Mitochondrial Inheritance Patterns Across a Cottonwood Hybrid Zone: Cytonuclear Disequilibria
and Hybrid Zone Dynamics
Author(s): Ken N. Paige, William C. Capman and Peter Jennetten
Source: Evolution, Vol. 45, No. 6 (Sep., 1991), pp. 1360-1369
Published by: Society for the Study of Evolution
Stable URL: http://www.jstor.org/stable/2409885
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Evolution,45(6), 1991, pp. 1360-1369
MITOCHONDRIAL INHERITANCE PATTERNS ACROSS A COTTONWOOE
HYBRID ZONE: CYTONUCLEAR DISEQUILIBRIA AND HYBRID
ZONE DYNAMICS
KEN N. PAIGE,' WILLIAM C. CAPMAN, AND PETER JENNETTEN
Institutefor EnvironmentalStudies,DepartmentofEcology,Ethologyand Evolution,
University
of Illinois, Urbana, IL 61801 USA
Abstract.-In this studywe examine the cytoplasmicinheritancepatternsof an interspecifichybridizingpopulation of Fremontand narrowleafcottonwoods,using mitochondrialDNA. Three
mitochondrialprobes showingpolymorphismswere used to distinguishbetween trees of known
nuclear inheritance.Every tree screened had only one cytoplasmicgenotype,eitherFremont or
narrowleaf.Thus, theseresultsdemonstratethatmitochondriaare uniparentallyinheritedin these
trees. Previous studies of the nuclear inheritanceof this interspecifichybridizingpopulation of
cottonwoodtreesindicatedan asymmetryin the frequencyof parentalgenes. Using mitochondrial
markerswe tested one hypothesispotentiallyresponsible for this asymmetricdistribution(i.e.,
treesof mixed genotypeswill be sterileor will not survive if theircytoplasmis derived fromone
or the otherparent).Our results,however,show thatboth Fremontand narrowleafmitochondrial
markersare foundin treeswith mixed nuclear genotypes.Thus, nuclear-cytoplasmicincompatibilitiesdo not appear to account forthe asymmetricdistributionof nucleargenotypeswithinthe
hybridswarm. An alternativeexplanation for the observed asymmetricdistributionof nuclear
genotypesis advanced. Althoughnuclear-cytoplasmicincompatibilitiesdo not appear to explain
the asymmetricdistributionof nuclear alleles withinthe hybridzone, nonrandom associations
between nuclear and cytoplasmicgenotypesdo exist. For example, all F1 hybridshad Fremont
mitochondrialgenotypes.Furthermore,backcrossesbetweenF1 hybridand narrowleaftreeshave
a higherthan expected proportionof heterozygousloci and a higherthan expectedproportionof
Fremont mitochondria.We propose that seeds, seedlings,or trees with high proportionsof heterozygousloci are at a disadvantage unless theyalso have the Fremontmitochondrialgenotype.
While it is generallydifficult
to inferdynamicprocesses fromstaticpatterns,studies such as ours
enable one to gain new insightsto the dynamicsof plant hybridzones. A hybridizationpatternof
decreasinglycomplex backcrossesas one proceedsfromhigherto lowerelevationwithinthehybrid
swarm,a residue of FremontcytoplasmicDNA withinthe pure narrowleafpopulation, and the
unidirectionalnature of these crosses suggestthat the narrowleafpopulation may be spreading
down the canyon and the Fremontpopulation receding.The eventual fateof the hybridzone, in
relationto these processes,is discussed.
Key words.- Cytonucleardisequilibria,Fremontcottonwood,hybridzone dynamics,hybridization,
mitochondrialinheritance,narrowleafcottonwood,nuclearinheritance,Populus angustifolia,Populusfremontii,restrictionfragment.
Received June29, 1990. Accepted February4, 1991.
With the advent of recombinantDNA
technologyand its use forthe identification
of restrictionfragmentlength polymorphisms(RFLPs) we now have thecapability
of addressing several questions that have
proven intractable using conventional
methodsof investigation(e.g.,geneticstudies ofnaturalpopulationsoflonglived plants
thatare difficult
to analyze by classical segregationapproaches; see Keim et al., 1989;
Paige et al., 1990). The currentsurgein the
use of moleculargenetictechniqueshas, for
example, provided new approaches to the
studyof hybridzones, withrecentattention
focusingon the dynamics of hybridzones
and the processes that have contributedto
theirpresentdistributionsand maintenance
(e.g., see Ferris et al., 1983; Millar, 1983;
Sage et al., 1986; Baker et al., 1989; Keim
etal., 1989; Rand and Harrison,1989). Most
3f these studies have focused on animals
(Hewitt, 1988). Plant hybridzones, however,have been much neglectedand studies
examining both cytoplasmic and nuclear
markersare even rarer.
