Studies on Genetic Variation and Introgression in the Allopatric and Sympatric Populations of Pinus rigida ~!ill.' Pinus serotina Michx.and Pinus taeda L.
D. ?.. Govindar~ju -~·" ~ / ··r-.. ;-: : ' "":. ,. ' I '
ABSTRACT
Pinus rigida Mill., _p. serotina rzichx. and ..P• taeda L. form a distinct geographical pRttern in ~he U. s. Yet, however, they hybri- dize freely in the contact zone forming a hybrid complex in the Cape May County of New Jersey and the Delmarva peninsula in Delaware. Pitch and pond pine have received a controversial taxonomic status because of their morphological similarities. Starch gel electrophoretic study is proposed here with the following objectives: a. To quantify
the gene frequencies within and among the allopatric, sympatric,nh~brid and intrcgressive populations of pitch, pond and loblolly pines. to exal!line the nature of intergradation between:the participating
species in the contact zone base1, ' the linkage values. c. to workout the genetic similarities between these species. and d. to arrive at a technique which could be used to locate ~nd to utilize "pure" parents in the tree breeding programs. ....
~ i , ' f: 't
TABL~ QE CO~TENTS
~esearch E1!m Pa.iii~. ~Ul!l\let' _ .• - ' .. '. Introduction 1
Experimental r'.aterial 6 Objectives 8
Material and Methods 8 Literature Cited Resources for the Project Personnel Facilities and Equipment Budget ~esearch Plan
Introduction: A case of multiple species hybrid complex in the genus Pinus was reported by Smouse (1972), and Smouse and Saylor (1973a & b). The .. complex includes Pinus rigid~, E• serotina, f.· taeda and f• elliotti. These species occupy different geographical regions in the Eas.tern United States forming a distinct distributional pattern (Fowells 1965). However, they also overlap and form a hybrid zone in the Cape t"tay County in New Jersey and in the Delmarva Peninsula in Delaware. Smouse and Sa=.or (1973 a) treated the hybrid complex. between~- rigida and P. serotin~ separately from the rest of the species in that area. They found a steep cline with regard to few characters in the hybrids· formed by P. rigi,da (Pitch pine) and f• serotina (Pond pine) in the ' ~~nsition zones. The steep cline ~~s interpreted as supporting a sys- tem of secondary intergradation.._rather than primary intergration. Nevertheless, they also stated there is" as yet no conclusive data available to resolve this question ". According to Smouse and Saylor, spe~iation eve·nts in Pitch and Pond pines might have occured during the Wisconsin glaciation. The glaciation probably cleaved a single taxon into two seperate entities. Subsequent~:·, convergence of these populations resulted in the present intergraded~opulation in the contact zone. Maynard Smith (1966) theo retically analysed a similar situation and concluded that the popula tions when inhabiting two distinct niches in heterogeneous environ ments, and mating is restricted to within niche, such populations will eventualy result in the formation two isolated sub-populations with stable polymorphisms. These sub-populations will also accumulate a series of modifier genes in order to adapt .to ':local ·condi tj:ons:.--t_ ..
