CIRCULAR 201 OTBR17 OCTOBER 1972 A Karujoujpie Study of Cypresses Indigenous to the ........................ Southwestern United States hUh AGRICULTURAL EXPERIMENT STATION/AUBURN R. Dennis Rouse, Director UNIVERSITY Auburn, Alobomo CONTENTS Page INTRODUCTION 3 REVIEW OF LITERATURE 4 MATERIALS AND METHODS 5 PRETREATMENT AND STAINING SCHEDULE CHROMOSOME MEASUREMENT AND DETERMINATION OF KARYOTYPE-6 RESULTS ---Chromosome Numbers 7 Chromosome Length 10 12 12 Position of Centromeres Secondary Constrictions DISCUSSION 14 SUMMARY 17 CITED LITERATURE 18 19 APPENDIX FIRST PRINTING 3M, OCTOBER 1972 A Karyotypic Study of Cypresses Indigenous to the Southwestern United States G. E. THOMAS and J. F. GOGGANS 1 INTRODUCTION IN improvement among several species of cypress indigenous to the 1964, Auburn University initiated a program of genetic tree southwestern United States. Its purpose was to develop varieties of cypress suitable for Christmas tree production in the Southeast. Part of this program has involved accumulation of basic cytogenetic information necessary to determine the inherent karyotypic variation within the particular group of species under study. Cypresses native to the United States have not been thoroughly examined karyotypically, and there are several possible reasons. First, the genus Cupressus has not been important commercially. Secondly, the difficulty of seed collection because of widely scattered and isolated natural stands, coupled with low seed viability and resultant poor germination, have undoubtedly been deterrents to investigation. A third reason might be the presence of numerous, long chromosomes, characteristic in general of all conifers, which renders separation, measurement, and identification of individual chromosomes extremely difficult. Finally, the problem of morphological differentiation of species has also precluded more extensive study. This study was undertaken in order to help clarify the karyotypes of Cupressus and to further delineate the natural variation 1 Research Associate and Professor, respectively, Department of Forestry. which occurs within the genus. It presents findings which corroborate the generally accepted haploid number of 11 for Cupressus and describes several variants which show the presence of one or more accessory chromosomes, heretofore rarely observed in woody plants. A total of seven species of Cupressus, including four varieties of C. arizonica and two varieties of C. goveniana, are represented. REVIEW OF LITERATURE Sax and Sax (13) first reported a basic number of n - 11 for the Cupressaceae. Their findings on the genera Thuja, Juniperus, and Chamaecyparis indicated that the chromosomes of all three were similar in morphology and more or less isobrachial (metacentric). Several studies since then have corroborated the basic number of n - 11 for the Cupressaceae and shed additional light on the karyotypes of Cupressus. In 1936, Numata and Yamashita (cited by Kanezawa (5)) reported 2n - 22 for Cupressus lusitanica var. benthamii. Camara and DeJesus (2) observed meiosis in C. lusitanica and reported a number of irregularities which included formation of univalents, multivalents, translocation rings, anaphase bridges, and fragments. They reported a basic number of n -- 11 chromosomes. Mehra and Khoshoo (8) found 11 chromosomes in gametophytic tissue of C. funebris and C. torulosa and 22 chromosomes in root tips of C. sempervirens. For the first two species, they reported a karyotype of 1 heterobrachial chromosome and 10 approximately isobrachial chromosomes. In addition, a satellite, which was "somewhat thicker" in C. funebris, was found in one of the median-submedian chromosomes of both species. For the diploid set of C. sempervirens, Mehra and Khoshoo observed two heterobrachial chromosomes, each of which carried a secondary constriction in its long arm. Finally, they observed meiosis in pollen mother cells of C. cashmeriana, C. lusitanica, and C. arizonica and reported a normal meiosis with formation of 11 bivalents in each of these species. The most extensive study of the karyotypes of Cupressus was reported by Hunzicker (4) who worked with root tips. He reported karyotypes for seven species which included, C. arizonica, C. funebris, C. glabra, C. lusitanica, C. macrocarpa, C. sempervirens, and C. torulosa. All of these showed a basic number of 2n - 22 except for C. glabra, which had an extra, small accessory chromosome that gave it a number of 2n =23. In addition, all species had median-submedian centromeres with the exceptions [4] of C. lusitanica var. lusitanica and C. torulosa, each of which had two heterobrachial chromosomes. Finally, he observed three types of secondary constrictions. The first of these divided one of the chromosome arms so that a linear satellite was formed (distal section of the arm longer than the proximal) and the second so that a rounded satellite was formed (distal section shorter than