ecological determinants of genetic diversity in an expanding population of the shrub myrica cerifera

10
Molecular Ecology (2004) 13, 1655–1664 doi: 10.1111/j.1365-294X.2004.02139.x © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. Ecological determinants of genetic diversity in an expanding population of the shrub Myrica cerifera DAVID L. ERICKSON,* J. L. HAMRICK †‡ and GARY D. KOCHERT *Laboratory of Analytical Biology, Smithsonian Institution, 4210 Silver Hill Road., Suitland, MD 20742, USA, Department of Plant Biology, Department of Genetics, University of Georgia, Athens, GA 30602, USA Abstract The ecological mechanisms that contribute to the acquisition of genetic diversity in an expanding population of the shrub, Myrica cerifera, on an island habitat were investigated. Genealogical reconstruction was used to assess the contribution of early reproductive colonists to subsequent recruitment. In addition, through determination of parentage, the source of recruiting seedlings was identified and the contribution of seed and pollen dis- persal into the colonizing sites was inferred. The relative contribution of different sources of gene flow was determined directly and an investigation was made into how variability in breeding patterns may have contributed to observed levels of genetic variability. It was expected that early colonists that could flower would contribute to subsequent recruiting cohorts, and that the limited number of such early reproductive colonists would lead to variance in mating success, inbreeding, or bottlenecks which could reduce genetic diversity and increase genetic differentiation among subsequent recruiting cohorts. Analyses of par- entage (with paternity exclusion probability > 95%) for all recruiting plants demonstrated that in fact, there was little contribution by the early reproductive colonists to subsequent cohorts, and that immigration from outside the study sites in the form of seed dispersal accounted for over 94% of the recruitment in the study plots, with pollen dispersal account- ing for less than 3% gene flow. No genetic bottleneck or evidence of reproductive skew in the recruiting cohorts were found, suggesting that propagule dispersal was from many source individuals in other established populations. Keywords: colonization, gene flow, genetic diversity, maternity, paternity Received 9 September 2003; revision received 6 January 2004; accepted 6 January 2004 Introduction The genetic structure of populations is of increasing interest to ecologists, particularly as researchers combine elements of evolutionary biology with ecology (Frankham 1995; Hamrick & Nason 1996; Lee 2002). The distribution of genetic variation within and among populations is import- ant because it can provide insights into the history of a population, and because the current levels and distribution of genetic variation can influence the future success of populations. Ultimately it is the mechanisms of ecology — dispersal, mating patterns and demographic change — that determine the genetic composition and structure of popu- lations and species (Loveless & Hamrick 1984; Ingvarsson 1997; Schnabel et al . 1998). Consequently, documenting the ecological mechanisms that affect genetic structure and variation is of importance to ecologists and evolutionary biologists. However, investigations into the distribution and levels of genetic variation often treat the observed pattern as a static quantity. Thus, how populations arrive at observed levels of genetic diversity is often unknown. This is particularly true with regard to the contribution of specific ecological determinants to the acquisition of genetic diversity. While it is possible to infer events such as bottlenecks or strong selection in the history of a popu- lation (Wade & McCauley 1988; Irvin et al . 1998; Leblois et al . 2000), reconstructing the ecological contributions of such phenomena as variance in mating success, the relative contribution of different sources of gene flow and inbreed- ing among related individuals, are often not investigated or are difficult to determine. It is therefore important to Correspondence: David L. Erickson. Fax: 301 238 3059; E-mail: [email protected]

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Page 1: Ecological determinants of genetic diversity in an expanding population of the shrub Myrica cerifera

Molecular Ecology (2004)

13

, 1655–1664 doi: 10.1111/j.1365-294X.2004.02139.x

© 2004 Blackwell Publishing Ltd

Blackwell Publishing, Ltd.

Ecological determinants of genetic diversity in an expanding population of the shrub

Myrica cerifera

DAVID L . ERICKSON,

*

J . L . HAMRICK

†‡

and GARY D. KOCHERT

*

Laboratory of Analytical Biology, Smithsonian Institution, 4210 Silver Hill Road., Suitland, MD 20742, USA,

Department of Plant Biology,

Department of Genetics, University of Georgia, Athens, GA 30602, USA

Abstract

The ecological mechanisms that contribute to the acquisition of genetic diversity in anexpanding population of the shrub,

