Anatomy of red spruce needles from forest decline sites in Vermont

Environmental and Experimental Botany, Vol. 28, No. 1, pp. 19~26, 1988.

009S-8472/88 $3.00 + 0.00 Pergamon Journals Ltd.

Printed in Great Britain.

A N A T O M Y OF R E D S P R U C E NEEDLES F R O M F O R E S T D E C L I N E SITES IN V E R M O N T A N N F. V O G E L M A N N and B A R R E T T N. R O C K

Jet Propulsion Laboratory, MS 183-501, California Institute of Technology, 4800 Oak Grove Drive, Pasadena, CA 91109, U.S.A. (Received 29 April 1986; accepted as submitted 11 June 1987) VOGELMANNA. F. and ROCK B. N. Anatomy of red spruce needlesfrom forest decline sites in Vermont. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 28, 19-26, 1988. Red spruce trees on upper elevation sites on Camels Hump Mountain, Vermont have shown severe decline symptoms since the mid-1960s. The anatomy of needles has been studied. Microscopic damage was seen primarily in mesophyll cells, characterized by cytoplasmic clumping and indiscernible chloroplasts. Damage increased over a single growing season and abnormal needle anatomy was found in 1985 and 1986 collections. There was a correlation of structural needle damage severity and field estimates of percentage foliar loss on low and high damage sites. INTRODUCTION

MATERIALS AND M E T H O D S

CAMELS H u m p M o u n t a i n , located in the G r e e n M o u n t a i n s of n o r t h e r n V e r m o n t , has been the site of l o n g - t e r m ecological investigations. SmCAMA et al. O~ a n d VOOELMANN et al. 117) have r e p o r t e d t h a t since 1965, the forests of this a r e a have shown decreases in basal area, density, biomass a n d vigor, a n d increases in mortality. T h e i r extensive d a t a base, the decline o f red spruce (Picea rubens Sarg.) a n d the high influx of air pollutants into this region/12/ m a k e Camels H u m p a useful location for studies on forest decline. T o date, little work has been published on a n a tomical studies of leaves from trees u n d e r g o i n g this decline. Exposure o f western pines to oxidants/2'3'9) a n d E u r o p e a n conifers to industrial pollutants/14'15/ has been shown to be correlated with cellular collapse, chloroplast d e g e n e r a t i o n a n d c y t o p l a s m i c distortions./1'15/ A n a t o m i c a l studies have been c o n d u c t e d on red spruce needles collected from various sites on Camels H u m p . W e present results from the a n a t o m i c a l assessment o f red spruce needles of different ages collected at different times over the 1985 a n d 1986 g r o w i n g seasons.

Site selection A n a t o m i c a l work used red spruce needles collected from trees on two sites on Camels H u m p , V e r m o n t . These sites h a d different levels of forest d a m a g e as m e a s u r e d b y foliar loss a n d m o r t a l i t y estimates. (18) Needles were collected from trees on a low d a m a g e site (12°/o red spruce foliar loss; 3.9% red spruce mortality) a n d a high d a m a g e site (76.0°/0 red spruce foliar loss; 24.20/0 red spruce mortality). T h e high d a m a g e site is on a west facing slope at ca 930 m elevation; the low d a m a g e site is on a northwest facing slope at ca 580 m elevation a n d is used as a " c o n t r o l " site. P i g m e n t analysis of needle tissues as well as g r o u n d based a n d aircraft-carried spectrometric measurements are in a g r e e m e n t with these differences in d a m a g e levels. (11)

Sample collection R e d spruce needles were collected from representative trees from each site on 1-2 J u l y 1985. S a m p l e d trees were tagged a n d a d d i t i o n a l collections were m a d e on 12-14 August 1985; 21 19

20

ANN F. VOGELMANN and BARRETT N. ROCK

23 June 1986; and 14-15 August 1986 from the same trees. Exposed (not shaded by overhanging branches), southwest facing branches located ca 5-6 m above the ground containing current year (first year) and 3 previous years' needles were cut from trees. Only needles growing alon~ the main axis of each branch were used. Current year and third year needles were collected separately. All needles collected were green and had no obvious chlorosis or flecking. Needles were collected twice in each growing season (1985 and 1986) from both study sites. First year needles are needles produced in the spring of the year in which they were collected and thus had not been exposed to winter conditions (except as overwintering buds). Older needles have undergone at least two winters. The first collections were made in late June (1985) or early July (1986), a time which is essentially still "spring" in high elevation forests in northern Vermont. First year needles had broken bud within a weel~ prior to collection and were still soft and pale green; needles from both sites were immature. The second collections of each year were made at both sites in mid-August. By this time hypodermal wall thickenings had formed in first year needles; needle color and firmness resembled those of older needles. Starch, which usually disappears at the onset of winter dormancy, was still abundant in the chloroplasts of all needles collected in mid-August. No frosts had occurred in the region in the time between summer collections.

