tracheids obtained from macerations according to the methods of Mauseth & Fujii (1994). Individual vascular bundles at least 5 cm in length were excised from the stipe and submersed in a 50 : 50 solution of 30% hydrogen per- oxide and 80% glacial acetic acid (Sigma-Aldrich, St. Louis, MO, USA), and heated to at least 110C for c. 2–3 d. IMAGEPRO was used to measure at least 50 tracheids per spe- Fig. 1 Examples of conduit diameter distributions in stipes belonging to six species of north-temperate and tropical terrestrial ferns with cies at ·200–400. some of the narrowest and broadest ranges in conduit sizes (n = 5–6 Anatomical data from conifer root and shoot xylem were stipes ± SD). Additional data can be found in Supporting collected from previously published work by Pittermann Information, Fig. S1 and Table S1.

2011 The Authors New Phytologist (2011) 192: 449–461 New Phytologist 2011 New Phytologist Trust www.newphytologist.com New 454 Research Phytologist

(a) (b) (c)

(d) (e) (f)

Fig. 2 Mid-segment cross-sections of six fern stipes, stained with toluidine blue, mounted in glycerol and photographed under a compound microscope. Bars, 1 mm. These species were chosen on the basis of their cavitation resistance and the arrangement of their primary xylem, beginning with species that exhibit the least cavitation resistant and integrated xylem and ending with those with the most resistant and

sectored xylem: (a) Psilotum nudum, P50 = )0.69 MPa; (b) Pteridium aquilinum, P50 = )1.05 ± 0.53 MPa; (c) Dicksonia antarctica, P50 = )0.96 ± 0.45 MPa; (d) Cheilanthes bonariensis, P50 = )3.18 MPa; (e) Lygodium flexuosum, P50 = )3.97 ± 1.88 MPa; and (f) Dryopteris arguta, P50 = )11.8 ± 7.6 MPa. The arrangement of L. flexuosum tracheids within the stele is unusual because parenchyma cells separate the conduits from one another. A thick ring of schlerenchyma fibers known as the hypodermal sterome (Rowe & Speck, 2005) is located just below the epidermis in all cross-sections. In addition to its mechanical role, the sterome may also aid in preventing water loss.

areas. The observed wide range in conduit diameter frequencies explains the significant standard deviations asso- ciated with mean and hydraulic tracheid diameter values reported in figures below. We generated vulnerability curves on stipes from all spe- cies examined to determine how susceptible ferns are to drought-induced cavitation given the tremendous variation observed in conduit dimensions and xylem stele arrange- ments (Fig. 2). Fig. 3 shows vulnerability curves in six species that span a broad range of cavitation resistances, from a P50 of –0.68 MPa in P. nudum to a surprising )11.8 ± 7.6 MPa (mean ± SD) in Dryopteris arguta. In several fronds of D. arguta, the P50 was extrapolated from the Weibull fits assigned to each vulnerability curve. Surprisingly, several D. arguta stipes showed < 60% loss of Fig. 3 The hydraulic response to increasingly negative water hydraulic conductivity at )9 MPa but had a tendency to potential in six ferns representing the most vulnerable and the most ) resistant species to drought-induced cavitation. Because of its short shred when subjected to 10 MPa xylem pressures in the stature, the vulnerability curve in Psilotum nudum stipes was centrifuge, so data at this pressure are not reported. obtained using the bench-dry method while other species were We observed no hydraulic or structural xylem trade-offs evaluated using the centrifugal method of Alder et al. (1997). associated with species’ cavitation resistance, in contrast to trends evident across a broad sampling of the conifer xylem conduit walls, both of which tend to lower hydraulic response (Fig. 4). Indeed, cavitation resistance in conifers conductivity, especially within the Pinaceae and north- comes at the cost of reduced conduit diameters and thicker temperate Cupressaceae (Pittermann et al., 2006a,b), but

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(a) (a)

(b)

(b)

(c)

Fig. 5 Conduit double-wall thickness plotted as a function of conduit hydraulic diameter in conifers (circles) and ferns (letters; see Table 1 for species abbreviations) (a). In contrast to conifers, we observed in fern conduits a strong, positive correlation between conduit hydraulic diameter and double-wall thickness. Surprisingly, 2 this scaling results in a low (t ⁄ b)h ratio (b) that exhibits no safety factor from implosion, even under very negative xylem pressures (ns, not significant).

