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Comparative karyotype analysis of four Douglas-fir Pseudotsuga menziesii (Mirb.) Franco provenances De-Vescovi, Maria Adela 1974

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COMPARATIVE KARYOTYPE ANALYSIS OF DOUGLAS-FIR PSEUDOTSUGA MENZIESII (MIRB.) FRANCO PROVENANCES by Maria Adela De-Vescovi B.Sc. Catholic U n i v e r s i t y of C h i l e , A p r i l , 1966. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Faculty of FORESTRY We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1974 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s thesis f o r sc h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Forestry The Uni v e r s i t y of B r i t i s h Columbia, Vancouver 3 , B.C., Canada ( i i ) ABSTRACT A comparative karyotype study was made of chromosomes i n root-t i p meristematic c e l l s of Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco germinants obtained from three Coastal (#1,3,22,) and one I n t e r i o r (#28) sources d i s t r i b u t e d through 8°04" l a t i t u d e . The somatic chromosome number i n a l l provenances was 2n=26 or x=13. The morpho-l o g i c a l index was found to be the most useful c r i t e r i o n f o r separating the f i v e metacentric, s i x submetacentric and two subtelocentric chromo-somes of the basic set. A c l i n a l increase from south to north i n chromo-some width (1.3), and volume (1.6), was noted among the chromosomes of the three Coastal provenances. The haploid chromosome complement of the Coastal provenance (#22) had 1.5 times more volume than the corresponding I n t e r i o r one (#28), substantiating previous r e s u l t s as f a r as v a r i a t i o n i n nuclear volume and DNA contents are concerned. Gasguet (#3) provenance showed more d i s s i m i l a r i t i e s regarding the length of the chromosome arms and secondary c o n s t r i c t i o n s than the chromosomes of the other three pro-venances . ( i i i ) TABLE OF CONTENTS Page L i s t of t a b l e s (v) L i s t o f f i g u r e s (vi) Acknowledgements ( v i i ) 1 . I n t r o d u c t i o n 1 2 . M a t e r i a l s and Methods 4 Seed samples 4 P r e p a r a t i o n of s l i d e s and p r i n t s 5 Measurements o f chromosomes and c a l c u l a t i o n methods 9 3 . Observat ions 15 Karyotype p a t t e r n 15 Chromosomal c o n s t r i c t i o n s and s p h e r i c a l p r o j e c t i o n i n Gasquet (#3) provenance 15 D i f f e r e n c e s between provenances 20 Morphometric v a l u e s 20 Shor t arm l e n g t h 20 Long arm l e n g t h 20 T o t a l l e n g t h 21 Width 21 Volume 23 Index v a l u e s : arm r a t i o , centromere i n d e x , r e l a t i v e l e n g t h , m o r p h o l o g i c a l index 23 Comparison w i t h p r e v i o u s work 24. (iv) TABLE OF CONTENTS (cont'd) Pages Differences within provenances 26 Covelo provenance (#1) • 26 Gasquet provenance (#3) 26 Forks provenance (#22) 26 Spokane provenance (#28) 27 4. Discussion 32 Chromosome number and karyotype pattern 32 Secondary c o n s t r i c t i o n s 33 Differences between r e p l i c a t e s 34 C l i n a l v a r i a t i o n from south to north i n width and volume i n the Coastal provenances 35 Differences i n chromosomal width and volume between Coast and I n t e r i o r provenances 35 5. Summary 40 References 41 (v) LIST OP TABLES Pages Table 1 . L o c a t i o n and year o f c o l l e c t i o n of provenances used i n the exper iment . Table 2 . M o r p h o l o g i c a l c a t e g o r i e s of chromosomes a f t e r Levan e t a l . Tab le 3 . Averages of morphometric and index v a l u e s of the f o u r provenances . Table 4 . A n a l y s i s of v a r i a n c e : shor t arm l e n g t h Table 5 . A n a l y s i s of v a r i a n c e : long arm length Table 6 . A n a l y s i s of v a r i a n c e : t o t a l l e n g t h Table 7 . A n a l y s i s o f v a r i a n c e : w i d t h Table 8 . A n a l y s i s of v a r i a n c e : volume Table- 9 : A n a l y s i s of v a r i a n c e : index v a l u e s Tab le 10 . R e l a t i v e chromosome l e n g t h and c o r r e l a t i o n c o e f f i c i e n t s f o r the four provenances (#1 ,3 ,22 ,28 , ) and t h e i r combined average as compared t o r e s u l t s of Sax and Sax (A) , Barner and C h r i s t i a n s e n (B), and Thomas and Ching (C) . Tab le 1 1 . Morphometric and index v a l u e s of Covelo (#1) p r o -venance. Tab le 12 . Morphometric and index v a l u e s o f Gasquet (#3) p r o -venance. Tab le 1 3 . Morphometric and index v a l u e s of Porks (#22) p r o -venance . Tab le 14. Morphometric and index v a l u e s of Spokane (#28) provenance. 12 18 20 21 21 22 23 24 25 28 29 30 31 (vi) LIST OF FIGURES Page Figure 1. Location of Douglas-fir provenances sampled. 6 Figure 2. Meristematic r o o t - t i p chromosomes of m i t o t i c metaphase from Gasquet (#3) provenance. 8 Figure 3. Schematic representation of measurements of chromosomes. 10 Figure 4. The 13 pa i r s of meristematic r o o t - t i p chromo-somes Gasquet (#3) provenance. 13 Figure 5. Idiogram of the four provenances. 14 Figure 6. Projection of s p h e r i c a l structure on the short 16 arm of chromosome number 3 i n Gasquet (#3) provenance. Figure 7. Chromosomes with secondary c o n s t r i c t i o n s and 17 d i f f e r e n t c o i l i n g patterns i n Gasquet (#3) provenance. Figure 8. Meristematic r o o t - t i p chromosomes of m i t o t i c 36 metaphase from Forks (#22), Coastal provenance. Figure 9. Meristematic r o o t - t i p chromosomes of m i t o t i c 37 metaphase from Spokane (#28), I n t e r i o r proven-ance . (vii) ACKNOWLEDGEMENTS The author wishes to express her gratitude for the guidance, advice and encouragement given by Drs. K.M. Cole, C.J. Marchant, and 0 . Sziklai throughout the course work, the research and the thesis preparation. The author also wishes to thank Miss L. Cowdell and Mrs. K. Hejjas for their individual assistance in computing, Miss M. Schmidt and Miss A. Adamovich for their assistance, and Mr. M. Paris for his photographic advice. Thanks also to Mrs. M.C. Sziklai for technical assistance in the various stages of research and the preparation and checking of the manuscript, and to Mrs. E. Jensen for the fi n a l typing of the manuscript. -1-1. INTRODUCTION Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, is the most im-portant and most valuable timber tree in Western North America. It comprises approximately sixty percent of the standing timber of Coast forests. It was also introduced in Europe by David Douglas in 1827, and since that time has played an important role in the reforestation of the Western and Central European countries. This species extends into different ecogeographical zones, from Kemano in the north of the Pacific Coast of British Columbia to Salmon Creek in the Santa Lucia Mountains in California. The most northerly limit of the species is in the Interior of B.C. at Takla Lake, and extends eastward to the Rocky Mountain range, then south into the Sierra Madre in Mexico ( L i t t l e , 1952). Douglas-fir presents a large variation pattern which is related to the wide range of the species. This observation on the intraspecific variation was mentioned by Larsen,(1956): "one has to travel very widely throughout the natural range of Douglas-fir in order to get an impression of differences in geographical type, but standing on one place one can, without moving a foot, see many individuals differing wide-ly in their structure; i t is often more d i f f i c u l t to pick out those that resemble one another. Although they belong to the same species and the same geographical type, yet they can be widely divergent in respect of their economic value in forestry. This can be observed everywhere. It does not matter i f one chooses in California a site in the Coast Range or in the Sierra Nevada, passes through Oregon and Washington, or in British Columbia selects a place on Van-couver Island or in the Rocky Mountains; everywhere one is bound to be impressed by the great individual variation of this tree species;" -2-Peace (1948) described cones collected from the northern part of the species and found the range in cone length from 3.4 to 8.4 cm. Tusko (1963) collected samples from 43 provenances in British Columbia and reported the range of variation in cone length as 3.2 to 9.3 cm. Sziklai (1967) found c l i n a l variation in cone and seed characteristics among the 91 provenances from California to British Columbia. Yao (1971) studied seed weight (1000 seed), cone scale morphology and germination percentage on 124 provenances collected from California, Oregon, Washington and British Columbia. His findings substantiated the pre-vious results regarding c l i n a l