and readily obtainable field fixative. Immersion in FAA occurred within 15 rain after branches were cut from trees. After returning to the laboratory, specimens were placed under vacuum (1.6-2.0 bar) for ca 30 min. The vacuum was released and reapplied periodically to remove air from the tissues and to aid penetration of the fixative. Total time in FAA was 24-48 hr; needles were then stored in 70% ethanol. Dehydration was through a graded ethanol series to tertiary butyl alcohol (TBA)./6~ Specimens were infiltrated over 4-5 days in TBA: mineral oil (1 : 1) and four changes of Paraplast Plus tissue embedding medium (Monoject Scientific Div., Sherwood Medical, St. Louis, MO). After each dehydration and infiltration step, samples were placed under vacuum (1.6-2.0 bar) for ca 1 hr. Prior to microtoming, hardened 15locks of Paraplast Plus containing specimens were cut to expose samples and soaked in a 2% aerosol O T (American Cyanamid Co., Wayne, NJ) solution for at least 72 hr to soften tissues./2) Transverse sections were sliced to 15 pm, stained with safranin and counterstained with fast green. (6) Sections were viewed and photographed through a Zeiss photographic microscope using Kodak Panatomic-X film. Three needles of each age class from each tree, from each collection, were examined. All mesophyll cells from a single transverse section were scored as normal or damaged as defined below. Serial sections were used to determine accurately cellular health. Student's t-ratios were calculated to determine statistical differences./~6~

Sample treatment

Individual needles were cut from branches into 1-2 mm segments and dropped directly into formalin-acetic acid-alcohol (FAA) fixative. Only middle portions of needles were used for this study. It is recognized that FAA fixation induces artifacts such as plasmolysis. However, prior to collecting needles a comparison was made between glutaraldehyde (3% in 0.05 M phosphate buffer at pH 6.9 for ca 18 hr at room temperature) and FAA (as outlined below) fixatives on red spruce needles. Both fixatives gave comparable fixation images with no apparent introduction of artifacts. Consequently FAA was selected for its convenience as an easily-transportable

RESULTS

The anatomy of spruce needles as described used as a basis for establishing cellular anatomical normality. Mesophyll cells were found to exhibit the greatest differences among our collections. Few cells other than mesophyll cells were found to exhibit damage symptoms. In most cases, epidermal and hypodermal cells appeared to be normal even though adjacent mesophyll cells were highly damaged. Resin canals and surrounding epithelial cells and stomatal guard cells also appeared to be normal. Endodermal cells and transfusion parenchyma cells occasionally exhibited damage symptoms, the

by MARCO(a) was

ANATOMY OF RED SPRUCE NEEDLES IN VERMONT severity of which reflected the severity of the damage to the mesophyll cells of the same needle. No damage was seen in xylem or phloem other than normal crushing of phloem cells resulting from needle growth. Examination of needle transverse sections revealed several different cytoplasmic conditions within mesophyll cells. We defined normal mesophyll cells as those with numerous chloroplasts and non-granular to uniformly finely granular cytoplasm (Fig. 1). Mesophyll cells exhibiting variations from these criteria were defined as damaged. Several damage categories were observed: 1--cytoplasm comprised of darkly staining thread-like structures (Fig. 2); 2--cytoplasm aggregated into darkly staining clumps (Fig. 2); 3--coarsely granular cytoplasm that appears uniformly stained (Fig. 3); and 4 cytoplasm condensed into darkly staining dense mass(es) (Fig. 4). In all damage categories chloroplasts are generally indiscernible. Each needle transverse section usually exhibited several different levels of mesophyll cell damage. Although the kinds of abnormalities were usually distinct and recognizable, they occasionally intergraded. Consequently, all damaged cells were lumped into a single category and mesophyll cells were scored as either normal or damaged. Mean values resulting from this scoring were normalized as percentages (Table 1). Other cellular characteristics such as cell wall distortions and apparent plasmolysis were noted but not studied in detail. The range of percentage normal mesophyll ceils was very wide, extending from a high of 85.9% for first year needles collected in June 1986 from the low damage site, to a low of 13.4% normal for third year needles collected in August 1985 from the high damage site (Table 1). Standard deviations around mean values were usually large. Percentage normal cells for each collection were compared to establish differences, if any, between sites, over the growing season, between first and third year needles, and between collection years (Table 2). In the between site comparison (Table 2, A) all of the mean percentage normal values for the low damage site are greater than the mean percentage normal values for the high damage site. This difference is statistically