'· . - ? - Accumulation of I I modifier genes ~ny continue to the point when sub-populations will become reproductively isolated fro'C'l one another (t,:ather 1955), .even tually forming distinct species (Dobzhansky 1951). Occasionally however;· reproductive isolati.-0n may be unnecessary for population differentiation. Environmental discontinuity can sele ct for physiological barriers to gene flow between adjacent populations. For example, Squillace and Bingham(l958) · recognized two ·well)lifferen"": tiated races of Western White Pine (Pinus monticola), in which, one population was growing on a dry site, while the at.her on a moist si te:11 in less than half a mile apart from each other in Northern Idaho. Con tinued selection for local edaphic conditons has kept the races gene tically apart in spite of gene (pollen) flow. ::. Similar situations have been reported under various situa~ions ( Antonovics 1968.,Brad shaw 1971, Ehrlich and Raven 1969). Small populations from the margins of a large ancestral populat ion in a given geographical area may shift rapidly from the original populations in their adaptive characters (Mayr 1963). Gottlieb (1973a) reported that a recent event has brought about speciation in Stephano
rneria exi&;1;R ssp. coronaria by altering its breeding sy.stem. The res- 1 tant species '}1alhuerensis' is highly self-pollinated as c:,pposed to its progenitor - an obligate out-crossing species. A comparison of allo zyme frequencies between these two species revealed that Stephanomeria exi&;1;a ssp.:cQronaria was poly~orphic at many loci; whereas, 'Malhuer ensis' was monornorphic for~ ~ajority of the loci. On the other hand, he found a different behavior in the e;enus Clarkta (Gottlieb 1974b). Clarkia bilob~ has been identified as the prof;i.nitor of Clarkia lingu- ;.,-. lata, and the interspecific hybrids are substantially sterile (1ewis - A 1973). The sterility has b9en ~ttributed to a tr3nslocation, several :Y, . ,· - 3 - ., '
, paracentric inversions, ·and also the presence of an extra chromosome in Clar~ia lingulata (Lewis and Roberts 1956). Unlike 'Halhurensis' Q. lingulatn showed substantial variability with regard to enzyme patterns (Gottlieb 1976). Mere physical contact between two species with overlapping flowe ring dates often lead to the formation of hybrids, but the parental -c,,,,,U s:pecie9..Astil 1. maintain their integrity • Evolu_tionary potentiallof interspecific hybrids in plants has long been recognized and discussed
( Grant 1963, Stebbins 1974). Smouse and Saylo~ (1973a &b) evaluated the hybrids between .f• rigida, f• taeda, .f• echinata and .f•, serotina •. They concluded that the " three entries (_f ... rigida..:serotina complex, . .f• taeda and .f.echinata) maintain their separate genetic and morphol- ogical integreties in syrnpatric populations inspite of the abilty to hybridize".
A hybrid upon a series··o-r--b,!'rc-k crosses with either of its parents
will yield an introgressed population:- (Anderson 1949) and may even ... ~ result in the formation of a new species (Anderson and Stebbins 1954). The resultant species should then theoretically possess reprodu:}ctive affinity to the more closely related p~Fental species. For instance,
Haller( from Stern and Roche 1974) report·ed that Pinus washoensis is the product of the introgression between P. jeffrei and P. nonderosa •
.f• washoensis hybridizes freely with f. nonderosa but not with .f• jef ~, indicating its genetic affinity with E• ponderosa. Further, hyb rids resulting through interspecific gene flow may so~etirnes even fun ction as selective sceives either to accu~ulate or elininate the alien genes. Heiser(l954) reported th~t the cultivated species of sunflower (Helianthus annus) expa.~de::i its geographical a?!lplititude by accumulating C genes fror.1 -'.3t least h;}lf a dozen species. Convrsely,,.. Stephens (1949) found sig?1i fic:rn t elir.:i.n4 tion of the donor germplasm through both male
and f3:::c) le c:u:ietes in tho b"lck-cross progenies of G. pirsutum ·x G. Hir- sutum X Q.barbadense). Levin (1975) also found evidence for the diff
erential fixa2,tion and the elimination of genes between the interspe cific hybrids of Phlox drummondiiand f• cusnidata. Extremely disturbed sites are also known to serve as suitable habi
tats for the survival of-- hybrids. While parental species thriving on the undisturbed sites. Stebbins,Matzge and Epling (194?) reported the occurance of hybrid swarms between Quercus marilandica and B• illicif .2lli on disturbed habitats. Whereas,the hybrids were rarely encount- ! ered in stands growing on soils typic~l for either species. Similar reports are also available on Betula (Froiland 1952, Clausen 1959).