variations. Numerous authors worked on the cytogenetics of Douglas-fir and results di f f e r regarding the haploid chromosome number and the chromo-some morphology. Sax and Sax (1933), Zenke (1953), Allen (1960), Barner and Christiansen (1962), Thomas and Ching (1968), El-Lakany (1969), re-ported n = 13. On the other hand, Langlet (1943), and Durrie-Vabre (1958) found the haploid chromosome number for Douglas-fir to be n = 12. No work has been done at the provenance level and no attempt has been made to investigate morphological chromosomal changes on a geogra-phical basis in Douglas-^fir. The objective of this study has been to describe chromosomal d i f f -erences between four provenances of Douglas-fir from California and Washington including the Coast and Interior region. This work also aimed at providing a comparative karyotypic description based on mor-phological characteristics. The samples used in this research were from the same provenances that El-Lakany and Sziklai studied' in a - 3 -previous work on nuclear volume and r e l a t i v e DNA content (1971), and i t was intended to co r r e l a t e the chromosomal c h a r a c t e r i s t i c s with t h e i r r e s u l t s . -4-2. MATERIALS AND METHODS Seed Samples Gone samples were c o l l e c t e d from t h e i r natural habitat i n 1966, 1968 and 1969 by the International Union of Forest Research Organi-zation (IUFRO). Root-tips from the seed samples were used for the current karyotype analysis of Douglas-fir. Unfortunately, haploid material was not ava i l a b l e since seedlings from d i f f e r e n t f a m i l i e s , planted out at Haney U.B.C. Research Forest i n 1970, w i l l not reach the stage of reproductive bud d i f f e r e n t i a t i o n for another 15 years. Although an average of f i v e trees were selected from each of the 31 provenances to represent each provenance i n t h i s study, (Table 1, and F i g . 1) only four provenances provided enough germinants f o r the cytogenetical studies, namely: Covelo (#1), Gasquet (#3), Forks (#22) and Spokane (#28). For each provenance three r e p l i c a t e s were selected and were designated r-p r2, and r-j f or each provenance. F i l l e d seeds were separated from the empty ones using X-ray fluoroscopy, according to the method described e a r l i e r by El-Lakany (1969). The seeds were soaked i n tap water for 24 hours at room temperature and then s t r a t i -f i e d at 0°-2°C. for two weeks. Germination of the seeds took place using the Jacobsen Germinator. Germinants were counted at 2-day i n t e r -v a l s . In a few provenances i t usually took only 4-5 days to obtain the germinants, whereas i n many other provenances the rate of germination was much lower or even no germination occurred during the 28 day period. -5-Preparation bf micro s l i d e s and p r i n t s In order to determine the most s a t i s f a c t o r y pre-treatment f o r spreading and shortening of the chromosomes a number of techniques were tested; c o l c h i c i n e , 8-hydroxyquinoline, paradichlorobenzene, cold treatment and monobromonaphthalene. Table 1. Location and year of c o l l e c t i o n of provenances used i n the experiment.  No. Provenance Range Province State * 3 » X o ' Year of C o l l e c t i o n 1. Covelo C C a l i f . 39 55 123 18 1968 2. Dunsmuir I C a l i f . 41 12 122 18 1968 3. Gasquet C C a l i f . 41 51 123 59 1968 4. Coquille C Oregon 32 12 124 10 1966 5. Waldport c Oregon 44 24 123 52 1966 6. Marion Forks I Oregon 44 30 122 00 1966 7. Pine Grove I Oregon 45 06 121 23 1966 8. Glenwood I Wash. 46 00 121 00 1966 9. Sequim Bay c Wash. 48 02 123 00 1966 10. Sloan Creek I Wash. 48 05 121 18 1966 11. Chilliwack c B.C. 49 06 121 42 1966 12. . Forbidden P l a t . c B.C. 49 40 125 09 1966 13. Courtenay c B.C. 49 41 125 03 1966 14. M e r r i t t I B.C. 50 04 120 51 1966 15. Nimpkish c B.C. 50 19 126 53 1966 16. Owl Creek I B.C. 50 20 122 43 1966 17. Barriere I B.C. 51 12 120 09 1966 18. Williams Lake I B.C. 52 06 122 02 1966 19. Stuie c B.C. 52 22 126 00 1966 20. Alexandria I B.C. 52 41 122 26 1966 21. Naselle c Wash. 46 22 123 44 1966 22. Forks c Wash. 47 59 124 24 1966 23. Humptulips c Wash. 47 19 123 54 1966 24. Cathlamet c Wash. 46 18 123 16 1966 25. Hoh River c Wash. 47 48 123 58 1966 26. Lake Crescent c Wash. 48 04 124 00 1966 27. W i l l a r d I Wash. 45 48 121 41 1966 28. Spokane I Wash. 47 28 117 12 1966 29. Packwood I Wash. 46 34 121 40 1966 30. Parkway I Wash. 47 02 121 34 1966 31. Twisp I Wash. 48 23 120 24 1966 Symbols i n the table above: T|» - North l a t i t u d e ( in degrees and minutes) . X - West longitude from Greenwich ( i n degrees and minutes). C - Coastal range of the species, usually west of the Coast or Cascade Mountains. I - I n t e r i o r range, usually east of Coast or Cascade Mountains. Fig- I- Location of Douglas-fir provenances sampled-- 7 -The combinat ion of c o l d and monobromonaphthalene t reatment proved to be the b e s t . R o o t - t i p s of germinants were e x c i s e d when they reached a l e n g t h of about 5 mm. l o n g and p r e t r e a t e d o v e r n i g h t a t 2 C i n d i s t i l l e d wate r . One drop of monobromonaphthalene was added to approx imate ly 2 c c . of d i s t i l l e d water f o r 3 hours a t room temperature f o r f u r t h e r s h o r t e n i n g o f the chromosomes and s p i n d l e i n h i b i t i o n thus p e r m i t t i n g more a c c u r a t e counts and measurements (O'Mara, 1948) . The m a t e r i a l was then t r a n s f e r r e d to Farmer ' s f i x a t i v e (3 :1 a b s o l u t e a l c o h o l r g l a c i a l a c e t i c ac id ) f o r about 24 h o u r s . M a t e r i a l was hydro lyzed i n IN HCl i n a 60° C oven f o r ten m i n u t e s , and then r i n s e d th ree t imes i n d i s t i l l e d water t o a r r e s t ' h y d r o l y s i s . I t was p l a c e d i n l e u c o - b a s i c f u c h s i n f o r one and a h a l f h o u r s , under dark c o n d i t i o n s . The m a t e r i a l was squashed i n a c e t i c a c i d . When s to rage was necessary be fo re e x a m i n a t i o n , s t o r e d m a t e r i a l was p l a c e d i n d i s t i l l e d water a t 0 ° - 2°C. S i n c e there was o n l y a s m a l l q u a n t i t y of r o o t - t i p s a v a i l a b l e , i n order to make a l a r g e number of s l i d e s from t h i s m a t e r i a l a d i s s e c t i o n s te reo microscope was used to s e l e c t the m e r i s t e m a t i c t i s s u e , ma in l y w i t h lOOx m a g n i f i c a t i o n . Approx imate ly 15 -20 s l i d e s were prepared from each r o o t - t i p ; a t o t a l o f f i v e hundred c e l l s were observed f o r each o f the four provenances . The s l i d e s were f r e q u e n t l y examined a f t e r every stage o f s q u a s h i n g , u n t i l the chromosomes appeared to be reasonab ly s e p a r a t e , w i t h i n the i n t a c t c e l l s . In some provenances a l a r g e number of c e l l s were found i n d i f f e r e n t s tages of m i t o t i c d i v i s i o n . The chromosomes were s e l e c t e d from those c e l l s whose n u c l e i were c l o s e s t t o metaphase s t a g e . - 8 -F i g . 2. Meristematic r o o t - t i p chromosomes at m i t o t i c metaphase from Gasquet (#3) provenance x 8000. Micro-photographs were taken when satisfactory separation of chromo-somes became apparent. An automatic camera "Orthomat photo unit" mounted on a Leitz Ortholux microscope was used, employing Agfa fine grain film (IFF) with relatively high contrast. The microphotographs to be used for measurements and analyses were printed and enlarged approximately 8,000 times from those cells that presented clear, well separated mitotic meta-phase chromosomes. Numerous pictures were taken from each c e l l in order to get a l l the chromosomes in sharp focus. Several times when the chromosomes overlapped on the ends, and i t was not possible to see them clearly, another kind of picture was taken using phase contrast and bright f i e l d attachments. The cells were again studied under the microscope, but now they were com-pared with the enlarged prints (Fig. 2). As a result of these manipulat-ions a clearer understanding of the overlapped chromosomes was achieved. From the selected pictures of each nucleus i t was possible to ob-serve details as to the position of the centromere, and in a few cases the secondary constrictions. From the series of photographs of each c e l l , three prints were selected in order to measure the chromosomes, namely one picture where the chromosomes were numbered from 1 to 26, and two other pictures on the reverse of which the individual chromosomes were outlined on a light table. Measurements of the chromosomes and calculation methods Using the above photographs a line was drawn in the middle of each chromosome extending from one end to the other, passing through the - 1 0 -centromere and r u n n i n g p a r a l l e l t o the s i d e s o f t h e chromosomes. The w i d t h l i n e s were drawn a t a p p r o x i m a t e l y e v e r y 2 mm. a l o n g t h e l e n g t h o f each chromosome p e r p e n d i c u l a r t o t h i s l o n g i t u d i n a l l i n e . The t o t a l l e n g t h was c a l c u l a t e d by a d d i n g each segment o f t h e l o n g i t u d i n a l l i n e . The c u r v e d chromosomes were measured by means o f a s e r i e s o f s t r a i g h t l i n e s a l o n g t h e a x i s o f t h e a r c . The measurement s t a r t e d f r o m one end o f t h e l o n g arm t o t h e c e n t r o m e r e , and from t h e c e n t r o m e r e t o t h e ex-treme end o f the s h o r t arm ( F i g . 