Fig. 4 The relationship between the xylem pressure causing a 50% are found in increasingly drought-resistant species, indicat- loss of hydraulic conductivity as a result of cavitation (P50) and ing greater conduit strength (Fig. 5; Hacke et al., 2001; xylem specific conductivity (a), conduit hydraulic diameter (b) and Pittermann et al., 2006a). However, conifer tracheids also conduit double-wall thickness (c) in conifers (circles) and ferns tend to exhibit more reinforcement than strictly necessary, a (letters; see Table 1 for species abbreviations). In contrast to trait interpreted as a safety factor protecting against implo- conifers, fern xylem showed no hydraulic or structural trade-offs 2 associated with P . ns, not significant. sion, which is shown by the relationship between the (t ⁄ b)h 50 2 ratio and species’ P50 (Fig. 5). By contrast, (t ⁄ b)h ratios in no such costs were apparent in fern xylem. We can, how- fern conduits are decoupled from cavitation resistance and ever, make the generalization that cavitation resistance in exhibit no safety factor from implosion. Thus, fern conduits ferns is on par with cavitation resistance in conifers. may show some degree of mechanical flexibility. The outlier Despite showing a broad range of P50 values, fern tracheid in Fig. 5 is P. nudum, a basal filicopsid fern, which, despite allometry is surprisingly different from conifer tracheids showing modestly reinforced conduits, is the least cavita- (Fig. 5). In conifers, the tracheid double-wall thickness to tion-resistant species sampled. 2 tracheid lumen diameter ratio (t ⁄ b)h is a proxy of tracheid Several studies indicate that photosynthesis in pterido- resistance to implosion, and this trait strongly correlates phytes is inherently constrained by the low hydraulic with cavitation resistance in the north-temperate Pinaceae efficiency characteristic of this group (Brodribb et al., 2005; 2 and Cupressaceae. In these two families, higher (t ⁄ b)h ratios Watkins et al., 2010). Although our photosynthesis and

2011 The Authors New Phytologist (2011) 192: 449–461 New Phytologist 2011 New Phytologist Trust www.newphytologist.com New 456 Research Phytologist

(a)

(b)

Fig. 8 Fern xylem supports similar leaf areas as conifer xylem despite relying on primary xylem with a total area that is two orders of magnitude smaller than that of conifers. The tight scaling between xylem and leaf area in ferns is likely to reflect the limited venation and low capacitance of fern leaves (Brodribb et al., 2005, 2007), a result consistent with the constant scaling between stipe length and leaf area observed in Polystichum munitum (Limm & Dawson, 2010). In the absence of the indeterminately growing fronds of Lygodium japonica, the r2 of the regression increases to 0.61 and P < 0.001. Conifers are indicated by circles and ferns by letters (see Table 1 for species abbreviations).

stomatal conductance rates agree with previous studies (see Fig. 6 Photosynthesis (a) and stomatal conductance (b) as a also Watkins et al., 2010), it is important to point out that, function of hydraulic conduit diameter in conifers (circles) and ferns across a broad sampling of ferns including climbing as well (letters; see Table 1 for species abbreviations). Cheilanthes as north-temperate species, photosynthesis and stomatal bonariensis (CB), an outdoor-grown desert fern, exhibited the conductance rates can equal or exceed those of evergreen highest rates of gas exchange, consistent with its thicker pinnae which presumably provide some degree of capacitance. The highest conifers (Fig. 6). We attribute this result in part to the photosynthesis and conductance rates in the conifers belong to ferns’ much wider and longer conduits which account for three deciduous species of Cupressaceae. ns, not significant. the surprisingly high hydraulic conductivity of fern xylem, and which can, in turn, support a broad range of stomatal conductance rates. Indeed, it is likely that the ferns’ large conduit volumes compensate for the ferns’ small functional xylem areas that are two orders of magnitude smaller than those of conifers, despite serving a similar amount of distal foliage (Figs 7, 8). If evolution acted as expected to increase hydraulic efficiency in the xylem-limited ferns, then P. aquilinum represents the pinnacle of hydraulic selection in fronds as it is the one species in our pool known to con- tain vessels in both fronds and rhizomes (not shown), with some vessels exceeding 20 cm in length (Fig. 7). The hydraulic consequences of the high-volume tracheids of ferns are readily apparent when xylem conductivity is expressed per leaf and functional xylem area, kL and kx, respectively, and compared with equivalent data in conifers (Fig. 9). The similar kL values in conifers and ferns support the finding that leaf-level function in both plant groups can Fig. 7 Average conduit length plotted as a function of conduit exhibit a similar range of assimilation and conductance rates diameter in conifers (circles) and ferns (letters; see Table 1 for (Fig. 6). species abbreviations). Assuming a cylindrical geometry, fern tracheid volumes are 8 to 60· greater than those of conifers. This Lastly, our data indicate that fern xylem can exhibit lower factor would be considerably higher in Pteridium aquilinum, where transport resistivity (rx, the inverse of kx) for a given conduit vessel length exceeds 20 cm. diameter than the xylem of the more derived conifer

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(a) and angiosperm lineages (Fig. 10; additional data from Pittermann et al., 2005). Put another way, when we compared the area-specific xylem resistivities of fern, conifer, vine, and ring- and diffuse-porous species with equivalent mean conduit sizes, fern xylem exhibited higher transport efficiencies than the xylems of the remaining plant groups with the exception of vines, which had a mean conduit diam- )2 eter of 150 lm and an rx of 13 MPa s m (Fig. 10).