the proximal section). The third type was described by Hunzicker as an "anucleolar secondary constriction," rarely seen, and possibly an artifact due to variations in the cell's environment. All of the species showed the first type of constriction while only C. lusitanica var. lusitanica and C. lusitanica var. ben- thaniji had the second type. MATERIALS AND METHODS Seeds used in the present study were collected in the southwestern and western United States during the summer of 1964 and included the following species and varieties: Population and species 1.C. arizonica Greene var. arizonica 2. C. arizonica Greene var. arizonica_ Location -- hihuahua, C Arizona Big Bend National Park, Texas Mexico 3. C. arizonica Greene var. arizonica_ 4. C. arizonica Greene var. arizonica 5. C. arizonica Greene var. arizonica. 6. C. arizonica Greene var. arizomica_ 7. C. 8. C. 9. C. 10. C. _Chiricahua Naional Monument, Greenlee County, Arizona Cochise Stronghold, Arizona Portal, Arizona, Cave Creek, Cochise County orizonica Greene var. arizonica____ -Pima County, Arizona, Bear Canyon arizonica var. glabra Sudw., Little ____Oak Creek Canyon, Arizona Gila County, Arizona var. glabra Sudw., arizonica _Guatay Mountain, San Diego County, guadalupentsis S. Wats.---------- Little_ California 11. C. arizonica var. stephensonii Wolf, Little-------------------12. C. sargentii Jepson, Little 13. C. macrocarpaHartw. 14. C. goveniana Cord. var. goveniana 15. C. goveniana Cord. var. pygmaea L em m on -------------------- -16. C. bakeri Jepson ---------------.__Cuyamaca Peak, San Diego County, California Cypress Creek, Monterey, California ___Monterey County, California _.Santa Cruz County, California -- 1 .C ba eiJp 18.A.---------------C. macnabiana Murr..--------_ o 19. C. arizonica var. nevadensis Abrams, Little ---------20. C. arizonica Greene var. arizonica-- _Mendocino County, California __Siskiyou County, California --Shasta County, California __Amador County, California --Kern County, California --Graham County, Arizona Each of the above populations has been fully described by Posey and Goggans (10). In the identification of species, the taxonomic treatment given by Little (6) has been used. [5] PRETREATMENT AND STAINING SCHEDULE Only somatic tissue obtained from root tip meristems was used in karyotype determination. It was originally intended to examine karyotypes from at least two seedlings from each of three parent trees from every 1 of the 20 populations, thus giving a minimum of six observations per population with an overall total of 120 observations. However, in a few instances poor germination coupled with inability to obtain a plate suitable for measurement resulted in fewer observations than the minimum of six per population. In other instances it was possible to obtain more than the minimum number, so the final results were based on a total of 172 measurements. Seeds from each of the populations were germinated either in petri dishes filled with moistened vermiculite and placed in a growth chamber or in trays filled with a sand/soil mixture and placed in a greenhouse. Root tips from newly germinated seedlings were excised when they reached about 5 mm. in length and placed in a solution of 8-hydroxyquinoline (0.3 g./liter) for 24 hours at 10°C to contract the chromosomes and to arrest cell division at metaphase (11). They were then fixed in Farmer's fixative (3 parts absolute alcohol: 1 part glacial acetic acid) for 24 hours, hydrolyzed in either IN HCL at 60°C or in 1 part absolute alcohol: 1 part concentrated HCL at room temperature for 10 minutes, and stained in Schiff's reagent (Feulgen) for 1-2 hours. After each root tip had been stained, it was put into 45 per cent acetic acid for 10 minutes, transferred to a microscope slide where it was immersed in a drop of acetocarmine, and squashed. CHROMOSOME MEASUREMENT AND DETERMINATION OF KARYOTYPE Microscopic slides were examined for cells which had mitotic chromosomes well spread out and, as nearly as possible, in one plane. At 1,125X magnification, such cells were examined and photographed using 35 mm. black and white film. The negatives were then mounted in slide holders and projected onto white paper so that the largest chromosomes measured approximately 6 centimeters. Each enlarged image was closely compared with the original cell viewed through the microscope so that each chromosome on the image could be outlined to show such details as the position of the centromeres, location of secondary constrictions, and location of ends that might be hidden through overlapping. [61] Chromosomes of a given cell were then arbitrarily numbered from 1-22, and individual arms were measured from their ends. Centromere regions were not included in the measurements, and when a chromosome arm was curved it was measured along a series of straight lines tangent to the arc described by the curve. The longer of the two arms of a given chromosome was