21

significant (P < 0.05) in all cases except for first year needles collected in July 1985. All collections exhibited a decrease in mean percentage normal cells over both growing seasons. Most of these were statistically significant (Table 2, B). The average percentage decrease over the growing season was greater for first year needles (18.7% for both 1985 and 1986 collections) than for third year needles (12.3% for both 1985 and 1986 collections). Although percentage normal cells in first year needles from both sites decreased over the growing season the decrease was greater at the high damage site (Figs 5-8). Mean percentage normal values ranged from a high of 85.9% to a low of 50.6% in June/ July collections and from 65.7 to 31.5% in August collections. Percentage normal cells from third year needles also decreased over the summer, from a range of 62.7-25.0% normal in June/July to a range of 53.5-13.4% normal in August. All collections had a trend of increasing cellular damage with increasing needle age (Table 2, C). Although the needles from the low damage site had a significantly higher percentage of normal cells than needles from the high damage site, needles from both sites exhibited significant increases in damage. Trends of change in cellular anatomy between needle collections from 1985 vs 1986 (Table 2, D) are less obvious than those already discussed. Data from 1985 and 1986 from the low damage site are not statistically significant although three of the four means are higher in 1986 than in 1985. DISCUSSION

Damaged mesophyll cells were characterized by cytoplasmic clumping and indistinct chloroplasts. These results agree with similar results reported by Son~I~ELIil4/for conifer [Picea abies (L.) Karst, and Pinus sylvestris (L.)] needles collected from industrial areas in Finland (fluoride, SO2 and H~S emissions) and with those of EVANS and MILLER/2'3/ from California pines (Pinus ponderosa Dougl. ex Laws, P. jeffreyi Grev. and Balf., P. coulteri D. Don and P. lambertiana Dougl.) damaged by ozone. Anatomical comparisons among various collections of red spruce needles revealed several trends: needles from the low damage site had a

22

ANN F. VOGELMANN and BARRETT N. R O C K

Table 1. Anatomical assessment of first (1) and third (3)year red spruce needlesfrom low damage (LD) and high damage ( HD) sites in Vermont. Each value is a mean ( +- one standard deviation) of 12 needles 1985 % Normal +1 SD % Damaged LD1 LD3 HD1 HD3 August LD1 LD3 HD1 HD3

June/ July

73.9 56.2 65.2 25.0 60.4 53.5 43.4 13.4

19.0 11.6 16.9 8.9 19.9 9.2 8.1 7.5

26.1 43.8 34.8 75.0 39.6 46.6 56.6 86.6

1986 No. cells 2255 2215 2100 1891 1917 2159 2178 1915

higher p e r c e n t a g e of n o r m a l cells t h a n those from the high d a m a g e site; a n d a n a t o m i c a l d a m a g e increased with time, both over a single growing season a n d with needle age. These trends occurred in collections m a d e in both 1985 a n d 1986. T h e most n o t e w o r t h y difference observed here is the decline of p e r c e n t a g e n o r m a l cells with time. First y e a r needles were meristematic when collected in J u n e / J u l y . P e r c e n t a g e n o r m a l cells in these needles were found to decrease m o r e t h a n those o f m a t u r e third y e a r needles over a single growing season. This is in a g r e e m e n t with results from a study on a c i d - r a i n - t r e a t e d white pine (Pinus strobus L.) where it was suggested that d e v e l o p i n g leaves m a y be more susceptible to acid rain d a m a g e t h a n m a t u r e leaves. (4) O u r d a t a ( T a b l e 2, Figs 5-8) indicate that d a m a g e can occur over a single g r o w i n g season. All m e a n p e r c e n t a g e n o r m a l values from collections m a d e in A u g u s t were less t h a n those from the J u n e / J u l y collections of the same year. This trend was consistent for both years of this study. A t present we have no d a t a r e g a r d i n g possible differences in needle health over the winter. T h e two d a t a sets from the different years c a n n o t be r e g a r d e d as successive since the collections are from different ages of needles. T h a t is, first a n d third y e a r needles from 1985 would be second a n d fourth y e a r needles in 1986. W e are c u r r e n t l y a n a l y z i n g these ages of needles from 1986, as well as second y e a r needles from 1985, to establish changes, if any, t h a t would have occurred over the 1985/1986 winter.