Recently,we conducteda study(Keim et
1
Thisworkwasinitiated
during
mytenure
as a postdoctoralfellowin the Department
of Biology,Uni- al., 1989) of nuclear inheritancein an inversity
ofUtah,SaltLake City,UT 84112 USA.
terspecifichybridizingpopulation of cot1360
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MITOCHONDRIAL
INHERITANCE PATTERNS
1361
tonwood treesusing RFLP markers.Here, forthe asymmetricdistributionof Fremont
we expand our efforts
to incorporatestudies and narrowleafnucleargeneswithinthehybrid population. Asymmetry,forexample,
of cytoplasmicinheritance.
could resultfromassortativematingdue to
AND
STUDY SITE, ORGANISMS,
phenologicaldifferences
in flowering,
a bias
OBSERVATIONS
in the population sizes of the two parental
Genetic studies of the interactionof two species,selectionforparticulargeneticcomspecies of cottonwood trees,Fremont cot- binations, nuclear incompatibilitiesinhibtonwood, Populus fremontii,and narrow- itingreproductivesuccessduringsome stage
leaf cottonwood,P. angustifolia,were con- of development,or nuclear-cytoplasmic
inducted in the Weber River drainage north compatibilities(leading to only one typeof
of Salt Lake City,Utah. One parentalpop- cytoplasmicinheritance(e.g.,narrowleaf)in
ulation,P. fremontii(Fremont),is located at hybridtrees;Asmussen et al., 1987).
lower elevations (below 1,310 meters),the
By determiningthe cytoplasmicinheriother,P. angustifolia(narrowleaf),is found tance of trees in the hybrid swarm with
at higherelevations (above 1,490 meters). known nuclear inheritancewe have been
These individualsgrowalong thebanks and able to: (1) establishthatmitochondrialinin the bottomlandsadjacent to the Weber heritanceis uniparentalin cottonwood;(2)
River. These two species can be easily dis- testone mechanismpotentiallyresponsible
tinguished by leaf morphology. Fremont for the observed distributionof genotypes
treeshave wide leaves, long petioles,and a withinthe hybridswarm (i.e., nuclear-cyfew serratedteeth. In contrast,narrowleaf toplasmic incompatibilities);(3) gain furtreeshave narrowleaves withshortpetioles therinsightinto the directionalityof reproand manysmall serratedteeth.The two pa- duction withinthe hybridswarm and; (4)
rentalpopulationsare connectedbya 13 km gain insightinto the dynamicsof this plant
overlap zone composed mostly of inter- hybridzone. To accomplish this,we have
mediatephenotypes(withintermediateleaf used RFLP markersto determinethe cymorphologies)and a few parentalsof both toplasmic inheritanceof individual trees,
types(i.e., a hybridswarm). It is, however, using mitochondrialDNA.
extremelydifficult
to distinguishbetweena
METHODS AND MATERIALS
pure narrowleafand a complex backcross
(BC2s, BC3s, etc.) to the narrowleafside
Mitochondrial inheritance was deterbased on morphologyalone (Paige et al., mined for40 treesof knownnuclearinher1990). RFLPs allow one to circumventthis itance. The geneticconstitutionof individproblem, yielding more precise genotypic ual trees (whethera pure parentaltype,an
information.Both treespecies are dioecious F1 hybrid,or a backcross) was determined
and wind pollinated.
by the use of nuclear recombinantDNA
Previous studies of the nuclear inheri- probes (see Keim et al., 1989 and Paige et
tance of this interspecific
hybridizingpop- al., 1990 fordetails). A largeportionof the
ulation of cottonwood trees indicated an nuclear data presented in this paper was
asymmetryin the frequencyof parental taken from Keim et al. (1989). We have
genes(Keim et al., 1989; Paige et al., 1990). since added additionalnucleardata, includIn individualsmakingup thehybridswarm, ing severalnew treesand severalnew markloci werehomozygousfornarrowleafalleles ers on previouslyscored trees.