designated the "a" arm; the shorter, the "b" arm (11). Presence and position of secondary constrictions were recorded whenever they occurred, and these were designated types 1, 2, and 3 in accordance with the descriptions offered by Hunzicker (4). Types 1 and 2 corresponded respectively to the linear and rounded constrictions found by him, while type 3 fitted his description of a diffuse anucleolar constriction, rarely seen and possibly an artifact. After all of the chromosomes of a cell had been measured, individual lengths were converted to relative values to permit comparisons between cells. This was done by computing average chromosome length for the cell and relating absolute lengths to this average (16): Relative length = Absolute length Average length X 100 Chromosomes were then arranged in descending order of total relative length, and homologous pairs were chosen by careful reexamination of the original microscope slide and matching of similar arm lengths. In determining pairs the shorter arm was considered to be of greater diagnostic value since it was less susceptible to stretching (11). Once paired, the lengths of matched arms were averaged in order to arrive at the haploid karyotype of each cell. Population mean relative lengths of each chromosome in the karyotype were calculated. The standard errors at the 5 per cent level were calculated for the population means of the longest and shortest chromosomes. RESULTS Chromosome Numbers Most karyotypes examined from each population possessed a diploid complement of 22 chromosomes with median to submedian centromeres (short/long arm ratio 0.5-1.0) which confirms results of previous cypress chromosome investigations. However, there were five populations, 4, 8, 10, 19, and 20, that exhibited some tendency toward abnormality in chromosome numbers. Within [7] each of these, individuals were found which possessed an extra chromosome (2n - 23), and in addition, in the samples from populations 8 and 20, one individual in each had 2n - 24 chromosomes. The aneuploid individual in the sample from population 10 possessed an extra chromosome which did not differ greatly in total relative length from the smallest member of the diploid set and could possibly be a trisomic. Table 1 presents the haploid karyotype of this abnormal seedling. The extra chromosome was identified through the pairing of similar arm lengths and could not be recognized by microscopic observation since it had no features to readily distinguish it from other chromosomes of similar length. TABLE 1. HAPLOID KARYOTYPE OF AN ANEUPLOID INDIVIDUAL 23) FROM POPULATION 10, PARENT 1. (2n - Chromosome Total relative length a Relative length* b 60 70 130 1 -------------------------------------51 61 112 2 --------------------------------------5 0' 59 1 09 3 --------------------------------------49 58 107 4------------------------------------48 57 105 5--------------------------------45 52 97 6--------------------------------------38 57 95 7--. 42 52 94 8----------------------------. 41 51 92 9-------------------------------. 36 51 87 1 0-------------------------------------32 49 81 11 -------------------------------------35 39 74 12 ----------------------------* Secondary constrictions of types 1 and 2 are indicated by small numbers adjacent to the arms in which they occur. ** Extra chromosome that is possibly the result of a simple duplication of an entire chromosome (a trisomic). 2 In all other cases of abnormality in chromosome numbers the extra individual(s) could be identified at a glance. They were approximately half the size of the smallest chromosome in the diploid set and had median to submedian centromeres. Figures 1 and 2 show these extra chromosomes while Appendix Table 1 gives the karyotypes of all seedlings that possessed them and Table 2 presents the average karyotype of these seedlings. Chromosomes observed in several cells were in the process of duplication, and in these instances the accessory chromosome(s) replicated in a regular fashion and apparently had fully functional centromeres. Figure 3 illustrates this by showing normal replication of an accessory chromosome. The remaining chromosome complement of these abnormal cells did not differ greatly from that of individuals with [8] ,t b. R 4 4 -0 44%0 A I"' Xea FIG. 1 Chromosomes of Cupressus arizonica var. arizonica from a seedling of 24). Arrows point to the extra chromosomes. population 20, parent 10; (2n r "9 t ci 4 l E ; 4, S H [91 LI FIG. 2. Chromosomes of Cupressus arizonica var. nevadensis from seedling of 2 23). Arrow points to the extra chromosome. population 19, parent 4; ( n (dip)loidl ttttttlcrs of 22 except lolt a etleoex of the tx o lott~est that tmiori t. For two tc itutllibeFs to Lax c a larger relati e letl chllmlONttl part'it trees, ino scedltis x eve to tll to Lax e torntl tttttlnitcs. fliese \x ti r 1)ttett 2, [)[ttlatiott 19 mtidl partnt .5 pop\\ 0111( utlationi 20. 