Myrica cerifera

, on an island habitat were investigated.Genealogical reconstruction was used to assess the contribution of early reproductivecolonists to subsequent recruitment. In addition, through determination of parentage, thesource of recruiting seedlings was identified and the contribution of seed and pollen dis-persal into the colonizing sites was inferred. The relative contribution of different sourcesof gene flow was determined directly and an investigation was made into how variabilityin breeding patterns may have contributed to observed levels of genetic variability. It wasexpected that early colonists that could flower would contribute to subsequent recruitingcohorts, and that the limited number of such early reproductive colonists would lead tovariance in mating success, inbreeding, or bottlenecks which could reduce genetic diversityand increase genetic differentiation among subsequent recruiting cohorts. Analyses of par-entage (with paternity exclusion probability > 95%) for all recruiting plants demonstratedthat in fact, there was little contribution by the early reproductive colonists to subsequentcohorts, and that immigration from outside the study sites in the form of seed dispersalaccounted for over 94% of the recruitment in the study plots, with pollen dispersal account-ing for less than 3% gene flow. No genetic bottleneck or evidence of reproductive skew inthe recruiting cohorts were found, suggesting that propagule dispersal was from manysource individuals in other established populations.

Keywords

: colonization, gene flow, genetic diversity, maternity, paternity

Received 9 September 2003; revision received 6 January 2004; accepted 6 January 2004

Introduction

The genetic structure of populations is of increasinginterest to ecologists, particularly as researchers combineelements of evolutionary biology with ecology (Frankham1995; Hamrick & Nason 1996; Lee 2002). The distribution ofgenetic variation within and among populations is import-ant because it can provide insights into the history of apopulation, and because the current levels and distributionof genetic variation can influence the future success ofpopulations. Ultimately it is the mechanisms of ecology —dispersal, mating patterns and demographic change — thatdetermine the genetic composition and structure of popu-lations and species (Loveless & Hamrick 1984; Ingvarsson

1997; Schnabel

et al

. 1998). Consequently, documenting theecological mechanisms that affect genetic structure andvariation is of importance to ecologists and evolutionarybiologists. However, investigations into the distributionand levels of genetic variation often treat the observedpattern as a static quantity. Thus, how populations arriveat observed levels of genetic diversity is often unknown.This is particularly true with regard to the contributionof specific ecological determinants to the acquisition ofgenetic diversity. While it is possible to infer events such asbottlenecks or strong selection in the history of a popu-lation (Wade & McCauley 1988; Irvin

et al

. 1998; Leblois

et al

. 2000), reconstructing the ecological contributions ofsuch phenomena as variance in mating success, the relativecontribution of different sources of gene flow and inbreed-ing among related individuals, are often not investigatedor are difficult to determine. It is therefore important to

Correspondence: David L. Erickson. Fax: 301 238 3059; E-mail:[email protected]

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© 2004 Blackwell Publishing Ltd,

Molecular Ecology

, 13, 1655–1664

document and quantify not only the levels and distribu-tion of genetic diversity in natural populations but alsoto understand the processes that generate the observedpatterns through reconstructing the acquisition of geneticdiversity.

A powerful tool in the reconstruction of the ecologicaldeterminants of genetic structure is genealogical recon-struction, or maternity and paternity analyses. Since theirdevelopment, analyses of maternity and paternity havedemonstrated the extent and magnitude of gene flow innatural populations (Meagher 1986; Broyles

et al

. 1994;Aldrich

et al

. 1998; Schnabel

et al

. 1998), the uncoupling ofdemographic and genetic patterns as a result of inbreedingand variance in fertility (Aldrich & Hamrick 1998), as wellas the contribution of different sources of gene flow, as inthe relative contribution of seed and pollen dispersal inplant populations (Dow & Ashley 1996). Yet the applica-tion of genealogical reconstruction has been limited to theexamination of phenomena within established populationsor population fragments (Dow & Ashley 1996; White

et al

.2002), and has not been used to investigate how geneticdiversity arrives in a population (although see Aldrich &Hamrick 1998). In addition, genealogical reconstruction istypically made for a single year or population, and thusthe temporal dynamics of paternity and maternity remainlargely unknown. The application of genealogical recon-struction to environments where recruitment is activeshould shed light on the ecological mechanisms that medi-ate the acquisition of genetic variation in populations.

In studies of genetic structure in plants, there have beensome efforts to reconstruct the ecological determinants ofgenetic structure, as in the relative contributions of seedand pollen dispersal in plant populations (Ennos 1994; ElMousadik & Petit 1996). The results of such investigationsdemonstrate that there is far less genetic structure (

F

ST

) atnuclear gene markers than at cytoplasm-based markers (i.e.chloroplast DNA), suggesting the predominance of pollengene flow relative to seed-mediated gene flow. However,these results are historical, and do not represent a contem-porary examination of population founding or expansionand hence suffer from the possibility that gene flow sub-sequent to establishment obscures patterns early in thepopulations’ history.

