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Annals of Botany 108: 347 –357, 2011 doi:10.1093/aob/mcr135, available online at www.aob.oxfordjournals.org Pushed to the limit: consequences of climate change for the Araucariaceae: a relictual rain forest family Catherine A. Offord* The Australian Botanic Garden, Mount Annan, Royal Botanic Gardens and Domain Trust, Mount Annan Drive, Mount Annan, NSW 2567, Australia * E-mail [email protected] Received: 4 January 2011 Returned for revision: 18 February 2011 Accepted: 11 April 2011 Published electronically: 3 July 2011 † Background and Aims Under predicted climate change scenarios, increased temperatures are likely to predispose trees to leaf and other tissue damage, resulting in plant death and contraction of already narrow distribution ranges in many relictual species. The effects of predicted upward temperatures may be further exacerbated by changes in rainfall patterns and damage caused by frosts on trees that have been insufficiently cold-hardened. The Araucariaceae is a relictual family and the seven species found in Australia have limited natural distributions characterized by low frost intensity and frequency, and warm summer temperatures. The temperature limits for these species were determined in order to help understand how such species will fare in a changing climate. † Methods Experiments were conducted using samples from representative trees of the Araucariaceae species occurring in Australia, Agathis (A. atropurpurea, A. microstachya and A. robusta), Arauacaria (A. bidwilli, A. cunninghamii and A. heterophylla) and Wollemia nobilis. Samples were collected from plants grown in a common garden environment. Lower and higher temperature limits were determined by subjecting detached winter-hardened leaves to temperatures from 0 to –17 8C and summer-exposed leaves to 25 to 63 8C, then measuring the efficiency of photosystem II (Fv/Fm) and visually rating leaf damage. The exotherm, a sharp rise in temperature indicating the point of ice nucleation within the cells of the leaf, was measured on detached leaves of winter-hardened and summer temperature-exposed leaves. † Key Results Lower temperature limits (indicated by FT50, the temperature at which PSII efficiency is 50 %, and LT50 the temperature at which 50 % visual leaf damage occurred) were approx. – 5.5 to –7.5 8C for A. atropurpurea, A. microstachya and A. heterophylla, approx. –7 to –9 8C for A. robusta, A. bidwillii and A. cunninghamii, and –10.5 to –11 8C for W. nobilis. High temperature damage began at 47.5 8C for W. nobilis, and occurred in the range 48.5– 52 8C for A. bidwillii and A. cunninghamii, and in the range 50.5–53.5 8C for A. robusta, A. microstachya and A. heterophylla. Winter-hardened leaves had ice nucleation temperatures of – 5.5 8C or lower, with W. nobilis the lowest at –6.8 8C. All species had significantly higher ice nucleation temperatures in summer, with A. atropurpurea and A. heterophylla forming ice in the leaf at temperatures .3 8C higher in summer than in winter. Wollemia nobilis had lower FT50 and LT50 values than its ice nucleation temperature, indicating that the species has a degree of ice tolerance. † Conclusions While lower temperature limits in the Australian Araucariaceae are generally unlikely to affect their survival in wild populations during normal winters, unseasonal frosts may have devastating effects on tree survival. Extreme high temperatures are not common in the areas of natural occurrence, but upward temperature shifts, in combination with localized radiant heating, may increase the heat experienced within a canopy by at least 10 8C and impact on tree survival, and may contribute to range contraction. Heat stress may explain why many landscape plantings of W. nobilis have failed in hotter areas of Australia. Key words: Hardiness, frost, high temperature, climate change, Araucariaceae, rain forest. IN T RO DU C T IO N With a predicted warming trend over the Earth’s surface, many plant species face an uncertain future. Climate projections for Eastern Australia based on the effects of atmospheric greenhouse gases of anthropogenic origin remaining at current or at increased levels indicate a substantial upward shift in temperature ranges (CSIRO, 2010). Coupled with changes in rainfall frequency and intensity, temperature shifts are likely to impact on plant growth and survival, and a high degree of regional variation is expected (IPCC, 2007). There is concern for Australian wet-tropical and sub-tropical forest taxa with restricted distributions that are already fragmented through past climate shifts. Such species should be considered for proactive management, given greater information on their vulnerability to climate change and response to proposed actions (Hilbert, 2007). Species of greatest concern are those that have narrow geographical ranges that will become dissociated from their presentday temperature envelopes, especially those species with a North – South range of ,300 km or an elevation range ,300 m (Westoby and Burgman, 2006). The Araucariaceae is an evergreen conifer family from temperate, tropical and sub-tropical regions of the Southern Hemisphere, although they once ranged into the Northern Hemisphere (Stockey, 1982). They are emergent rain forest species that once occupied vast distribution ranges, peaking in # The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] * Boland et al. (2006). † Australian Bureau of Meteorology. ‡ From data recorded at one site 1998– 2006. 42.2 8C; –8.3 8C; 0.3 d; 38.4 d (Mudgee)†; 35.9 8C; – 8.2 8C; 0 d; 17.3 d (Nullo)†; 34.9 8C; –1.8 8C‡ 0– 1/24– 26 8C; moderate to high frost incidence† 900 m Norfolk Island pine Wollemi pine Araucaria heterophylla (Salisb.) Franco (cultivated source, 29 8,167 8, 30 m) Wollemia nobilis W.G.Jones, K.D.Hill, J.M.Allen (950218, 32 8S, 149 8E, 900 m) 18– 19/23 –2 8C; no frost† Hoop pine Araucaria cunninghamii Aiton ex A.Cunn (932671, 28 8S, 153 8E, 30 m) 0 –300 m 5– 10/28 –32 8C; low to moderate frost (up to 30 p.a.)