1 Loxx ex t it Itiax Itc that I irtlier txaittitiiotn 22 since otlx txx o of the Needtex tal sotle itftli idlItals xxitli 2t hug(s obtaitned froit eac] pJaretnt wxte eamiinedl. k: ntS( 111 \nizosio\In~ (ii 23 anii~i2 l li 24) iix ('Iii ujili I n , inu '1o h ( ;I ti constrictions of tvpcs I. ?, and 3 arc indicated I,\ Secondal adiaccnt to the arm, in \01ich the\ appeared most fie lnentlc. "m all ,ores n (.111 iOoIIIc(,). oil smaill innniii ii FIG. 3. Photomicrograph that shows a supernumerary chromosome of a seedlinig from population 19, parent 4 which has duplicated in a regular manner. Arrow points to teextra chromosome. Chromosome Length lar c andi thelr( are peculliar fealturles abou~lt lilt chromollsomelis [101 cypress, neither one of these criteria was met except for one or two chromosomes in each set which carried a secondary constriction which can be used as a distinguishing marker. Differential contraction, small differences in pretreatment period, bending of chromosome arms, and inability in given instances to obtain plates with the chromosomes all in one focal plane further complicate the situation. However, from the data gained in the present study it was possible to obtain a reasonably reliable estimate of the basic karyotype of each population based on the average relative lengths of the chromosomes from the various progenies. Haploid karyotypes of each population and standard errors of the population means of the longest and shortest chromosomes are presented in Appendix Table 2. No important differences in relative chromosome lengths between populations could be detected. Karyotypes were all very similar, therefore they were combined and the average karyotype for the Cupressus populations studied is presented in Table 3. In examining 152 cells from seedlings which had karyotypes of 2n - 22, eight cells had longest chromosomes that were abnormal. In each case they were approximately 15 units of relative length greater than the average lengths of the longest chromosomes of the populations to which the seedlings belonged. These instances were thought to be a result of differential contraction or stretching of the chromosomes in question and not to be reflections of differences in karyotype between parents or between populations. This conclusion seems valid since measurements of additional cells from the same seedlings revealed karyotypes more near the average. TABLE 3. AVERAGE HAPLOID KARYOTYPE OF Cupress'us POPULATIONS STUDIED.* Chromosome 1 2 3 4 5 6 7 8 9 -- - - - - - - - - - - - - - - - - - - -. 10 11 --- Total relative length 130 116 -110 Short/long arm ratio .91 .90 .88 .86 .83 .82 .81 .82 .8 0 .76 .76 106 102 98 -95 -92 88 84 79 Refer to Appendix Table No. 2 for relative lengths of arms and locations of constrictions. [11] Position of Centromeres All chromosomes of cypress examined had their centromeres in median to submedian positions as shown by the short/long arm ratios in Table 3. Differences in the position of the centromere between numerically equal chromosomes of the various populations were not large and probably reflected errors in measurement rather than actual morphological variation. Secondary Constrictions Three types of secondary constrictions were identified, two of which occurred frequently enough to be taken as regular features of the karyotypes in which they occurred. The first type of constriction (type 1) occurred in the progeny of each of the various parents, regardless of population, and was most probably a nucleolar organizer region in function. It appeared as a distinct narrow band located approximately 16 relative units from the centromere, Figure 4, or occasionally as a long, tenuous strand (possibly a result of stretching during preparation of the tissue) which terminated in a linear satellite that was approximately 30 relative units in length. Most of the time the constriction appeared to be located in the short arm of the chromosome. Because of the phenomenon of chromosome reversals as described by Simak (14), in which chromosomes of nearly equal length can be misoriented in a karyotype description because of differential contraction and bending, it was not possible to accurately assign the constriction to any one chromosome of the haploid set. Similarly, because of reversals of order in chromosome arms of nearly equal length, it occasionally occurred in the long arm. In Figure 5 and Appendix Table 2 the type 1 constriction is shown in the karyotype position where it occurred most frequently. The second type of constriction (type 2) had the appearance of being the opposite of the first type and could be the result of an inversion if the possibility of misorientation because of arm reversals is considered. It was generally a distinct narrow band located approximately 30 relative units from the centromere. It occurred about half of the time in the long arm and the other half of the time in the short arm. Distal to the constriction, a