Discussion The greater xylem specific conductivity observed in the (b) xylem of fern stipes relative to conifer xylem can be attrib- uted in part to the ferns’ longer and wider tracheids. Large lumen diameters reduce the frictional resistance to water transport that arises from conduit walls, while increasing conduit length reduces the frictional resistance attributable to pit membrane crossings from one conduit to the next (Sperry et al., 2006). In ferns, high-volume conduits were presumably free to arise because the xylem serves solely in a hydraulic capacity, in contrast to the double-duty xylem of conifers where tracheids not only deliver water to the can- opy but also support it (Pittermann et al., 2006a,b). This support function reduces conifer tracheid diameter and length, and frequently calls for extra wall thickening, as Fig. 9 Leaf (a) and xylem (b) specific conductivities plotted as a function of hydraulic conduit diameter in conifers (circles) and ferns observed in compression wood (Spicer & Gartner, 2002). (letters; see Table 1 for species abbreviations). Species’ transport In ferns, however, the presence of the hypodermal sterome efficiency is a function of xylem conduit diameter in both conifers (Fig. 2) relaxes the support constraints imposed upon the and ferns. The large discrepancy in kL values between Lygodium tracheids, such that high-volume conduits function solely in flexuosum and Lygodium japonica can be attributed to the the efficient transport of water. An analogous situation indeterminately growing habit of the fronds, which varied between 0.5 and 1.5 m in length. occurs in the xylem of woody angisperms, where thick- walled fibers constitute the structural matrix that supports the canopy, leaving the hydraulic function to the much longer vessels (Hacke et al., 2001; Sperry et al., 2006), as well as in leaves, which are supported by turgor pressure and collenchyma. In conifers, root xylem is also released from the support function and characteristically exhibits tracheids that are longer, wider and more conductive (Pittermann et al., 2006a). The primary xylem of conifer seedlings and juvenile shoots is developmentally most anal- ogous to fern xylem, but whether or not the structure– function patterns of primary tracheids resemble those of conifer roots or ferns is unknown. Fern xylem may be hydraulically efficient, but it is pri- mary xylem nonetheless and is subject to limits imposed by a basal developmental program. First, the overall structure of ferns is constrained by the lack of a vascular cambium, which gives rise to lateral branching and secondary xylem (Rowe & Speck, 2004; Spicer & Groover, 2010). The Fig. 10 Xylem specific resistivity plotted as a function of hydraulic absence of wood imposes a fundamental limit on fern size conduit diameter in conifers (closed circles), angiosperms (open and canopy development even in tree ferns, which raise circles) and ferns (letters; see Table 1 for species abbreviations). Some conifer and angiosperm data were re-plotted from the original their relatively modest canopy by virture of anomalous sec- comparison of tracheid vs vessel transport efficiency in Pittermann ondary growth courtesy of roots and fibers. Secondly, ferns et al. (2005). possess much smaller xylem areas in the absence of a cam-