According to Grant (1971), hybrids can survive on a .distubed site bee-·· ause" somewhat hybridized habitat can accomodate some of the recom
bination products of a natural hybridization''• e I Se~ral hypotheses could be adduced to explain the evolutionary
intricacies of species.~contact zones, hybrids and intriDgressed P9·P
II ulat1ons of forest tree species. Reviews on other organisms are found ' in Dobzhanzky (1951,,1970), Mayr(l963) a.nd Stebbins (1974). Supposing that a marginal population (Carson 1955, Dobzhansky 1956, Levin 1970)
becomes specialized to occupy a diffent niche; then,the newly formed population forms a continuous cline or primary intergradation with the
. ' former., one. at the tonstricted contact zrne'. On the other hand, if a pop- ulation diverges into two sub- populations and converges again should
theoretically result in secondary inte~gradation. A swarm or discon tinuous clines is possible upon contact of two distinct species. The refore, it is reasonable to argue that primary or secondary intergeadatioi wil~¬ only reflect the nature and degree of fixation and infiltra tion of genes from one species to the other but also the evolutionary time scale as well. - 5-
Specializatimn of sub-populations adapting to different niches vrill lead- to stable polymorphisms and linkage equilibria (Maynard .:: Smith 1966).,, Since " gene frequencies change toward a stable equili
brium condition corrersponding to R local ~aximum in mean adaptive value" (Lewontin and Kojina 1960).· However, when two. populations meet and hybridize, the stable linkage equilibrium would be disturbed;resu- 1 ting in··: linkage diseqtiili bria~ Therefore;·-the'·deg:ree of disequili- . I
bria not only indicate the genetic affinity betv,een the participating . I populations but also, the time since they have been in contact. In the case of either a population founded by a few colonizing individuals or a small population emigrating from a large one, a sub
stantial number of alleles will be lost due to the 'bottleneck effect•. The loss of alleles will be directly proportional to the size of bott- o leneck(Nei and Roychoudhury 1976), and new species developed rr.m small founder populations may di verge rapidly as opposed to :~ · the outgro wths of large geographic races which seem to be conservative( Grant 1963). Therefore, a founder population may be ad.vantageous -from the standpoint of its effect in accelarating the rate or evolution. Upon expansion such a population may result in secondary intergradation;. and s~high linkage disequilibria are intuitively reasonable. Howe- w ver, population originating as out groths and showing prinary inter- . ,.. . . gradation may not be as advantageous in increas:1ng the overal11. gene tic variability at the species level. Ideally then, a measure of gen etic variability in any population is a reflection of the bottleneck sizes of the ancesral population,~n1 the span of ti~e, since the differentiation has occured •. An elegant /demonstration of such possi
oili ties hP-.s been reported by Cars0::1 (1976). on Hawaiian Drosophilidae.
I·11 wriiu.·, ~ h0 found dir frequencies in various populations from the different islands. The proposed research involves an examination of the nature of gene complexes and linkage disequilibria in the 4llopatric, sympatric, hyb rid and introgressive populations of pines as reflected by ecological events. It will also at-tempt to account for the evolutionary aspects of multiple species hybrid complex in forest -t~ee species; specific ally in the genus Pinus. This genus seems appropriate for such a study of its because~very conservative genetic nature ( P,,o.