3). F i g . 3 S c h e m a t i c p r e s e n t a t i o n o f measurement o f chromosomes. The w i d t h was c a l c u l a t e d by m e a s u r i n g , a d d i n g and t h e n a v e r a g i n g , t h e h o r i z o n t a l l i n e s a l o n g t h e l e n g t h o f t h e chromosome. Once a l l t h e chromosomes were measured on t h e p r i n t s , t h e s h o r t arm l e n g t h ( p ) , t h e l o n g arm l e n g t h ( q ) , t h e t o t a l l e n g t h ( T ) , and t h e w i d t h (w) were c a l c u l a t e d . R e l a t i v e chromosome u n i t l e n g t h was c a l c u l a t e d as: i n d i v i d u a l t o t a l chromosome le n g t h average l e n g t h of chromosomes x 1 0 0 . -11-The relative chromosome length was calculated in relation to the length of the longest individual chromosome. The maximum value was 1, assigned to the longest chromosome. The calculation of the arm ratio was based on long arm length (q) over short arm length (p). For the centromere index (C.I.) the following formula was used (Stephenson et a l . 1972): C.I. = lOOp (p+q) The morphological index (M.I.) was calculated according to Giannelli and Howlett (1967) as follows: M.I. = (p/q)* (p+q) • Chromosomes volume calculations were based on the assumption that the cross section of the chromosome is a ci r c l e , (Belling 1928, Svardson 1945, Manton 1950 and Bajer 1959) and the chromosome is a cylinder. After obtaining the average diameter: w = + w^  + ....wn n the surface of the cross section W^TT was calculated. This value was 4 then multiplied by the total length of the chromosome. The length alone was not the main criterio.nfforppairingtthevhomol-ogous chromosomes (e.g. Fig. 4 - chromosome pairs numbers 1 and 6). The centromere position and index values assisted in matching of the homo-logous chromosomes. The morphometric values (short arm, and long arm length, total length, width and volume) and furthermore, the index values (arm ratio, centromere index, relative length, and morphological index) -12-provided the basic c r i t e r i a for cytological differences which accompany the geographical variations of the four provenances. The chromosomes were designated to morphological categories using tha classification of Levan et a l . (1964) (Table 2 ) . Table 2. Morphological categories of chromosomes after Levan et a l . Centromere position Arm ratio Chromosome designation Median sensu strictu 1.0 M metacentric o Median region m •rH u 1.7 u d Submedian region sm <D submetacentric 3.0 O o Subterminal St T—l subtelocentric 7.0 0) •M Terminal region t acrocentric Terminal sensu strictu 00 T telocentric Finally, an idiogram was constructed and the chromosomes were arranged in descending order of relative length each with their centro-mere on the horizontal line and their short arm located above the line (Fig. 5). © (1 ® ® ® I! ® ti © (I ® 1 1 ® ( 1 ® f 1 © 1 1 ® 1 1 1 — 1 Fig.4.The 13 p a i r s of meristematic roo t -t i p chromosomes of Douglas-fir Gasquet(#3)provenance. F i g . 5 . Idiogram of the.four provenances. Short Arm Centromere Long Arm 1 3 22 28 Provenance numbers. -15-3. OBSERVATIONS Karyotype pattern The haploid chromosome number con s i s t e n t l y appeared to be n = 13 i n a l l c e l l s studied from the four provenances (#1, #3, #22 and #28). Using Levan et a l . (1964) c l a s s i f i c a t i o n i t was possible to i d e n t i f y f i v e metacentric, s i x submetacentric and two subterminal chromosomes (Fig. 5). Chromosomal c o n s t r i c t i o n s and a spherical p r o j e c t i o n i n Gasquet (#3)  provenance  In the present study, several chromosomes i n provenance #3, Gasquet, showed prominent secondary c o n s t r i c t i o n s as clear-cut gaps (Fig. 6 and 7). Smaller secondary c o n s t r i c t i o n s were seen as l i g h t - s t a i n i n g transverse bands. The appearance and the p o s i t i o n of the secondary c o n s t r i c t i o n was not constant f o r a l l the c e l l s studied. In several s l i d e s one chromosome was seen i n which the centromere was represented by two p a r a l l e l f i l a -ments and the secondary c o n s t r i c t i o n was one sin g l e filament. In the same s l i d e the centromere of other chromosomes appeared l i k e a single s t r a i g h t thread structure. In s t i l l other chromosomes t h i s connecting thread had a twisted shape. A structure of t h i s kind has not previously been reported i n Douglas-fir chromosomes. -16-A unique abnormality observed i n th i s study was the pr o j e c t i o n of a t i n y s p h e r i c a l structure o f f the side of one arm i n chromosome number 3 i n Gasquet (#3) provenance. The chromosome appeared to be the shortest i n Gasquet provenance compared to other chromosomes i n the other three provenances. Fi g . 6 P r o j e c t i o n of spherical structure on the short arm of chromosome number 3 i n Gasquet (#3) provenance. -17-Figure 7. Chromosomes with secondary constrictions and different coiling patterns in Gasquet (#3) provenance. a) secondary constriction as single filament b) centromere as two parallel filaments c) secondary constriction as two paral l e l filaments Table 3. Averages of morphometric and index values of the four provenances Morphometric values T. F N r. T H S H O R T A R M L 0 N G \ R M (mm.) . T n T A T. Chrom. 1 3 22 28 T o t a l Av. 1 3 22 28 Tota l Av. 1 3 22 28 Tota l Av. Pa ir 0 1 29 10 23.47 26 93 24 .43 103.93 25.95 37.70 26 25 32 62 32.23 128.80 32.20 66.80 49.75 59.55 56 .67 232 77 58 19 2 24 .65 20.88 22 45 21 43 89 41 22.35 30.77 24 07 30 08 30.67 115.59 28.90 55.42 44.95 52.53 52 .10 205 00 . 51 25 3 21 .10 18.77 21 33 21 30 82 50 20.63 26.12 23 52 24 22 25.90 99.76 24.94 47.22 42.28 45.55 47 .20 182 25 45 56 4 20 48 19.17 " 19 88 19 07 78 60 19.65 23.40 21 00 23 70 23.65 91.75 22.94 43.88 40.17 43.58 42 .72 170 35 42 59 5 18 72 17.70 18 07 17 60 72 09 18.02 23.00 20 26 22 35 22.13 88.10 22.03 41.72 38.32 40.42 39 .73 160 19 40 05 6 12 38 9.27 .12 98 10 80 45 43 11.36 25.92 23 40 23 30 24.98 97.60 24.40 38.30 32.67 36.28 35 .78 143 03 35 76 7 10 82 9.23 10 95 10 10 41 10 10.28 23.70 20 20 22 50 22.48 88.88 22.22 34.52 29.43 33.45 32 .58 129 98 32 50 8 10 83 8.88 10 60 10 33 40 64 10.16 22.18 18 73 21 07 20.25 82.23 20.56 33.02 27.62 31.67 30 58 122 89 30 72 9 10 03 8.38 8 65 Q 05 36 11 9.03 21.52 17 87 21 47 20.38 81.24 20.31 31.55 26.25 30.12 29 43 117 35 29 34 10 9 25 7.42 9 18 8 10 33 95 8.49 20.33 10 03 19 62 19.43 76.41 19.10 29.58 24.45 28.80 27 53 110 36 27 59 11 9 20 6.57 8 67 7 95 32 39 8.10 18.55 16 17 17 80 18.35 70.87 17.72 27.75 22.73 26.47 26 30 103 25 25 81 12 4 97 3.52 5 60 3 93 18 02 4.51 18.47 16 45 17 57 18.82 71.31 17.83 23.23 19.97 23.17 22 75 89 12 22 28 13 3 52 2.82 3 17 3 50 13 01 3.25 16.23 12 13 16 83 14.63 59.82 14.96 19.75 14.95 20.00 18 13 72 83 18 21 • To ta l 185 05 156.08 178 46 167 59 687 18 171.80 307.89 257.44 293 13 293.90 1152.36 288.09 492.73 413.66 471.58 461 50 1839. 37 459. 85 Av. 14 23 12.01 13 73 12 89 13 22 13.22 23.68 19 80 22.55 22.61 22.16 22.16 37.90 31.84 36.28 ' 35 50 35.37 35. 37 -W I D T H V 0 L U M E (mm ) r I 3 22 28 Tota l Av. 1 3 22 28 Tota l Av. 3 09 3 33 3 52 3.00 12.94 3.24 496.25 423.11 578.66 402.73 1900 75 475 19 2 90 3.15 3 62 2.86 12.53 3.13 364.69 339.05 543.51 351.97 1599 22 399 81 2 91 3 37 3 90 3.25 13.43 3.36 314.28 374.36 541.36 390.54 1620 54 405 14 2 92 3 21 3 88 3.50 13.51 3.38 293.90 323.88 518.71 413.11 1549 60 387 40 2 90 3 15 3 86 2.88 12.79 3.20 275.73 296.12 474.96 258.95 1305 76 326 44 2 99 3 58 3 87 3.05 13.49 3.37 269.54 326.77 434.92 264.74 1295 97 323 99 -3 01 3 43 3 69 3.11 13.24 3.31 245.99 267.52 359.95 248.95 1122 41 280 60 2 94 3 43 3 83 2.96 13.16 3.29 225.09 251.30 370.27 211.30 1057 96 264 49 2 96 3 33 3 76 3.07 13.12 3.28 217.11 225.56 334.58 221.12 998 37 249 59 3 09 3 40 3 76 3.09 13.34 3.34 221.03 220.51 325.07 211.35 977 96 244 49 3 24 3 51 4 04 3.27 14.06 3.52 230.35 217.62 343.08 220.66 1011 71 252 93 2 91 3 32 4 11 3.25 13.59 3.40 155.16 177.42 315.76 190.14 838 48 209 62 3 09 3 43 3 80 3.04 13.36 3.34 151.98 135.42 235.13 137.29 659 82 164 96 38 95 43 63 49 64 40.33 172.56 43.14 3461.12 3578.65 5275.96 3522.85 15938.55 3984 64 3 00 3 36 3 82 3.10 3.32 3.32 266.24 275.28 413.53 270.99 306 51 306 51 cont. , Table 3 (cont'd). Averages of morphometric and index values of the four provenances I n d e x v a l u e s A R M R A T I 0 CENTROMERE INDEX RELATIVE LENGTH MORPHOLOGICAL INDEX Chrom. P a i r 1-1 3 22 28 T o t a l A v . 