small, rounded satellite nearly 11 relative units in length was formed, Figure 6. The position of type 2 in the idiogram of Figure 5 and in Appendix Table 2 was handled in the same manner as for type 1. Type 2 did not occur as frequently as did type 1 and was not [12] 0 FIG. 4. band. Photomicrograph that shows type 1 constriction as a distinct narrow 3M FIG. 5. Haploid idiogram of population 1 [ 1:,31 at presen(It at al~l illsmpe 'HI f)(ronil popI)I atiotis 1.3 m( 15 XX hich nIIH bec ilidicatiX c of a karyotx pic (lifirei beH( pophilat(is. III )tweend moost cases it ocdcl-rce in dct tjutction wXith t\ pcd 1, andt~ rlatiX (I fc.v (I iffcrditds it potlto the basis of thle t\ pes of secotilti cm(istrictioo s wXhichi tileX carried. In samtples froti popt latiotis 14 mtid 19, x pe 2 cons1trictiois XX(t 5 foltidl almi ost excitisiX (IX ill the Iotiest chrintith(Itii XX11kb is Icss sttsce1)tiIhlc to th. Iii these tXXo idlentificationi (rrlls because of its (4t(cater Icti case(, the 1 )ositioni 1IetXX cell sd(llt 4 coitld 1)c discrnd l of thic \ ittiotis p)are I Its vv itIIin of the tv pc 2 c011stricttins lpossibbl rclected actual popu)f t i on di If crcices. Tllc third t\ pc of cot striction ( t\ pc :3) as ia difl sc 1hand occasiotlix0 on i jot is mi mbilees of the hlaploid set isi alix (h11-isoois tes or 8 aid il t it lia S (I bee anI artifact 1 ichdoccltnr( c(HI 1) ft IX ceal lid t (01itriictit, ii It 4 6c 0 100 FIG. 6. Photomicrographs of type 2 and type 3 constrictions. DISCUSSION 0stmall aidc55)-N (1(0)11115011 is ilt 4 Fl te presetnce of 1)n e or tXo AI the 2t) poputlationts of c\ press5 examlined~ is untitc itcteestint ini fact thlat 1B-dhrf(Ittos()tcs htaX c ildet tareix ol)5I9V (d tcX\ of thll ill oo(1\ species. Nlhlra and~ imX at 7 olbseredl 13cliontosottic> XX for the first tithie iniXXoo(1\ anios( etuts. XX liilc Savior and Sit 1 doastal led(md1(( ( Sequoia sc iliy lIi ((ils oblsenX cd thetii ill 112) [1I1t1 rens), a hexaploid species. The only other case of their appearance in conifers that is known to these authors is in Cupressus glabra as reported by Hunzicker (4). Thus the present study confirmed Hunzicker's findings and indicated that B-chromosomes may occur in cypress more extensively and in larger numbers than previously reported. The accessory individuals reported in this paper were classified as B-chromosomes even though meiosis had not been observed because they fit the generally accepted definition of supernumeraries very well. Darlington (3) describes supernumerary chromosomes as having certain unique properties. They are generally small, vary in numbers among different individuals, occur in both odd and even numbers, tend toward neutrality in that they exhibit no apparent effect on the external morphology of the plant, and do not pair with members of the normal complement, or A-set, at meiosis. Furthermore, they are variable in regard to heterochromatin content and range from being completely devoid of it to being nearly completely heterochromatic. Our observations indicated that B-chromosomes of cypress may be largely devoid of heterochromatin since examination of interphase nuclei of individuals with extra chromosomes revealed no heteropycnosis. It remains to be seen whether their presence in any way alters the phenotypic expression of the plant or affects its fertility. Various seedlings of known female parents from population 20 were planted in 1966 and 1967 as part of progeny studies. There appeared to be wide variation within the population sample in a number of traits, including form, color, and branch angle. Whether or not this variation was correlated with the presence of B-chromosomes is not known. That supernumeraries are not necessarily genetically inert has been documented by a number of studies, and Darlington (3) states that "- they produce small, less specific effects and are the active basis of quantitative variation." The matter of origin of supernumeraries in cypress is one for speculation until further studies, especially of meiosis, can be conducted. It is known, however, that B-chromosomes may be derived from the normal chromosome complement of a plant in at least three ways (1,3, and 15). First, they may arise through irregular meiotic pairing involving translocations and non-disjunction. Secondly, they could originate by fragmentation across the [15] centric region. The third possibility would involve crossing-over in inversion-heterozygotes coupled with inclusion of a centric fragment in a germ line which already has normal chromosomes of the same kind. According to Darlington (3), the second mechanism, that of fragmentation across the centric region, would be recognizable by examination of root tip mitoses wherein varying numbers of supernumeraries would be seen. This phenomenon would be observed since fragmented centromeres are usually incapable of regular movement. In cypress, no evidence of varying numbers of B-chromosomes