~IV\- , Fowler and Wilson 1976) and the fact that it has remained in the Northeast for at least 100 generations (Ledig Personal comm. 1977) ExEerimental material: Tha svr.tpatric hybrid complex involving Pinus ri12:da,_ P. serotina and P. taeda,which occur in the Cape May County of New Jersey,and the I. Delmarva peninsula in Delaware,and its allopatric populations will form chief components in the study. Pitch pine (Pinus rigida Mill.) ranges from ·southern quebec to northern Georgia and from central Kentucky t~he coast of Maine. Ledig and Fryer(l974) reported a great phenotypic amplitude with re gard to its height and form. It grows as a prostrate shrub on Fire Island and a scrubby bush in the pine plains and attains about 30 rn in the Pine Barrens or New Jersey. P0 nd pine (Pinus serotina M1chx.) ranges from southern New Jersey to :central Florida. It is a small to medium siz_ed tree usually found in wet boggy sites. Botanical 1.y, Pond pine has been described as a southern counter:::part of pitch pine (Harlow and Harrar 1969), a sub species(Clausen 1939; Smouse and Saylor 1973a) and a disti-nct species (Little, Little and Doolittle 1967). Lob:olly pine (Pinus t~eda L.) occupies a geographical range 9xtending f'!'o,~ e,'3.st Texas to so:1 the!'n ~ew Jersey, including northern .. . - 7 - . - . 0,. ' r' Florida. It isAmedium to large sized tree growing on a wide range or soil types and a variety of species mixtures (Fowells 1965). Several clines have been reported in pitch and pond pine. A continuous cline froi Massachussetts to northern ~lorida and an int~mediate cline in New Jersey with regard to needle length has been described by Clausen (1939). Ledig and Fryer (197~) found 100% cone serotiny in th(/popula tions from the pine barrens and this feature de~resed clinally north and southwards. They also hypothesised that cone serotiny may be mo- I nogeniclly selected by the frequently occuring fires in the pine bar¥ens.· All of ·; these species have commercial importance. Hybrids between·, pitch and loblolly pine exceed both parents in volume growth. Mass · ~ production of this cross is underway in the u. s. (Little and Trew 1976) and produced in large scale in Korea(Hyun 1974). Pond pine is used for pulp production in the South, wn~le loblolly pine is one of the !!lajor timber yielding· tree--·species in the U. s. Techniques have been developed for starch gel electrophoresis using megagar::etophy;ee by Ledig and Guries (?ersonal comm. 1977) as a _part of their genecolo g1ca1 :i. studies in pitch pine. Their results in dicate the occurance of several discrete constellations of electroph oretic variants as well·as phenotypic characters in the .studied popula tions •• The genetic variability alre~dy demonstrated in these three species could be further quantified and augmented by the proposed re search. A ~e31th of valuable infor~ation is expected from this study on the chosen species and their hybrid complexes. T·echniques for seed processing, seedling raising, el~ctrophoresis ·and statistical proced ares have been developed. - 8 - Objectives: ·, : .. Tbe primary objective is to detect whether the introgression between pitch and pond pine ·is of primary or secondary origin in tne transition zone. Existance of linkage equilibrium would indicate primary inteTgradation,and disequilibrium the secondary intergradation. Also,to locate the linked loci among the participating species in the transition zone and to investigate the a.mount of linkage disequilibr ium in the transition zone. The second objective is to find out whe ther the gene frequencies in the allopatric populations are different or.intermediate from that of the sympatric populations in the trans1- e tion zone. Third, an assessment of gentic distance!between these three ~ . . . o?i species basedAthe gene frequ~ncy data. The ultimate objective would be to arrive at a solution to locate alien genes in a given genome. The results could be used in identifying and isolating "pure" parents, thereby avoidi~g contamination in the- establishment.of seed orchards which will be very valuable in various breeding programs. Vaterials -artd Yethods: Three allopatric populations of pitch, pond and loblolly pine will be located -·i·n: their respective geographical distributional areas. ~hese populations will be chosen in such a way that,there is no ge~e·flow a~ong the selected stands. The selected population will consist or 40-50 trees. At least 50 cones from each tree will be collected and stored separately. Four sy!':'lpatric populationc consisting P-11.the three species and the their hybrids will be