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bium than conifers, despite supporting an equivalent may exhibit variable porosity that corresponds to species’ amount of foliage and similar rates of gas exchange resistance to air entry air-seeding, as previously mentioned; (Figs 8,9). Hence, the observed frond allometry in Fig. 8 is and the integration of the vascular bundles, specifically the the result of a limited vascular system that supplies water in frequency of conduit contact between two or more vascular sufficient quantities for transpiration, but probably lacks the bundles, affects the rate at which air spreads through the buffer against fluctuations in water availability that capaci- xylem. Empirical and theoretical work on woody plants has tance provides in woody plants. The reliance on primary shown that hydraulically integrated vascular networks are xylem for water transport may explain why ferns exhibit associated with increased hydraulic conductivity, but at the more rapid stomatal closure than angiosperm leaves cost of more rapid spread of air-seeding-induced embolism (Brodribb & Holbrook, 2004). (Zanne et al., 2006; Loepfe et al., 2007; Schenk et al., The extent to which pit membrane characteristics con- 2008). Looking across the stelar patterns of the sampled tribute to fern hydraulic efficiency is largely undetermined. fern species (Fig. 2), we suspect that cavitation resistance in However, the few studies that addressed the relative contri- these plants is controlled by a similar, spatially determined butions of pit and lumen resistance in fern xylem indicate vascular arrangement that may inhibit or facilitate the that pit membranes in fern tracheids account for 36 to 47% spread of air through the conduit network depending of total tracheid resistance (Calkin et al., 1985; Schulte on conduit-to-conduit or bundle-to-bundle contact. For et al., 1987). By contrast, conifer and angiosperm pit example, hydraulically integrated xylem belonging to membranes confer considerably greater resistance to flow, P. aquilinum and P. nudum was the most vulnerable to accounting for c. 64 and 56% of conduit resistance, respec- cavitation, while xylem belonging to the cavitation-resistant tively (Wheeler et al., 2005; Pittermann et al., 2006b; D. arguta and P. cretica was packaged in a limited number Sperry et al., 2006). Whether or not reduced pit membrane of spatially separated (sectored) vascular bundles (Fig. 2). resistance in ferns is attributable to greater porosity and ⁄ or Variation in P50 values was greater in species with sectored a higher fraction of conduit pit area remains to be seen, but xylem, presumably because of irregular and as of yet un- some evidence suggests that fern pit membranes not only characterized bundle-to-bundle connectivity over the length may be quite porous, but may extend along the full length of the sampled stipe segments. A combination of methods of the tracheid wall (Carlquist & Schneider, 2007). including dye perfusions and high-resolution computed Selection has been shown to act on pit membrane area as tomography (HRCT) could be used to construct three- well as porosity, and both traits are thought to have a pro- dimensional renderings of variable xylem patterns along the found effect on hydraulic efficiency and cavitation by the length of the frond and as well as in different species entry of air, otherwise known as ‘air-seeding’ (Choat et al., (Schulte et al., 1987; Brodersen et al., 2010). 2008; Jansen et al., 2009; Lens et al., 2011). Certainly, Perhaps a more fundamental question than what controls greater pit area and membrane porosity should compensate air-seeding in ferns is what selects for cavitation resistance for xylem comprised of single-celled conduits lacking the in these overwhelmingly mesophylic plants? Watkins et al. low-resistance torus-margo pit membrane (Pittermann (2010) show that tropical epiphytic ferns are more cavita- et al., 2005; Sperry et al., 2006). Interestingly, the homo- tion resistant than terrestrial species, a result consistent with geous pit membranes found in the tracheids of vesselless the epiphytes’ lower midday water potentials. In our assort- angiosperms including the Amborellaceae, Winteraceae and ment of terrestrial ferns, the issue may also be complicated Trochodendrales exhibit considerably higher porosity than by ecology and life history strategy. For example, those of eudicots, a finding that is consistent with the low, P. aquilinum is a drought-deciduous fern, and exhibits conifer-like area-specific pit resistance in this basal lineage xylem with high hydraulic conductivity and low cavitation (Hacke et al., 2007). Similar to these basal dicots, fern resistance. This hydraulic ‘boom and bust’ strategy is similar xylem exhibits hydraulic conductivities that are at least 38· to the phenology and physiology of deciduous ring-porous greater than predicted for tracheids lacking conifer-type or riparian trees. By comparison, D. arguta is a semi-perennial torus-margo pits (Pittermann et al., 2005; Hacke et al., species that retains fronds for at least 2 yr, during which it 2007). Whether Botrichium dissectum, an Ophioglossid fern endures the dry, mediterranean summer climate of coastal with torus-margo pit membranes (Morrow & Dute, 1998), California. In addition to drought stress, cavitation resist- exhibits comparable hydraulic efficiencies that are closer to ance in ferns is probably associated with frond longevity. conifers or ferns remains to be seen. An extreme example of this is L. flexuosum, the fronds of Recent work indicates that pit membrane features control which grow indeterminately to lengths well beyond 2 m. cavitation resistance in both conifers and angiosperms Although this species is native to mesic, subtropical habi- (Christman et al., 2009; Pittermann et al., 2010; Lens tats, its stipe is surprisingly cavitation resistant, presumably et al., 2011; Delzon et al., 2010), but what controls cavita- to ensure water transport to distal pinnae. Whether or not tion resistance in ferns? Currently, we can entertain one or a reproductive, spore-bearing fronds show alternative hydrau- combination of two possibilities: the fern pit membrane lic strategies is an open question.