or were heterozygous(both narrowleafand
MitochondrialDNA Extraction
Fremontfragmentswere observed). In genetically mixed individuals, however, no
DNA forthe preparationof a mitochonhomozygous Fremont alleles were found. drial DNA library(for detectingpolymorThus treesin thehybridswarmappeared to phic loci) was extractedfroma narrowleaf
be eitherF1 hybridsor backcrossesbetween cottonwood cell suspension tissue culture
hybridsand narrowleafparents.No progeny (prepared by Jill Roth). Eightyml of cells
could be attributedto hybrid-hybrid
crosses were homogenized in 10 ml of extraction
or backcrossesto Fremont.
buffercontaining25 mM Tris (pH = 8.0),
There are several possible explanations 0.3 M Mannitol, 1% PVP 40, 0.1% BSA,
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1362
KEN N. PAIGE ET AL.
1mM beta-mercaptoethanoland 3 mM
EDTA. Homogenizationwas carriedout using a tight-fitting,
motor driven Teflon insertin a glass homogenizer.
Following homogenization, debris was
removed by centrifugation
at 1,000 x g for
10 minutes[chromosomaland plastid contamination was significantlyreduced by
carefullydecantingthe supematantand repeatingthe centrifugation
(Lansman et al.,
1981; Schmitz, 1988)]. Mitochondriawere
pelleted fromthis second low speed supernatant by centrifugation
for 20 minutes at
15,000 x g. Next, the mitochondrialpellet
was resuspendedin 2 ml of buffer(25 mM
Tris (pH = 8.0), 0.3 M Mannitol, 10 mM
MgCl2). A camel hair paintbrushwas used
to facilitateresuspensionof the mitochondrial pellet.
After resuspending the mitochondrial
pellet,DNAse I (100 ,ug/ml)
was added and
the mixturewas incubated at 37?C for 1
hour to remove nuclearDNA. The reaction
was stoppedbyaddingEDTA to a finalconcentrationof 100 mM. The temperatureof
the mixturewas elevated to 6 5?C, to inactivate the DNAse, and the mitochondria
were lysed by the addition of sodium dodecyl sulfate(finalconcentration,1% SDS)
and digestedfor 1 hour with proteinaseK
(20 ,ug/ml)
to inactivatenucleases. To each
1.2 g/mlof lysate in TSE [lOmM Tris.Cl
(pH 8.0), lOOmM Nacl, lmM EDTA (pH
8.0)], 200 ,ugof Hoechst II dye was added
togetherwith8.7 g of solid cesium chloride.
This mixturewas centrifugedfor40 hours
at 20?C at 36,000 rpmin polyallomertubes.
The gradientwas visualized underUV light
and the GC rich,mitochondrialDNA band
was removed.Afterdialysisin a Tris-EDTA
buffer(two times in lOmM Tris, 0.5mM
EDTA, then once in lOmM Tris, 0.05mM
EDTA) to remove the cesium chloride,this
DNA was used to prepare the mitochondrial DNA library.
pylthio-B-D-Galactoside). Bacterial colonies containingplasmids with insertswere
selected and grownovernightin LB broth
(tryptone,yeast-extract,NaCI). Bacteria
were lysed and the plasmids were isolated.
Insertswere cut out fromthe vector,using
HinDIII, and isolated bygel electrophoresis
in low meltingagarose (Sea Plaque), diluted
in 5X sterilewater(volume to weight)and
boiled to denaturethedouble-strandedDNA
fragment(Langridgeet al., 1980; Keim and
Shoemaker, 1988). Radioactively labeled
probes were then prepared fromthese inserts by hybridizing a random primer
(pd(N)6,Pharmacia) to thedenaturedstrand
of DNA and synthesizingDNA containing
32p
labeledcytosine.
Probes were screenedagainstDNA from
known pure narrowleafand Fremont cottonwoods(treesthathad been screenedwith
between 25 and 35 nuclear markers):FollowingDNA extraction(fromleafmaterial)
and purification,
DNA fromthesetreeswas
digested with restrictionenzymes and the
fragments
wereseparatedaccordingto their
molecularsize bygel electrophoresisin agarose gels. The separated fragmentswere
transferred
to nylonmembranes(Southern,
1975) and then hybridizedwith the radioactively labeled probe, after which the
membrane was analyzed by autoradiography(see Keim et al., 1989 fordetails). Useful mitochondrialclones showing a polymorphism between the two parental tree
species were used againstindividual hybrid
or parental trees withinthe population to
determinetheirpatternof inheritance.
