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Ecology |
2 Department of Biology, Duke University, Durham, North Carolina 27708 USA 3 Nicholas School of the Environment and Earth Sciences, Duke University, Durham, North Carolina 27708 USA
Received for publication 10 September 2007. Accepted for publication 11 January 2008.
ABSTRACT
In the conifer genus Juniperus (Cupressaceae), many species are increasing rapidly in distribution, abundance, and dominance in arid and semiarid regions. To help understand the success of junipers in drier habitats, we studied hydraulic traits associated with their water stress resistance, including vulnerability to xylem cavitation, specific conductivity (KS), tracheid diameter, conduit reinforcement, and wood density in stems and roots, as well as specific leaf area (SLA) of 14 species from the United States and the Caribbean. A new phylogeny based on DNA sequences tested the relationships between vulnerability to cavitation and other traits using both traditional cross-species correlations and independent contrast correlations. All species were moderately to highly resistant to water-stress-induced cavitation in both roots and shoots. We found strong phylogenetic support for two clades previously based on leaf margin serration (serrate and smooth). Species in the serrate clade were 34–39% more resistant to xylem cavitation in stems and roots than were species in the smooth clade and had 
35% lower KS and 39% lower SLA. Root and stem resistance to cavitation and SLA were all highly conserved traits. A high degree of conservation within clades suggests that hydraulic traits of Juniperus species strongly reflect phylogenetic history. The high resistance to cavitation observed may help explain the survival of junipers during recent extreme droughts in the southwestern United States and their expansion into arid habitats across the western and central United States.
Key Words: juniper • Juniperus • phylogeny • vulnerability to cavitation • xylem cavitation • xylem embolism
The environment and evolutionary history are important forces shaping the hydraulic properties that determine how plants respond to water shortages. During water stress, negative pressure in the xylem can induce cavitation through the aspiration of air bubbles into a functional xylem conduit from a neighboring air-filled conduit (Zimmermann, 1983
). Cavitation results in conduits that can no longer transport water, eventually leading to stomatal closure (Sperry and Pockman, 1993), reduced photosynthesis (Jones and Sutherland, 1991), and potentially mortality (Tyree and Sperry, 1988). Vulnerability to water-stress-induced cavitation is an index of the maximum seasonal water stress in the field and provides insight into drought tolerance (Linton et al., 1998; Brodribb and Hill, 1999; Pockman and Sperry, 2000; Pratt et al., 2007). Physiological traits relating to drought tolerance are influenced by the evolutionary history of species and will likely influence survival in new habitats. By combining physiological data and molecular hypotheses, we investigated evolutionary history and hydraulic traits in the genus Juniperus (Cupressaceae), a generally drought-tolerant and invasive group of >60 conifer species (Adams and Demeke, 1993; Adams, 2004; Little, 2006).
Many Juniperus species in North America are invading drier habitats and increasing in abundance where they already grow by surviving droughts that other conifers cannot. In Africa, Asia, Europe, and the Caribbean, many Juniperus species are considered threatened or endangered, in many cases from habitat loss or degradation, agriculture, or extraction (Adams, 2004). In the western hemisphere, however, many of the species in section Sabina are expanding their distributions into relatively dry, lower-elevation habitats to the point of being "weedy" (Adams and Demeke, 1993; Jackson et al., 2002). For example, in the last 60 years J. occidentalis populations have encroached exponentially into lower-elevation grasslands and shrublands in the northwestern United States during severe droughts (Miller et al., 1994). In the southwestern United States, while co-occurring piñon pines (Pinus spp.) have experienced massive die-offs as a result of severe droughts in 1996 and 2002, juniper species have suffered little mortality and are increasing in dominance (Breshears et al., 2005; Mueller et al., 2005). Because of both the recent, widespread expansion of Juniperus species and their ability to survive severe droughts, we studied 14 New World juniper species to better understand their success in dry environments.
Juniperus species are among the most resistant (least vulnerable) species in the world to water-stress-induced xylem cavitation (Maherali et al., 2004), which along with conduit wall structure is one of the most important hydraulic traits determining drought tolerance in plants (Tyree and Ewers, 1991). The more resistant the xylem is to cavitation, the more negative the water potential the plant can sustain, and the stronger the conduit wall must be (Hacke et al., 2001). Within a plant, roots are generally more vulnerable than stems to water-stress-induced cavitation (Jackson et al., 2000). Species with smaller, thicker leaves have lower specific leaf area (SLA; leaf area/leaf mass) and generally occur in more stressful environments. Previous studies revealed correlations between SLA and maximum hydraulic conductivity and water availability (Cavender-Bares and Holbrook, 2001; Wright et al., 2001). The ratio of sapwood area to leaf area (AS/AL) has also been shown to increase with habitat aridity (Cavender-Bares and Holbrook, 2001). For Juniperus species then, we expected greater resistance to cavitation to be correlated with higher conduit wall strength, lower SLA, and higher AS/AL.