chosen from ,, Cape ~ay County in New Jersey and the Delmarv."J. peninsula in ·Del:nwre (Smouse ;md Saylor 1973a &b). - 9 - Forty to 50 cones will be collected from at.least 100 trees or each ; population. Seeds from individual trees will be separately exracted and stored according to the stand~rd procedure outlined by Schopenme yer(l975). However, pitch pine seeds from different populations and the results obtained by Led~g and Guries(Unpublished report) on the gene frequencies will be utilized to establish a base line in the current study. Methods to excise and to extract enzymes from the pine megagame tophyte have been standardized by Ledig and Guries based ·on the met4 hods outlined by Conkle(l972). The method basically involves separa tion or the megagametophyte and the embryo by removing the seed coat of a germinating seed. The excised megagametophyte will be used as the enzyme source. Each megagametophyte wil1 be homogenized in extraction buffer( 0.5 :'! phosphate pH7.o with 0.005 ml mercaptoethanol added to per ml. of buffer). Extract will be absorbed by paper wicks and the same will be place1 in the hori_zontal gel slabs for electrophoretic analyses • Methods for best resolution of the enzymes _in pitch pine are ava.... . - lable (Ledig and Guries Unpublished Data.). If necessary, the procedures · will be manipulated accordint5 to the methods outlined by Selander .!.!!l•, (1969); Bre'.'ler (1970); Shaw and Prasad (1970). The use of I:1egagametop hytes provides an additional advantage in the genetic anlyses, for it provides a direct estimate of the allelic frequencies. The theoretical expectations and the departure from the Hardy Weinberg equilibrium will be calculated from the obtained allelic fre quencies. Mean heterozy~ote deviation will-be calculated by the:folloWing: (Hobs - H exp) :) = ·::right (1965). - 10 - Mean sampling variance of an allele frequency will be estimated as: . Cavalli-Sforza and Bod~er (1971) PC 1 • - P~ -n Where, Pis the allele frequency and n is the mean number of alleles over samples. Gene admixture in the hybrid populations can, also be calculated by Berstein's formulae. This has been largely used to estimate the ' I admixture of genes in human populations. According to this frmulae: If 'm':.is the proportion of genes at a particular locas in a hybrid population (H) which is derived from a population (p) \Vhich has mis geniated with an immigrant population (I}, and .if !.:.<1' is the gene frequency, then~. I and therefore, If the gene in questien is absent from the immigrant population then Emery (197b) Linkage disequlibrium values between any two species will be cal-. culated after Cavalli -Sforza and Bodmer (1971, P.69). Supposing two populations mix in the proportion of m to 1-m and the two loci are no longer independent; assuming that the participating popualtions /are in equilibrium foT two alleles at each of two linked loci. ~ ' Let 1the frequencies of alleles A and A at the first locus be 1 2 r, and q in tte first population and P ~nd 1 in the second. Simila- 1 1 1 1 rly,let p ,q~ ~nd P ,0, be th3 corcesponding frequencies or alleles 2 2 2 - 11 - B1and B2 at the second locus. The frequencies of the foru ga12etes A B , A ·, A 3 , and A B in the mixed population are then 1 1 1 a2 2 1 2 2 ·-m t--,l"a..-t c,-in> r, P:l , '\'I\.,," c1. + c,- 'l'n) P, q:\ , 'hl't', ~ .., c, -?n) 'i~ P.1. &\'n The measure of association between the loci is thus l) = [~ ~,l'-2. + (1-rm)l'1i>:a..)['h\~1'h--+ (1-rm)q,q1) '. - [~I\ °v2--t (1-~) i>1'h][~t,l'-z.i" (i-~)q,V.z.]., 01" ~(,-'\'AX,tw1\'-2. q,~z.·-\- P,P. 'Vi '\t~ - \'('1/J.. ~, f.- f192-t';L"11) D= :: ""' (\-"MX,~1 ~ 1 - P1 \ 1)(h.. q:a.-- P2C\i.1-)• .. Therefore ~~ ~(1-m)(pl - Pl)(p2 - P2) Nhich is zero 0!'1.ly if p1 = F1 Rnd p~=P2,. that is·, if either· or_ both of IJ the gene fre'1:.11::.:1cies a.re the s3me in two popul"l.tions. Genetic identity between any two populations will be calculated by Nei's forrnulae(l972) or normalized genetic identity (I). According to this method, supposing x and y are the frequencies of the 1 th allele in populations x and y respectively then the genetic d~_::;t:ince betwe_en x Rnd y is then defined as .. D ;:; - loe; I e Literature cited:· Anderson, E. 1949. Introgressive Hybridization. John Wiley. New York. N. Y • ------• , and G. L. Stebbins. 