In this study a population was examined that wasactively expanding into new habitat, where it was possibleto examine the process of colonization as it occurred. Inparticular, the shrub

Myrica cerifera

was investigated as itinvaded newly formed island habitat. Active recruitmentwas observed throughout the study period, and earlycolonists were also observed to flower at a young age(3 years). Early established colonists, or those present whenthe study began, were considered to be active participantsin the recruitment process by mating either with each otheror with plants in other areas of the population to generate

subsequent recruiting cohorts. Our expectation was thatthere would be the opportunity for an initial bottleneckin genetic diversity within cohorts as a result of matingamong a limited number of early colonists. Levels of geneticdiversity were expected to correlate with demographicchange such that increases in population size would resultin increases in genetic diversity within the colonizingstudy sites. Within our study sites not only was the patternof genetic variation investigated, but changes in geneticdiversity were tracked over time to reflect the dynamicprocess of colonization. A suite of genetic markers wasused, in conjunction with genetic analyses to deduce theecological mechanisms that mediated the observed pat-terns of genetic change, and in particular, genealogicalreconstruction was used to infer patterns of seed and pol-len dispersal as contributors to gene flow among recruitingcolonists. It was further possible to reconstruct matingpatterns, such that deductions could be made on the extentto which inbreeding and variance in fertility among repro-ductive colonists contributed to patterns of the acquisitionof genetic diversity within recruiting cohorts. In this way,it was possible document the ecological processes that con-tribute to the acquisition of genetic diversity in an expand-ing population.

Materials and methods

Study species

Myrica cerifera

L. (Myricaceae) (or also

Morella cerifera

) isa dioecious evergreen shrub that may reach 7–10 m inheight, and is commonly found in coastal or disturbed inlandsites throughout the southeastern United States (Radford

et al

. 1968). It flowers from April to May, and its minuteflowers are clustered into small, 5–10-mm long catkins insimilar fashion to other members of the oak superfamily(Fagales) of which it is a member (Manos & Steele 1997).It is probably wind pollinated as most members of Fagalesare wind-pollinated and because no adaptations for insectpollination are apparent. The fruits of

M. cerifera

are small,

4-mm diameter, single-seeded drupes that are coveredby a resinous waxy substance from which the species takesits common name, wax myrtle. Fruits mature in early autumnand are eaten by migratory bird species, particularly

Dendroica coronata

, suggesting that long-distance, animal-mediated seed dispersal is possible. If not eaten, the fruitstypically remain attached to the plant, facilitating seedcollection, although germination rates rapidly decline after9 months (Erickson personal observation).

Study site

The study site was Hog Island, a member of the VirginiaBarrier Island system, and part of the Virginia Coast Reserve

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, 13, 1655–1664

Long-Term Ecological Research site (VCR LTER) (Fig. 1).It lies approximately 11 km off the coast of Virginia, USA,within a chain of similar islands that punctuate the easterncoast of the USA. Hog Island, like all barrier islands, ismobile (Hayden

et al

. 1995), with a current northeastwardmovement of

6 m/year. Constant accretion of sand resultsin new habitat being washed ashore, as well as a constantdune-building process, which provides the habitat intowhich colonization may occur.

Myrica cerifera

is a commonspecies on the island (Young

et al

. 1995). The high frequencyof storm disturbance and the low elevation of the islandinhibit the invasion of classical maritime forest species butdo not significantly deter the growth of

M. cerifera

.Three study plots were established, termed T1, T2 and

T3, at the eastern edge of the island (150 m from thehigh-tide line), where

M. cerifera

was beginning to establish

(Fig. 1). Few plants were present when the plots wereestablished, with the expectation that colonization over theduration of the study would increase population size. Aparallel set of plots was established even closer to the coast-line (

50 m from the high-tide line), in a zone of vegetationdominated by grasses and where no

M. cerifera

seedlingswere present at the beginning of the study, but no seed-lings were observed to establish in those sites during thecourse of our study.

Study design

Plot size in the colonizing sites was 20 m

×

20 m. In 1995,42, 56 and 39 shrubs were tagged in colonizing plots T1,T2 and T3, respectively (Table 1). In addition to the 20 m

×

20 m plots, a 10-m buffer zone was established around eachof the three colonizing sites in 1996, thereby expanding eachof the 20 m

×

20 m core plots to 40 m

×

40 m. The bufferzone was established to increase the scale at which matingcould be inferred among colonists that might contributeto colonization. Only individuals that flowered as of 1996were collected within the buffer zone. Seedling recruitsused in the analysis of parentage and genetic diversityfrom the 1996 and 1997 cohorts were collected only fromthe initial 20 m

×

20 m core plots. Tissue samples were notcollected until the 1996 field season, since sampling ofplants from the 1995 cohort could have resulted in mortal-ity, hence altering the natural patterns of reproduction andrecruitment. Therefore collections were made in Septemberof 1996 and 1997 and the samples were returned to the Uni-versity of Georgia for analysis. The numbers of individualscollected from the three core plots at each study site, aswell as the number of individuals collected in the bufferzones, are given in Table 1.