On the one hand, the ability of Juniperus species to survive in xeric environments today may arise from a single evolutionary event, an adaptation to a prehistoric climate in a common juniper ancestor. Alternatively, a trait such as high resistance to cavitation may have arisen multiple times in different lineages. In comparative studies, inference about adaptation is hampered by the statistical nonindependence of species (Felsenstein, 1985b). Consideration of phylogenetic relationships among species accounts for this nonindependence because closely related species may have similar phenotypes due to descent from a common ancestor. For example, in a study of six conifer species, Piñol and Sala (2000) found a trade-off between sapwood-area specific conductivity and resistance to cavitation only when two groups were considered separately, pines and nonpines. In another study, Jacobsen et al. (2007) found that sapwood-area specific conductivity, hydraulic vessel diameter, and maximum vessel length in 26 angiosperm species were correlated with resistance to cavitation in standard cross-species correlations but not when phylogenetic relationships were considered. These studies demonstrate the potential importance of evaluating trends between vulnerability to cavitation and climate or hydraulic traits in a phylogenetic framework. When analyzing traits, phylogenetic information plays a crucial role in distinguishing between evolutionary convergence (i.e., homoplasy, distantly related species that are phenotypically similar) and evolutionary conservatism (closely related species that are phenotypically similar). Comparative methods can also help identify relationships between pairs of continuously varying traits that reflect correlated evolution (e.g., Preston and Ackerly, 2003).
We used two types of comparative evolutionary analyses in this study: (1) an index to quantify levels of convergent evolution for single traits and (2) a test for correlated evolutionary change between pairs of traits. We investigated the degree of convergent or conserved evolution in single traits using the Quantitative conVergence Index (QVI; Ackerly and Donoghue, 1998). Because evolutionary correlations between pairs of traits can point to potentially adaptive associations or trade-offs, we also examined correlations among hydraulic traits and between hydraulic traits and habitat moisture availability. One particularly strong tool when comparing functional correlations of continuously varying traits among taxa is the use of phylogenetically independent contrast (PIC) correlations (Felsenstein, 1985b). Independent contrasts use reconstructions of ancestral traits to identify statistically independent comparisons among species. The differences are calculated between the trait values of sister taxa in a bifurcating phylogenetic tree. Then, the differences of each trait in the trait–trait pairs or trait–environment pairs of interest are used in correlations. For both types of comparative analyses, a phylogeny is required.
Because a DNA sequence-based phylogeny for the Juniperus species in this study did not previously exist, another goal of this study was to generate a phylogenetic hypothesis for the 14 Juniperus species under study (12 American, two Caribbean; all of which are members of section Sabina; Fig. 1). Within section Sabina, species have been divided into informal series based on leaf-margin serration, with smooth margin species mainly in eastern and northern North America, and serrate margin species mainly in western and southern North America and Central America (Gaussen, 1968; Adams and Demeke, 1993; Adams, 2004). We chose to base the phylogeny on the ITS region of nuclear ribosomal DNA, owing to its utility in numerous phylogenetic studies (e.g., Baldwin et al., 1995; Liston et al., 1999). We also used the nuclear second intron of LEAFY because it has been shown to be variable and phylogenetically useful in other taxa (Oh and Potter, 2003, 2005), including other gymnosperms (e.g., Won and Renner, 2003). We created a phylogenetic hypothesis using sequence variation in both ITS and LEAFY to examine the phylogenetic structure of physiological traits.
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) showed that higher vulnerability to cavitation was correlated with decreasing xylem density and stem mechanical strength, with and without taking into account phylogeny. Exploring the relationship between phylogenetic history and interspecific variation in water relations may help us to explain the distributions and the successful survival of Juniperus species after invasion into rangelands or during extreme drought. It may also offer a more general evolutionary perspective on physiological and ecological traits associated with water transport. MATERIALS AND METHODS
Taxon sampling and molecular markers
The study species have distributions ranging from the Mojave Desert of California (most arid, J. californica) to the Blue Mountains of Jamaica (most mesic, J. lucayana; Fig. 1). With the exception of the low-growing circumboreal shrub J. communis (section Juniperus), all North American junipers belong to Juniperus section Sabina (the third section, Caryocedrus, is monotypic; Adams, 2004). The Juniperus species in section Sabina are believed to be of relatively recent origin (Adams and Demeke, 1993). Section Sabina is divided into two informal series based on denticulation of leaf margins (as seen at 40x magnification): serrate (denticulate) and smooth (entire) margins (Appendix 1; Gaussen, 1968; Adams and Demeke, 1993; Adams, 2004). In addition to the 12 North American species, we added two Caribbean species to increase the number of taxa with smooth leaf margins in the study.
We sampled 14 Juniperus species within section Sabina to create a phylogeny based on DNA sequences (Fig. 1). Two accessions were sampled for each species (see Appendix 1 for collection information), and one accession of J. communis was used as an outgroup because it is a member of a separate section (section Juniperus; Adams and Demeke, 1993). All accessions were vouchered as herbarium specimens and deposited at the Duke University Herbarium in Durham, North Carolina, USA (Appendix 1). Total genomic DNAs were extracted from fresh or silica dried material using a DNeasy Plant Mini Kit according to manufacturer's protocols (Qiagen, Valencia, California, USA).