1 3 22 28 Tots i l Av 1 3 22 28 Tot a l A v . 1 3 22 28 T o t a l A v . 1 . 1 3.2 1. 13 1 22 1.33 5 .00 1 25 43 37 4 7 . 0 7 45 25 43 10 178 79 44 70 0 .96 0 . 96 0 .96 0 98 3 . 86 0 .97 51 91 44 47 49 21 4 3 . 2 1 188 80 4 7 . 2 0 2 1 28 1. 17 1 34 1.45 5 .24 1 31 4 4 . 2 9 4 6 . 5 6 42 84 40 95 174 64 43 66 0 . 8 0 0 87 0 .85 0 90 3 .42 0 .86 44 59 39 09 39 23 3 6 . 5 6 159 47 39 .87 3 1 25 " 1 . 27 1 15 1.22 4 . 8 9 1 22 44 62 4 4 . 4 6 45 63 45 03 180 79 45 20 0 .69 0 82 0 .74 0 82 3 07 0 .77 38 15 33 79 40 16 3 8 . 8 2 150 92 3 7 . 7 3 4 1 15 1 10 1 22 1.28 4 . 7 5 1 19 46 72 4 7 . 7 2 45 38 44 61 184 43 46 11 0 .64 0 78 0 . 7 0 0 74 2 86 0 . 7 2 38 50 36 66 36 64 3 4 . 4 9 146 29 36 .57 5 1 24 1 17 1 28 1.28 4 . 9 7 1 24 44 83 4 6 . 5 2 44 43 44 29 180 07 45 02 0 .61 0 74 0 . 6 5 0 69 2 69 0 .67 34 01 33 .06 32 71 31 .62 131 40 3 2 . 8 5 6 2 13 2 55 1 87 2 .33 8 .88 2 22 32 41 2 8 . 3 6 36 38 30 19 127 34 31 84 0 .56 0 63 0 . 5 8 0 62 2 39 0 . 6 0 18 35 12 94 21 05 15 .49 67 83 16 .96 7 2 23 2 21 2 08 2 .27 8 .79 2 20 31 33 3 1 . 3 9 32 71 30 90 126 33 31 58 0 . 5 0 0 57 0 .54 0 57 2 18 0 .55 15 76 13 46 16 32 14 .66 60 20 15 .05 8 2 09 2 16 1 99 1.98 8 .22 2 06 32 72 31 .94 33 71 33 74 132 11 33 03 0 .48 0 54 0 .51 0 53 2 06 0 . 5 2 16 17 13 13 15 98 15 .61 60 85 15 .22 9 2 20 2 18 2 51 2 .30 9 .19 2 30 31 75. 31 .87 .28 97 30 59 123 18 30 80 0 .46 0 52 0'.49 0 51 1 98 0 . 5 0 14 83 12 32 12 21 13 .11 52 47 13 .12 10 2 23 2 33 2 16 ' 2 . 42 9 .14 2 29 31 31 3 0 . 4 4 31 85 29 43 123 03 30 76 0 .43 0 48 0 .47 0 48 1 86 0 .47 13 46 10 65 12 34 1 1 . 5 0 47 95 11 .99 11 2 03 2 59 2 09 2 .33 9 .04 2 26 33 27 2 9 . 1 3 32 76 30 25 125 41 31 35 0 .41 0 44 0 .43 0 46 1 74 0 . 4 4 13 82 9 28 13 27 11 .41 47 78 11 .95 12 3 88 4 83 3 48 5 .01 1 7 . 2 0 4 30 21 41 17 .96 24 56 17 24 81 17 20 29 0 .34 0 40 0 .38 0 40 1 52 0 . 3 8 6 33 4 31 7 50 4 . 7 6 22 90 5 .73 13 4 70 4 45 5 34 4 . 3 0 18 .79 4 70 17 75 19 .31 15 82 19 52 72 40 18 10 0 .29 0 30 0 .33 0 32 1 24 0 . 3 1 4 28 3 49 3 76 4 . 3 8 15 91 3 .98 T o t a l 27 72 29 14 27 73 29 .50 114 .10 28 53 455 78 4 5 2 . 7 3 461 34 439 84 1809 69 452 .42 7 .17 8 05 7 .63 8 02 30 .87 7 .76 310 16 266 64 300.38 275 .62 1152 81 2 8 8 . 2 0 A v . 2 13 2 24 2 13 2 .27 2 .19 2 19 35 06 3 4 . 8 3 35 .49 3 3 . 8 3 34 .80 34 .80 0 .55 0 62 0 . 5 9 0 62 0 60 0 . 6 0 23 86 20 51 23 11 - 2 1 . 2 0 22 17 22 .17 -20-Differences between provenances Morphometric values Short arm length The average short arm length of the 13 chromosomes i n c e l l s of the four provenances was 13.22 units (LI459jinm).(Table 3) . The largest value was 25.95 units (2.93 juml) for #1 chromosome, while the smallest one was 3.25 units (0.37 pra.) for #13 chromosome. The differences among the four provenances were not s i g n i f i c a n t (Table 4). Table 4. Analysis of variance; short arm length SourceDF DF Sun SO Sum sq Me in SMean sq F F Loc 3 111.97 37.322 0.69 NS Error 152 8244.7 54.241 To t a l 155 8356.7 NS = non s i g n i f i c a n t * = s i g n i f i c a n t at 5 percent confidence l e v e l ** = s i g n i f i c a n t at 1 percent confidence l e v e l Underlined values are NS Long arm length For Average long arm length of the 13 chromosomes i n c e l l s of the four provenances was 22.16 units (2.52 ;im.) (Table 5). The highest value was 32.20 units (3.66 Jim.) f o r #1 chromosome and the lowest value was 14.96 units (1.70 jjm.) f or #13 chromosome. The long arm length of chromosomes from Gasquet (#3) was s i g n i f i c a n t l y shorter than the long arm length of the other three provenances. (Table 5). - 2 1 -Table 5. Analysis of variance; long arm length Source DF Sum sq Mean sq F Loc 3 320.96 106.99 3.63* Error 152 4475.1 79.44? Total 155 4796.1 Duncan's test. #3 #22 - #28 #1 19.80 22.55 22.61 23.68 Total length The average total length of the 13 chromosomes in cells of the four provenances was 35.37 units (4.02 | i m . ) (Table 3). The largest value was 58.19 units (6.61 pm.) for #1 chromosome while the smallest one was 18.21 units (2.07 pra.) for chromosome #13. The differences in total length of the chromosomes in cells of the four provenances were not significant (Table 6). Table 6. Analysis of variance- total length Source DF Sum sq Mean sq F Loc 3 776.28 258.76 1.73 NS Error i 152 22752. 149.68 Total 155 23528. Width The average total width of the 13 chromosomes in cells of the four provenances was 3.32 units (0.38 pm) (Table 3). The largest value was 4.11 units (^;0.47 |im.) for chromosome #12 in Forks (#22) provenance, while the smallest one was 2.86 units (0.33 jum.) for chromosome #2 in Spokane (#28) provenance. - 2 2 -The average chromosome w i d t h i n c r e a s e d from south to n o r t h i n the th ree C o a s t a l provenances (#1, #3, #22). C o a s t a l Provenances #1 #3 #22 w i d t h 3 .00 3.36 3.82 S i g n i f i c a n t d i f f e r e n c e s were found among the widths of the chromo-somes i n some of the f o u r provenances . (Table 7) Table 7. A n a l y s i s of v a r i a n c e ; w i d t h Source DF Sum sq Mean sq Loc 3 15.652 5.2173 ** 52.27 E r r o r 152 15.171 0.99812D-01 T o t a l 155 30.823 Duncan's Test #1 3 .00 #28 3 .10 #3 3.36 #22 3.82 The chromes;omevi'.widith of the I n t e r i o r provenance (#28) was s i g n i f i -c a n t l y l e s s than i n the cor responding C o a s t a l one (#22) on the same l a t i t u d e . C o a s t a l I n t e r i o r Provenance #22 #28 3.82 3 .10 "^23-Volume hs. seen in Table 3, a significant south to north c l i n a l trend was also observed for the three Coastal provenance chromosome volume averages. This is a trend in the same direction as was noted previous-ly for the width. The average volume for the Interior provenance was significantly smaller than the corresponding Coastal provenance (Table 8). Coastal Provenances Interior Provenance #1 #3 #22 #28 Volume 266.24 275.28 413.53 270.99 Table 8. Analysis of variance; volume Source DF Sum sq Mean sq F Loc 3 0.59720D 06 0.19907D 06 16.87** Error 152 0.17939D 07 11802. Total 155 0.23911D 07 Duncan's test #1 #28 #3 #22 266.24 270.99 275.22 413.53 Index .values The index values (arm ratio, centromere index, relative length and morphological index) were not significantly different among the chromo-somes of the four provenances (Table 9). The morphological index appeared to be the most useful criterion for classifying metacentric, submetacentric and subterminal chromosomes, since the morphological categories of the 13 chromosomes were clearly separated in each of the four provenances studied. -24 -Table 9. Analyses of variance; index values Arm ratio Centromer Source DF. Sum sq Mean sq F Loc 3 0.61237 .0.20412 0.15 NS Error 152 205.09 1.3493 Total 155 205.70 e index Relative Loc 3 57.472 19.157 0.21 NS Error 152 13980. 91.972 Total 155 14037. 91.972 Length Morpholog Loc 3 0.11594 0.38646D-01 1.02 NS Error 152 5.7361 0.37738D-01 Total 155 5.8521 ical index Loc 3 288.21 96.069 0.44 NS Error 152 33165. 218.19 Total 155 33454. Comparison with previous work . It is usually d i f f i c u l t to compare chromosome length directly with previous results since the different treatments could alter the chromo-some length to various degrees. However, using the relative chromosome length, the effects of different treatments are largely eliminated. R e l a t i v e chromosome lengths were reported previously i n three works on Douglas-fir (Sax and Sax 1933, Barner and Christiansen 1962,and Thomas and Ching 1968) and these values were compared to the r e s u l t s of the present study (Table 10). The simple c o r r e l a t i o n c o e f f i c i e n t s were calculated between the four provenances and the previous information. The figures from the present study coincided with the e a r l i e r work. The cl o s e s t c o r r e l a t i o n was found with Sax and Sax's work, and le a s t with r e s u l t s of Barner and Christiansen. Table 10. Relative chromosome unifetieng L l3hcand ' eG(a.rrelation c o e f f i c i e n t s f o r the four provenances (#1,3,22,28) and t h e i r combined average as compared to r e s u l t s of Sax and Sax (A), Barner and Christiansen (B), Thomas and Ching (C). Chrom. Prov. #1 S3#3 #22 #228 Av. #28A BAv. C A B C number Y l Y2 Y 3 Y 4 Y 5 X l x 2 X 3 1 176. 