* South: 1– 7/27– 30 8C; North: 16– 33 8C; low to moderate frost incidence* 150– 1200 m 0 –1000 m 44.2 8C; –2.8 8C; 0 d; 0.3 d (Tewantin)†; 36.7 8C; – 0.6 8C; 0 d; 0.3 d (Atherton)† 41.7 8C; –6.2 8C; 1.1 d; 19.3 d (Dalby)†; 41.2 8C; –4 8C; 0.2 d; 0.3 d (Imbil)† 36 8C; – 3.5 8C; 0 d; 7.7 d (Dorrigo)†; 37.7 8C; 0.8 8C; 0 d; 0 d (Bundaberg)†; 40.6 8C; 7 8C; 0 d; 0 d (Coen)† 28.4 8C; 6.2 8C; 0 d; 0 d (Norfolk Aero)† 13– 19/30 –32 8C; low frost incidence* 0 –900 m Queensland Kauri Bunya pine 36.7 8C; –0.6 8C; 0 d; 0.3 d (Atherton)† 10/30 8C; low to moderate frost incidence* 600– 1000 m Bull Kauri 38.1 8C; –5 8 C; 0 d; 1.6 d (Herberton)† 900– 1500 m Blue Kauri Agathis atropurpurea B.Hyland (971022, 17 8S, 145 8E, 1,140 m) Agathis microstachya Bailey and C.T.White (20010050, 17 827’S, 145 8 38’E, 740 m) Agathis robusta (F. Muell.) Bailey (86429, 25 8S, 153 8E, 10 m) Araucaria bidwilli Hook. (960623, 26 8S, 151 8E, 1100 m) 10/30 8C; low to moderate frost incidence* Temperature range (min/max monthly average); frost Altitude range Common name Species (accession, lat, long, alt of provenance) diversity in the Jurassic and declining towards the end of the Cretaceous (Miller, 1977). Perhaps as late as 140 000 years ago, the Araucarian forests were still a significant component of the Australian vegetation (Kershaw, 1994). With the drying and increased variability of the climate of the Australian continent, ranges contracted significantly so that by the late Pliocene, the Araucarian forests in south-eastern Australia were replaced by a mosaic of sclerophyllous forests and open vegetation, and localized cool temperate rain forests (Gallagher et al., 2003). By the late Pleistocene, Araucaria extinctions or range contractions had occurred in north-eastern Australia (Kershaw, 1994). Several species are severely restricted in distribution, especially Wollemia nobilis of which ,100 trees grow in the wild, and Araucaria heterophylla, found naturally only on Norfolk and Phillip Islands. Agathis atropurpurea and Agathis microstachya are also very limited in distribution. Agathis robusta, Araucaria cunninghamii and Arauacaria bidwilli are relatively more widely distributed and are found from sea level to high altitude (Table 1). Low temperatures are thought to determine the latitudinal and altitudinal limits of woody plants (Larcher, 2005) including Australian rain forest species (Dodson and Myers, 1986). Episodic, low temperature events, such as frosts, are more likely to influence plant distributions than average minimum air temperatures (Loik et al., 2004). Under climate change, frosts are likely to be more frequent in some areas and less frequent in others (Inouye 2000). The impacts of frost range from cellular to ecosystem in scale (Saxe et al., 2001; Loik et al., 2004). Ice can enter the leaf through stomata or hydathodes, or freezing nucleation of intrinsic water can occur (Pearce, 2001). Within a leaf, freezing occurs as a wave, and ice may travel at 3 – 47 mm s21, originating in the xylem vessels and tracheids (Hacker and Neuner, 2007) of the mid-vein, and spreading rapidly into the leaf (Ball et al., 2002). The freezing of apoplastic water in the leaf is generally characterized by a sharp increase in temperature known as the exotherm – the release of latent heat – with slow recovery to lower temperatures. The length of recovery is related to the position of freezing in the leaf, i.e. the leaf margin – quicker – and near the mid-vein – more slowly. Frost, resulting in the formation of ice crystals in plant tissue, may lead to plant death, or may at least reduce the functioning of sensitive plant parts such as buds, ovaries and leaves. Plants may therefore be killed outright or become predisposed to insect or fungal attack leading to plant death (Inouye, 2000). Structural differences between acclimated and nonacclimated leaves have been documented. In Eucalytus pauciflora, for example, tissue death is caused by the formation of intracellular ice in the cambium and phloem, with dehydration of cells generally leading to irreversible tissue damage. In contrast, acclimated leaves are characterized by the formation of extracellular ice in expansion zones in the mid-vein, resulting, largely, in restoration of the original functions of the leaves on thawing (Ball et al., 2004). Leaf death may retard plant growth/regrowth, and the ultimate effect of leaf death and stem survival would need to be investigated in order to determine fully the temperature limits of vulnerable species. Araucariaceae species are generally considered to be tolerant of mild frost. Visual inspection of frozen plant parts of Southern Hemisphere conifers for browning indicated that Lowest and highest recorded temperatures at nearest weather station(s); mean days .40 8C; and ,0 8C Offord — Temperature limits for relictual rain forest Araucariaceae TA B L E 1. Altitude, temperature and frost incidence in the natural range of Australian Araucariaceae species 348 Offord — Temperature limits for relictual rain forest Araucariaceae A. bidwillii was the most frost resistant of the Araucariaceae tested, with damage not occurring until – 10 8C for this species, while damage occurred at – 7 8C for A. australis and at – 5 8C for A. excelsa and A. cunninghamii, the least resistant (Sakai et al., 1981). High temperatures also limit plant growth and species distribution. The effects of high temperatures on plants are often more complex than those caused by cold temperatures and are, typically, photoinactivation (which may be reversible), followed by irreversible destruction of the photosynthetic apparatus, such as by disconnection of photosystem II (PSII) reaction centres and biomembrane lesions (Berry and Bjorkman, 1980) which is irreversible. Consequently, measurements of heat damage, and cold damage for that matter, should be made after reversible damage has been repaired, usually after several days to 1 week (see, for example, Krause et al., 2010). In