Amplification of ITS (ITS 1 intron, 5.8s exon, and ITS 2 intron) was accomplished using ITS4 (White et al., 1990) and ITS5a (Stanford et al., 2000) primers. Amplification of ITS used 0.1 µl Qiagen Taq DNA polymerase, 5 µl Q solution, 2.5 µl 10x buffer, 2.5 µl 10 mM dNTPs, and 1.25 µl each of the two 10 µM primers in a final volume of 25 µl. The PCR amplification conditions were as follows: an initial 2 min at 97°C, followed by 30 cycles of 1 min at 97°C, 1 min at 48°C, and 45 s at 72°C, and a final extension cycle of 7 min at 72°C.
Amplification of the second intron of LEAFY was initiated by using the degenerate primers LFY1 and LFY2 described by Oh and Potter (2003). LEAFY is a nuclear homeotic gene that regulates the establishment of floral meristem identity and flowering time in Arabidopsis (Weigel, 1995; Blázquez et al., 1997). The gene is found in all plants (Frohlich and Parker, 2000; Himi et al., 2001). The gene was duplicated on the lineage leading to seed plants, so although one copy was lost in angiosperms, two copies are present in gymnosperms: LEAFY and NEEDLY. We confirmed that the allele amplified was LEAFY and not NEEDLY by comparing our sequences with LEAFY and NEEDLY gymnosperm sequences in GenBank. We then designed Juniperus-specific primers LFY5J (ATG TTC AGC ACG TCG CAA AGG) in the second exon and LFY4J (TTG TCG ATA TGA CCT ACA CCA G) in the third exon of LEAFY. Amplification of LEAFY used 0.2 µL Phusion polymerase (Finnzymes, Helsinki, Finland), 10 µL 5x Phusion buffer, 4 µL 10 mM dNTPs, and 2 µL each of the two 10 µM primers in a final volume of 50 µL. The PCR amplification conditions were as follows: an initial 30 s at 98°C, followed by 40 cycles of 10 s at 98°C, 30 s at 55°C, and 90 s at 72°C; and a final extension cycle of 7 min at 72°C.
Amplified products were purified using the Qiagen Qiaquick purification protocol. Purified products were sequenced directly with the automated sequencing methodology of the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, California, USA). Sequencing primers for ITS were ITS4 and ITS5a plus Juniperus-specific primers ITS2J (GCT ACA TTC TTC ATC GTG GC) and ITS3J (GCC ACG ATG AAG AAT GTA GC). Sequencing primers for LEAFY were ITS4J and ITS5J. Products were cleaned in Sephadex G-50 (fine) Centri-Sep spin columns (Princeton Separations P/N 901, Adelphia, New Jersey, USA), dried under vacuum, and run on either an ABI 373, 377, or 9700 Automated Sequencer at the Duke University Department of Biology Sequencing Facility. Raw sequences were assembled and edited using the program Sequencher version 3.1.1 (Gene Codes, Ann Arbor, Michigan, USA) and manually aligned using the program MacClade version 4.06 (Maddison and Maddison, 2003).
Molecular phylogenetic analyses
Separate and combined parsimony analyses of ITS and LEAFY nucleotide sequences along with any potentially informative insertion/deletion characters (indels) derived from each alignment were conducted using the program PAUP* version 4.0b10 (Swofford, 2002). Indels were considered further and coded as binary characters when their length was uniform in all taxa sampled, whereas those of variable length where exact placement was uncertain were excluded. Settings in PAUP* were as follows: all characters equally weighted, uninformative characters excluded, 1000 random-sequence addition replicates, saving all shortest trees under ACCTRAN optimization, tree bisection-reconnection (TBR) branch swapping, STEEPEST DESCENT off, MULTREES on, and COLLAPSE branches if maximum length is zero (AMB-). Support for nodes resolved in the strict consensus of the most parsimonious trees for each data set was evaluated with bootstrap (BS) analyses (Felsenstein, 1985a). Bootstrap analyses were conducted using PAUP* with TBR branch swapping on 1000 bootstrap replicates, saving all trees. Bootstrap values of >85% support were considered strong, 70–84% moderate, 50–69% weak, and <50% lacking. Incongruence between data sets, specifically for taxa with conflicting phylogenetic placement, was evaluated using the criterion of >70% BS (Mason-Gamer and Kellogg, 1996). Initial analyses were performed using multiple accessions (2–6) per species. Then we randomly selected one accession per species to produce the tree used for comparative analyses. Overlaying of continuous characters onto the phylogeny was done using Adobe (San Jose, California, USA) Illustrator CS based on results from the program Mesquite version 1.06 (Maddison and Maddison, 2005).
Field sites and plant material for vulnerability curves
We collected stems and roots from 14 Juniperus species for vulnerability curves (Fig. 1, Table 1 for location and climate information for all sites). We collected all plant material before 1100 hours local time to minimize water stress. Stems and roots typically consisted of 3–8 growth rings. Branches were collected from the northern aspect (except for J. lucayana, where tree height limited branch selection) of the bottom third of the canopy. Canopy heights varied from 2 to 5 m for the American species and from 3 to 10 m for the Caribbean species. Branches and roots were selected to have diameters of 0.7–1.1 cm and a minimum length of 30 cm free of side branches. From each adult tree, we collected one branch and one root, for a minimum of six branches and six roots (although in some cases, only four roots were possible) per species. We collected the first roots encountered from a depth of 10–30 cm. Material was cut longer than needed (>4 cm at each end) so that embolism caused by severed conduits did not reach the segments used for vulnerability curves. Branches or root segments were immediately enclosed with damp paper towels, triple-bagged in plastic bags, and placed in a cooler to inhibit dehydration during transit to the University of New Mexico, Albuquerque, New Mexico, USA, or Duke University, Durham, North Carolina, USA. Vulnerability curves were initiated in the laboratory within 3 d of collection. For J. flaccida from Texas, J. lucayana from Jamaica, and J. barbadensis from St. Lucia, samples were sealed with damp paper towels and mailed overnight to Duke University for processing within 4 d of collection.