156. 164. 160. 165. 148. 139. 144. 2 146. 141. 145. 147. 145. 135. 135. 136. 3 125. 133. 126. 133. 129. 123. 128. 128. 4 116. 126. 120. . 120. 120. 121. 123. 128. 5 . 110. 120. 111. 112. 113. 111. 122. 120. 6 101. 103. 100. 101. 101. 108. 95. 99. 7 91. 92. 92. 92. 92. 96. 90. 92. 8 87. 87. 87. 86. 87. 91. 89. 87. 9 83. 82. 83. 83. 83. 81. 86. 83. 10 78. 77.. 79. 78. 78. 81. 86. 80. 11 73. 71. 73. 74. 73], 79. 76. 77. 12 61. 63. 64. 64. 63. 69. 76. 69. 13 52. 47. 55. 51. 52. 54. 56. 57. C o r r e l a t i o n c o e f f i c i e n t s 0 .977 0.993 0.987 0.990 0.989 1.000 0 .945 0.987 0.965 0.975 0.969 0.969 1.000 0 .958 0.995 0.977 0.984 0.980 0.983 0.992 •1.000 -26-Differences within provenances Covelo provenance (#1) Uniform morphometric and index values were obtained as can be seen i n Table 11 and the s t a t i s t i c a l analyses did not indi c a t e any differ e n c e between the three r e p l i c a t i o n s . Gasquet provenance (#3) Table 12 shows that the short arm length measurements were not s i g -n i f i c a n t l y d i f f e r e n t between r e p l i c a t e s but the long arm length i n r ^ d i f f e r s , s i g n i f i c a n t l y from r ^ and r 2 . The average long arm length i n r 3 was 1 5 . 4 7 units ( 1 . 7 6 j im.) while r-^ and r 2 were very s i m i l a r , 2 1 . 9 8 and 2 1 . 9 7 units r e s p e c t i v e l y ( 2 . 5 0 pm. and 2 . 5 0 urn.) As a r e s u l t of these differences i n r ^ the t o t a l length was 2 5 . 0 0 units ( 2 . 8 4 urn.) which i s d i f f e r e n t and smaller than r ^ and r 2 ( 3 4 . 6 1 and 3 5 . 8 2 units or 3 . 9 4 and 4 . 0 7 ym. r e s p e c t i v e l y ) . The values of width were s i g n i f i c a n t l y d i f f e r e n t from each other i n a l l three r e p l i c a t i o n s , r 3 providing the shortest and widest i n d i -v i d u a l chromosome with 3 . 55 width units ( 0 . 4 0 ym.). No s i g n i f i c a n t differences were found i n volume or i n the four index values. Forks provenance (#22) The short arm length, the t o t a l length and the index values did not d i f f e r amongst the three r e p l i c a t i o n s i n Forks provenance (#22) Table 1 3 . However, the long arm length i n r ^ was s i g n i f i c a n t l y greater than r ^ and r 2 . The width values were s i g n i f i c a n t l y d i f f e r e n t i n rj_, r 2 and r ^ . The volume of chromosomes i n was s i g n i f i c a n t l y smaller than r^ and -27-Spokane provenance (#28) As can be seen i n Table 14, only one c h a r a c t e r i s t i c , the width, showed any s i g n i f i c a n t d i f f e r e n c e between the three r e p l i c a t i o n s . In t h i s case, r ^ was s i g n i f i c a n t l y d i f f e r e n t from r\ and r 2 . Table 11. Morphometric and index values of Covelo (#1) provenance. Morphometric values Chrom. Pair D L E N G T H (mm) SHORT ARM WIDTH (mm) VOLUME (mm ) r 3 r l 1 2 3 4 5 6 7 3 9 10 11 12 13 35.35 25.00 22.45 19.75 18.45 13.60 11.45 12.30 10.95 9.25 9.55 4.55 3.40 28.60 27.75 20.95 21.00 20.00 11.90 10.45 10.05 10.95 9.05 8.70 4.65 3.20 23.35 21.20 19.90 20.70 17.70 11.65 10.55 10.15 8120 9.45 9.35 5.70 3.95 29.10 24.65 21.10 20.48 18.72 12.38 10.82 10.33 10.03 9.25 9.20 4.97 3.52 39.20 33.00 27. 10 23.95 22.40 25.05 24.20 22.35 21.55 19.75 17.25 18.15 15.30 41.80 30.45 26.20 24.85 22.80 27.45 24.05 22.60 20.50 21.05 20.30 19. 70 16.25 32.10 28.85 25.05 21.40 23.80 25.25 22.85 21.60 22.50 20.20 18.10 17.55 17.15 37.70 30.77 26.12 23.40 23.00 25.92 23.70 22.18 21.52 20.33 18.55 18.47 16.23 74.55 58.00 49.55 43.70 40.85 38.65 35.65 34.65 32.50 29.00 26.80 22.70 18. 70 70.40 58.20 47.15 45.85 42.80 39.35 34.50 32.65 31.45 30.10 29.00 23.75 19.45 55.45 '50.05 44.95 42.10 41.50 36.90 33.40 31.75 30.70 29.65 27.45 23.25 21.10 66.80 55.42 47.22 43.88 41.72 38.30 34.52 33.02 31.55 29.58 27.75 23.23 19.75 3.06 2.77 3.06 3.04 3.14 3.07 2.96 3.01 2.85 3.03 3.00 2.71 2.74 2.81 2.71 2.92 2.76 2.91 2.89 2.90 2.97 3.11 3.01 3.40 3.14 2.99 3.41 3.23 2.74 2.97 2.65 3.02 3.17 2.85 2.92 3.22 3.33 2.87 3.55 3.09 2.90 2.91 2.92 2.90 2.99 3.01 2.94 2.96 3.09 3.24 2.91 3.09 548.33 348.53 363.22 317.64 315.49 285.79 244.84 246.01 207.56 208.54 188.93 130.88 U0.26 435.22 335.65 314.85 273.22 283.67 258.63 229.22 226.28 238.36 213.70 262.75 184.10 136.52 505.20 409.88 264.77 290.84 228.02 264.21 263.91 202.98 205.40 240.86 239.37 150.40 209.15 14SS.75 1094.06 942.84 831.70 827.18 308.63 737.97 675.27 651.32 663.10 691.05 465.47 455.93 496.25 364.60 314.23 293.90 275.73 269.54 245.99 225.09 217.11 221.03 230.35 155.16 151.93 Total Av. 196.05 137.25 171.85 15.03 14.40 13.22 185.05 14.23 309.25 318.00 296.40 23.79 24.46 22.80 307.89 23.68 505.30 504.65 468.25 38.87 33.82 36.02 492.73 37.90 J38.44 2.96 38.52 2.96 -39.93 3.07 38.95 3.00 Duncan ' s* 3516.02 3392.17 3475.08 270.46 260.94 267.31 10333.36 266.24 3461.12 266.2 Index values ARM RATIO CENTROMERE INDEX RELATIVE LENGTH MORPHOLOGICAL INDEX r l r 2 r 3 Av. r 1 2^ r 3 Av. r l r 2 r 3 Av. r X 2 r 3 Av. 1 11 1 47 1 .38 1 32 47 45 40 57 42 09 43.37 0 93 0 96 0.98 0 96 67 22 48 17 40 33 51.91 1 35 1 11 1 .37 1 28 42 95 47 64 42 29 44.29 0 72 0 80 0.89 0 80 43 94 53 04 36 78 44.59 1 21 1 26 1 .27 1 25 45 27 44 38 44 21 44.62 0 62 0 65 0.79 0 69 '41 05 37 70 35 71 38.15 1 23 1 19 1 04 1 15 45 18 45 80 49 17 46.72 0 54 0 63 0.74 0 64 36 04 38 74 40 72 38.50 1 22- 1 15 1 .36 • 1 24 45 16 46 72 42 61 44.83 0 51 - 0 59 0.73 0 61 33 64 37 54 30 86 34.01 1 84 2 37 2 19 2 13 35 19 30 43 31 61 32.41 0 48 0 54 0.65 0 56 20 98 17 06 17 02 18.35 2 13 2 32 2 23 2 23 32 15 30 23 31 60 31.33 0 45 0 47 0.59 0 50 16 87 14 99 15 42 15.76 1 82 2 31' 2 14 2 09 35 49 30 72 31 95 32.72 0 43 0 45 0.56 0 48 19 07 14 52 14 92 16.17 1 99 1 87 2 74 2 20 33 72 34 82 26 72 31.75 0 41 0 43 0.54 0 46 16 51 16 80 11 19 14.83 2 14 2 33 2 22 2. 23 31 91 30 09 31 94 31.31 0 36 0 41 0.53 0 43 13 58 12 94 13 87 13.46 1 81 2 34 1 94 2. 03 35 64 30 00 34 16 33.27 0 34 0 40 0.48 0 41 14 84 12 43 14 18 13.82 4 42 4 13 3 08 3. 88 20 15 19 55 24 54 21.41 0 29 0 33 0.41 0 34 5 69 5 75 7 55 6.33 4 60 5 13 4 36 4. 70 18 04 16 39 18 81 17.75 0 23 0 27 0.37 0 29 4 16 3 83 4 86 4.28 26 87 28. 98 27 32 27. 72 468 30 447. 35 451. 70 455.78 6 31 6. 93 8.26 7 17 333. 59 313. 51 283 41 310.16 2 07 2. 23 2 10 2. 13 36 02 34. 41 34. 75 35.06 0 49 0. 53 0.64 0 55 25. 66 24. 12 21 80 23.86 Chrom. Pair . # 1 2 3 4 5 6 7 8 9 10 11 12 13 Total Av. Duncan Table 12. Morphometric and index values of Gasq'uet (#3) provenance. Morphometric values Chrom. Pair D L E N G T H (mn) SHORT ARM LONG ARM TOTAL WIDTH (ITO) VOLUME Oral ) 1 2 3 ' 4 5 6 7 S 9 10 11 12 13 r 3 ?2 r 2 Total 24.00 19.65 19.05 21.15 18.45 9.55 10.15 10.60 9.60 8.00 6.80 3.90 3.05 23.50 26.40 22.15 22.00 20.90 10.80 10.05 9.35 9.10 7.90 7.50 2.80 2.60 17.90 16.40 15.10 14.35 13.75 7.45 7.50 6.70 6.45 6.35 5.40 3.85 2.80 23.47 20.88 18.77 19.17 17.70 9.27 9.23 8.88 8.38 7.42 6.57 3.52 2.82 25.75 25.15 25.30 23.25 24.50 24.25 22.40 19.95 20.25 19.05 19.60 20.15 16.10 31.95 29.60 28.00 24.00 23.15 27.70 22.35 20.30 18.60 18.20 16.00 16.10 9.60 21.05 17.45 17.25 15.75 14.20 18.25 15.85 15.95 14.75 13.85 12.90 13.10 10.70 26.25 24.07 23.52 21.00 20.62 23.40 20.20 18.73 17.87 17.03 16.17 16.45 12.13 49.85 45.00 44.35 44.40 42.95 33.80 32.55 30.55 29.85 27.05 26.40 24.05 19.15 60.45 56.00' 50.15 46.00 44.05 38.50 32.40 29.65 27.70 26.10 23.50 18.90 12.20 33.95 33.85 32.35 30.10 27.95 25.70 23.35 22.65 21.20 20.20 18.30 16.95 13.50 49.75 44.95 42.28 40.17 38.32 32.67 29.43 27.62 26.25 24.45 22.73 19.97 14.95 3.34 3.30 3.46 3.49 3.30 3.51 3.31 3.29 3.10 3.31 3.48 3.70 3.20 3.00 2.67 3.18 2.85 2.86 3.50 3.27 3.17 3.31 3.37 3.27 2.85 3.6A 3.65 3.47 3.48 3.28 3.30 3.74 3.70 3.83 3.58 3.52 3.77 3.40 3.44 3.33 3.15 3.37 3.21 3.15 3.58 3.43 3.43 3.33 3.40 3.51 3.32 3.43 436.43 384.87 417.28 423.68 366.56 327.63 280.22 258.69 225.69 232.55 425.93 312.95 399.01 294.09 283.23 369.67 271.30 234.40 237.59 232.06 406.98 319.33 306.80 253.88 233.58 283.00 251.05 260.61 213.39 196.91 1269.34 1017.15 1123.09 971.65 888.37 980.30 802.57 753.90 676.67 661.62 258.10 120.28 153.89 154.01 126.70 125.55 532.27 406.26 4:3.11 339.05 374.36 323.59 296.12 326.77 267.52 251.30 225.56 220.51 251.65 196.71 204.51 652.87 217.62 177.42 135.42. Tota l Av. Duncan 1 s 64.15 12.63 180.05 13.85 124.00 9.54 156.08 12.01 285.70 21.98 285.55 201.05 21.97 15.47 _ * * 257.44 19.80 449.95 465.60 34.61 35.82 325.05 413.66 25.00 31.84 143.79 3.37 40.94 • 46.16 3.15 3.55 i ** 43.63 3.36 4017.56 3503.92 3214.48 10735.96 309.04 269.53 247.27 275.28 357S.65 275.28 Index values ARM RATIO CENTROMERE INDEX RELATIVE LENGTH MORPHOLOGICAL INDEX