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a Natural Science Division, Pepperdine University, Malibu, California 90263-4321; and b Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48823-1312
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Ceanothus • chaparral • electrical conductivity • fluorescein diacetate • maximum fluorescence • Rhus (Malosma), supercooling • variable fluorescence
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drought has been shown to be an important factor contributing to the mortality of seedlings of chaparral shrubs (Frazer and Davis, 1988; Thomas and Davis, 1989). However, drought rarely causes mortality among adults (Riggan et al., 1994; Portwood et al., 1997). In contrast, at some sites in southern California freezing can cause total shoot dieback in Rhus laurina (= Malosma laurina) three or more years in a row (Misquez, 1990). Such repeated shoot mortality can result in the death of the entire plant in this species (Mooney, 1977).
Susceptibility of chaparral shrubs to freezing injury could be as important as fire and water stress in limiting the distribution and abundance of this important vegetation type (Langan, Ewers, and Davis, 1997). Chaparral shrubs are found growing in areas that possess temperatures well below freezing during winter months. We established two study sites that are only 4 km apart but have a 10°C difference in minimum air temperature (Fig. 1). The "warm" site at Malibu rarely has air temperatures below 0°C; the "cold" site at Cold Creek Canyon frequently reaches a seasonal minimum in air temperature of -8° to -12°C (Fig. 1; cf. Langan, Ewers, and Davis, 1997). Furthermore, at night leaf temperatures can drop several degrees lower than air temperatures due to radiational heat loss (Larcher and Bauer, 1981). The terms "warm site" and "cold site" refer to seasonal minimum temperatures. Maximum daily temperatures are often greater at the inland than at the coastal site. Langan, Ewers, and Davis (1997) reported a difference in freezing tolerances among species of chaparral shrubs growing side by side at the cold site and suggested that such differences may influence distribution patterns.
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Widespread wildfires during the fall of 1993 offered an opportunity to address the same questions for seedlings. Seedlings of most chaparral species occur in greatest numbers during the first year following fire. The seeds of many chaparral species require fire to germinate (Keeley, 1987). Seedling mortality due to water stress during the first summer drought after wildfire has been investigated in chaparral shrubs (Frazer and Davis, 1988; Thomas and Davis, 1989; Williams, Davis, and Portwood, 1997), but low temperature damage to chaparral seedlings has not been previously examined. This prompted the questions, (4) do seedlings have less resistance to low temperature damage than adults of the same species? and (5) how much do the seedlings acclimate to cold temperatures in comparison to adults?
We worked with two pairs of congeneric species of chaparral shrubs. We chose Rhus laurina and R. ovata because, after a naturally occurring freezing event at the cold site, we observed differences in survival of these congeneric species. Rhus laurina experienced complete shoot dieback and leaf death, while R. ovata at the same site was apparently uninjured (Langan, Ewers, and Davis, 1997). Both Rhus species have the capability to regenerate from a root crown after a stress that kills the aboveground parts (James, 1984). We selected Ceanothus spinosus and C. megacarpus because they co-occur with R. laurina and R. ovata at the warm site, and because C. spinosus is a sprouter after shoot removal by fire or freezing, whereas C. megacarpus is a nonsprouter (Thomas and Davis, 1989). Nonsprouters of Ceanothus are more drought tolerant than co-occurring congeneric sprouters (Davis, Kolb, and Barton, 1998). This leads to the question, (6) are the leaves of a nonsprouter more freezing tolerant than those of a co-occurring congeneric sprouter? This last question is particularly significant because previous studies suggest that stem xylem is more susceptible to freezing injury among sprouters (Langan, Ewers, and Davis, 1997).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seedlings
Since most chaparral seedlings germinate only after fire, seedlings could not be found at the exact same sites as for adults, so nearby sites comparable in elevation and in distance from the ocean were used. Rhus laurina seedlings were taken from Malibu Canyon across the street from Pepperdine University and just east of warm site A (Fig. 1). The rest of the coastal seedlings were taken from Las Flores Canyon and Hume Road, Los Angeles County (~165 m, warm site B). Inland, cold site seedlings were taken from the hiking path off Cold Canyon road (~420 m, cold site B, Fig. 1).