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Despite the high cavitation resistance exhibited by a observed in the xylem of variably dehydrated fronds number of fern species, the tracheid allometry of ferns is (J. Pittermann & E. Limm, unpublished), and it is not considerably different from conduits in conifers and woody unreasonable to suspect that, in the very cavitation-resistant angiosperms. In north-temperate conifers, the trend toward species, the reductions in hydraulic conductivity with increased implosion resistance comes courtesy of wall thick- progressively more negative water potentials may be ness:lumen diameter ratios that increase with P50, but do attributable to conduit distortion, rather than embolism. so by reducing tracheid diameter rather than fortifying The aforementioned HRCT method is nondestructive, and the tracheid walls (Pittermann et al., 2006a; Fig. 5). could provide a window into the intricacies of fern vascular Consequently, reduced transport efficiency constitutes the function in response to drought and rewatering in a live fundamental hydraulic trade-off associated with cavitation plant (Brodersen et al., 2010). resistance in north-temperate conifers (Pittermann et al., The extent to which we understand variation in xylem 2006a,b; Fig. 4). These safety–efficiency trade-offs are also structure and function across tracheophytes will affect the apparent at the level of the pit, whereby increased cavitation inferences we draw about the evolution of transport tissue resistance is associated with reduced pit conductivity across across both extinct and extant lineages, including the a range of conifer species and within a single tree Lycopodiales, Sellaginales and perhaps even some bryo- (Pittermann et al., 2006b, 2010; Domec et al., 2008). By phytes. For example, empirically based biophysical models of contrast, we found no hydraulic or structural ‘investment’ xylem performance have provided important insights into the trade-offs in vulnerable vs cavitation-resistant ferns, similar xylem function of the earliest land plants as well as the more to the results of Watkins et al. (2010). This is consistent derived seed ferns, pro-gymnosperms and other lineages with with the weaker relationship between vessel diameter and tracheid-based xylem (Wilson et al., 2008; Wilson and P50 observed in angiosperms, and the probabilistic role that Fischer, 2010; Wilson & Knoll, 2010). These models pit porosity plays in controlling cavitation resistance in di- suggest that, far ahead of the appearance of heteroxylous cots (Wheeler et al., 2005; Hacke et al., 2007; Christman angiosperm xylem, xylem evolution in ancient plants was et al., 2009; Lens et al., 2011). Across the pteridophytes, consistently guided by selection for hydraulic efficiency, the costs of drought resistance may be reflected in the cavitation resistance and, eventually, mechanical support. increased production of osmolytes and proteins that protect Our data show that extant ferns have succeeded in master- living tissues from low water potentials rather than xylem ing these three essentials, although not in the manner one structure per se (Proctor & Tuba, 2002). would predict based on the structure–function model In ferns, larger conduit diameters scale directly with derived from woody plants. The physiological resilience greater wall thickness (Fig. 5). Interestingly, despite the of ferns may go a long way toward explaining why they unusually thick tracheid walls found in several ferns, the have persisted across nearly all parts of the globe despite 2 relationship between species’ P50 and the conduit (t ⁄ b)h several mass extinctions and the intense competitive pres- ratio falls directly on the predicted conduit implosion line – sure exerted by angiosperms (Schneider et al., 2004; a dramatic departure from the pattern observed in conifer Schuettpelz & Pryer, 2009; Watkins et al., 2010). tracheids. Conduit implosion thresholds are computed without k, the safety factor from cell collapse, so our data Acknowledgements indicate two things: first, ferns do not allocate any more resources to their xylem than is necessary to maintain a We are grateful to the NSF (IOS-1027410; J.P.) and Save 2 circular geometry, and second, the conduit (t ⁄ b)h ratio the Redwoods League (E.L. and J.P.) for supporting this cannot be used to infer cavitation resistance in these plants. research. C.R. received funding through an NSF REU grant Fern tracheids may exhibit some degree of flexibility to tol- and from the Save the Redwoods league. M.A.C. was sup- erate bending or torsion stress associated with life in the ported by NSF-IBN-0743148 to John Sperry (University of understory or to facilitate the climbing habit. To wit, the Utah). The conifer data were collected with support from largest tracheids in the climber L. flexuosum are located in the Miller Institute for Basic Research (UC Berkeley; J.P.) the stele center, that is, the neutral axis region, which expe- and assistance from Todd Dawson. We wish to thank Lucy riences minimal tensile and compressive stress (Niklas, Lynn, Stephanie Ko and Shervin Bastami for contributing 1992). Also, flexible tracheids may accommodate shape to the xylem anatomical measurements, and Duncan Smith change in response to the tension imposed on them by the and John Sperry for kindly helping with the silicone injec- water column. A good example of this phenomenon is tion method. Holly Forbes, Mona Bourrell and the staff at transfusion tracheids in conifer leaves and needles. These the University of California Botanical Garden (Berkeley, unlignified tracheids flex and collapse under negative xylem CA) and the San Francisco Botanical Garden kindly pro- pressure, presumably to avoid cavitation and to facilitate vided access to plant material, as did Jim Velzy and Denise rapid refilling (Cochard et al., 2004; Brodribb & Polk (UC Santa Cruz), who also assisted with propagation Holbrook, 2005). Similarly distorted tracheids have been and glasshouse space. We also thank three anonymous