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). Hydraulic conductivity (Kh, kg•m•MPa-1•s-1), defined as the mass flow rate of water through a segment divided by the pressure gradient along the segment, was measured under a gravity-induced pressure head of 5–6 kPa with deionized and filtered (0.22 µm) water using an electronic balance connected to a computer. Before treatment, each segment was measured for initial Kh (hereafter referred to as Khmax). In pilot studies using J. virginiana from North Carolina and J. ashei from Texas, we found either negligible (<4%) native embolism or, in some segments, conductivity declines after flushing with deionized, degassed, and filtered (0.22 µm) water for 30 min at 40 or 50 kPa. We therefore did not flush segments in this study. Other studies have also reported that flushing decreases hydraulic conductivity in conifers (Sperry and Tyree, 1990; Pittermann et al., 2006) and angiosperms (Wang et al., 1992; Macinnis-Ng et al., 2004). Not flushing segments theoretically could result in underestimates of loss of hydraulic conductivity. However, in our pilot studies, flushing decreased hydraulic conductivity, perhaps as a result of no detectable native embolism (Pittermann et al., 2006) or of torus aspiration in conifers.
Specific hydraulic conductivity (KS, kg•m-1•MPa-1•s-1; a measure of conducting efficiency and sapwood permeability) was calculated as Khmax divided by the cross-sectional xylem area of the segment (AS; determined as the mean area of both ends of the segment). Active xylem area was not determined by staining, which may result in overestimated functional xylem area and underestimated KS. Leaf-specific hydraulic conductivity (KL, kg•m-1•MPa-1•s-1) was calculated as Khmax divided by the distal leaf area supported by the segment (AL). Leaf area was determined from a projected leaf area to leaf dry mass ratio, or specific leaf area (SLA), for each species based on a subsample (>25% of total leaf material) of leaves per branch. The ratio of sapwood to leaf area (AS/AL, unitless; Tyree and Ewers, 1991) was calculated as the cross-section of conducting tissue (m2) divided by leaf area distally supported (m2). The median value of mean annual precipitation (MAP) for each species was taken from Thompson et al. (1999), or from nearby weather stations for J. arizonica, J. lucayana, and J. barbadensis (Table 1).
Vulnerability to xylem cavitation
Although both freezing and water stress can induce cavitation, we focus here on water-stress-induced cavitation in juniper stems and roots. (See Willson and Jackson, 2006, for data on freezing-induced cavitation in a subset of the study species.) The relationship between embolism, quantified as a decrease in Khmax, and xylem tension and can be described in a "vulnerability curve." We used the centrifugal force method on stem and root segments (Pockman et al., 1995). Stem and root segments were spun to negative xylem tension, inducing cavitation and reducing hydraulic conductivity. The loss of hydraulic conductivity was plotted as a percentage loss (PLC) from Khmax against the corresponding xylem tension to generate a vulnerability curve using the following equation: PLC = (1 – Ktreated/ Khmax) x 100.
Xylem conduit dimensions and wood density
To examine the relationship between vulnerability to cavitation and conduit diameters, we measured tracheid diameters on segments used for vulnerability curves. Transverse sections (20–30 µm thick) were cut from stem (N = 6) and root (N = 4–6) segments using a sliding microtome (American Optical, Buffalo, New York, USA). The sections were stained with toluidine blue, then rinsed in deionized water and mounted in glycerol on a glass slide. Images along each of 3–4 radial axes approximately 90 degrees apart were captured at a magnification of 100x for roots and 200x for stems for one section per stem or root with a Nikon CoolPix 990 digital camera (Nikon, Melville, New York, USA) mounted on a light microscope (Nikon E400). Along each of the 3–4 radial axes, enough images were taken to cover the outer one-quarter of each radial axis. A minimum of 1000 tracheids per section was analyzed. After these images were downloaded to a computer, we measured lumen area of all intact tracheids with image analysis software (Scion Image v.β4.02 for Windows, Scion, Frederick, Maryland, USA). Because the tracheids were more square than circular or rectangular in cross section, tracheid diameter (d, in µm) was determined to be equal to the side of a square with an area equal to the lumen area. To determine the functional significance of conduit size distribution, we also calculated the hydraulically weighted mean diameter (dh, in µm) for each segment as 
d5 / d4 (Pockman and Sperry, 2000).