a study of Australian rain forest species, Cunningham and Read (2006) found that seedlings of tropical species have a greater heat tolerance (51– 55 8C) than temperate species (48 – 51 8C), but considerably less tolerance than many semiarid sub-tropical woody plants (50 – 60 8C). These ranges represent the temperatures at which there was 50 % visible leaf damage (LT50). They also found that the range of temperatures within which all species tested maintained at least 50 % of their initial photosynthetic efficiency were consistently in the order of 56– 60 8C, regardless of their lower and upper limits. Techniques used to determine the effects of temperature on plant organs, include: visual assessment of damage (e.g. Sakai et al., 1981; Deans et al., 1995), measurement of fluorescence (e.g. Neuner and Buchner, 1999; Zhang et al., 2003) or electrical impedance (e.g. Zhang et al., 2010), and fractal analysis (Mancuso et al., 2004). Electrolyte leakage, measured by relative conductivity, is one of the most widely used methods for assessing low temperature effects as it is based on release of 35 30 349 cell contents due to cell wall hydrolysis and is easily measured (see, for example, Sutinen et al., 1992; Strimbeck et al., 2007). The ability to measure temperature tolerance adequately using this technique is variable, however, and depends on the species, the material used and the conditions of testing. It is, therefore, often appropriate to use a number of techniques to make assessments (Mancuso et al., 2004), especially when comparing species that are not well characterized for temperature tolerance. Given the limited natural distributions of Australian Araucariaceae and the increased need for knowledge of temperature limits to manage wild stands, forestry and horticultural specimens, the temperature tolerance range of seven species was investigated using plants from a common garden environment. Of particular interest was the question of whether highaltitude tropical species are more likely to be tolerant of high temperatures, while being more sensitive to frost, than more temperate species. M AT E R I A L S A N D M E T H O D S Plant material and site temperatures Leaf samples were taken in late winter (21 August 2006) from Araucariaceae species (Table 1) grown at The Australian Botanic Garden, Mount Annan, NSW Australia (34 805’S, 150 847’E). All plants had been grown from seed, were sexually mature and were between 9 and 14 years old. During winter 2006, the nearest weather station, Camden, recorded 21 separate frost events and several minimum night temperatures of – 4 8C in the months preceding the tests (Fig. 1). Material from the same trees was collected on 16 February 2007 following peak summer temperature exposure. The lowest temperature for Camden since recording commenced No. days < 0º C 2006 No. days > 35º C 2006 Max 2006 Min 2006 Max since 1943 Min since 1943 Temperature (ºC) 25 20 15 10 5 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Month F I G . 1. Average monthly minimum and maximum temperatures in 2006 and since 1943, and number of events of daily minimum or maximum temperatures below 0 8C or above 35 8C, for Camden NSW in 2006 (Australian Bureau of Meteorology). 350 Offord — Temperature limits for relictual rain forest Araucariaceae in 1943 was –6 8C (12 July 2002) and the highest was 45 8C (30 January 2003). To test the difference between the nearest Bureau of Meteorology (BoM) screen temperature and the air temperature within the plant canopy, Thermocron iButton temperature loggers (OnSolution, Sydney, Australia) were placed on several different trees of A. atropurpurea, with three replicates located on the eastern and three on the western sides of the trees. Temperatures were logged every 15 min for 13 d in late February 2009. The radiant heat factor was determined as the average difference between temperatures on the eastern and western sides of the trees. This factor was then used to estimate the upper screen temperature limits for each species. FvFm and visual rating Leaf samples (detached moistened terminal branch sections, 150 mm in length) were harvested from three trees per species. Samples (three samples per tree) were collected from the eastern side of the tree at a height of 1.5 – 3 m above ground level. They were stored overnight prior to use in sealed plastic bags at 3 8C (winter collections) and at 21 8C (summer collections) to dark adapt and re-oxidate. Winter-hardened samples were lightly ‘sandwiched’ in the resealable plastic bag and placed into a variable-controlled temperature freezer (Engel 40, Engel Australia). The temperature was slowly lowered by 3 8C h21 to the target temperature, and was held at that temperature for 2 h to ensure equilibration of the temperature throughout the samples, before being slowly raised back to room temperature. The target temperatures ranged from 0 to – 17 8C in 2 or 3 8C increments. Sealed plastic bags containing summer temperature-exposed samples were placed in a water bath and exposed for 30 min to temperature treatments ranging from 25 to 63 8C in 3 8C increments. All samples were then stored in the plastic bags at room temperature for 2 weeks. Chlorophyll fluorescence was measured using a MINI-PAM photosynthetic yield analyser (Walz, Germany). Yield (Fv/Fm) was calculated to determine PSII damage after the treatments. A visual rating of damage was made based on a scale of 0 (no visible damage) through to 5 (all leaves, stems and buds brown or necrotic). Both measurements were made 7 d after treatment, as more immediate measurements give less reliable results (Cunningham and Read, 2006; Buchner and Neuner, 2009). Ice nucleation temperature A thermocouple (Walz Ni-CrNi, diameter 100 mm) was attached using Micropore tape (3M Company) to the abaxial side of a moist detached leaf in the mid-laminal position. The leaf was placed in a resealable plastic bag and placed into a variable-controlled temperature freezer. The temperature was slowly lowered