between root tip cells was found, so it is unlikely that the extra chromosomes originated in this fashion. Also, all of the B-chromosomes observed in this study had mediansubmedian centromeres which were fully functional, an impossibility if their origin was by centric fragmentation. The first and third possible origins then remain as more likely candidates, and there is some direct and indirect evidence that one or both phenomena may be functioning in some species of cypress. The direct evidence is that presented by Camara and DeJesus (2) for C. lusitanica in which they reported irregularities in meiosis that included formation of univalents, multivalents, translocation rings, anaphase bridges, and fragments. The indirect evidence concerns the findings of the present authors with regard to the two kinds of secondary constrictions, types 1 and 2. Measurements of relative length of the chromosome arms having these constrictions indicated that one could be the inversion of the other. In a number of instances, only one member of each type could be found in a given cell. For pairing purposes it was assumed that the constriction of the missing member merely could not be seen and that the two types of constrictions were located on non-homologous chromosomes. There were a few cells which seemed to uphold this contention by showing two chromosomes with one type of constriction and one with the other, Figure 7. Out of 172 cells examined, only 4 or 5 expressed this condition, but the possibility of an inversion involving a satellited chromosome should not be discarded without further investigation. Furthermore, samples from two populations, 13 and 15, showed no sign of the type 2 constriction, and in all seedlings of these samples, both homologues of the type 1 constriction were clearly visible. Taking into account the limitations imposed by technique on a study such as this, the measurements of relative length revealed [16] FIG. 7. Photomicrogroph which shows that type 1 and type 2 constrictions apparently occur on nan-homologous chromosomes. It( u t sa i t ( d. e if nes rei i t le t I -\ot Iciisi wtI io\)tu h it r iii> id-ct 1nd Iaitri i( ;tin po ui ploultions citied at t(hid liIc cii s on~\ a Tihis desi not menuth liiii i snai thrciniie 1 st 4 (st l sial and~tl id nu tcI spet sc irt nic5 I'rhriissciit IfourO samptso sdifelr tiep euf ')s'inltsp of oifi dil coi;nsricioh~in4 preseii or ( t absienc tsI( '? costrie ictin ( c hs \il ithose(I froliI I "1(m c ;tnd 9 thei preencealmt k iltelw~ sented. No important differences in relative chromosome lengths could be detected between any of the species or varieties, and all chromosomes examined had median-submedian centromeres. Nearly all species and varieties of cypress investigated showed the presence of both a linear and a rounded satellite in two apparently non-homologous members of the haploid set. However, C. macrocarpa and C. goveniana var. pygmaea differed in that the presence of the rounded satellite could not be detected. Finally, certain individuals of C. arizonica var. arizonica, C. arizonica var. glabra, C. arizonica var. nevadensis, and C. guadalupensis had one or two accessory chromosomes which in all but the case of C. guadalupensis appeared to be B-chromosomes. LITERATURE CITED (1) BURNHAM, CHARLES R. 1962. Discussions in Cytogenetics. Burgess Publishing Company, Minnesota. 375 pp. (2) CAMARA, A. AND ALCINDA DEJESUS. 1946. Ur estudo citologico de Cupressus lusitanica Miller. Agronom. Lus. 8(2):95-122. (3) DARLINGTON, C. D. 1956. Chromosome Botany. Allen and Unwin, London. (4) HUNZICKER, JUAN H. 1961. Estudios cromosomicos en Cupressus y Libocedrus (Cupressaceae). Revista de Investigaciones Agricolas 15(2) :169-184. (5) KANEZAWA, R. 1949. A list of chromosome numbers in woody plants I. Gymnosperms and II. Monocotyledons. La Kromosomo, 5-6:249260. (6) LITTLE, E. L., JR. 1953. Checklist of Native and Naturalized Trees of the United States. USDA Agr. Handbook No. 41. 472 pp. (7) MEHRA, P. N. AND K. S. BAWA. 1968. B-chromosomes in some Himalayan hardwoods. Chromosoma 25:90-95. (8) MEHRA, P. N. AND T. N. KHOSHoo. 1956. Cytology of Conifers I and II. J. of Genetics. London and N.Y. 54(1):165-180. (9) PEDERICK, L. A. 1967. The structure and identification of the chromosomes of Pinus radiata. Silvae Genetica 16:69-77. (10) PosEY, C. E. AND J. F. GOGGANS. 1967. Observations on species of cypress indigenous to the United States. Auburn Univ. (Ala.) Agr. Exp. Sta. Cir. 153:20 pp. (11) SAYLOR, L. C. 1961. A karyotypic analysis of selected species of Pinus. Silvae Genetica 10:77-83. (12) SAYLOR, L. C. AND HOLLY A. SIMONS. 1970. Karyology of Sequoia sempervirens: Karyotype and accessory chromosomes. Cytologia 35(2):294-303. (13) SAx, K. AND H. J. SAX. 1933. Chromosome numbers and morphology in conifers. J. Arnold Arboretum 14:356-375. (14) SIMAK, J. 1962. Karyotype analysis of Larix decidua Mill. from different provenances. Medd. Skogsforskn. Inst. Stockh. 