Chrom. Pair. >} r 2 1 2 3 4 5 6 7 8 9 10 11 12 13 1.08 1.31 1.35 1.10 1.35 2.57 2.26 1.89 2.13 2.40 3.15 5.26 5.68 1.12 1.12 1.32 1.09 1.12 2.57 2.25 2.18 2.07 2.33 2.19 5.75 3.70 1.18 1.07 1.15 1.10 1.04 2.50 2.13 2.41 2.33 2.19 2.44 : 3.47 3.97 1.13 1.17 1.27 1.10 1.17 2.55 2.21 2.16 2.18 2.33 2.59 4.83 4.45 48.12 44.09 42.95 47.63 42.93 28.23 31.16 34.70 32.17 29.47 25.79 16.13 15.92 47.16 47.16 43.74 47.83 47.42 28.04 30.92 31.53 32.91 3d. 37 31.99 15.12 21.31 45.92 48.44 46.68 47.70 49.20 28.82 32.08 29.59 30.54 31.49 29.60 22.62 20.69 47.07 46.56 44.46 47.72 46.52 28.36 31.39 31.94 31.87 30.44 29.13 17.96 19.31 0.94 0.85 0.84 0.84 0.82 0.64 0.62 0.58 0.57 0.51 0.50 0.46 0.36 0.98 0.91 0.81 0.74 0.71 0.62 0.52 0.48 0.45 0.42 0.38 0.31 0.20 0.97 0.84 0.80 0.75 0.70 0.64 0.58 0.56 0.53 0.50 0.45 0.42 0.34 0.96 0.87 0.82 0.78 0.74 0.63 0.57 0.54 0.52 0.48 0.44 0.40 0.30 46.37 53.92 33.12 44.47 35.51 49.95 31.81 39.09 33.39 39.67 28.32 33.79 40.39 42.17 27.42 36.66 32.34 39.77 27.06 33.06 13.31 15.01 10.49 12.94 14.75 14.57 11.05 13.46 16.23 13.66 9.51 13.13 14.15 13.55 9.27 12.32 11.36 11.33 9.26 10.65 9.16 11.02 7.66 9.28 4.65 3.29 4.98 4.31 3.63 3.30 3.53 3.49 Total Av. 31.62 2.43 28.81 2.22 26.98 2.03 29.14 2.24 439.29 33.79 455.50 35.04 463.37 35.64 D u n c a n ' s • 452.73 34.83 8.53 0.66 7.53 0.58 8.08 0:62 8.05 0.62 275.24 21.17 311.21 23.94 213.48 16.42 266.64 20.51 T a b l e 13. M o r p h o m e t r i c and i n d e x v a l u e s o f F o r k s (#22) p r o v e n a n c e . M o r p h o m e t r i c v a l u e s Chrom. P a i r t L E N G T H (mm) SHORT ARM WIDTH (mm) VOLUME (mm ) *2 1 2 3 4 5 6 7 8 9 10 11 12 13 26 .60 23 .30 17.65 15.95 15.45 15.55 9 .45 10 .40 9 .10 8 .55 8 .05 4 . 9 5 3 .55 31.35 24 .80 26 .65 25.25 22.85 13.15 13.25 11.90 9 .40 10.85 10.25 5 .55 3 .15 22.85 19.25 19.70 18.45 15.90 10.25 10.15 9 .50 7 .45 8 .15 7 .70 6 .30 2 .80 26.93 22.45 21.33 19.88 18.07 12.98 10.95 10.60 8 .65 9 .18 8 .67 5 .60 3 .17 26.65 25.15 22 .60 21.95 19.05 20.85 19.40 19.15 20 .15 18.15 16 .20 13.90 15. 25 34 .15 29 .90 21 .15 21.05 20.80 17.70 22 .15 18.05 18.25 17.90 16.90 18.85 18.65 37.05 35 .20 28.90 28 .10 27.20 31. 35 25.95 26 .00 26.00 22.80 20 .30 19.95 16.60 32.62 30.08 24.22 23.70 22.35 23.30 22.50 21.07 21.47 19.62 17.80 17.57 16.83 60.75 53.20 38.80 37.00 36.25 33.25 31.60 28.45 27.35 26.45 , 24.95 23.80 22.20 68 .40 60 .00 55.55 53.35 50.05 44 .50 39.20 37.90 35.40 33.65 30.55 25.50 19.75 49 .50 44 .40 42 .30 40 .40 34.95 31.10 29.55 28.65 27.60 26.30 23.90 20. 20 18.05 59.55 52 .53 45 .55 4 3 . 5 8 40 .42 36.28 33.45 31.67 30.12 28.80 26.47 23.17 20.00 3.68 3 .78 4 . 3 9 4 .28 4 .33 3 41 3 47 3 52 644 37 624.82- 466 .79 1735.98 ' 57S .66 3 65 3 42 3 62 595 42 628.40 406 .71 1630.53 -" 543 51 3 75 3 57 3 90 . 585 93 614.72 423 44 1624.09 541 .36 3 96 3 40 3 88 533 40 657 .19 365 54 1556.13 ' 518 71 3 76 3 50 3 86 532 99 555.60 336 28 1424.87 - 474 96 3 80 3 22 3 87 547 89 503 .43 253 44 1304.76 434 92 3 64 3 40 3 69 403 94 408 .24 267 68 1079.S6 359 95 3 92 3 31 3 83 407 75 457.39 245 66 1110.80 370 27 3 65 3 43 3 76 378 91 370.67 254 16 1003.74 334 5S 4 02 3 41 3 76 308 18 426 .97 240 07 975.22 325 07 3 94 3 50 4 04 428 11 371.83 229 30 1029.24 343 OS 4 28 3 47 4 11 390 25 366.33 190 71 9 4 7 . 2 9 ' .315 76 3 81 3 21 3 80 333 72 225.67 146 00 7 0 5 . 3 9 . ; -235 13 49. 59 44 . 31 4 9 . 64 6090. 84 6211 .23 3825. 78 16127.67 5-275. 96 3. 82 * 3. 41 3. 82 468. 53 477 .79 294. k 29 405 .84 413 . 53 T o t a l A v . D u n c a n 68 .55 12 .97 203 .40 16.03 158.45 12 .19 178.46 13.73 258.45 19 .88 275.50 345.40 21.19 26.57 293.13 22.55 444 .05 34.16 553.80 416 .90 471 .58 42 .60 32.07 36.28 55.01 4 . 2 3 Index v a l u e s ARM RATIO CENTROMERE INDEX RELATIVE LENGTH MORPHOLOGICAL INDEX Chrom. P a i r . r 3 1 2 3 4 5 6 7 8-9 10 11 12 13 1.29 1.29 1.20 1.35 1.40 1.15 2.35 1.74 2.05 2.11 2.12 1. 20 1.43 1.09 1.12 1.22 2 .39 1.97 2 .19 2 .78 2 .11 1.99 3.82 3 .62 5 .26 5 .27 1.17 1.31 1.15 1.19 1.23 2.06 1.92 2 .03 2.71 2.27 2 .15 2 .99 5 .50 1.22 1.34 1.15 1.22 1.28 1.87 2 .08 1.99 2.51 2.16 2 .09 3 .48 5.34 43 .72 43 .79 4 5 . 4 9 43 .07 42 .55 46 .70 29.95 36.56 33.33 32.32 32.25 20.84 16.01 4 5 . 8 0 41 .36 4 7 . 9 8 47 .34 4 5 . 5 5 29 .54 33.83 31 .41 26 .54 32.32 33.73 21 .73 15.96 46 .23 43 .37 46 .57 45 .72 45 .19 32.89 34.34 33.17 27.03 30.91 32.29 31.12 15.48 45 25 0 97 0 99 0 92 0 96 47 32 57 87 42 44 49 21 42 84 0 85 0 87 '0 83 0 85 41 45 42 27 33 98 39 23 46 68 0 62 0 80 0 79 0 74 32 38 51 22 36 87 39 23 45 38 0 59 0 77 0 75 0 70 28 0'3 47 93 33 96 36 64 44 43 0 58 0 73 0 65 0 65 26 92 42 04 29 17 32 71 36 38 0 53 0 64 >0 58 0 58 29 21 18 66 15 29 21 05 32 71 0 50 0 57 ' 0 55 0 54 13 48 20 01 15 46 16 32 33 71 0 46 0 55 0 53 0 51 16 39 17 34 14 21 15 98 28 97 0 44 0 52 0 52 0 49 13 64 12 80 10 20 12 21 31 85 0 42 0 49 0 49 0 47 12 64 12 58 11 81 12 34 32 76 0 40 0 44 0 45 0 43 11 88 15 42 12 50 13 27 24 56 0 38 0 37 0 38 0 38 6 25 7 09 9 16 7 50 15 82 0 35 0 29 0 34 0 33 4 22 3 75 3. 31 3 76 461 34 7. 05 8 03 7. 78 7 63 283. 80 348 98 .268. 36 300. 38 35. 49 0. 55 0. 62 0. 60 0. 59 21.83 26. 85 20 .64 23. 11 T o t a l A v . D u n c a n 1 s l_ 27.13 2.09 28 .38 2 .18 27 .68 2 .13 27 .73 2 .13 466 .58 35 .89 453 .09 34.85 464.31 35.72 Table 14. Morphometric and index values of Spokane (#28) provenance. Morphometric values L E N G T H (mm) SHORT ARM LONG ARM TOTAL WIDTH (mm) VOLUME (mm ) Chrom. Pair 1} r 1 r 2 r 3 Av r 1 r 2 r 3 Av. r 1 r2 r 3 Av. r l r 2 r 3 Av. r l r 2 r 3 Total Av. 1 21 50 28 65 23 15 24 43 27 95 33.25 35.50 32.23 49 45 61 90 58 65 56 67 2 83 2.88 -3.28 3 00 310.44 402 44 459 32 1208 20 402 73 2 17 10 24 20 23 00 21 43 27 75 30.75 33.50 30.67 44 85 54 95 56 50 52 10 2 47 2.59 3.52 2 86 214.58 290 53 550 91 1055 91 351 97 3 18 70 22 65 22 55 21 30 23 30 27.05 27.35 25.90 ' 42 00 49 70 49 90 47 20 3 49 2.95 3.32 3 25 401.39 339 43 430 81 1171 63 390 54 4 17 20 20 55 19 45 19 07 • 22 50 23.65 24.80 23.65 39 70 44 20 - 44 25 42 72 3 25 3.67 3.57 3 50 329.06 467 09 443 18 1239 33 413 11 ' 5 16 40 19 20 17 20 17 60 20 95 22.85 22.60 22.13 37 35 42 05 39 80 39 73 3 04 2.54 3.06 2 88 270.58 212 81 293 47 776 85 25S 95 6 9 55 11 65 11 20 10 80 24 40 25.30 25.25 24.98 33 95 36 95 36 45 35 78 2 79 3.30 3.07 3 05 207.64 316 02 270 56 794 22 264 74 7 9 70 11 20 9 40 10 10 22 45 22.80 22.20 22.48 32 15 34 00 31 60 32 58 2 80 3.02 3.51 3 11 198.46 242 69 305 70 746 85 248 95 8 10 10 11 00 9 90 10 33 19 70 21.55 19.50 20.25 29 80 32 55 29 40 30 58 2 85 2.57 3.45 2 96 189.85 169 21 274 83 633 89 211 30 9 7 90 10 35 8 90 9 05 20 05 20.70 20.40 20.38 27 95 31 05 29 30 29 43 2 66 3.35 3.19 3 07 154.85 273 95 234 55 663 35 221 12 10 8 05 8 85 7 40 8 10 17 75 20.50 20.05 19.43 25 80 29 35 27 45 27 53 2 68 3.48 3.11 3 09 145.44 279 60 209 02 634 06 211 35 11 8 00 8 10 7 75 7. 95 17 00 19.20 18.85 18.35 25 00 27 30 26 60 26 30 3 25 3.20 3.36 3 27 207.88 219 02 235 08 661 93 220 66 12 3 80 3 50 4 50 3. 93 17 85 18.90 19.70 18.82 21 65 22 40 24 20 22 75 2. 89 3.58 3.27 3 25 141.66 225 59 203 18 570. 43 190 14 13 3 95 2 65 3 90 3. 50 12 30 13.35 18. 25 14.63 16 25 16 00 22 15 18 13 3. 39 2.41 3.33 3 04 146.57 72 70 192 59 411. 86 137 29 Total 151 95 182 55 16S 30 167. 59 273 95 299.85 307.95 293.90 425 90 482 40 476 25 461 50 38. 39 39.54 43.04 40 33 2918.40 3511 04 4139 20 10568. 57 3522 85 Av. 11 69 14 04 12 95 12. 89 21 07 23.07 23.69 22.61 32 76 37 11 36 63 35 50 2. 95 3.04 3.31 3 10 224.49 270 08 313 40 270. 99 270 99 Duncan 1 Index values ARM RATIO CENTROMERE INDEX RELATIVE LENGTH MORPHOL OGICAL INDEX r l r 2 i •3 Av. r l "•2 r 3 Av r l r 2 r 3 Av. r l r 2 r 3 Av. 1 30 1 16 1 54 1 33 43 47' 46 32 39 52 43. 10 0 98 0 97 0 98 . 0 98 38.04 53 34 38 25 43.21 1 62 1 23 1 46 1 45 38 13 43 93 40 73 40. 95 0 89 0 86 0 95 0 90 27.64 43 25 38 79 36.56 1 25 1 21 1 21 1 22 44 49 45 40 45 19 45. 03 0 83 0 78 0 84 0 82 33.71 41 61 41 14 38.82 1 36 1 16 1 31 1 28 43 42 46 44 43 97 44. 61 0 79 0 69 0 74 0 74 30.35 38 41 34 71 34.49 1 29 1 21 1 34 1 28 43 88 45 65 43 35 44 29 0 74 0 66 0 67 0 69 29.24 . 35 34 30 29 31.62 2 56 2 17 2 27 2 33 28 13 31 62 30 82 30 19 0 67 0 58 0 61 0 62 13.29 17 02 16 17 15.49 2 34 2 04 2 42 2 27 30 12 32 95 29 64 30 90 0 64 0 53 0 53 0 57 13.89 16 70 13 38 14.66 1 97 1 99 1 97 1 98 33 86 33 67 33 68 33 74 0 59 0 51 0 .50 0 53 15.28 16 61 14 93 15.61 2 57 2 02 2 32 2 30 28 18 33 37 30 22 30 59 0 55 0 49 0 .49 0 51 .11.01 15 53 12 78 13.11 2 22 2 32 2 73 2 42 31 18 30 15 26 96 29 43 0 51 0 46 0 .46 0 48 11.70 12 67 10 13 11.50 2 15 2 40 2 45 2 33 32 01 29 64 29 10 30 25 0 50 0 43 0 .45 0 46 11.77 11 52 10 94 11.41 4 69 5 40 4 95 5 01 17 59 15 79 18 33 17 24 0 43 0 35 0 .41 0 40 4.61 4 15 5 53 4. 