To examine the relationship between vulnerability to cavitation and construction costs, we measured the reinforcement of conduit walls against implosion and a related trait, wood density. For pairs of tracheids with diameters within 3 µm of dh, we measured maximum span across a tracheid (B) and thickness of the double wall between the pair (T) using Scion Image. Conduit wall reinforcement, (T/B)2, was measured using digital images of sections of stems and roots used for determining d and dh (Hacke et al., 2001). The (T/B)2 value was determined for a minimum of 50 tracheid pairs per stem or root. We measured a related parameter, wood density (D; dry mass per fresh volume, g•cm-3) on the same segments used for vulnerability curves (N = 4–6). Segments 2.5 cm long were cut from stems and roots without heartwood. The bark was peeled away, and fresh volume was determined as the volume of a cylinder. The segments were then oven-dried at 75°C for 72 h to obtain dry mass.
Statistical analyses
The tension inducing a percentage loss of conductivity (PLC) of 50% (P50) was estimated by fitting vulnerability curves using an exponential sigmoidal function: PLC = 100 / {1 + exp[a (
– b)]}, where is tension, b is P50, and a is proportional to the slope of the vulnerability curve (Pammenter and Vander Willigen, 1998). Vulnerability curves were fit to data from stems and roots using the nonlinear mixed model procedure (NLINMIX), which did not require log transformation of the data, in the program SAS 9.1 (SAS Institute, Cary, North Carolina, USA). Significant differences in P50 and other traits were determined with one-way ANOVA for species or two-way ANOVA for tissue type (stem, root), species and tissue type x species effects using the program JMP IN 5.1 (SAS Institute). Multiple comparisons were made using Tukey's honestly significant difference (HSD) using JMP IN 5.1.
Comparative methods: single trait analyses
One comparative method used in this study quantifies the degree of evolutionary conservatism or convergence, or phylogenetic signal, in single continuous traits. For single hydraulic traits, we determined the quantitative convergence index (QVI; Ackerly and Donoghue, 1998), which quantifies the amount of convergent evolution in each trait over the phylogeny based on linear parsimony methods, using CACTUS 1.13 software (Comparative Analysis of Continuous Traits Using Statistics; Schwilk, 2001; Schwilk and Ackerly, 2001). The QVI varies from 0, for highly conserved traits, such that phenotypically similar species are closely related, to 1, for highly convergent traits such that similar phenotypes are distantly related. Significant levels of conserved or convergent evolution were determined using the same program based on randomization methods to test for levels of homoplasy that are greater or less than expected due to chance. Significance testing was accomplished in CACTUS by comparing the calculated QVI to that under the null model of no relationship between character values and the phylogeny (i.e., character values are randomly shuffled across the tips of the phylogeny).
Comparative methods: correlations between traits
We used two methods to determine correlations between pairs of continuous traits, both with and without considering phylogenetic relationships among taxa. Phylogenetic independent contrast (PIC) correlations were used to test for correlated evolutionary change among pairs of traits (Felsenstein, 1985b). PICs are calculated as the difference between values in both traits of the sister taxa at terminal or internal nodes, such that n taxa result in n – 1 contrasts. Correlations are then determined through the set of differences. Independent contrasts were standardized to meet the assumptions of parametric statistics by dividing the contrast value by the branch lengths as determined by the number of changes along each branch. PIC correlation coefficients and significance levels were determined using CACTUS 1.13. We also calculated Pearson correlation coefficients for cross-species correlations, without considering phylogeny, using JMP IN 5.1.
RESULTS
Phylogenetic analyses
When analyzed separately (Appendices S1 and S2, see Supplemental Data with online version of this article) or together (Fig. 2), both ITS and LEAFY produced strong support for the division of the 14 species into two clades, one with the nine western U. S. species in the serrate leaf margin clade, and the other with five species in the Caribbean and eastern United States, plus the western U. S. species J. scopulorum, in the smooth leaf margin clade. The ITS sequence data were 1119 bp long and yielded 86 informative characters, and the LEAFY sequence data were 1052 bp long and yielded 112 informative characters. Eight ITS indels (insertions or deletions of base pairs in the DNA sequence) and 12 LEAFY indels were coded as binary characters. Because there was low bootstrap (BS) support for the areas of disagreement between the two sets of sequence data, we combined the ITS and LEAFY sequences for each individual (Mason-Gamer and Kellogg, 1996). Parsimony analysis of 198 informative characters in the combined ITS and LEAFY data set yielded a single tree (Fig. 2). In the serrate margin clade, J. californica is basal to the other eight species, and J. ashei-J. flaccida-J. deppeana form a clade that is sister to a J. arizonica-J. pinchotii-J. monosperma-J. osteosperma-J. occidentalis clade (Fig. 2). In the smooth margin clade, J. virginiana and J. virginiana var. silicicola form a highly supported clade (97% BS), which is sister to a weakly supported J. scopulorum-J. lucayana-J. barbadensis clade (65% BS). Strong support (100% BS) for both the serrate and smooth leaf margin clades (Fig. 2) confirmed previous taxonomic division into informal series based on leaf margin serration (Gaussen, 1968) and random amplified polymorphic DNAs (RAPDs; Adams and Demeke, 1993). We noted some topological differences between our results and the trees produced by Little (2006) in a broad-level study of the Cupressaceae clade. Specifically, Little (2006) placed J. californica as sister to J. osteosperma based on ITS and chloroplast DNA sequences; however, only five taxa are common to both studies, thus a more complete phylogenetic study of the genus, including intraspecific sampling, is needed to resolve this discrepancy.