Sample collection
Adults
Five individuals of each species growing in the same vicinity were tagged and labeled as plants 1 through 5. These individuals represented mature healthy adults. For Rhus laurina and R. ovata, paired leaves that were fully expanded and matured, 6–10 nodes from the branch apex, were collected from each individual of each species. One leaf was used for the measurement of fluorescence, Fv/Fm, and visual score and the other leaf to perform conductivity and vital staining tests at 1° intervals at progressively lower temperatures. Further details on sampling for using four methods to determine the temperature of 50% change in activity or cell death (LT50) are as described elsewhere (Boorse, 1995; Boorse et al., 1998).
Seedlings
Rhus laurina seedlings were large enough to collect leaves as described for adults. Paired leaves were collected for each temperature (N = 5).
Both Ceanothus spinosus and Ceanothus megacarpus had small seedlings, but they were found in clusters. For both of these species five clusters were labeled 1–5. Each seedling was about the same size as the stem segments used for the adults, and so the entire shoots of seedlings, instead of stem segments, were collected from the cluster for each experiment.
Rhus ovata seedlings were smaller than other seedlings and were found in much lower densities and rarely in clusters. Thus, seedlings of R. ovata were not tagged and labeled but were collected as they could be found and randomly placed into samples 1–5.
Freezing treatment
A datalogger was used to control the cooling of excised leaves, branchlets, and seedlings in 0.5 mL of water placed in a test tube, at a rate of 1°C/h, a realistic rate for nighttime winter cooling in the chaparral (Langan, Ewers, and Davis, 1997). The datalogger read thermocouples on three of the leaves in the cooling chamber and calculated an average leaf temperature. It then sent an appropriate voltage to a chiller filled with a 1:1 mixture of ethylene glycol and water, to cool leaves at the desired rate (Boorse et al., 1998). All samples were seeded with ice at 0°C to insure freezing without supercooling. Samples were then removed at 1°C intervals, starting at 0°C down to a minimum of -20°C, depending on species and season.
Viability test
The four viability tests, (1) electrical conductivity (electrolyte leakage into a bathing solution), (2) photosynthetic fluorescent capacity (Fv/Fm), (3) percentage of palisade mesophyll cells stained by fluorescein diacetate (vital stain), and (4) visual score of leaf color (Munsell color chart), were performed by following the methods of Boorse (1995) and Boorse et al. (1998). Thus there were four different estimates of LT50 for each experiment.
Statistical analysis
The measurements of the four viability tests for each sample were plotted against temperature. From these plots a linear regression was used to calculate LT50, the temperature at which a 50% change in activity occurred (increase in visual score and electrical conductivity; decrease in Fv/Fm and vital stain) for each individual. An unpaired Student's t test was used to compare LT50 values of the same species located at the two different locations, in summer vs. winter and as seedlings vs. adults, at P < 0.05. The sample size in all cases was N = 5. A one-way ANOVA followed by a Fisher Protected Least Significant Difference test was used to compare the four different methods of estimating leaf viability at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison between species
Rhus laurina was significantly more cold sensitive than the other three species. The LT50 ranged from about -5.0°C for R. laurina in the summer to -16°C for R. ovata in the winter. Rhus laurina had relatively high LT50 values at both the warm and cold sites, in summer and winter (Fig. 2) and for seedlings as well as adults (Figs. 3, 4). For the warm site in the summer, results were very similar for Ceanothus megacarpus vs. C. spinosus (Fig. 4).
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Comparison between summer and winter
Both at the warm and cold sites, R. ovata had lower LT50 values in winter than in summer (Fig. 2). This was true for all four viability tests (P < 0.01 for all tests). The difference in LT50 between summer and winter was 4°C at the warm site vs. 6°C at the cold site (Fig. 2).
Rhus laurina had lower LT50 values in winter vs. summer only at the cold site, with LT50 values 3°C more negative in the winter (P < 0.0001 for all tests) (Fig. 2). At the warm site for R. laurina, there was no significant difference in LT50 in summer vs. winter (range in P > 0.2 to 0.6).
At the cold sites, R. ovata and R. laurina seedlings as well as adults underwent some acclimation, as indicated by significantly lower LT50 values in the winter vs. summer (Fig. 3). All three tests for R. ovata seedlings produced LT50 values that were significantly more negative in the winter (P < 0.01 for all three tests) with an acclimation of ~2.5°C (Fig. 3). For R. laurina seedlings at the cold site, both electrical conductivity and vital staining produced LT50 values that were significantly lower in the winter compared to summer (range in P of < 0.001 to 0.02). However, these two tests for seedlings of R. laurina indicated acclimation of only 1.5°C at the cold site, and the other two tests showed no significant difference between winter and summer (Fig. 3).