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reviewers whose insightful comments helped improve the vulnerability curves? A test with the Cavitron spinning technique Plant, manuscript. Cell & Environment 33: 1543–1552. Delzon S, Douthe C, Sala A, Cochard H. 2010. Mechanism of water- stress induced cavitation in conifers: bordered pit structure and function References support the hypothesis of seal capillary-seeding. Plant, Cell & Environment 33: 2101–2111. Alder NN, Pockman WT, Sperry JS, Nuismer S. 1997. Use of centrifugal Domec J-C, Lachenbruch B, Meinzer F, Woodruff D, Warren JM, force in the study of xylem cavitation. Journal of Experimental Botany 48: McCulloh K. 2008. Maximum height in a conifer is associated with 665–674. conflicting requirements for xylem design. Proceedings of the National Bailey IW, Tupper WW. 1918. Size variation in tracheary cells: I. A Academy of Sciences, USA 105: 12069–12074. comparison between the secondary xylems of vascular cryptogams, Gibson AC, Calkin HW, Raphael DO, Nobel PS. 1985. Water relations gymnosperms, and angiosperms. American Academy of Arts Sciences and xylem anatomy of ferns. Proceedings of the Royal Society of Edinburgh Proceedings 54: 149–204. 86B: 81–92. Bateman RM, Crane PR, DiMichele WA, Kenrick PR, Rowe NP, Speck Hacke UG, Sperry JS, Feild TS, Sano Y, Sikkema EH, Pittermann J. T, Stein W. 1998. Early evolution of land plants: phylogeny, physiology 2007. Water transport in vesselless angiosperms: conducting efficiency and ecology of the primary terrestrial radiation. Annual Review of and cavitation safety. International Journal of Plant Sciences 168: Ecology, Evolution, and Systematics 29: 263–292. 1113–1126. Brodersen CR, McElrone AJ, Choat B, Matthews MA, Shackel KA. Hacke UG, Sperry JS, Pockman WT, Davis SD, McCulloh KA. 2001. 2010. The dynamics of embolism repair in xylem: in vivo visualizations Trends in wood density and structure are linked to prevention of xylem using high-resolution computed tomography. Plant Physiology 154: implosion by negative pressure. Oecologia 126: 457–461. 1088–1095. Jacobsen AL, Pratt RB, Ewers FW, Davis SD. 2007. Cavitation resistance Brodribb TJ, Feild TS, Jordan GJ. 2007. Leaf maximum photosynthetic among 26 chaparral species of southern California. Ecological rate and venation are linked by hydraulics. Plant Physiology 144: Monographs 77: 99–115. 1890–1898. Jansen S, Choat B, Pletsers A. 2009. Morphological variation of Brodribb TJ, Holbrook NM. 2004. Stomatal protection against hydraulic intervessel pit membranes and implications to xylem function in failure: a comparison of co-existing ferns and angiosperms. New angiosperms. American Journal of Botany 96: 409–419. Phytologist 162: 663–670. Jansen S, Choat B, Vinckier S, Lens F, Schols P, Smets E. 2004. Brodribb TJ, Holbrook NM. 2005. Water stress deforms tracheids Intervascular pit membranes with a torus in the wood of Ulmus peripheral to the leaf vein of a tropical conifer. Plant Physiology 137: (Ulmaceae) and related genera. New Phytologist 163: 51–59. 