1954. Hybridization as an evol- lutionary stimulus. Evolution 8: 378-388. Antonovics, J. ·1968a. · Evolution in closely adjacent plant populations v. Evolution or self- fertility. · Heredity 23:219-:-_238. Bradshaw, A. D. 1971. Plant evolution in extreme environments. in R. Creed ed., Ecological Genetics and Evolution. 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Introgressive hybridization in red spruce and black spruce. Tech. Rep. No. 4. Faculty of forestry Jniv. of Toronto. Kuller, C.H. 1952. Ecological control of hybridization in Quercus~ A factor in the mechanism of evolution. Svolution 6: 147-161. !. ' ::ei, r-:. 1972. Genetic distance between populations. Amer. Natur • . , 106: 283-29<. ,, 1·•. ' T. Maruyama, and R. Chakraborty. 1975. The bottleneck effect and genetic variability i~ populations. Evolution 29: 1-10. Prager, E.M., D.P. Fowler,-·and A.C. Wilson. 1916. Rates of .evolution in Conifers (pinaceae). Evolution 30: 637-649. Schopmeyer, c.s. 1974. Seeds of woody plants in the United States. USDA For. Ser. Agriculture Handbook No. 450. 883p. Selander, R.K., N.H. Smith, S.Y. Yang, W.E. Johnso·n, and J.B. Gentry 1 1971• Biochemical polymorphism and systematics in the genus Peromyscus I. Variation in the old field mouse (Peromyscus nolionatus) in~- Genet VI. Univ. Texas Publ., 7130: 49-90. Shaw, c.R~, and R. 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Selective cli~ination of donor parent genotype in intersp~cific .. - Wright, s. 1965. The interpretation of population structure by F-.statistics with special regard tJ syster.is of mating. Evolution 19: 395;...420. I· DISMAL SWAMP JATIONAL WILDLIFF.; REFUGE MANA~ENT STUDY TRANSMITTAL ~oposal Permit# Amendment ------(assigned by refuge) -_ Final Report Progress Report - Time Period From ----- To ----- Title of Study c;; T'-'l:>~e;s ~ ~ A.y..p,: ...,-'Pe-1:(:.. J..J-,;;, <;;'Ct'Je &~ s;, \"9: \J"L~tt~ c:F- 1\~~ ~,:t.bk >• £· S.~n.lJI... t"s:HP 3'-.1~e4>f\... Project Supervisor '"'J)f2..· JA11.e-..s A· ~U'.X:..J.J'N- As..s.oc- t>e.cf-~sc~ ------Name Title J)tft>T· d?- \?.c~r 'KLl"'ics.~ ~'-'~'S~i:,, ~P:3>1\ E'-R:,~~· ~~""' Mailing Addressf-&,&;x tosr,-Plsc.J()'1/0/j AJ.J,#l8S'/-, ,•. J ~g.~05. Person(s) Conducting Field Work On The Refuge: f ~ ~ A;l:> . <;n..;t) ~ 6,Y'C(ttude ~~ Name Title Agency or Institution Cooperators (other agencies or individu~s assisting in project) r ·T. L~q., A~f;oe. ? ~of· __)'"--kt.. __ o_r· __ u____ ~ ____ v_· --- Name Title Agency or Institution Si natures /)::,,..\,.) <:;; 'Z-.5 c:::::::::--...,-==---"--'N,J,... Akj, 0€::F ~-i~tt I ~-,y Submitted By Date f01t 1:> fV >f . .,._. {-?U:;!H.,._j ·),...\Jc;,_ \! t I :,, q • Project Supervisor Date Cooperator (other than Refuge) Date A rovals Submit all transmittals and reports in duplicate. All management study proposals must confor:m to the outline provided by the refuge office. (OVER) FORM ~3 LRE 1,,. -l UNITED STATES DEPARTMENT OF THE INTERIOR Permit number Sta. No. to be credited U.S. Fish and Wildlife Service 1 -1 -1. 515 Contract number ______National Wildlife Refugei------SPECIAL USE PERMIT D Purpose (Specify in detail privilege requested, or units of products involved) allo Description (Specify unit numbers; metes and bounds; or other recognizable designations) f'uge r ds in pers ta 'cl.e - C • Amount of fee $ ______If not a fixed fee payment, specify rate and unit of charge: ______ D Full payment D Partial payment-Balance of payments to be made as follows: Record of Payments Special Conditions 1. Collectio fr i restricted to those teri s r spec· s defined· this pe · t. 2. Ve ·cular 3. Offic at ds. 4. s. o bef re ager. This permit is issued by the U.S. Fish and Wildlife Service, and accepted by the undersigned, subject to the terms, covenants, obligations, and reservations, expressed or implied therein, and to the conditions and require ments appearing on the reverse side. Permittee (Signature) Issuing Officer (Signature an;t title) '· , fu,ge