Comparison between adults and seedlings
At the warm site in the summer, two to three of the viability tests indicated that seedlings of R. laurina, C. megacarpus, and C. spinosus were more sensitive to freezing than adults located in Malibu in summer (Fig. 4). This was not the case for R. ovata, where the visual score indicated that seedlings were less sensitive than adults with the other two tests showing no significant difference for seedlings vs. adults.
At the cold site in the summer, R. laurina had more resistant seedlings than adults for three viability tests (Fv/Fm, vital stain, visual score), range in P < 0.005 to 0.008) (Fig. 3). In contrast, there was no significant difference between the LT50 values of R. ovata seedlings and adults (Fig. 3).
In the winter, seedlings located at the cold site were more sensitive to freezing than were the adults. Rhus ovata seedlings have significantly higher LT50 values than those of adults according to all three viability tests (range in P < 0.0001 to 0.02). The LT50 for R. ovata seedlings was ~3°C higher than for adults (Fig. 3). For R. laurina, all viability tests produced less negative LT50 values for seedlings than for adults, but only vital stain and visual score were significantly lower (range of P < 0.0001 to 0.007). For R. laurina the seedling LT50 was only ~1°C higher than for the adults (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison between species
Malibu is located in a coastal exposure of the Santa Monica Mountains where nighttime temperatures are ameliorated by the influence of the Pacific Ocean. Rarely do temperatures drop below 0°C in Malibu (the warm site), whereas just 4 km inland, at Cold Creek (the cold sites), without the coastal influence, minimum seasonal temperatures reach -8° to -12°C during winter nights (Fig. 1). There is also a striking change in chaparral distribution over the 4-km transect. While the codominants in Malibu are R. laurina and C. megacarpus (Frazer and Davis, 1988; Thomas and Davis, 1989), neither species are common at the inland, cold site. Both species are restricted to hill tops and ridge crests at the cold site, with R. ovata and C. crassifolius (LT50 = -18°C, unpublished data) predominant in the valleys (Langan, Ewers, and Davis, 1997). The hill–valley effect results from a thermal ground inversion where cold air drains into the valleys at night. Langan, Ewers, and Davis (1997) have monitored leaf temperatures of R. laurina leaves on a hill vs. R. ovata leaves in an adjacent valley ~150 m apart, at the cold site. They found during a radiation freeze at night that the leaf temperatures were 3.1°–3.8°C colder in the valleys than on the ridge crest (hill top = -5°C, valley = -9°C).
This distribution pattern and thermal gradient observed in the field are consistent with our findings that R. laurina is more susceptible to freezing injury than R. ovata. It is also consistent with the observation that both R. laurina and R. ovata are less susceptible to freezing injury at Cold Creek than in Malibu, especially during winter months.
The nonsprouter, C. megacarpus, had similar LT50 values as the sprouter C. spinosus. However, C. spinosus generally extends into more northerly aspects and higher altitudes in the Santa Ynez Mountains (Nicholson, 1993) and into cold valleys in the Santa Monica Mountains than C. megacarpus (S. D. Davis, personal observation). This pattern may result from the sprouting ability of C. spinosus, which allows survival after infrequent, low temperature extremes. Furthermore, Rhus laurina (sprouter) with a high LT50 extends lower into cold valleys than C. megacarpus, possibly because of R. laurina's vigorous resprouting ability (cf. Langan, Ewers, and Davis, 1997).
Comparison between warm and cold sites
The most sensitive species, R. laurina, showed no difference in its resistance to freezing temperatures from the warm site to the cold site. This is despite the fact that the cold site reaches a minimum temperature that is up to 10°C colder than the warm site. This lack of ecotypic variation may represent genetic limits of adaptability, restricting R. laurina's distribution to warmer sites. Indeed, Misquez (1990) found strong correlation between minimum yearly temperatures and the abundance of this species at various sites throughout its range in California and Mexico. Also, pioneer citrus growers successfully used Rhus laurina as an indicator species of where it was safe to plant orchards.