RESULTS
Probes
Plasmid clones preparedfromthecottonwood mitochondrial DNA library were
screened forpolymorphismsby hybridizationwithsoutherntransfers
ofDNA restriction digestsof narrowleafand FremontcotLibraryConstruction
and Screening
tonwood.All 30 probesidentifiedmulticopy
Mitochondrial DNA was digested with DNA (most autoradiogramscould be deHindlIl, ligated into the plasmid pBS + veloped afteran hour whereas the nuclear
(StrategeneInc.) and transformed
intocom- probes revealed fragmentsfrom the same
petent cells, (E. coli DH5alpha, Bethesda digestsonly aftermany days (7-10) of exResearch Laboratories). Cells were thense- posure). Three probes were found to be
lectedforgrowthon ampicillinon indicator polymorphicand identifiedfragments
ofdifplates containingX-gal and IPTG (Isopro- ferentlengthsin the narrowleafand Fre-
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MITOCHONDRIAL
INHERITANCE
A
0
0
E
0?
cq Co q
_
o
Q
9.5-
7.6-
B
c
_
0
2E
*0
LL
Z1.
cn$
6:
=xz 0*i
N
gvs
*,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~'
;n
0.99FIG. 1. Two mitochondrialDNA probes used in
determiningthematernalinheritanceofindividualcottonwood trees. Probes M30 (A) and M 10 (B) were
hybridizedwithfragmentsof total DNA digestedwith
BglII (A) and EcoRI (B). "Fremont" DNA was from
tree F; "narrowleaf"DNA was fromtree S (Table 1).
Remaining tree samples were obtained fromall three
zones; the pure narrowleafand Fremontzones (A) and
the hybridzone (B).
A
o
1363
PATTERNS
montDNA preparations
(Fig. lA and B).
In each case, the polymorphism
was observedwhenDNA was digestedbyone particularenzyme;otherenzymesfailedto reveal a difference.
This was in striking
contrast
toourpreviousexperience
withnuclear RFLP markersin whichmostpolymorphisms
appearedto be theresultofrearrangement,resulting in changes in
fragment
lengthsthatwereobservedusing
severaldifferent
restriction
enzymes(Keim
et al., 1989).Data in Figure1 showthatall
polymorphic
mitochondrial
probesidentifiedtheDNA oftreesas eitherFremontor
narrowleaf.
As expectedforcytoplasmic
inheritance,
no cases occurredin whichboth
typesof markerswerefoundin the same
tree.Trees989 and HM-1,forexample,had
been shownto be nuclearhybrids,
heterozygousforall nuclearmarkers
examined[26
for989, 17 forHM-1; Fig.2B and D (Keim
et al., 1989)],butbothtreeshad onlyFremontmitochondrial
markers(Fig. 2A and
C).
Inheritance
Cytoplasmic
Resultsof examiningall of thetreesfor
whicha patternof nuclearinheritance
has
B
c~~~~~~~~~~~~~~~~~
3
E2
IL
Z
'
U.
9.57.8-
Z
a
10.0-
3.9-
C
oC
E
2
3
.
0f
19
Db
0~~~~~~~~~~~~~~~
c
W
7.69.5~~~~~~~~~~~~~~~.
N
co
0I
?
0
a
cm
0co 0I
E
CD
I0
LE a.
E
Zo 00
co C4
I
8
aC
E
8.5-
5.4
FIG. 2. A comparison
of mitochondrial
(A and C) and nuclearmarkers
(B and D). Trees989 and HM-1,
forexample,arenuclearhybrids,
forall nuclearmarkers
examined(B andD). Bothtrees,however,
heterozygous
haveonlyFremont
mitochondrial
markers
forcytoplasmic
(A andC). As expected
no casesoccurred
inheritance,
in whichbothtypesofmarkers
werefoundin thesametree.
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1364
KEN N. PAIGE ET AL.
TABLE 1. Nuclear and mitochondrialgenotypesof individual cottonwoodtreesalong the Weber River north
of Salt Lake City,Utah. The nuclear genotypeof each tree is presentedby the number of markersexamined
and classifiedas Fremont(F) only,hybridor heterozygotic
(H), or narrowleaf(N). Trees weredesignatedas either
pure parentaltypes(all markershomozygous Fremontor narrowleaf,respectively,F1 hybrids(all loci heterozygous),or as a backcross(e.g., BCl -25% Fremont,75% narrowleaf,BC2-12.5% Fremont,87.5% narrowleaf
etc.). Mitochondrialgenotypesare presentedas eithernarrowleaf(N) or Fremont(F).