by 3 8C h21 and the mid-lamina temperature was recorded every 10 s using a logging device. For A. cunninghamii and A. heterophylla, the thermocouple loop was placed around a leaf (‘needle’) and taped in place. Exotherms (sharp rises in temperature, with a slow recovery to the freezer temperature), which show the temperature of ice nucleation within and between the cells of the leaf, were observed and recorded for 9 – 12 leaves of each species. Data analysis Using the Fv/Fm and visual rating data, the temperatures at which significant damage occurs, the FT50 (50 % reduction in Fv/Fm of leaf ) and LT50 (50 % lethal damage to leaf ) were estimated as the inflection point ( parameter C) of eqn (1): Y = [A/1 + eB(x−C) ] + D (1) where Y ¼ Fv/Fm or visual rating, A ¼ range of values, B ¼ slope at the temperature of inflection, C ¼ inflection point, D ¼ minimum value. The logistic regression curves were also used to estimate LTi (temperature at which damage was initiated) and LT100 (temperature at which 100 % tissue damage occurred) to show the range over which damage occurred to that species. Using FT50 and LT50 values for the individual trees, the species were compared by analysis of variance (ANOVA) and the treatment means compared by least significant difference (l.s.d.) using the Genstat (Lawes Agricultural Trust, 2007) package. Tspan, the temperature tolerance range, was calculated for each species by taking the difference between upper and lower FT50 and LT50 values. Exotherm data were analysed by ANOVA and treatment means compared by l.s.d. R E S U LT S Leaf damage The leaf damage variables, Fv/Fm and visual damage rating, were significantly correlated for all species at both low (Table 2) and high temperatures (not shown; P , 0.001). The strong relationship is reflected in the agreement between the LTi, LT100 and LT50 values (Fig. 2) for the respective species. In the lower temperature range, leaf damage was initiated at approx. – 7.5 to – 8 8C in W. nobilis, at – 6 to – 6.5 8C in A. cunninghamii and at – 4 to – 5 8C (P , 0.05) in the other species. Arauacaria heterophylla was the only species to show some difference between FTi and LTi (–5.5 and – 3.5 8C, respectively), which may be the result of difficulty in measuring fluorescence on the needles of this species. The visual rating is TA B L E 2. Correlations between FT50 and LT50 temperature values (R 2 values) Species Agathis atropurpurea Agathis microstachys Agathis robusta Araucaria bidwillii Araucaria cunninghamii Araucaria heterophylla Wollemia nobilis *** P , 0.001. for low R2 0.90*** 0.84*** 0.70*** 0.82*** 0.82*** 0.80*** 0.76*** Offord — Temperature limits for relictual rain forest Araucariaceae 351 Upper temperature (ºC) 60 55 50 45 Wollemia nobilis Araucaria heterophylla Araucaria cunninghamii Araucaria bidwillii Agathis robusta Agathis microstachya Agathis atropurpurea Wollemia nobilis Araucaria heterophylla Araucaria cunninghamii Araucaria bidwillii LTi LT50 LT100 Agathis robusta FTi FT50 FT100 Agathis microstachya 0 –2 –4 –6 –8 –10 –12 –14 –16 –18 –20 –22 Agathis atropurpurea Lower temperature (ºC) 40 F I G . 2. Ranges within which leaf damage occurs to Australian Araucariaceae species at low and high temperatures (+s.e.). FTi, FT50 and FT100 are the temperatures at which initial, 50 % and 100 % reduction in Fv/Fm occurred; LTi, LT50 and LT100 are the temperatures at which initial, 50 % and 100 % lethal leaf damage occurred. likely to be a more accurate measure of leaf damage in this species. Maximum estimated damage occurred at – 12.5 to – 14 8C in W. nobilis, at – 10 to – 11 8C in A. cunninghamii and somewhere between – 7.5 and – 12 8C for the other species. Again, A. heterophylla was damaged at consistently higher temperatures than the other species (– 7.5 to – 8 8C). Agathis atropurpurea, A microstachya and A. heterophylla were the least cold tolerant, with average FT50 values in the range of approx. – 5.5 to – 7.5 8C. Agathis robusta, A. bidwillii and A. cunninghamii were damaged in the range –7 to – 9 8C, and W. nobilis did not show significant damage until – 10.5 to – 11 8C. In the high temperature experiment, FT50, mirrored by slightly higher LT50 values of W. nobilis occurred at significantly (P , 0.05) lower temperature (approx. 47.5 8C) than for A. bidwillii and A. cunninghamii (48.5 – 52 8C) which was lower than A. robusta, A. microstachya and A. heterophylla (50.5– 53.5 8C). The initial and 100 % values generally mirrored this pattern. It can clearly be seen that in the upper temperature range, FT50 was consistently lower than LT50, with the obverse pattern in the lower temperature range (Fig. 2). The Tspan, indicating the temperature tolerance range of each species, was consistent between the two variables (Table 3). All species had a temperature tolerance range between 58 and 61 8C. TA B L E 3. The temperature tolerance span (Tspan) for Australian Araucariaceae (from upper and lower LT50 estimates determined by FvFm and visual rating, Fig. 2) Tspan Fv/Fm Tspan visual rating 58 59 61.5 58.5 57.5 58.5 58.5 58 59.5 60 59 59 59.5 59 Agathis atropurpurea Agathis microstachys Agathis robusta Araucaria bidwillii Araucaria cunninghamii Araucaria heterophylla Wollemia nobilis Canopy vs. screen temperature The effect of localized radiant heat is demonstrated in Fig. 3. Meteorological screen temperatures recorded approx. 1 km away from the trees at Camden (BoM) on the observation days were consistent with the temperatures recorded in the shaded canopy of the trees. The exposed western side of the trees had consistently higher temperatures. Temperature differentials between the eastern and western parts of the canopy of 5 –10 8C were recorded on the hotter days (Fig. 3). Using a 10 8C radiant heat factor, estimates of upper temperature 352 Offord — Temperature limits for relictual rain forest Araucariaceae Screen Eastern side of tree Western side of tree 40 Temperature (ºC) 35 30 25 20 1 2 3 4 5 6 7 8 Day 9 10 11 12 13 F I G . 