51:1-21. [18] (15) (16) SWANSON, CARL P. 1957. Cytology and Cytogenetics. Prentice-Hall, Inc., New Jersey. 596 pp. THOMAS, C. E. AND K. K. CHING. 1968. A comparative karyotype analysis of Pseudotsuga menziesii Mirb., Franco, and Pseudotsuga wilsoniana Hayata. Silvae Genetica 17:138-143. APPENDIX APPENDIX TABLE 1. HAPLOID KARYOTYPES OF INDIVIDUALS 0 EXTRA CHROMOSOMES WITH Population 4, Parent 11-based on two observations. (2n = 23) Chromo- relativebrelative Total Relative length b a length some 127 2-----------120 1---------66 62 592 Population 8, Parent 15-based on one observation. (2n = Chromosome 1 23) Total ength Relative length 61 58 54 2 3 141 113 74 57 67 56 3-----------113 4------------ 61 111 5-____ 107 57 6-__----_-_ 100 57 7------------ 52 96 8 ---------94 51 9-----------91 52 10 ----------- 49 84 11 ----------83 47 12" ------ 43 28 501 50 43 44 43 39 35 36 15 4 5.____ 6 .________101 7 100 8 94 9 90 10 90 11 83 12 28 110 108 105 58 56 63 53 511 42 59 55 47 42 45 47 49 50 49 41 40 35 15 13 Population 19, Parent 4-based on four observations. (2n = 23) Population 19, Parent 2-based on four observations. (2n= 23) 6 Chromo- Total soe relative length 1-----2-----3-----129 117 114 Belative length a bsoe a bsoe 68 61 62 61 56 52 Chromo1-----2-----3------ Total relativeb length 130 120 113 Relative length a b 67 642 56 541 4-----5______ 108 105 56 59 59 521 46 4 -----5------ 111 104 57 57 54 47 6 .___-_ 7------ 102 99 96 92 89 82 53 53 56 51 50 47 49 46 40' 41 39 35 10 ------ 8.__-_9------ 6 _-____ 7-----8-----9-._____ 101 99 95 91 5~4 57 51 50 50 47 47 42 443 11------ 12______ 10 ----- 87 11 -----84 41 37 37 36 19 17 12 -----32 [19] 17 15 (Gont.) APPENDIX TABLE 1. (Cont.) Population 20, Parent 5-based on four observations. (2n = 23) Population 8, Parent 15-based on one observation. (2n = 24) Chromo- Total soerelative soe length 1_ 23456789. 10. 11_ 12_ 139 116 112 109 105 102 97 94 88 87 82 37 ChromoRelative length a bsoe-relativea bsm 73 61 61 56 56 53 50 54 49 49 49 24 66 551 51 53 49 49 47 40 39 38 33 13 Total length 133 128 114 112 110 103 101 97 91 87 86 34 Relative length a b 68 64 60 59 62 59 60 50 52 51 47 18 65 641 3 4 5_ 6 7- 8 1011 12 54 53 48 44 41 47 39 36 39 16 Population 20, Parent one observation. (2n 8-based on 24) = Population 20, Parent 10-based on one observation. (2n - 24) Total Chromo- relative some length 2 -140 133 119 114 110 100 99 96 90 86 77 Relative length a b 74 70 63 64 57 59 52 50 51 57 41 66 73 56 Chromo - Total soe relative soe length 44 1 2 3. Relative length a b 140 72 68 117 61 56 541 113 59 501 112 59 53 5-53 5 108 51 57 41 44 105 61 47di 7103 55 48 8. 46 97 8. 53 44 939 989 55 34 29., 10_ 87 48 39 36 1187 52 35 38 24 14 12_ 41 12-24 17 4 Secondary constrictions of types 1, 2, and 3, are indicated by superior numbers adjacent to the arms in which they occur. ° ° Small accessory chromosome(s). [2,0] APPENDIX TABLE 2. Population 1 HAPLOID KARYOTYPES OF POPULATIONS 1-20' Population 2 long ar .93 .90 .93 .84 .87 .78 .79 .77 .82 .75 .74 TotalShort! Chro-Toa Relative length b mosome relativea h a length 1---2-34-- TotalShort! Chro-Toa Relative length mooerelativeb lengtharatio long ar ar ratio .94 .90 1__________ 131 2 __________ 114 3__________ 110 4 ._________ 105 5.________ 101 6_________ 98 7__________ 95 68 60 572 57 54 55 53 63 54 53 48' 47 433 126 114 110 105 65 60 57 57 572 61 54 57' 48 1.00 .84 8_________ 94 9 89 10 _____ 84 11 _____ 80 sxlongest* 53 49 48 46 * 42 41 40 36 34 =1.4410 5----6----7----8----910 11- - 102 99 96 93 90 85 81 sX longest 54 53 52 50 48 47 - 45 45 48 41 40 37 34 0.8498 .79 .83 .83 .79 .80 .77 .72 s~ shortest =0.7894 Population 3 Chro-Toa Relative length mooerelative a b osmelength 1----2----TotalShort! S. long arm ratio .89 .89 .91 .88 .82 .77 .86 .79 .69 .77 .75 Chro-Toarltv shortest =0.8461 Population 4 RShort! Total a 70 61 59 mosome rltv length 1____ 2 _____ 3 _____ Relative length b 63 55 511 long 132 115 3----4----5_----- 109 107 102 6 ----7 ----8 -----9 -----10----- 11! 99 95 93 89 83 77 ---- 70 61 57 57 56 56 51 52 53 47 44 62 54 522 133 116 110 arm ratio .90 .90 .86 50 461 43 44 413 36 36 33 85 10 ----- 5 4 1__________ 552 55 5___-__ 100 97 52 6______ 54 95 7 50 8.____ 93 50 89 9 49 5 45 45 41 43 39 36 .91 .82 .87 .76 .86 .78 .73 11 _____ s- longest =4.0797 s. shortest - 3.5862 43 33 76 s longest =1.8864 sx shortest =2.2047 Population 6 .77 Population 5 mosome length Total Chro- relative Relative length a b 63 69 132 116 60 56 111 59 522 57 49' 106 47 101 54 44 54 98 42 53 95 42 50 92 40' 49 89 35 44 84 78 44 34 s; ongest =1.1546 sx shortest - 0.9043 TotalShort/ long arm ratio Chro-Toa Relative length mosome relative b a length 72 59 131 59 59 118 46 60 106 46 58 104 47' 54 101 52 46 98 53 45 798 92 49 43 38 88 50 8910. 86 36 50 11_ 45 37 82 sX longest =0.3500 sX shortest - 2.5000 1 2 34 5[21] 1oalShort! long arm ratio .82 1.00 .77 .79 .87 .88 .85 .88 .76 .72 .82 1. 