*
Tree
Nuclear genotype
(F:H:N)
Mitochondrial
genotype
(N or F)
Cross
Tree
F
20
H14
001
012
033
9-17
HM- 1
989
1994
1997
1979
1981
1934
1935
H1
H2
H6
H12
H3
37:00:00
16:00:00
05:00:00
16:00:00
16:00:00
16:00:00
18:00:00
00:17:00
00:26:00
00:05:00
00:05:00
00:03:01
00:13:02
00:03:01
00:03:01
00:11:02
00:10:03
00:12:04
00:06:04
00:05:08
F
F
F
F
F
F
F
F
F
F
F
F
F
N
F
F
F
F
N
F
F
F
F
F
F
F
F
F1
F1
F1
F1
BC1
BC1
BC1
BC1
BC1
BC1
BC1
BC1
BC2
11
18
H9
H10
1007
1992
999
1008
1025
1019
G
S
13
48
T15
996
1011
1012
1023
3200
Nuclear genotype
(F:H:N)
00:07:19
00:07:13
00:03:04
00:04:07
00:04:11
00:01:03
00:02;09
00:02:13
00:01:04
00:01:10
00:00:23
00:00:29
00:00:16
00:00:16
00:00:16
00:00:19
00:00:07
00:00:05
00:00:12
00:00:16
Mitochondrial
genotype
(N or F)
Cross
F
N
N
N
N
N
F
N
N
N
N
N
N
F
N
N
N
N
N
N
BC2
BC2
BC2
BC2
BC2
BC2
BC3
BC3
BC3
BC4
N
N
N
N*
N
N
N
N
N
N
Complex backcross.
been determinedare summarized in Table
1; the cytoplasmicinheritanceis presented
as eitherF (Fremont) or N (narrowleaf).It
is immediatelyapparentthatthe mitochondrial inheritanceof backcross trees in the
hybridswarmcan be eitherFremontor narrowleaf,ruling out one hypothesiswhich
contends that nuclear-cytoplasmicincompatibilitieslead to backcross trees of only
one type of cytoplasmic inheritance(e.g.,
narrowleaf)and an asymmetryin the distributionof nuclear genes.
Nonetheless, our data do suggestan interdependencebetween nuclear and cytoplasmic inheritance.All of the F, hybrid
trees(i.e., 50% F/50% N nuclearDNA) that
we have looked at so far,forexample, have
inheritedFremontmitochondria(Table 1).
This includes the well established hybrids
989 and HM-1 as well as 1994 and 1997
(five markers each). Furthermore,among
treesthatare the resultof a singlebackcross
betweenan F, hybridand a pure narrowleaf
tree (i.e., BC,s), those with FremontmitochondrialDNA have a higherthanexpected
proportionof heterozygousloci (80% insteadof50%; X2 = 23.40, df= 1,P < 0.0001).
These include4 treeseach ofwhichhas been
analyzed formore than 10 nuclear markers
and 2 trees analyzed for only 4 nuclear
markerseach. Two otherputativebackcross
1 trees have narrowleafmitochondria,but
foronly one of these,H 12, has the nuclear
inheritancebeen analyzed extensively(inH12 does not contain as higha
terestingly,
proportionofheterozygousnuclearmarkers
as do the majorityofbackcross I s thathave
been analyzed extensively).The otherone,
1934, has been analyzed foronly fournuclear markers. In addition, a higherthan
expected proportionof these randomlyselected F, and BC, treeshave inheritedFremont mitochondria(82% instead of 50%;
X2 = 4.46, df = 1, P < 0.025). This pattern
furthersupportsthe idea that nuclear and
cytoplasmicinheritanceare interdependent
(e.g., varied interactionsbetween products
of nuclear and mitochondrial genotypes
could have epistatic effectson fitnessresultingin cytonucleardisequilibria; see Asmussen et al., 1987).