1139–1146. Kenrick P, Crane PR. 1997. The origin and early evolution of plants on Brodribb TJ, Holbrook NM, Zwieniecki MA, Palma B. 2005. Leaf land. Nature 389: 33–39. hydraulic capacity in ferns, conifers and angiosperms: impacts on Kolb KJ, Sperry JS. 1999. Differences in drought adaptation photosynthetic maxima. New Phytologist 165: 839–846. between subspecies of sagebrush (Artemisia tridentata). Ecology 80: Calkin HW, Gibson AC, Nobel PS. 1985. Xylem water potentials and 2373–2384. hydraulic conductances in eight species of ferns. Canadian Journal of Lens F, Sperry JS, Christman MA, Choat B, Rabaey D, Jansen S. 2011. Botany 63: 632–637. Testing hypotheses that link wood anatomy to cavitation resistance Carlquist S. 1988. Comparative wood anatomy. Berlin, Germany: Springer- and hydraulic conductivity in the genus Acer. New Phytologist 190: Verlag. 709–723. Carlquist S, Schneider EL. 2001. Vessels in ferns: structural, ecological Limm EB, Dawson TE. 2010. Polystichum munitum (Dryopteridaceae) and evolutionary significance. American Journal of Botany 88: 1–13. varies geographically in its capacity to absorb fog water by foliar uptake Carlquist S, Schneider EL. 2007. Tracheary elements in ferns: new within the redwood forest ecosystem. American Journal of Botany 97: techniques, observations and concepts. American Fern Journal 97: 1121–1128. 199–211. Loepfe L, Martinez-Vilalta J, Pinol J, Mencuccini M. 2007. The relevance Choat B, Cobb AR, Jansen S. 2008. Structure and function of bordered of xylem network structure for plant hydraulic efficiency and safety. pits: new discoveries and impacts on whole-plant hydraulic function. Journal Theoretical Biology 247: 788–803. New Phytologist 177: 608–626. Mauseth JD, Fujii T. 1994. Resin-casting: a method for investigating Choat B, Drayton WM, Brodersen C, Matthews MA, Shackel KA, Wada apoplastic spaces. American Journal of Botany 81: 104–110. H, McElrone AJ. 2010. Measurement of vulnerability to water stress- McCulloh K, Sperry JS, Lachenbruch B, Meinzer FC, Reich PB, Voelker induced cavitation in grapevine: a comparison of four techniques S. 2010. Moving water well: comparing hydraulic efficiency in twigs and applied to a long-vesseled species. Plant, Cell & Environment 33: trunks of coniferous, ring-porous, and diffuse-porous saplings from 1502–1512. temperate and tropical forests. New Phytologist 186: 439–450. Christman MA, Sperry JS, Adler F. 2009. Testing the ‘rare pit’ hypothesis Meyer-Berthaud B, Scheckler SE, Wendt J. 1999. Archaeopteris is the for xylem cavitation resistance in three species of Acer. New Phytologist earliest known modern tree. Nature 398: 700–701. 182: 664–674. Moran R. 2008. Diversity, biogeography and floristics. In: Ranker TA, Cichan MA. 1985. Vascular cambium and wood development in Haufler CH, eds. Biology and evolution of ferns and lycophytes. Carboniferous plants. I. Lepidodendrales. American Journal of Botany Cambridge, UK: Cambridge University Press, 367–383. 72: 1163–1176. Morrow AC, Dute RR. 1998. Development and structure of pit Cichan MA. 1986. Vascular cambium and wood development in membranes in the rhizome of the woody fern Botrichium dissectum. Carboniferous plants. IV. Seed plants. Botanical Gazette 147: 227–235. IAWA Journal 19: 429–441. Cochard H, Froux F, Mayr S, Coutand C. 2004. Xylem wall collapse in Neufeld HS, Grantz DA, Meinzer FC, Goldstein G, Crisosto GM, water-stressed pine needles. Plant Physiology 134: 401–408. Crisosto C. 1992. Genotypic variability in vulnerability of leaf xylem to Cochard H, Herbette S, Barigah T, Badel E, Ennajeh M, Vilagrosa A. cavitation in water-stressed and well-irrigated sugarcane. Plant Physiology 2010. Does sample length influence the shape of xylem embolism 100: 1020–1028.