3. Maximum daily temperatures measured in late February 2009 within the canopy of Araucariaceae trees grown at Mount Annan Botanic Garden compared with the closest meteorological screen temperature (Campbelltown-Mount Annan, Australian Bureau of Meteorology, 2009). limits for the species were determined using LT50 values (Table 4). Maximum BoM screen temperatures ranged from 38.5 8C for W. nobilis to 43.5 8C for A. microstachya and A. heterophylla. TA B L E 4. Predicted upper meteorological screen temperature limits based on LT50 (temperature at which leaves sustained 50 % damage; based on visual rating) (Fig. 2) +10 8C radiant heat factor (Fig. 3) Upper screen temperature limit (8C) Ice nucleation temperature All species showed a greater resistance to low temperature exposure when the plants were winter-hardened (Table 5). Ice nucleation occurred in leaves of A. heterophylla at only – 2.5 8C in unhardened plants, but winter-hardened leaves withstood similar temperatures to the other species, indicating that some species are naturally more susceptible to ice nucleation when in the unhardened state. Ice nucleation did not occur in winter-hardened A. microstachys until – 6.2 8C and in W. nobilis until – 6.8 8C (Table 5). DISCUSSION Measuring thermal tolerance There was generally good agreement between the two techniques used to determine leaf damage caused by low and high temperatures. Fv/Fm is thought to overestimate the lower temperature tolerance (Neuner and Buchner, 1999) and underestimate the upper temperature tolerance (Bigras, 2000), the magnitude of which was similar in each of the species examined here (Fig. 2) and by Cunningham and Read (2006). Both Fv/Fm and visual rating can, therefore, be taken as reliable measures of leaf damage due to temperature in these species. Fv/Fm values have been shown to correspond to whole plant damage and mortality due to freezing (Percival and Henderson, 2003). Several studies have identified chlorophyll fluorescence (Fv/Fm) as the most useful from an array of more conventional techniques used to study cold (MacRae Agathis atropurpurea Agathis microstachys Agathis robusta Araucaria bidwillii Araucaria cunninghamii Araucaria heterophylla Wollemia nobilis 43 43.5 42.5 42 41 43.5 38.5 TA B L E 5. Exotherm temperature of summerand winter-hardened detached leaves of the seven Australian Araucariaceae species Species Agathis atropurpurea Agathis microstachys Agathis robusta Araucaria bidwillii Araucaria cunninghamii Araucaria heterophylla Wollemia nobilis Summer (8C) Winter (8C) –3.1ab –4.6cd –4.9d –3.8ac –3.5a –2.5b –4.6c –6.6a –6.2ac –5.7bc –5.5b –5.5b –5.7bc –6.8a Means within each column with the same letter are not significantly different by l.s.d. (P . 0.05). Summer and winter means were significantly different for each species. et al., 1986; Perks et al., 2004) as well as heat tolerance (Neuner and Pramsohler, 2006). The ability of this technique to reflect irreversible tissue damage is because reversible PSII damage may occur to a certain threshold beyond which regulatory processes cannot cause a reversal and the Offord — Temperature limits for relictual rain forest Araucariaceae measurement is only detecting irreversible damage, which happens rapidly past that critical point (Perks et al., 2004), as estimated by FT50. The degree of hardening and the sensitivity of the organ to temperature would vary greatly in natural environments and so these estimates, taken at the most acclimated point, reflect only the possible thresholds for these species and not absolute values. Further, these temperature tolerance values and rankings should be used as indicators of relative frost tolerance, as frost tolerance varies greatly according to degree of cold acclimation (‘hardening’) and provenance of the plant (Read and Hill, 1989; Larmour et al., 2000). Frost severity in the field depends on microtopography and exposure to the sky (see King and Ball, 1998). While such factors may affect the thermotolerance range of species, the comparison of wellacclimated plants from a common garden environment is likely to be the best guide to the relative vulnerability of the species to temperature extremes. This is borne out by the consistent temperature span of the species tested here which varied by no more than 3.5 8C (Table 3) regardless of the estimates for minimum or maximum temperatures. In their natural environment, it is the ability of these species to acclimate that will determine their response to climate change. What are the temperature limits of Australian Araucariaceae? This study confirms that Australian Araucariaceae are mildly frost tolerant and may survive temperatures in the range –5 to – 10 8C, with W. nobilis appearing to be the most frost tolerant and the least heat tolerant. Agathis atropurpurea, A. microstachya and A. heterophylla were the least cold tolerant and the most heat tolerant species. It might be expected that A. bidwillii would be more cold tolerant than A. cunninghamii, based on the lower temperatures experienced in the wild (Table 1), but in this study the two species appear to have similar low and high temperature responses and, along with A. robusta, are the next most cold tolerant of the species tested. These results are consistent with their current locations and climatic regimes: W. nobilis, for example, occurs the furthest south and at an altitude of 900 m, while A. heterophylla occurs on an island devoid of frost. The A. bidwillii provenance used was from a low altitude site, whereas the A. cunninghamii was originally collected from a high altitude location (Table 1). This does not explain why Sakai et al. (1981) found A. bidwilli able to tolerate – 10 8C and A. cunninghamii – 5 8C. Araucaria bidwillii and A. cunninghamii are the most widespread of the Australian Araucariaceae, and it is likely that there are differences between provenances that were not specifically tested in either study. Further examination of the differences between provenances would reveal the extent of temperature