We have also foundthatas theproportion
of homozygousnuclearinheritanceincreases (in BC2s, BC3s, and BC4s) the frequency
of treeswithnarrowleafcytoplasmicinheritance also increases(8 of 11); such a result
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MITOCHONDRIAL
FremontZone
204
033 4
1283m
Overlap Zone
0014
012
1295m
H
l
H10
H12
1329m
-,
I
999.4
1a007
9-17o
e
/
,
/,
NarrowleafZone
H-6
1934
H14-4
Riverdale
1935-4
1979-4
l~~~~981l4
1992
19944
~1381m ~1997-4
~~1433m
/'
~~~1369m
Hml
18-2
1365
INHERITANCE PATTERNS
320
F4
9894
996
996
7
Peterson
1008
1011
1012
1019
1023
1025
T15
A
1582m
Moga
G
484
FIG. 3. The geographical
distribution
and cytoplasmic
inheritance
ofindividualcottonwood
patterns
trees
alongtheWeberRivernorthof Salt Lake City,Utah. Sitesof treesare indicatedby arrows.Mitochondrial
inheritance
is indicatedbythepresence(Fremont)
orabsence(narrowleaf)
ofa triangle.
The hybrid
(oroverlap)
zoneis represented
a distance
bytheshadedareabetweenRiverdaleand Peterson(sites3 through
7), covering
ofapproximately
13 km.
is not significantly
differentfrom the expected distribution(73% narrowleafversus
80%; x2 = 0.375, df = 1, P > 0.50).
Furthermore,nuclear-cytoplasmic disequilibriumvalues (D = 0.0463, D1 = 0.093,
D2= - 0.093, D3 = 0; Pearson x2 Goodnessof-Fit test = 86.72, df = 1, P < 0.0001)
calculated fromthe statisticsof Asmussen
et al. (1987) are consistentwith these results, indicatinga nonrandom association
among nuclear and cytoplasmicgenotypes.
Overall,bothcytoplasmicgenotypeswere
found throughoutthe overlap zone (Fig. 3,
Table 1). Clearly,cytoplasmicallyinherited
markersfromeach parentalpopulationhave
successfullypenetratedthe hybridswarm,
reachingto the boundaries of the overlap
zone (treesH10, H 12, 999, 1994) or beyond
(tree 48).
Tree 48, in particular,representsan interestinganomaly (Fig. 1) in thatit has Fremont mitochondriadespite its nuclear inheritance(all 16 nuclear markersanalyzed
have been narrowleaf;(see Table 1). Moreover, it is geographicallysituated in what
we believed to be a population of pure narrowleaftrees(Fig. 3). The factthatFremont
mitochondriacan be foundin this narrowleaf population suggeststhat some of these
may in factbe complex backcrosses.
DISCUSSION
Mitochondrial
Markers
We have isolated probes froma preparation of DNA purified from organelles
treatedwithDNAse. Probes fromthis G-C
richorganelleDNA identifyonlymulticopy
DNA. Three polymorphisms (restriction
fragment
lengthpolymorphisms)werefound
that distinguishedbetween trees of known
Fremont and narrowleaf nuclear inheritance (for this screen we used DNA from
trees whose nuclear genotypeshave been
tested with between 25 and 30 markers).
These three probes were used to screen a
hybridswarm of cottonwood trees located
between genetically pure populations of
Fremontand narrowleafcottonwoodalong
the Weber River in northernUtah. Every
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1366
KEN N. PAIGE ET AL.
treethatwe have screenedhas had onlyone
cytoplasmic genotype,either Fremont or
narrowleaf.As withmostotherplantspecies
that have been looked at so far (e.g., see
Conde et al., 1979; Schmitz, 1988), no evidence of heterozygouscytoplasmicinheritance was found.Thus, aside fromacquiringa unique tool fortestinghypotheses,these
resultsare of particularimportancein that
we have demonstratedthat mitochondrial
inheritanceis uniparental in cottonwood.
To date, mitochondrialinheritancehas been
established for only a limited number of
plantspecies (Schmitz,1988). Furthermore,
since mitochondriaare maternallyinherited
in mostangiosperms(e.g.,see Schmitz1988;
Stine et al., 1989), it is likely that mitochondria are also maternallyinheritedin
cottonwood (although,as yet,untested).
UnidirectionalIntrogressionand
Nuclear-Cytoplasmic
Incompatibility
In a previous studyit was foundthatnuclear genotypesof individual trees within
the hybrid swarm were consistentwith a
ofnucleargenes
unidirectionalintrogression
fromthe Fremontpopulation into the narrowleaf population (Keim et al., 1989).