New Phytologist (2011) 192: 449–461 2011 The Authors www.newphytologist.com New Phytologist 2011 New Phytologist Trust New Phytologist Research 461

Niklas KJ. 1985. The evolution of tracheid diameter in early vascular plans Sperry JS, Hacke UG, Pittermann J. 2006. Size and function in conifer and its implications on the hydraulic conductance of the primary xylem tracheids and angiosperm vessels. American Journal of Botany 93: strand. Evolution 39: 1110–1122. 1490–1500. Niklas KJ. 1992. Plant biomechanics: an engineering approach to plant form Sperry JS, Hacke UG, Wheeler JK. 2005. Comparative analysis of end and function. Chicago, IL, USA: University of Chicago Press. wall resistivity in xylem conduits. Plant, Cell & Environment 28: Page CN. 2002. Ecological strategies in fern evolution: a neopteridological 456–465. overview. Review of Palaeobotany and Palynology 119: 1–33. Spicer R, Gartner BL. 2002. Compression wood has little impact on the Pittermann J. 2010. The evolution of water transport in plants: an water relations of Douglas-fir (Pseudotsuga menziessii) seedlings despite a integrated approach. Geobiology 8: 112–139. large effect on shoot hydraulic properties. New Phytologist 154: Pittermann J, Choat B, Jansen S, Stuart SA, Lynn L, Dawson T. 2010. 633–640. The relationships between xylem safety and hydraulic efficiency in the Spicer R, Groover A. 2010. Evolution of development of vascular cambia Cupressaceae: the evolution of pit membrane form and function. Plant and secondary growth. New Phytologist 186: 577–592. Physiology 153: 1919–1931. Stewart WN, Rothwell G. 1993. Paleobotany and the evolution of plants. Pittermann J, Sperry JS, Hacke UG, Wheeler JK, Sikkema EH. 2005. Cambridge, UK: Cambridge University Press. The torus-margo pit valve makes conifers hydraulically competitive with Taylor TN, Taylor EL, Krings M. 2009. Paleobotany: the biology and angiosperms. Science 310: 1924. evolution of fossil plants. Oxford, UK: Academic Press. Pittermann J, Sperry JS, Hacke UG, Wheeler JK, Sikkema EH. 2006a. Tyree MT, Zimmermann MH. 2002. Xylem structure and the ascent of sap. Mechanical reinforcement against tracheid implosion compromises the Berlin, Germany: Springer. hydraulic efficiency of conifer xylem. Plant, Cell & Environment 29: Veres JS. 1990. Xylem anatomy and hydraulic conductance of Costa Rican 1618–1628. Blechnum ferns. American Journal of Botany 77: 1610–1625. Pittermann J, Sperry JS, Wheeler JK, Hacke UG, Sikkema EH. 2006b. Watkins JE, Holbrook NM, Zwieniecki MA. 2010. Hydraulic properties Inter-tracheid pitting and the hydraulic efficiency of conifer wood: the of fern sporophytes: consequences for ecological and evolutionary role of tracheid allometry and cavitation protection. American Journal of diversification. American Journal of Botany 97: 2007–2019. Botany 93: 1265–1273. Wheeler JW, Sperry JS, Hacke UG, Hoang N. 2005. Intervessel pitting Pockman WT, Sperry JS, O’Leary JW. 1995. Sustained and significant and cavitation in woody Rosaceae and other vesseled plants: a basis for a negative water pressure in xylem. Nature 378: 715–716. safety vs. efficiency trade-off in xylem transport. Plant, Cell & Proctor MC, Tuba Z. 2002. Poikilohydry and homoihydry: antithesis or Environment 28: 800–812. spectrum of possibilities? New Phytologist 156: 327–349. Wilson JP, Fischer WW. 2010. Hydraulics of Asteroxylon mackei, an early Robinson RC, Sheffield E, Sharpe JM. 2010. Problem ferns: their impact Devonian vascular plant, and the early evolution of water transport and management. In: Mehltreter K, Walker LR, Sharpe JM, eds. Fern tissue in terrestrial plants. Geobiology 9: 121–130. ecology. Cambridge, UK: Cambridge University Press, 255–310. Wilson JP, Knoll AH. 2010. A physiologically expicit morphospace for Rothwell GW, Karrfalt EE. 2008. Growth, development and systematics tracheid-based water transport in modern and extinct seed plants. of ferns: does Botrychium S.L. (Ophioglossales) really produce secondary Paleobiology 36: 335–355. xylem? American Journal of Botany 95: 414–423. Wilson JP, Knoll AH, Holbrook NM, Marshall CR. 2008. Modeling Rowe N, Isnard S, Speck T. 2005. Diversity of mechanical architectures in fluid flow in Medullosa, an anatomically unusual Carboniferous seed climbing plants: an evolutionary perspective. Journal of Plant Growth plant. Paleobiology 34: 472–493. Regulation 23: 108–128. Zanne AE, Sweeney K, Sharma M, Orians CM. 2006. Patterns and Rowe N, Speck T. 2004. Plant evolutionary forms; an ecological and consequences of differential vascular sectoriality in 18 temperate tree and evolutionary perspective. New Phytologist 166: 61–72. shrub species. Functional Ecology 20: 200–206. Saldana A, Lusk CH, Gonzales WL, Gianoli E. 2007. Natural selection on ecophysiological traits of a fern species in a temperate rainforest. Evolutionary Ecology 21: 651–662. Supporting Information Schenk HJ, Espino S, Goedhart CM, Nordenstahl M, Martinze-Cabrera HI, Jones CS. 2008. Hydraulic integration and shrub growth form Additional supporting information may be found in the linked across continental aridity gradients. Proceedings of the National online version of this article. Academy of Sciences, USA 105: 11248–11253. Schuettpelz E, Pryer KM. 2009. Evidence for a Cenozoic radiation of ferns in an angiosperm-dominated canopy. Proceedings of the National Fig. S1 Additional vulnerability curves (bench dry (BD) Academy of Sciences, USA 106: 11200–11205. and centrifuge method). Schneider H, Schuettpelz E, Pryer KM, Cranfill R, Magallon S, Lupia R. 2004. Ferns diversified in the shadow of angiosperms. Nature 428: Table S1 Additional conduit diameter data (minimum and 553–557. l Schulte PJ, Gibson AC, Nobel P. 1987. Xylem anatomy and hydraulic maximum diameter in m) for the species surveyed in the conductance of Psilotum nudum. American Journal of Botany 74: study 1438–1445. Sperry JS. 1993. Winter xylem embolism and spring recovery in Betula Please note: Wiley-Blackwell are not responsible for the cordifolia, Fagus grandifolia, Abies balsamea, and Picea rubens. In: content or functionality of any supporting information Borghetti M, Grace J, Raschi A, eds. Water Transport in Plants under Climatic Stress. Cambridge, UK: Cambridge University Press, 86–98. supplied by the authors. Any queries (other than missing Sperry JS. 2003. Evolution of water transport and xylem structure. material) should be directed to the New Phytologist Central International Journal of Plant Sciences 164: S115–S127. Office.

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