In contrast, R. ovata at the cold site had a significantly lower LT50 than at the warm site, suggesting possible ecotypic variation in the two populations. To fully test this hypothesis would require reciprocal transplant experiments between the two sites.
Comparison between summer and winter
Both R. laurina and R. ovata at the cold site demonstrated a downward shift in LT50 between summer and winter months, suggesting cold hardening (seasonal acclimation to freezing). However, R. laurina only shifted by 3°C at the cold site between summer and winter, and underwent no acclimation at the warm site. In contrast, R. ovata acclimated at both sites, 6°C at the cold site and 4°C at the warm site, providing further evidence of possible ecotypic variation between the two populations. The inability of R. laurina to acclimate as much as R. ovata may contribute to the limited distribution of R. laurina into colder regions. In contrast, R. ovata is widely distributed, occurring even at sites in southern California with minimum temperatures below -20°C, e.g., in the San Jacinto Mountains (unpublished data).
Comparison between adults and seedlings
At the warm site, seedlings for three out of four species were more sensitive to freezing than the adults. This is consistent with other studies in Mediterranean-type climates where seedlings were also found to be particularly sensitive (Larcher, 1981; Sakai and Larcher, 1987). Rhus ovata seedlings at the warmer site in the summer were the only exception, but only the visual score showed seedlings to be significantly more resistant to low temperatures than adults. One possible reason for this disparity was the low availability of R. ovata seedlings at the warm site, forcing us to select much smaller, shallow-rooted seedlings than was the case for the other three species. It is thus likely that the sampled R. ovata seedlings were more drought stressed than the other seedlings, although water potential measurements were not made.
In summer, differences between seedlings and adults of R. laurina at the cold site were opposite to those at the warm site. Rhus laurina seedlings had lower LT50 values than adults at the cold site but higher values than adults at the warm site. It should be pointed out that sampling at the cold site was done later in the summer than at the warm site and thus seedlings at the cold site were actually under drier, hotter conditions than at the coast. In general, seedlings sustain much drier and hotter conditions than adults during dry summer months (Frazer and Davis, 1988; Thomas and Davis, 1989). This increase in thermal and water stress for seedlings at the inland site may have resulted in a general stress response that also enhances their resistance to freezing. The winter months are the wettest season so all plants, seedlings, and adults were hydrated. This would release any resistance that seedlings had developed to thermal stress and water stress in summer. An interesting experiment would be to irrigate seedlings in summer to see whether their elevated cold resistance persists.
In the winter, results were consistent with previous studies of seedling vs. adult vulnerability to freezing reported for other ecosystems (Sakai and Larcher, 1987). Seedlings were consistently more sensitive to freezing temperatures than were the adults. These data indicate a very vulnerable stage that could influence the distribution of chaparral species. Rhus ovata seedlings acclimated more than the R. laurina seedlings at the cold site, which may explain why R. laurina is so rare at inland locations. Neither the R. laurina adults nor the seedlings undergo as much acclimation as R. ovata.
To conclude, our tests have shown that different chaparral species have different resistance to low temperatures in seedlings as well as in adults and differences in the ability of their leaves to acclimate. These results, combined with our observations of chaparral distribution patterns, strongly indicate that freezing could be a major force in the distribution of chaparral species.
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FOOTNOTES |
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4 Author for correspondence: Natural Science Division, Pepperdine University, Malibu, CA 90263-4321 [davis{at}pepperdine.edu
; telephone: (310) 456-4321; FAX: (310) 456-4785].
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REFERENCES |
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Nicholson, P. 1993. Ecological and historical biogeography of Ceanothus (Rhamnaceae) in the transverse ranges of southern California. Ph.D. dissertation, University of California, Los Angeles, CA.
Portwood, K. A., F. W. Ewers, S. D. Davis, J. S. Sperry, and G. C. Adams. 1997. Shoot dieback in Ceanothus chaparral during prolonged drought—a possible case of catastrophic xylem cavitation. Bulletin of the Ecological Society of America 78: 298.
Riggan, P. J., S. E. Franklin, J. A. Brass, and F. E. Brooks. 1994. Perspectives on fire management in Mediterranean ecosystems of southern California. In J. M. Moreno and W. C. Oechel [eds.], The role of fire in Mediterranean-type ecosystems, 140–162. Springer-Verlag, New York, NY.
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