There are several possible explanationsfor
the observed distribution.Differencesin
flowering
phenology,forexample,could account for the observed distributionof genotypesin the hybridswarm.This hypothesis, however, appears unlikely because
significantoverlap in phenologieshas been
observed (Keim et al., 1989). Assortative
matingdue to a bias in populationsize (Barton and Hewitt, 1985) is also an unlikely
explanation for the observed asymmetric
distribution of genotypes in the hybrid
swarm. When trees were selected from a
regionof the hybridzone immediatelyadjacent to the Fremont population (where
backcrossingto Fremont should be likely)
no homozygous (FF) alleles were found in
hybridindividuals, suggestingthat hybrid
by hybridcrosses or backcrosses between
hybridand Fremonttreesfailedto produce
viable offspring
(Keim et al., 1989).
Thus, perhapsthe mostparsimoniousexplanationforunidirectionalgene flowis genetic incompatibility.For example, asymmetry in crosses may be due to an
incompatibilitybetween the cytoplasmic
genomeofone parentaltypeand thenuclear
genome of the other (i.e., trees of mixed
genotypeswill be sterileor will not survive
if their cytoplasm is derived from one or
the other parent; Asmussen et al., 1987).
Examples of such a mechanism mightinclude hybrid dysgenesis (as observed in
Drosophila [Kidwell et al., 1977; Bingham
et al., 1982; Engels 1983; Anxolabehere et
al., 1988)] or regulatorysystemdifferences
governing transcription and translation
(Whitt et al., 1977). Our results,however,
show thatbothFremontand narrowleafmitochondrialmarkersare foundin treeswith
mixed nuclear genotypes(in particular,in
treesthat are productsof backcrossingone
or more timesto narrowleafparents).Thus,
nuclear-cytoplasmicincompatibilities do
not appear to account for the asymmetric
distributionofnucleargenotypeswithinthe
hybridswarm.
Alternatively,there may be incompatibilities among nuclear genes that could account forthe asymmetryin the distribution
of nuclear genes withinthe hybridswarm
(Barton and hewitt, 1985). Incompatibilities, forexample, mightarise when homozygous Fremontalleles are introducedinto
a geneticbackgroundcontainingnarrowleaf
alleles. This idea is consistentwith hand
pollinationexperimentsshowingthatbackcrossesto Fremontcan producesome viable
seed, but thatseedingsare developmentally
abnormal,ultimatelydyingat an earlystage.
In contrast,backcrosses to narrowleafresulted in the productionof typical,healthy
(Keim et al., 1989).
offspring
Disequilibria
Cytonuclear
Although, nuclear-cytoplasmic incompatibilities do not appear to explain the
asymmetricdistributionof nuclear alleles
withinthe hybridzone, nonrandom associations between nuclear and cytoplasmic
genotypeswerefoundto exist(usingthedisequilibrium statisticsof Asmussen et al.,
1987). The relative proportionof narrowleaf and Fremont nuclear markers found
withinindividual treesin thehybridswarm
suggeststhat among treeswith a high proportion of heterozygous(NF) loci, nuclear
inheritancemay depend upon cytoplasmic
inheritance.For example,all fourrandomly
selectedF, hybridtreeswere foundto have
Fremont mitochondrialgenotypes(HM-1,
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MITOCHONDRIAL
INHERITANCE PATTERNS
989, 1994, 1997; theprobabilityofselecting
fourF, hybridsall withFremontcytoplasm
by chance alone is only 1 out of 16, a relativelyrareevent).More strikingis a higher
loci
ofheterozygous
thanexpectedfrequency
in those progenyof backcrossesbetweenF,
hybridand narrowleaftreesthat have Fremont mitochondrial genotypes (80% insteadof50%; X2 = 23.40, df= 1,P = 0.000 1).
Two explanations can be proposed forthe
excess of heterozygoticnuclear loci found
in treeswiththeFremontmitochondrialgenotype:It is possible thathomozygousnarrowleafnuclear loci are incompatiblewith
FremontcytoplasmicDNA. This explanation-involving the incompatibilityof nuclear and cytoplasmicgenes-appears unlikely, because Fremont mitochondrial
markersare found in trees with extremely
highproportionsofhomozygousnarrowleaf
nuclear loci (e.g., trees999 and 48). Moreover, these trees do not appear to have
unique combinationsofnuclearalleleswhen
compared to othernarrowleaftrees(see, for
example, Fig. 4 in Keim et al., 1989).