76 3 11 5 07 4 72 4 30 24 40 16 61 17 56 19 52 0 33 0 25 0 .30 0 32 5.22 3 18 4 73 4.38 28 43 29 43 30 69 29 50 438 86 451 59 429 07 439 84 8 45 7 56 8 .01 8 02 245.75 309.33 271 77 275.62 2 19 2 26 2 36 2 27 33 76 34.74 33 01 33 83 0 .65 0 58 0 .62 0 62 18.90 23 .80 20 91 21.20 l „ _ Chrom. P=iir. it 6 7 8 9 10 U 12 13 TDtal Av. -32--4. DISCUSSION Chromosome number and karyotype pattern Pinaceae family has the haploid chromosome number of 12. However, Pseudotsuga menziesii appears to be an exception since n = 13 has been reported by t h i s current study as well as other i n v e s t i g a t o r s . In a l l r o o t - t i p meristematic c e l l s examined of the four provenances (#1, #3, #22 and #28) of Pseudotsuga menziesii the haploid chromosome number has been constantly observed as 13. These findings are i n agree-ment with e a r l i e r studies on Douglas-fir. The same haploid chromosome number was reported by other authors using m i t o t i c material (Sax and Sax 1933, Thomas and Ching 1968, El-Lakany 1969) and meiotic tissues (Zenke 1953, A l l e n 1960, Barner and Christiansen 1962 and Livingston 1971). A few reports have shown the haploid chromosome number to be 12. (Langlet 1943, and Durrie-Vabre 1958). This could be explained by tech-n i c a l problems or i t may have been assumed that Pseudotsuga menziesii would generally have the same chromosome number as the re s t of the Pinaceae family (n = 12). In the present study, using the c l a s s i f i c a t i o n of arm r a t i o of Levan et a l . (1964), f i v e metacentric and s i x submetacentric chromosomes were found i n the Douglas-fir provenances, corresponding to previous findings of the authors l i s t e d above. T e l o c e n t r i c chromosomes have a terminal centromere i n sensu s t r i c t u following the same c l a s s i f i c a t i o n of Levan et al_. , and short arms appeared c o n s i s t e n t l y i n the shortest chromosomes of the four provenances, giving arm r a t i o s from 3.48 -33-to 5.34. These arm ratios are within the range assigned to subterminal chromosomes, therefore, these two chromosomes are classified as subtelo-centric in the present study. Secondary constrictions Observations on chromosomal segments separated from the main body of the chromosome, usually by one secondary constriction, are important in karyotype studies. Classifications by Battaglia (1955) have been devised distinguishing micro-, macro-, linear-, and pseudosatellites but references to conifers are less defined, less consistent and more sporadic. Contradictory observations, El-Lakany (1969), Durrie Vabre (1958) , Thomas-Ching (1968), on secondary constrictions of Douglas-^fir could be attributed to the small chromosomes size, the d i f f i c u l t i e s in obtaining a clear separation and definition of chromosomes. Constrictions were observed in the current study where cla r i t y of chromosome definition was considered to be good. Several chromosomes in Gasquet (#3) provenance showed structures very much like secondary constrictions, this area of the chromosome presenting these achromatic regions was narrower than the rest of the chromosome.^ Thesetconstric-tions were located to the distal end of the chromosome arm but the length of the s a t e l l i t e portion, distal to the constriction varied i n length (Fig. 7). - 3 4 --It. i s - i n t e r e s t i n g t o note t h a t i n Gasquet (#3) provenance, where d i f f e r e n t k i n d s of c o n s t r i c t i o n s and s p h e r i c a l p r o j e c t i o n s were o b -s e r v e d , the chromosomes appeared to be s h o r t e r than i n the o ther p r o -venances (#1, #22, #28). The condensat ion of the s t a i n e d segment was seen i n the s h o r t e n i n g of the chromosomes, and the excess o f heterochromat in might have accumulated as a s p h e r i c a l p r o j e c t i o n . T h i s might suggest t h a t the d i s t r i b u t i o n of the hete rochromat in v a r i e s i n chromosomes from d i f f e r -ent provenances . D i f f e r e n c e s between r e p l i c a t e s I n a l l f o u r provenances the s i g n i f i c a n t d i f f e r e n c e s between the r e p l i c a t i o n s i n d i c a t e t h a t the chromosomes may not have been a t the same stage of m i t o t i c metaphase. T h i s may a l s o be the case w i t h provenances #3 and #22 i n which s i g n i f i c a n t d i f f e r e n c e s were observed f o r w i d t h v a l u e s . On the o ther hand, provenance #1 was q u i t e u n i f o r m , not showing any s i g n i f i c a n t d i f f e r e n c e s between the r e p l i c a t e s . T h i s cou ld be c o n -s i d e r e d a r e s u l t of optimum phase s a m p l i n g . The index v a l u e s i n every provenance showed no s i g n i f i c a n t d i f f e r e n c e s between r e p l i c a t e s and t h i s f a c t was a good i n d i c a t i o n of the u n i f o r m i t y of the exper imenta l m a t e r i a l u s e d . No t rend was de tec ted on the bases of arm l e n g t h and t o t a l l e n g t h , when provenances from d i f f e r e n t g e o g r a p h i c a l l o c a t i o n s were compared. However, a d e f i n i t i v e c l i n a l t rend was apparent i n chromosome w i d t h and volume. -35-C l i n a l v a r i a t i o n from south to north i n width and volume i n the  Coastal provenances. The southerly provenance, Covelo #1, had the smallest average chrom-osome width of 3.00 units (0.34 urn.). This value increased to 3.36 units (0.38 yum.) f o r Gasquet (#3) and 3.82 units (0.43 ^ um.) for the most north-e r l y provenance Forks (#22) . (Table 3) A s i m i l a r northerly increasing c l i n a l trend was also noted f or the basic chromosome volume f o r the Coastal region provenances #1, #3, and #22 with 266.24 u n i t s , 275.28 units and 413.53 units r e s p e c t i v e l y . (Table 3) Differences i n chromosomal width and volume between Coastal and  I n t e r i o r provenances. There was also a noticeable d i f f e r e n c e between the I n t e r i o r (#28) and the nearest Coastal (#22) provenances, with regard to the width and volume of the chromosomes. The values were s i g n i f i c a n t l y lower f or the I n t e r i o r than f or the Coastal provenance; e.g. for width 3.10 units (0.35 urn) as compared with 3.82 u n i t s (0.43 pm.) and f o r volume 270.99 units compared with 413.53 u n i t s . (Table 3).and F i g . 8 and 9) These observations coincide with numerous e a r l i e r observations.on the d i f f e r e n c e s between Coastal and I n t e r i o r provenances. The previous studies concentrated mainly on morphological, p h y s i o l o g i c a l andfrecently on nuclear c h a r a c t e r i s t i c s . L i t t l e (1952) mentioned that Murrayy was the f i r s t to notice d i f f e r e n -ces between Coastal and I n t e r i o r provenances of Douglas-fir as early as 1896. Clear morphological d i f f e r e n c e s between Coast and I n t e r i o r provenances -36-F i g . 8. Meristematic r o o t - t i p chromosomes at mi t o t i c metaphase from Forks (#22), Coastal provenance x 8000. F i g . 9. Meristematic r o o t - t i p chromosomes at m i t o t i c metaphase from Spokane (#28), I n t e r i o r provenance x 8000. -38-have been described for seed c h a r a c t e r i s t i c s (Allen 1960, Robinson 1963 and Dunlap 1964), cone scale and bract c h a r a c t e r i s t i c s (Yao 1971) , needle thickness, cone diameter and cone s p e c i f i c g r a v i t y (Tusko 1963). There are also d i f f e r e n c e s between Coastal=and I n t e r i o r provenances i n the r a t e of germination (Allen and Bientjes 1954, A l l e n 1961) . El-Lakany and S z i k l a i (1971) explored the v a r i a t i o n i n nuclear volume and r e l a t i v e DNA content from the protoderm of the embryos of 11 Coast and 10 I n t e r i o r provenances. In t h e i r l a t e r study (1973), another 31 provenances were included. For both c h a r a c t e r i s t i c s d ifferences were obvious f o r Coastal and I n t e r i o r sources. Furthermore a c l i n a l increase of 2.1 from south to north i n nuclear volume and 1.8 i n DNA content was established. Similar differences were reported by Miksche (1968) for white spruce and jack pine. He found the differences between highest and lowest DNA content as 1.6 and 1.5 r e s p e c t i v e l y for the two species studied. There has been no s a t i s f a c t o r y explanation for t h i s phenomemon i n nuclear volume and DNA content. Stebbins (1966) hypothesized that the large chromosomes and the high DNA content i n d i c a t e an increase i n the number of genes but not i n the number of gene-controlled metabolic processes. Miksche (.1968) put forward three explanations; (1) l a t e r a l m u l t i -p l i c i t y of chromosome strands, or polynemy, or possible d i f f e r e n t i a l polynemy; (2) d u p l i c a t i o n or d e l e t i o n processes which, r e s p e c t i v e l y , could cause lengthening or shortening of chromosomes; or (3) s e r i a l -39-r e p e t i t i o n of encoded DNA base sequences by a "master" - 'slave' process. The present study regarding the chromosome complements of Douglas-f i r substantiates the previous findings as f a r as the i n t r a s p e c i f i c var-i a t i o n i s concerned. I t has been found i n the samples used i n t h i s work that the volume of haploid chromosome complement of the Coastal pro-venance Forks (#22) was 1.5 times larger than the corresponding Inter-i o r one Spokane (#28). The northerly increase i n chromosome width of 1.3 and i n volume of 1.6 was c l e a r l y noted (Table 3)and F i g . 