tolerance in such widespread species. It has been suggested that species with a wide geographic and altitudinal spread, such as Nothofagus cunninghamii, have a wider thermal tolerance than species with a narrower distribution (Howard and Ashton, 1973). The consistency of the temperature tolerance range for each of the species tested (57.7 – 61.5 8C, Table 3), and for other Australian rain forest species studied by Cunningham and Read (2006) which have similar temperature tolerance ranges of about 60 8C, indicates that the wide 353 temperature tolerance observed in N. cunninghammii, for example, is due to differences in temperature response among provenances (Read and Busby, 1990). According to the scheme of Larcher (2005), the Australian Araucariaceae may be classified as ‘tropical – typical of species from mountain forests’ (leaf threshold injury temperatures – 4 to – 12 8C) through to ‘temperate climate with mildwinter’, otherwise classified as Southern Hemisphere conifers (– 10 to – 20 8C). Threshold injury temperatures (Fig. 2) for W. nobilis place this species in the upper end of tropical and, possibly, the lower end of the Southern Hemisphere conifer category which experiences mild winters. This is consistent with its southerly location and exposure to lower winter temperatures (Table 1). While there were significant differences among the species in response to high and low temperatures, all were within the effective tolerance range of temperate and tropical species of around 60 8C (Cunningham and Read, 2006), regardless of which technique was employed (Fig. 2). The Fv/Fm and visual rating together indicate that the leaves of winterhardened Australian Araucariaceae species are critically damaged (as estimated by LT50) at temperatures below – 5 8C and above – 11 8C (Fig. 2). This indicates that these species are suitable for the zone described by Larcher (2005) as having ‘episodic frosts with temperatures down to – 10 8C’, which includes most of Australia, with the exception of the coastal tropics and the alpine areas. Rarely would these species experience temperatures in wild populations as low as these estimated LT50 values (Table 1), but the temperatures causing initial damage may be occasionally reached (Fig. 2). Winter cold resistance in evergreen Southern Hemisphere species has been shown to be imparted by tolerance to extracellular ice formation (Neuner and Bannister, 1995). Many temperate conifers avoid freezing-induced xylem cavitation by having relatively small volume solute conduits compared with deciduous species (Sperry and Sullivan, 1992; Feild and Brodribb, 2001). Wollemi pine, for example, is an evergreen conifer that is considered to be temperate in origin (Offord and Meagher, 2001) and has narrowly constricted xylem structure at its branch base (Burrows et al., 2007), and thus presumably has a high xylem pressure which would decrease the chances of stem cavitation occurring due to freezing of xylem sap. Among the Araucariaceae, W. nobilis has the most restricted xylem vessels (Burrows et al., 2007; Brodribb and Feild, 2000), and its Huber value (xylem transverse sectional area per leaf area unit) is among the lowest recorded for Gymnosperms (Burrows et al., 2007). This would suggest that the Wollemi pine is the most adapted of these species to avoidance of frost damage. Other functions of this xylem restriction may include the facilitation of branch shedding (Burrows et al., 2007), or hydraulic control in response to drought and minimization of cavitation (Brodribb and Feild, 2000). An investigation of the xylem characteristics across the Australian Araucariaceae, as suggested by Pittermann and Sperry (2003), is greatly warranted. Such a study may reveal much about the evolution of the tracheid diameter and its relationship to cavitation avoidance, and, in the context of this current study, to the water relations and growth capacity of these species under climate change. 354 Offord — Temperature limits for relictual rain forest Araucariaceae What does this mean for cultivation? Minimum survival temperature (‘low temperature tolerance’, ‘cold tolerance’, ‘frost hardiness’, ‘freeze resistance’ or ‘frost resistance’) is often the main, if not the only, determinant of cultivation zones of many species (DeGaetano and Shulman, 1990). Determination of climatic zones for cultivation is often based on climatic information of natural populations and rarely on physiological response, and many species may be tolerant of temperatures outside their natural range. Ice nucleation temperature proved a useful guide for determining the relative freezing temperature of the leaves acclimated at high and low temperatures (Table 5). While it did not detect major differences between the species, it is likely that this is an accurate estimation of the true freezing temperature of the detached acclimated leaf (Bannister, 2007). The marked difference experienced between winter-hardened and summer-exposed leaves indicates that these Araucariaceae species, along with many others studied (see for examples Larcher, 2005), undergo acclimatization to lower temperatures brought about by gradual exposure to sub-zero temperatures. The relative inability of ice nucleation temperature to differentiate among different cold-hardened Araucariaceae species may be explained by the ability of their photosynthetic apparatus to recover or protect the leaf from damage, even after the apoplastic freezing point, as can be measured by Fv/Fm. It is, therefore, the ability of the species to recover from freezing events that determines their relative cold hardiness. Species that are ice intolerant have ice nucleation temperatures similar to their lethal damage values, while ice-tolerant species can survive temperatures lower than their ice nucleation temperature (Bannister, 2007). Wollemi pine has an ice nucleation temperature 3.5 – 4.2 8C lower than its LT50, suggesting that leaf tissue of this species is ice tolerant to some degree when winter