% Normal +1 SD % Damaged 85.9 62.7 50.6 47.1 65.7 49.2 31.5 25.7

12.8 17.2 20.6 12.1 21.2 17.6 10.8 26.3

14.1 37.3 49.4 52.9 34.3 50.8 68.5 74.3

No. cells 2261 2149 2182 2124 2399 2073 2151 2079

Comparisons were also m a d e between different aged needles ( T a b l e 2). O u r observation that needle d a m a g e increases with increasing needle age has also been r e p o r t e d for Jeffrey pine (Pinus jeffreyi) a n d Ponderosa pine (P. ponderosa) exposed to ozone (19) a n d for Ponderosa pine exposed to various air pollutants. (~°/ T h e trends discussed r e g a r d i n g a n a t o m i c a l differences are the same for both collection years. T h e r e does not a p p e a r to be a n y obvious increase or decrease in p e r c e n t a g e n o r m a l cell values in 1986 relative to 1985. T h e r e is some indication that percentage n o r m a l values at the low d a m a g e site m a y have increased in 1986 b u t no a p p a r e n t trends were seen at the high d a m a g e site. A d d i t i o n a l years of study are needed to a c c u r a t e l y assess these trends over time. A certain a m o u n t of cellular d a m a g e is expected as a n o r m a l c o m p o n e n t of aging. However, we feel t h a t the types of a n a t o m i c a l d a m a g e observed a n d the degree of d a m a g e occurring over the relatively short time period of a single growing season is not normal. O u r results s u p p o r t those of others that red spruce in the northeast is declining. (5,7,13,17)

Acknowledgements--The research described in this paper was carried out by the Jet Propulsion Laboratory, California Institute of Technology, under contract with the National Aeronautics and Space Administration. We appreciate the advice and editorial assistance of Drs James Vogelmann and Richard Klein in the preparation of this manuscript.

24

FIGS 5-8. Transverse sections of first year red spruce needles collected in early July 1985 (Figs 5, 6) and mid-August 1985 (Figs 7, 8): D = dense cytoplasmic mass, M = mesophyll cell, VA = void area. 5. Normal needle from low damage site. Lack of wall thickenings in hypodermal cells and thin mesophyll cell walls indicate needle is immature. 6. Normal immature needle from high damage site. 7. Needle from low damage site. Cytoplasmic clumping can be seen in some mesophyll cells. 8. Needle from high damage site. Mesophyll cells exhibit marked damage: clumping (arrows), dense cytoplasmic mass formation and large void areas along cell walls; chloroplasts are still discernible in some cells. Scale bar = 70 #m (Figs 5-7) or 65 #m (Fig. 8).

23

FIOS 1-4. Transverse sections of red spruce needles showing mesophyll cells: C = chloroplast, CW = cell wall, EN = endodermis, EP = epidermis, H = hypodermis, N = nucleus, R C = resin canal, S = stomatal complex, T = transfusion tissue. 1. Normal mesophyll cells having distinct chloroplasts and non-granular cytoplasm. 2-4. Portions of needles having damaged mesophyll cells, chloroplasts generally indiscernible. 2. Cytoplasm aggregated as thread-like structures (arrows) or as coarse clumps (*). 3. Coarsely granular, uniformly stained cytoplasm (arrow). 4. Cytoplasm condensed into darkly staining dense masses (arrows). Figures 1 3 are first year needles. Figure 4 is third year needle. Figure 1 is from low damage site. Figures 2-4 are from high damage site. All needles from August 1986 collection. Scale bar = 48 #m.

~

~A

Z ~ P ~ Z Z ~

I+ I+ I+ I+ I+ I+ I+ I+

I÷ I÷ I÷ I÷ I~ I+ I+ I÷

.....