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Comparative methods: correlations between traits
In most cases, we found relationships between stem or root KS and other traits that were significant using both cross-species and PIC analyses (Table 5). Stem KS was negatively correlated with AS/AL and positively correlated with SLA and MAP in the cross-species analysis (Table 5). These relationships weakened somewhat in the PIC analysis. Root KS was negatively correlated with AS/AL and positively correlated with SLA and MAP in the PIC analysis, but not in the cross-species analysis (Table 5).
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Traits observed in nature often reflect both the adaptation of species to their present habitats and the legacy of traits found in species' ancestors. Overall, all 14 Juniperus species in our study are more resistant to xylem cavitation compared to taxa in previous studies (e.g., Maherali et al., 2004). Juniperus californica had an extrapolated P50stem of –22.0 MPa, one of the most resistant species ever reported (along with other members of Cupressaceae; e.g., Actinostrobus acuminatus, Brodribb and Hill, 1999). Such resistance to cavitation could be particularly important because the recent expansion of junipers during the past two centuries has occurred during a period of increasing aridity (Miller and Wigand, 1994). The high resistance to water-stress-induced cavitation in Juniperus might help explain the successful survival of junipers during drought and after their expansion into drier environments.
The 14 Juniperus species in this study separated into two groups on the basis of their phylogeny and hydraulic traits, coinciding with taxonomic divisions based on leaf margin serration (Figs. 2, 5, 6). The most striking feature of the phylogeny produced in this study is the strong support for the taxonomic divisions into informal series based on leaf margin serration: serrate or smooth margins (Fig. 2; Gaussen, 1968; Adams and Demeke, 1993; Adams, 2004). Our results were similar to those of Adams et al. (2006) for southwestern U. S. junipers in that J. osteosperma and J. occidentalis formed a clade, and J. californica was distinct among the serrate species. The smooth margin species in this study, J. virginiana, from the eastern United States, J. virginiana var. silicicola from the coastal, southeastern United States, and J. scopulorum from the Rocky Mountains of the western United States, formed a clade with J. lucayana from Jamaica (also found in Cuba and the Bahamas) and J. barbadensis from St. Lucia (Fig. 2). This supports the suggestion that Caribbean junipers appear to have arisen from an ancestor of J. virginiana or J. scopulorum from the Appalachian-southeastern U. S. region rather than from the junipers in southern Mexico and Guatemala, which belong to the serrate margin series (Adams, 1995). When averaging species within series, the serrate margin Juniperus species, common in the more arid western United States, were more resistant to xylem cavitation but had lower KS than the smooth margin Juniperus species (Table 2, Fig. 5). In general, conduit diameters (d and dh) were narrower in the serrate margin species than in the smooth margin species, but the differences in KS, d, and dh were only significant in roots and not in stems (Table 2). Specific leaf area (SLA) and mean MAP for the serrate series were lower than for the smooth series (Table 2, Fig. 6). This pattern of traits associated with clade membership leads us to suggest that physiological traits related to hydraulic architecture yield a strong phylogenetic signal in Juniperus.
By examining single traits across a phylogeny, we established that several hydraulic traits gave strong phylogenetic signals in Juniperus. Root P50 (QVI = 0.39) was more highly conserved than stem P50 (QVI = 0.53, Table 3, Fig. 5). The most highly conserved trait was SLA (QVI = 0.19, Table 3, Fig. 6). SLA has been shown to decline along gradients of decreasing moisture and/or nutrient availability (Cunningham et al., 1999). In accordance, SLA was highest in the two tropical Juniperus species, next highest in the two eastern U. S. species, intermediate in the southwestern U. S. species, and lowest in the Mojave Desert species (Fig. 6). High phenotypic and ecological similarity between closely related species implies that some traits have changed little since a divergence from a common ancestor or that parallel evolutionary changes have occurred since divergence. The lack of change in evolutionary lineages could be due to (1) lack of genetic variation upon which selection can act, (2) insufficient time since an evolutionary divergence, or (3) stabilizing selection that maintains ancestral traits (Ackerly, 2003; Caruso et al., 2005). At least some Juniperus species have likely arisen recently because Bermuda's soil was formed only during the first interglacial period of the Pleistocene (Cox, 1959). Although these reasons cannot be distinguished here, we propose that because phylogenetic branch lengths were relatively short, speciation may be relatively recent, which could explain why Juniperus species show a high degree of conservatism in vulnerability to cavitation and other hydraulic traits.
Interestingly, species more resistant to xylem cavitation, such as many in the serrate series, tended to have greater differences between stem and root P50 values than less resistant species (Fig. 4). Notably, several species had no significant differences in stem and root vulnerability (Figs. 3, 4). Similarly, Linton et al. (1998) found no difference in vulnerability to cavitation in stems and roots of J. osteosperma. In general, however, roots were more vulnerable than stems when all species are considered (mean P50stem = –10.6 ± 0.5 MPa, mean P50root = –8.0 ± 0.3 MPa, P < 0.001; Fig. 3). Roots are often more vulnerable to cavitation than stems in most angiosperms and conifers (Linton et al., 1998; Kavanagh et al., 1999; Jackson et al., 2000; Maherali et al., 2004, Maherali et al., 2006; McElrone et al., 2004). A possible reason for why root xylem is more vulnerable than stem xylem, according to the vulnerability segmentation hypothesis, is because xylem tension is highest in peripheral organs such as leaves and minor twigs, so embolism occurs first in peripheral organs (Zimmermann, 1983). Cavitation in the roots that are more easily replaced may protect stem xylem from further water loss and possible cavitation. Roots are also likely partly buffered from the most extreme water potentials faced by stems and leaves. In addition, greater vulnerability in roots may be less harmful than in stems because roots are typically lower in construction cost, as indicated by wood density and (T/B)2 (Table 2), and they may have the opportunity for refilling of emboli by root pressure or other mechanisms (Sperry, 1995).