hardened and is the most cold tolerant of the Australian Araucariaceae. In terms of low temperature tolerance, visual rating places these species in Plant Hardiness Zone 2 for Australia (the tablelands of south-east Queensland, New South Wales and Victoria) (Dawson, 1991). For A. atropurpurea, A. microstachya, A. heterophylla and possibly A. bidwillii, this equates to USA plant hardiness Zone 9 (– 6.6 to – 3.9 8C) (US National Arboretum, 1990); for A. robusta and A. cunninghamii Zone 8b ( –9.4 to – 6.6 8C); and, for W. nobilis Zone 8a ( – 9.4 to – 12 8C). Care should be exercised when using temperature as the determinant of plant ‘hardiness’, however, as soil moisture and other factors also influence the suitability of a species for a location (McKenney et al., 2007). Threshold injury temperatures for Southern Hemisphere conifers (from temperate climates with mild winters) indicate that Australian Araucariaceae might withstand minimum temperatures in the range – 5 to – 25 8C, depending on the organ affected and the timing of the frost event (Sakai et al., 1981; Larcher, 2005). The results from this present study indicate that all species tested were within this range when winter hardened, but had a consistent pattern of response between summer and winter. Where plants are gradually exposed to lower and lower temperatures, many species become increasingly tolerant of low temperatures and may be induced to harden beyond the normal range experienced in the wild. Cultivated species are often grown at the limits of their tolerance. All the species tested showed greater cold temperature tolerance during colder weather when compared with peak summer, the difference being in the order of between 1 and 3.5 8C depending on the species. While the specimen trees used in this study were from reasonably protected positions, trees of A. robusta, A. heterophylla and W. nobilis growing in exposed positions in high light exhibit symptoms of photoinhibition – yellowing characteristic of chlorophyll bleaching and leaf drop (Dungan et al., 2003) – in several locations at The Australian Botanic Garden, Mount Annan. While screen temperatures of –4 8C are common at this location, it is likely that wind-chill and localized temperatures, especially on exposed hillsides, may be much lower than recorded screen temperatures. In the case of A. robusta, and A. heterophylla in particular, the temperatures are likely to drop below the threshold for damage in winter. In the case of W. nobilis, at least, canopy temperatures were recorded that exceeded the critical temperatures for damage, and this may account for the commonly reported loss of this species in high light –high temperature situations in Australia, reinforcing the advice that this species be grown in protected positions (Offord and Meagher, 2006). . . . And under climate change? Ancestors of the extant conifer family Araucariaceae evolved .200 million years ago and peaked in abundance in the Jurassic (approx. 165 million years ago) (Stockey, 1982). Extant species have higher optimal growth temperatures than other temperate species and generally occur at lower elevations than North American conifers which are often adapted to subalpine conditions (Hawkins and Sweet, 1989). In New Zealand, there is evidence that Kauri survived both warming and cooling periods, through at least 20 glacial events (Mildenhall, 1980). Having survived many climatic regimes, extant Araucariaceae appear to possess the ability to adapt to changes in temperature over long periods of time. Short-term changes, such as anthropogenic climate change, however, are likely to affect significantly the ability of these species to survive. The results of this study suggests that, under current climate change scenarios, high temperatures, rather than low temperatures, are likely to be a major determinant of future species distribution in natural populations. Evidence that low temperatures are not currently limiting these species in the wild is that most LTi values and all LT50 values (Fig. 2) were lower than the lowest recorded temperature values (Table 1). Although low temperatures are likely to be somewhat limiting in combination with high light, poor soil, low rainfall and other factors that contribute to water stress (Sun and Sweet, 1996), upward shifts in air temperature are likely to damage leaves and other plant parts significantly (Allen et al., 2010). For example, the highest recorded canopy temperature at Wollemi pine site 1 (see Offord and Meagher, 2001 for general climate description) over an 8 year period (1998– 2006) was 34.8 8C (unpublished data). Given a localized radiant heat factor in the canopy of up to 10 8C (Fig. 3; Offord — Temperature limits for relictual rain forest Araucariaceae Larcher, 2000; Barker et al., 2005), little damage is likely to result. If overall air temperatures in Australia were to be in the order of 5 8C higher in 60 years time (CSIRO, 2010), however, significant damage may result due to localized canopy temperatures of approx. 49 8C, and the LT50 for this species may therefore be reached. In cultivation, it is very likely that this critical temperature is often reached or exceeded in many situations, which may explain plant loss in otherwise ideal situations. While the evidence shows that Wollemi pine is the Araucariaceae species most vulnerable to upward temperature shifts, other species may also be affected to some degree. All species have an optimal thermal range for photochemical activity. For example, measurement of Quercus ilex electron transport rates show that, for this species, the optimal leaf temperature range is 15– 35 8C and temperatures on the margins and outside this range result in leaf stress (Larcher, 2000). Warming per se may not compromise growth and survival for all species and may in fact enhance growth potential for some species, particularly deciduous species (Way and Oren, 2010). However, tropical rainforest species may be more vulnerable than temperate rainforest