~

o

~ ~

A

~A

~A

~

~

I+1+1+1÷1+1+1÷1+ PY9 . . . .

e~

N"

.

~

.

.

AAA AAA PPP

A ~ b

~

-~ ~ ~

c~ r_~ ~ ~

~ •

~.~ A r.~

.

I÷1÷1÷1÷1+1+1+1+

.

>

gq

0

AA . . . .

~AAAA~ AA~O~A A

I+ I+ I+ I+ I+ I+ I+ I+

I+ I+ I+ I+ I+ I+ I+ I+

~..~

~ .

~ .

• ~.~,

~',,e

© Z

Z

0

0

> Z >

26

ANN F. VOGELMANN and BARRETT N. R O C K

REFERENCES 1. CARLSON C. E. and GILLIGAN C . J . (1983) Histological differentiation among abiotic causes of conifer needle necrosis. USDA For. Ser. Res. Pap. INT-298. Intermt. For. Range Exp. Stn., Ogden, UT. 2. EVANS L. S. and MILLER P. R. (1972) Comparative needle anatomy and relative ozone susceptibility of four pine species. Can. J. Bot. 50, 1067-1071. 3. EVANSL. S. and MILLER P. R. (1975) Histological comparison of single and additive O~ and SO2 injuries to elongating Ponderosa pine needles. Am. J, Bot. 62, 416~421. 4. HAINES B., STEFANI M. and HENDRIX F. (1980) Acid rain: threshold of leaf damage in eight plant species from a southern Appalachian forest succession. Water Air Soil Pollut. 14, 403~/~07. 5. HORNBECKJ. W. and SMITH R. B. (1985) Documentation of red spruce growth decline. Can. J. For. Res. 15, 1199-1201. 6. JOHANSEN D. A. (1940) Plant microtechnique. McGraw-Hill, New York. 7. JOHNSONA. H . and SICCAMAT. G. (1984) Decline of red spruce in the northern Appalachians: assessing the possible role of acid deposition. Tappi J. 76, 68-72. 8. MARCO H. F. (1939) The anatomy of spruce needles. J. Agric. Res. 58, 357-368. 9. MILLER P. R. and EVANS L. S. (1974) Histopathology of oxidant injury and winter fleck injury on needles of western pines. Phytopathology 64, 801806. 10. RICE P. M., CARLSON C. E., BROMENSHENKJ. J., GORDON C. C. and TOURGANEAUP. C. (1986) Basal injury syndrome of Pinus needles. Can. J. Bot. 64, 632-642.

11. ROCK B. N., VOGELMANNJ. E., WILLIAMS D. L., VOGELMANNA. F. and HOSHIZAKIT. (1986) Remote detection of forest damage. BioScience 36, 439-445. 12. SCHERBATSKOYT. and BLISSM. (1984) Occurrence of acidic rain and cloud water in high elevation ecosystems in the Green Mountains of Vermont. Transactions of the APCA Specialty Conf., The Meteorology of Acidic Deposition, 16-19 Oct. 1983, SAMSON P. J. and HARTFORD C. T. eds, Air Pollution Control Ass., Pittsburgh. 13. SICGAMA T. G., BLISS M. and VOGELMANN H. W. (1982) Decline of red spruce in the Green Mountains of Vermont. Bull. Torrey Bot. Club. 109, 162-168. 14. SOIKKELI S. (1981a) Comparison of cytological injuries in conifer needles from several polluted industrial environments in Finland. Ann. Bot. Fennici 18, 47-61. 15. SOIKKELIS. (1981b) The types of uhrastructural injuries in conifer needles of northern industrial environments. Silva Fennica 15, 399-404. 16. SOKAL R. R. and ROHLF F. J. (1981) Biometry, Second edn. W. H. Freeman, San Francisco. 17. VOGELMANN H. W., BADGER G., BLISS M. and KLEIN R. M. (1985) Forest decline on Camels Hump, Vermont. Bull. Torey Bot. Club. 112, 274287. 18. VOGELMANNJ.E. and ROCKB. N. (1986) Assessing forest decline in coniferous forests of Vermont using NS-001 Thematic Mapper Simulator data. Int. J. Remote Sensing 7, 1303-1321. 19. WILLIAMSW. T. (1980) Air pollution disease in the California forests. A base line for smog disease on Ponderosa andJeffrey pines in the Sequoia and Los Padres National Forests, California. Envir. Sci. Technol. 14, 179-182.