When levels of convergent evolution are low and conservatism are high, there are often discrepancies between cross-species and evolutionary correlations (Ackerly and Donoghue, 1998). A deep divergence, coupled with reduced divergence within the descendant lineages can explain correlations among traits using traditional cross-species correlations that diminish with PIC correlations. In contrast, traits with a higher degree of homoplasy (e.g., convergent evolution) tend to have less discrepancy between cross-species and PIC correlations. Not surprisingly then, stem and root P50 were both significantly correlated with SLA in the cross-species correlation but not in the PIC correlation (Fig. 7), since stem and root P50 and SLA are all highly conserved traits in Juniperus (Figs. 5, 6). For stem P50, the significant PIC correlation suggests that evolutionary increases in vulnerability to cavitation in stems are associated with evolutionary decreases in (T/B)2 and wood density (Fig. 7). For root P50, it appears that large differences in trait values between species in the serrate and smooth series caused the PIC correlations to be insignificant. It should be acknowledged that PIC analyses can be sensitive to phylogenetic uncertainty, branch lengths, and nonrandom species sampling (Ackerly, 2000), although other recent studies also found inconsistencies between cross-species and PIC correlations of cavitation resistance and wood density or (T/B)2 (Jacobsen et al., 2007; Pratt et al., 2007). A lack of correlated evolution between cavitation resistance and (T/B)2 may point to other anatomical traits, such as fibers in the angiosperm species Ceanothus crassifolius or Cercocarpus betuloides as suggested by Jacobsen et al. (2007), that contribute to resistance to implosion.
Our results are consistent with a growing body of evidence suggesting a lack of a trade-off between safety from xylem cavitation and efficiency of xylem transport. Zimmermann (1983) hypothesized that there was a "safety vs. efficiency" trade-off in xylem such that wider conduits were more efficient in water transport yet more vulnerable to xylem cavitation. Although evidence for such a correlation between P50 and KS has been shown to exist in studies of single communities (e.g., Pockman and Sperry, 2000; Martínez-Vilalta et al., 2002), studies comparing numerous species show little or no evidence for the trade-off (Tyree et al., 1994; Hacke and Sperry, 2001; Maherali et al., 2004) or no trade-off when phylogenetic relationships are taken into account (Maherali et al., 2006, Jacobsen et al., 2007). Still other studies have shown that the trade-off exists at the level of the individual plant; i.e., that roots are more vulnerable than stems yet roots have higher KS (Hacke and Sperry, 2001; Willson and Jackson, 2006). We found no relationship between P50 and KS within stems or roots using either standard cross-species or PIC correlations (Fig. 7). According to the Hagen–Poiseuille relationship, flow through a conduit is related to its diameter to the fourth power (Tyree and Sperry, 1989). In conifers, hydraulic conductivity is largely a function of conduit diameter and length, whereas vulnerability likely depends on strength or flexibility of the torus-margo complex in the pits between adjacent tracheids (Sperry and Tyree, 1990; Hacke et al., 2004), so efficiency and safety from cavitation are not necessarily coupled.
Surprisingly, despite the assumption that high resistance to cavitation may be costly in mesic environments, we found relatively high resistance to cavitation even in the two tropical species, J. lucayana and J. barbadensis (Fig. 3). Soil conditions can exert a strong influence over water availability (Carlquist, 1975), which might explain why some species showed more resistance to xylem cavitation than might be expected due to climate alone. For example, J. ashei has a range with the highest MAP values of the serrate series, yet is one of the most resistant species (Table 1, Fig. 3). Additionally, J. barbadensis experiences the second highest MAP of all 14 species, yet is more resistant than all of the smooth series species and many of the serrate series species (Table 1, Fig. 3). Juniperus ashei occurs on the Edwards Plateau in central Texas, typically on limestone bedrock with very little soil, often <10 cm depth (Jackson et al., 1999). Similarly, J. barbadensis is endemic to the summit of the Petit Piton on the Caribbean island of St. Lucia, where it occurs in rocky, poorly developed soil alongside succulent species and cacti. The fact that Juniperus species typically occur on limestone or rocky substrates or otherwise arid microsites, even in mesic environments, may help explain the relatively poor correlations of P50 with MAP for all species (Table 4).