An alternativeexplanation suggeststhat
seeds, seedlingsor treeswith high proportions of heterozygousloci are at a disadvantage unless theyalso have Fremontmitochondria. This explanation is consistent
with the observed distributionof nuclear
and cytoplasmicalleles in F, or firstbackcross progeny.Trees withan equal proportion of heterozygousto homozygous loci
(predictedfora firstbackcross),forexample, are rare. Furthermore,highlyheterozygous trees with Fremont cytoplasm(hybrid F, trees,or backcrosstreessuch as HI
or 198 1) are extremelyrobustand relatively
pest free(see Whitham, 1989; Paige et al.,
1990). This suggeststhatheterosismightbe
expressed when heterozygousnuclear loci
are foundin conjunctionwithFremontmitochondria.If that were the case, progeny
of a singlebackcrosswitha preponderance
of heterozygousloci in Fremontcytoplasm
mighthave a selectiveadvantageovereither
parentaltypewithinthe hybridzone.
These resultsare also consistentwiththe
nuclear-cytoplasmicdisequilibrium statistics of Asmussen et al. (1987) that support
the hypothesisof a strongdirectionalityto
interspecific matings (Fremont females
crossingwith narrowleafmales; assuming
mitochondriaare maternallyinheritedin
1367
cottonwood) and hybridsbackcrossingto
only one of the two parentaltypes(to narrowleaftrees,which is also consistentwith
the resultsof Keim et al., 1989).
HybridZone Dynamics:Evolutionary
Implications
As withnucleargenes,Fremontmaternal
into the narrowleaf
genes are introgressing
population.Previousstudieshave suggested
thattheacquisitionofFremontnucleargenes
intothenarby unidirectionalintrogression
rowleafpopulation may allow these genotypesto become betteradapted to lowerelthe
evations,thehybridswarmrepresenting
vanguard of advancing narrowleafgenotypes (Keim et al., 1989). The acquisition
of Fremontcytoplasmicgenes may also allow thesegenotypesto compete more effectively with Fremont on its home ground,
especially if Fremont mitochondrialgenes
promotehybridvigor.The observedlinkage
disequilibrium between cytoplasmic and
nuclear alleles would also allow the persistence of Fremont nuclear and mitochondrial alleles in the hybridswarm.Thus, the
Fremontcontributionto the hybridpopulation would not be diluted as rapidlyand
could thereforebe of selective advantage
when encroachingFremontterritory.
to inferdyWhile it is generallydifficult
namic processes fromstaticpatterns(Endler, 1977, 1982, 1983; Rand and Harrison,
1989) studies such as ours also enable one
to gain new insightsto thedynamicsofplant
hybridzones. Our results,forexample,suggestthatthe narrowleafpopulation may be
spreading down the canyon and the Fremont population receding.A hybridization
patternof decreasinglycomplex backcrosses as one proceeds fromhigherto lower elevation withinthe hybridswarmwould be
consistentwithsuch a pattern.This appears
to be the case; complex backcrosses,BC3s
and BC4s, for example, are found at high
elevationsiteswithinthehybridswarmand
primarilyBC1s at low elevationsitesnearest
pure Fremont(SC = 0.56, P < 0.0001, N =
40). Thus, the distributionof backcrosses
suggeststhat the Fremontpopulation may
have been prevalentat higheraltitudes in
the past. As the narrowleaf population
spreads and descends (perhapsas the result
of the introgressionof Fremontgenes) the
zone of overlap and the hybridswarmalso
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1368
KEN N. PAIGE ET AL.
descends. Such a patternwould also be consistentwitha residue of Fremontcytoplasmic DNA withinthenarrowleafpopulation,
as we have found.An examinationof several randomlychosen treeswithinthe narrowleafpopulationresultedin thediscovery
of a tree with Fremont mitochondriadespite a nuclear inheritancepatternof narrowleaf (i.e., tree 48; Figs. 2, 3). We are
presentlyseekingfurthersubstantiatingevidence by examiningtrees withinthe narrowleaf population for Fremont cytoplasmic inheritance and by searching for
additionalindependentevidence oftheprevious existence of Fremont at higherelevations (e.g., a pollen profile).
T. G. Whitham,NIH grantES-01498 to K.
G. Lark and a Biomedical Research Support
Grant S07 RR07092 to K. N. Paige. This
researchwas also supportedby NSF grants
BSR-8907686 to T. G. Whithamand K. N.
Paige, BSR-8917556 to K. N. Paige, and
Biomedical Research Support Grant NIH
RR-7030 to K. N. Paige. We thank K. G.
Lark and T. G. Whitham fortheirhelp in
startingthe project and for comments on
the manuscript.We also wish to thank G.
Fullerand J.Power fortechnicalassistance.
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