8 and 9). Further studies are needed to analyze and to describe the chromo-somal structures on a wide population basis and to evaluate them i n terms of a d a p t a b i l i t y to s p e c i f i c environmental conditions i n Douglas-f i r . These studies should concentrate also on the banding patterns of chromosomes to elucidate the d i s t r i b u t i o n of heterochromatin, and through t h i s understanding provide the basic information needed for further g e n e t i c a l studies. -40-5. SUMMARY A comparative karyotype study of Douglas-fir germinants from four d i f f e r e n t provenances i n C a l i f o r n i a and Washington Coast #1, #3, #22, and I n t e r i o r #28 was conducted using r o o t - t i p meristematic t i s s u e . A somatic chromosome 2n=26 or x=13 was observed i n a l l provenances. The homologous chromosome p a i r s were i d e n t i f i e d using the arm r a t i o , centromere index, r e l a t i v e length and e s p e c i a l l y the morpho-l o g i c a l index. These values also provided the basic information for c l a s s i f y i n g the Douglas-fir chromosomes as f i v e metacentric, s i x sub-metacentric and two subt e l o c e n t r i c . Various structures resembling secondary c o n s t r i c t i o n s and one sp h e r i c a l p r o j e c t i o n were noticed i n Gasquet (#3) provenance. A c l i n a l v a r i a t i o n of chromosome width and volume was noted among the three Coastal provenances with increasing values toward north through the 8°04' l a t i t u d e . Chromosome width and volume of the I n t e r i o r provenance were s i g -n i f i c a n t l y smaller:than the width and volume of the corresponding Coastal one. These r e s u l t s substantiated the previous findings of c l i n a l v a r i a -t i o n i n nuclear volume and DNA content i n Douglas-fir. More studies are needed to be i n i t i a t e d on the broad geographical b a s i s , including samples from the e n t i r e range of t h i s species, to analyze, describe and understand the s i g n i f i c a n c e of t h i s v a r i a t i o n . -41-REFERENCES A l l e n , G.S. 1960 A method of d i s t i n g u i s h i n g Coastal and I n t e r i o r Douglas-fir seed. U.B.C. Faculty of For. Res. Note #28, 3p. 1961 Testing Douglas-fir seed f o r provenance. Proc. Int. Seed Test. Assn. 26 (3): 388-403. and W. B i e n t j e s . 1954 Studies on Coniferous tree seed at the U n i v e r s i t y of B r i t i s h Columbia. For. Chron. 30 (2) : 184^.196. Bajer, A. 1959 Change of length and volume of m i t o t i c chromosomes i n l i v i n g c e l l s . Hereditas 45: 579-596. Barner, H. and Christiansen, H. 1962 The formation of p o l l e n , the p o l l i n -a t i o n mechanism, and the determination of the most favourable time f o r c o n t r o l l e d p o l l i n a t i o n i n Pseudot-suga menziesii. Silvae Genetica 11: 89-102. B a t t a g l i a , E. 1955 Chromosome morphology and terminology. Caryologia 8: 179-187. B e l l i n g , J . 1928 Contraction of chromosomes during maturation d i v i s i o n i n L i l i u m and other p l a n t s . Univ. C a l i f o r n i a , Publ. Botany 14: 335-343. Dunlap, L.H. 1964 D i f f e r e n t i a t i o n of Coastal and I n t e r i o r provenances using morphology of seed. U.B.C. Fac. For. B.S.F. Thesis 41pp. Durrie-Vabre^ 1958 Chromosomes de Pseudotsuga d o u g l a s i i . Carr. CR. Acad. S c i . P a r i s 246: 3660-3663. El-Lakany, M.H'. 1969 Studies on the e f f e c t s of i o n i z i n g r a d i a t i o n on some Western Coniferous species. Ph.D. t h e s i s . The Univer-s i t y of B r i t i s h Columbia, Vancouver. 250pp. El-Lakany, M.H. and 0. S z i k l a i . 1971 I n t r a s p e c i f i c v a r i a t i o n i n nuclear c h a r a c t e r i s t i c s of Douglas-fir. Advancing F r o n t i e r s of Plant Sciences 28: 363-378. El-Lakany, M.H. and 0. S z i k l a i . 1973 Further in v e s t i g a t i o n s of i n t r a -specif i c v a r i a t i o n i n DNA contents of Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco. The Egyptian J . of Gen. and Cyt. V o l . 12 #2, Ju l y 1973. 345-354. - 4 2 -G i a n n e l l i , F . and H o w l e t t , R .M. 1967 The i d e n t i f i c a t i o n df the chromo-somes of the E group (16-18 Denver ) . An a u t o r a d i o -g r a p h i c and measurement s t u d y . C y t o g e n e t i c s 6 : 4 2 0 - 4 2 5 . L a n g l e t , 0 . 1943 Om v a r i a t i o n e n hos t a l l e n (Pinus s y l v e s t r i s L.) och dess samband med k l i m a t e t . Svenska SkogsvFor . T i d s k r . 32 : 8 7 - 1 1 0 . L a r s e n , C . S . 1956 Genet i cs i n S i l v i c u l t u r e . O l i v e r and Boyd, London. Levan , A . , K. Fredga and A . A . Sandberg. , 1964 Nomenclatura f o r the cent romer ic p o s i t i o n on chromosomes. H e r e d i t a s , Lund 5 2 , 201. L i t t l e , E . L . , J r . 1952 The genus Pseudotsuga ( D o u g l a s - f i r ) i n Nor th A m e r i c a . L e a f 1 . West. B o t . A p r i l 1 8 1 - 1 9 6 . L i v i n g s t o n , G .K. 1971 The morphology and behaviour of m e i o t i c chromo-somes of D o u g l a s - f i r . S i l v a e G e n e t i c a 20 ( 3 ) : 7 5 - 8 2 . Manton, I. 1950 The s p i r a l s t r u c t u r e of chromosomes. B i o l . Rev. 2 5 : 4 8 6 - 5 0 8 . M i k s c h e , J . P . 1968 Q u a n t i t a t i v e study o f i n t r a s p e c i f i c v a r i a t i o n of DNA per c e l l i n P i c e a g l a u c a and P inus b a n k s i a n a . Can. J . Genet . C y t o l . 1 0 : 5 9 0 - 6 0 0 . O 'Mara , J . G . 1948 A c e t i c a c i d methods f o r chromosome s t u d i e s a t prophase an and metaphase i n mer is tems. S t a i n Technology 2 3 : 2 0 1 - 2 0 4 . Peace , T . R . 1948 The v a r i a t i o n o f D o u g l a s - f i r i n i t s n a t i v e h a b i t a t (Pseudotsuga t a x i f o l i a B r i t . s y n . Pseudotsuga d o u g l a s i i C a r r . ) F o r e s t r y 22 : 4 5 - 6 1 . Rob inson , B .A . 1963 V a r i a t i o n i n seed c h a r a c t e r i s t i c s of D o u g l a s - f i r i n B r i t i s h Co lumbia . U . B . C . F a c u l t y For B . S . F . Thes i s 68pp. Sax , K. and H . J . Sax 1933 Chromosome number and morphology i n the C o n i f e r s . J o u r n . A r n o l d Arboretum 14 : 3 5 6 - 3 7 5 . S t e b b i n s , G . L . 1966 V a r i a t i o n and e v o l u t i o n i n p l a n t s . Columbia U n i v . P r e s s , N.Y. 643pp. Stephenson, E . M . , E . S . Robinson and N.G. Stephenson. 1972 K a r y o t y p i c V a r i a t i o n w i t h i n the Genus Le iopelma (Amphibea Anura) Can. J . Genet . C y t o l . 14: 691-702. - 4 3 -Svardson , G. 1945 Chromosome s t u d i e s on Salmonidae. Med. f r . S t a t . Unders . o . F o r s o k s a n s t . f . S o t v a t t . 2 3 : 1 - 1 5 1 . S z i k l a i , 0 . 1967 V a r i a t i o n of D o u g l a s - f i r i n i t s n a t u r a l h a b i t a t . Paper p resented a t I n t e r n a t i o n a l S c i e n t i f i c C o n f e r -ence , Z v o l e n , C z e c h o s l o v a k i a , 11pp. Thomas, G. and K . K . C h i n g . 1968 A comparat ive karyotype a n a l y s i s o f Pseudotsuga m e n z i e s i i (Mi rb . ) F r a n c o , and Pseudotsuga  w i l s o n i a n a (Hayata) . S i l v a e G e n e t i c a 1 7 ' ( 4 ) : 138 -143 . Tusko, F . F . 1963 A study of v a r i a b i l i t y i n c e r t a i n D o u g l a s - f i r p o p u l a -t i o n s i n B r i t i s h Co lumbia . U .B .C . Dept . of B i o l , and B o t . P h . D . T h e s i s 173 p p . Yao, C. 1971 Geographic v a r i a t i o n i n seed w e i g h t , some cone s c a l e measurements and seed germinat ion o f D o u g l a s - f i r , Pseudotsuga m e n z i e s i i (Mi rb . ) F r a n c o . M.F . T h e s i s , U . B . C . T h e s i s , U .B .C . F a c . F o r . 88pp. Zenke, U. 1953 t a S t u d i e s on the course of m e i o s i s i n Pseudotsuga  t a x i f o l i a B r i t t o n . Z e i t s c h r i f t f u r F o r s t g e n e t i k und F o r s t p f l a n z e n z u c h t u n g V o l . 2 , #5: 9 6 - 1 0 2 . 

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