Because reduced hydraulic efficiency is not consistently observed as a cost of resistant xylem, and all species are not equally resistant to xylem cavitation, there are presumably other trade-offs that are associated with xylem resistance to cavitation. Water in xylem is under tension, making conduit walls subject to bending stresses between a functional, water-filled conduit at negative pressure and an adjacent embolized, air filled conduit at atmospheric pressure (Hacke et al., 2001). Conduit collapse caused by high tension during water stress has been demonstrated in needles in Pinus (Cochard et al., 2004). Bending stress is related to the thickness of the double wall (T) between the conduits and the maximum conduit span (B) (Hacke et al., 2001). Resistance to cavitation in conifers and angiosperms is highly correlated with the ratio of the double wall thickness to span, (T/B)2 (Hacke et al., 2001). Because wood with thick walls in relation to conduit diameter [high (T/B)2] will also likely have high wood density, wood density is also related to P50 (Hacke et al., 2001). In our 14 Juniperus species, we found significant relationships between increasing resistance to cavitation and both increasing (T/B)2 and wood density (Fig. 7). In roots, the cross-species correlations were significant but the PIC correlations were not, and the opposite pattern occurred in stems (Fig. 7). Jacobsen et al. (2007) found in stems of 26 chaparral species that P50 and (T/B)2 were correlated with standard cross-species correlations, but not with PIC correlations. It appears that P50 and (T/B)2 may be functionally linked but may not display correlated evolutionary change. Jacobsen et al. (2007) suggested that selection may not act as long as (T/B)2 remains above the threshold necessary to resist implosion. The cost of having higher wall thickness in relation to span, while affording greater resistance to xylem cavitation, results in higher wood density and therefore greater metabolic costs and reduced growth rate (Enquist et al., 1999; Hacke et al., 2001).
In addition to a trade-off between vulnerability to cavitation and wood reinforcement, we found strong evidence for a significant trade-off in greater wood reinforcement (i.e., higher (T/B)2 and wood density) at the expense of reduced hydraulic conductivity in roots. In our study, lower root KS was strongly associated with higher wood density and (T/B)2, and both Pearson and PIC correlations were nearly identical (Table 5). A correlation was not evident for stems. Similarly, Pittermann et al. (2006) found a trade-off between greater mechanical reinforcement and reduced hydraulic efficiency in a study of 18 conifer species, including two Juniperus species. In their study, increases in (T/B)2 (and therefore wood density) were associated with reductions in conifer tracheid diameter rather than increases in wall thickness. The decreased tracheid diameter is therefore responsible for the reduced hydraulic conductivity. As in our study, Pratt et al. (2007) found in nine Rhamnaceae species that (T/B)2 in angiosperm vessels was significantly inversely correlated to KS in roots but not in stems. Also like our study, their correlations were strikingly similar in both standard cross-species correlations as well as PIC correlations. Tracheids function in both water transport and mechanical support. We may have seen a strong tradeoff in Juniperus roots but not in stems because roots have a role more limited to water transport, whereas stems have a greater mechanical support role to resist forces of wind, snow, and gravity.
Despite any associated costs, the high resistance to cavitation demonstrated here likely enables juniper species to survive drought conditions. Although the Juniperus species studied here were highly resistant compared to other tree species, the phylogenetic divergence of serrate and smooth leaf margins also divides the group physiologically and ecologically (Figs. 2, 5, 6). The division of our 14 Juniperus species into taxonomic series based on leaf margin serration (serrate or smooth) was well supported by the phylogeny based on ITS and LEAFY sequence variations (Fig. 2; Appendices S1, S2 [see Supplemental Data with online version of this article]). The two series were also distinguished in their ecophysiological traits, with the serrate group more resistant to xylem cavitation (Fig. 5). Vulnerability to cavitation in stems and roots, as well as SLA, showed evolutionary conservatism over the phylogeny (Figs. 5, 6). The divergence event between the two series has likely influenced the subsequent success of each group. The high resistance to cavitation seen in Juniperus may help explain their successful survival during the recent extreme droughts in the southwestern United States and why they have been able to expand into arid habitats across the western and central United States over the last century (e.g., Breshears et al., 2005; Mueller et al., 2005).
Appendix 1. Collection data for species analyzed for both ITS and LEAFY DNA sequences for phylogenies shown in Fig. 2 and Appendices S1 and S2 (see Supplemental Data with online version of this article). Vouchers for the 14 study species were deposited at the Duke University Herbarium, Durham, North Carolina, USA. Collection sites: NF = National Forest; NG = National Grassland; NM = National Monument; NP = National Park; SP = State Park.
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FOOTNOTES
1 This research was supported by an NSF Graduate Research Fellowship, an NSF Doctoral Dissertation Improvement Grant (no. 0308937), a Duke University Latin American and Caribbean Studies Travel Grant, and Duke University Biology Department grants to C.J.W. and an NSF CAREER grant (no. 97-3333) and an Andrew W. Mellon Foundation grant to R.B.J. The authors thank W. Pockman for lending his expertise and laboratory at the University of New Mexico; R. Addington, R. Banks, W. Cook, T. Crocker, S.-H. Oh, C. Moura, P. Selmants, and D. Willson for help in the laboratory or field; K. Ogle for help with fieldwork and statistical analyses; H. Maherali for helpful discussion; and the National Park Service, Jamaica Conservation and Development Trust, and St. Lucia Department of Forestry for access to field sites. They also thank members of the Jackson and Manos laboratories, A. Zanne, and two anonymous reviewers for helpful comments on the manuscript. 
4 Author for correspondence (e-mail: jackson{at}duke.edu)
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P< 0.10; *P< 0.05; **P< 0.005.