2. 3. 4. 5_ 6 78 9_ 10 11- .91 .93 .88 .86 .87 .81 .79 .84 .82 .71 .77 (Cont.) APPENDIX TABLE 2.(Cont.) Population 7 TotalShort! Chro-Toa Relative length mooerelative b a mooelength 1__________ 130 2 _________ 118 3._________ 110 4 _________ 105 Population 8 long arm ratio .88 .90 .90 .88 .89 .84 .80 Chro- TtlRelative leng hlort! mosome relative gth lon length a b rarmo 69 62 58 56 61 56 52 492 5._________ 102 54 6 --------- 57 98 7 ._________ 94 51 48' 41 1 2 345- .72 .80 .75 .70 6_ 78 910 11 433 8.--------- 51 92 41 9 --------- 49 88 39 10--------84 48 36 11 --------80 47 33 s- longest=2.9711 sYshortest 1.5275 Population 9 TotalShort! Chro- Total Relative length b mosome relative length a b I _________ 129 2 ._________ 116 3 127 115 111 105 101 98 96 92 89 87 80 s, longest 67 61 59 56 55 55 53 53 50 47 46 =2.0403 60 54 522 49' 46 43 43 393 .90 .89 .88 .88 .84 .78 .81 .74 39 40 34 0.7046 .78 .85 .74 s; shortest Population 10 long ar arm .87 .87 .80 ChroRelative length mooerelative b a mooelength 2 3 45_ 678910_ 11. TotalShort! long ratio .90 arm .84 .80 4.____ 5.____ 6.____ 7.____ 8----- _____ 69 62 61 60 54 492 1. 127 67 60 116 110 63 61 53 492 110 107 102 99 95 92 57 56 54 52 49 50' 46 45 43 42 34 433 .88 .82 .83 .83 .88 9.____ 10_____ 11----- 88 86 78 46 52 44 34 .91 .65 .77 sr longest =4.0987 sZ shortest =2.5905 Population 11 TotalShort! Chro-Toa Relative length long mosome relative arm b a length ratio 1 132 67 65 .97 2. 119 63 56 .89 3. 108 58 502 .86 56 49 .88 4. 105 5 101 55 46' .84 6. .87 99 53 46 7 94 53 41 .77 8.9 93 51 423 .82 85 49 36 .73 10. 84 46 38 .83 11 80 44 36 .82 s. 106 56 501 103 56 47 55 43 98 96 54 42 9251 41 38 88 50 85 48 37 48 34 82 sx longest =2.0428 sT Zshortest =0.8818 Population 12 Relative len b 68 63 59 58 56 .89 .84 .78 .78 .80 .76 .77 .70 moso mooerelative lengtharm a Chro-T 1. 2_ 3-4 5- 130 117 109 106 103 62 54 502 48 471 hlort ln ratio .91 .86 .85 .83 .84 6_ 78_ 10_ s; shortest longest =4.2064 =1.8393 [22] 54 45 99 433 96 53 92 52 40 41 90 49 83 47 36 31 75 44 sX longest =1.3627 sX shortest =1.7573 .83 .81 .77 .84 .77 .70 (Cont.) APPENDIX TABLE 2. (Cont.) Population 13 TotalShort! Chro- Total Relative length mosome relative length a b 1__________ 132 2.________ 113 3 _________ 111 4._________ 105 Population 14 long arm ratio .94 .85 .91 .88 Chro -Toa relative Relative length mosome length a h 1 2._ 3. 128 116 109 105 102 TotalShort! long ratio arm .88 .90 .85 .84 .79 68 61 58 56 64 52 53 491 5_________ 102 6 --------98 7__________ 95 8 _________ 93 55 56 51 51 49 49 .42 47 44 423 39 36 .75 .86 .82 .80 .73 .85 4. 78 9 68 61 59 57 57 602 55 50 48' 45 5- 99 96 92 55 53 48 49 45 52 44 43 40 .80 .81 .83 .73 .78 .80 10-----11 _____ 9----- 88 85 10 80 44 36 sXlongest =1.7872 s Ishortest =0.7222 Population 15 .82 11_ 88 85 80 sYlongest 40 36 35 =1.8846 s2 shortest =0.8459 Population 16 Chro - Total Relative length mooerelative mooelength a h 1 2_ 345 129 116 109 106 67 63 58 56 62 53 51 50 lnlongt arm ratio .93 .84 .88 .89 .82 .78 .78 .84 .76 .71 .72 TotalShort! Chro-Toa Relative length mosome relative length a h 1 2. 3 4 6_ 7-89 10. 11 _ 130 117 69 61 61 56 long arm ratio .88 .92 110 108 103 98 58 59 56 54 522 49 47' 44 .90 .83 .84 .81 7 10_ 11 461 102 56 55 43 98 96 54 42 423 50 92 88 50 38 84 49 35 79 46 33 sAlongest=1.2384 s1 shortest=1.3079 Population 17 96 93 51 50 45 43 .88 .86 90 84 80 49 47 44 41 37 36 2.4757 .84 .79 .82 s- longest s-. shortest =0.5947 Population 18 TotalShort! Relative length Chromooerelativeb long ar .93 .90 .89 .84 1 2 --133 116 108 105 101 97 95 length aratio --- TotalShort! Chro -Toa Relative length mooerelative h a mooelength long arm ratio .91 69 61 57 57 54 54 52 64 55 51 48' 1 2 3. 126 47 47 43 .87 .87 .83 4 5 116 110 106 101 98 66 62 57 57 56 52 60 54 53 49 45' 46 .87 .93 .86 .80 .88 7_ 10 _ 91 90 84 80 49 52 47 44 42 38 37 36 .86 .73 .79 .82 10_ 11_ 7 8. 9. 96 93 53 52 43 41 .81 .79 .82 .79 sXlongest - 1.6225 sx shortest =0.9128 [23] 49 40 89 37 47 84 47 34' 81 sx longest =1.2292 s. shortest =1.2292 .72 (cont.) APPENDIX TABLE 2. (Cont.) Population 20 Short/ long arm ratio .91 .97 .93 Population 19 Short/ Chro- Total Relative length long relative mosome length a b arm ratio 1__________ 128 2__________ 114 3__________ 109 Chro- Total Relative mosome length a 1_______ 128 2 120 3 110 b 61 59 532 672 60 57 61 54 52 .91 .90 .91 67 61 57 4_____.. 104 5__________ 102 6__________ 99 7__________ 96 8_.... 94 9_.... 90 10_.... 85 11 _... 81 55 55 53 54 50 51 48 45 492 47 46 42 443 39 37 36 .89 .85 .87 .78 .88 .76 .77 .80 4________ 109 5 102 6 101 7_______ 94 8_.... 91 9_.... 86 10_.... 82 11_.... 78 59 58 54 55 50 48 46 44 50 44 47 39 41 38 36 34 3.8441 1.1546 .85 .76 .87 .71 .82 .79 .78 .77 sXlongest = 1.1758 s; shortest = 0.9573 sx longest = s- shortest - Secondary constrictions of types 1, 2, and 3 are indicated by small numbers adjacent to the arms in which they occur. ** sstandard deviation of the mean.