species as they are often operating with limited heat tolerance ranges and may be less adaptable to upward shifts (Cunningham and Read, 2003). Another serious consequence of higher overall temperatures might be a lack of, or insufficient, cold hardening. It is not yet known what effect climate change will have on the frequency, severity and distribution of frost events; however, it is likely to drive extinctions or influence geographical distributions, through primary and second order effects (Inouye, 2000). Frost experienced by unhardened plants is likely to be catastrophic; if freezing occurs unseasonally, plants are more at risk (Loik et al., 2004; Augspurger, 2009). It is not known how long it takes Araucariaceae species to acquire frost hardiness; however, the acquisition of low temperature tolerance through mild incremental exposure is well documented (Odlum and Blake, 1996; Zhang et al., 2003). It is known that, in some species, sugar concentration plays a role in frost hardiness (Leborgne et al., 1995) while no relationship is found in others (Zhang et al., 2003). Ice nucleation temperature in this study was useful in demonstrating the large difference between cold-hardened and unhardened plants. Unhardened, ice nucleation temperatures of all Australian Araucariaceae species are much higher than winterhardened tissues (Table 5). This indicates a level of cold acclimation that has developed in response to exposure to lower temperature, resulting in morphological and biochemical changes, such as accumulation of carbohydrates and amino acids (Alberdi and Corcuera, 1991). Interestingly, the greatest difference between the two contrasting seasons was observed for A. heterophylla (a difference of 3 8C). This might indicate that this species has a greater capacity for thermal acclimation than the other species, suggesting a temperate origin and explaining its ability to acclimate to a variety of cultivated situations, regardless of its relatively high low-temperature tolerance threshold. Phenological development is important to the severity of frost damage (Miranda et al., 2005); damage to the leaves/ stem can be exacerbated by pruning and regrowth of leaves 355 prior to frosting events (Chappell et al., 2006). Climate variations, such as those projected under increasing greenhouse gas models, may well shift the timing of cold events, such that unhardened plants are exposed to low temperatures outside normal seasons. This may lead to reversible or irreversible damage and will be a determinant of where a species may be grown. The direction and extent of temperature change are difficult to predict. It is certain that Australian Araucariaceae species cannot be grown in very cold climates without protection and, while sharp downwards shifts are not anticipated, the species are more likely to be affected by subtle shifts, i.e. early, late or severe and repeated frosts. Frost de-hardening during spring may also render plants vulnerable to subsequent frosts, especially as warmer weather may have hastened new leaf growth (Saxe et al., 2001). Conservation implications Given the already generally limited natural distributions of Araucariaceae, these results suggest that climate change, specifically temperature, rainfall and seasonality changes, will increase the vulnerability of these species which are already at the limits of their distribution and generally few in number. The relatively naturally rare southern, more temperate Australian Araucariaceae, i.e. A. heteorphylla and W. nobilis, are already more restricted in their distribution than the more widely distributed more tropical species. Of the Araucariaceae that occur in Australia, A. robusta, A. bidwilli and A. cunninghamii are likely to be the most adaptable, displaying diversity and a range of ecotypes across their distributions (Whitmore and Page, 1980; Graham et al., 1996; Peakall et al., 2003: Pye and Gadek, 2004), which may extend to variability in temperature extreme tolerance as it does to the optimal temperature range for photosynthetic efficiency (Hill et al., 1988). The role of ‘refugia’ in persistence of these species under climate change is dependent on what the refugia will provide. Wollemia nobilis is currently housed in microrefugia (Ashcroft, 2010), in deep, protected gorges which may, under climate change, warm more than the surrounding areas due to their elevation, the effect of which may be mitigated by cooling because of topographic shelter (Ashcroft, 2008). Predicting the role of refugia may be problematic for these species, and conservation actions, such as ex situ conservation of Wollemi pine, for example (NSW Department of Environment and Conservation, 2006), may therefore provide refuge in the face of uncertainty (Rull, 2009). In the face of climate change, moving a species to a cooler environment but maintaining connection with essential or reliant biota and edaphic requirements of the species is one option for ensuring species survival (Westoby and Burgman, 2006). More information is required to determine the effects of temperature changes, in combination with other influential growth parameters, particularly the effects of drought, on the ability of these species to survive or adapt, and therefore to predict accurately and confidently future distributions (Beaumont et al., 2005) and to plan appropriate management strategies. Westoby and Burgman (2006) discuss future management options for species threatened by climate change, such as the ‘do nothing approach’, translocation to a more 356 Offord — Temperature limits for relictual rain forest Araucariaceae suitable location and, preferably, minimizing warming. The thermotolerance ranges of species, such as those determined in this study, are useful parameters for this process even though these data represent just snapshots. AC KN OW LED GEMEN T S Patricia Meagher and NSW Parks and Wildlife staff are thanked for site climate data collections. 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