1 2 3 4 5

Fig. 1 Field map of Hog Island. The upper left inset locates thefield site to the mid-eastern seaboard of the United States. Theupper left inset shows the chain of barrier islands off the coast ofthe Maryland–Virginia coastline, of which Hog Island is a memberand the central inset shows the shape of Hog Island with thedistribution of Myrica cerifera and the location of the field sites onthe Island.

Table 1 The number of individuals collected from the threecolonizing sites

PlotCore plot(≤ 1995)

Buffer(≤ 1995) Males Females 1996 1997 Total

T1 46 35 27 26 39 118 238T2 56 175 107 91 13 6 250T3 37 30 31 28 9 108 184Total 139 240 165 145 61 232 672

Numbers from the < 1995 cohort represent those individuals that survived from 1995 to 1996. The buffer zones were established in 1996 and represent only those individuals that were flowering at that time. The 1996 and 1997 cohorts represent all newly arrived individuals within the core plot in the intervals of 1996–97 and 1997–98, respectively. Males and females are the number of flowering males and females within the core and buffer plots of each site.

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Molecular Ecology

, 13, 1655–1664

Genetic markers

Each tissue sample was returned to the University ofGeorgia and prepared for allozyme analysis and DNAextraction. Protocols for allozyme extraction and analysisfollow the procedures of Mitton

et al

. (1979), with tissuesamples crushed in an enzyme extraction buffer (phosphate–PVC buffer as in Mitton

et al

. 1979) and separated on 10%starch gels for 3–5 h. Five polymorphic allozyme loci wereused: phosphoglucose-isomerase (Pgi), phosphoglucose-mutase 1 and 2 (Pgm1 and Pgm2), shikimic dehydrogenase(Skdh), and diaphorase (Dia). Fifteen other loci were screenedfor polymorphism but showed little or no variation or werenot interpretable. The five allozyme loci did not have sufficientvariation for use in genealogical reconstruction, so a set ofmicrosatellite markers was developed to increase the re-solution of genetic variability within the population and toallow for high levels of discrimination among individuals.

DNA for microsatellite analysis was extracted fromsamples by one of two methods. The first was a modifiedversion of the standard centyltrimethylammonium bro-mide (CTAB) extraction method of Doyle & Doyle (1990),in which 0.5 g of ground tissue is mixed with 750 mL ofextraction buffer (0.2

m

Tris–HCl, 0.05

m

ethylenediamine-tetraacetic acid, 2

m

NaCl, 1% polyvinylpyrolidone (PVP))and 0.1 mL of 10% Sarcosyl, followed by incubation at65

°

C for 30 min. Contaminants are removed with CVAG(24 : 1 chloroform : isoamyl alcohol) after which the DNAwas precipitated by incubation in isopropanol at

20

°

Covernight. The resulting DNA was used directly in polymer-ase chain reactions (PCR) as described below. The secondmethod was the Plant-Mini DNA

©

extraction kit by Qiagen,which was particularly successful at removing excess

carbohydrates and secondary chemicals from the DNAsample. Samples prepared by both methods producedadequate, repeatable results, although the latter was faster.

Following extraction, DNA samples were used in PCRamplification of the three microsatellite markers. Ampli-fication conditions were identical for all primers as follows:one cycle of 5 min at 95

°

C, 40 cycles of 33 s at 94

°

C, 33 s at54

°

C and 1 min at 70

°

C, followed by one cycle of 5 min at70

°

C. Thirty seconds was added to the extension time foreach additional primer pair added, thus for amplificationof all three primers together the 70

°

C extension timewould be 2 min. The PCR reaction buffer used was: 1

×

PCRbuffer (supplied by Perkin Elmer), 1.8 m

m

MgCl

2

, 0.1 m

m

dNTPs (or 0.25 m

m

if multiplexing two primers and0.3 m

m

if multiplexing with three primers), 0.75 U Ampli-taq Polymerase from Perkin Elmer, 30 n

m

each for 3

and 5

primers (with the 3

primer labelled with

γ

32

P) and H

2

O toa final volume of 0.02 mL, with approximately 50 ng DNAas template. After PCR, alleles were visualized by poly-acrylamide gel electrophoresis on 5.5% acrylamide (28.5 : 1.5acrylamide : bisacrylamide) in a 1

×

Tris-Borate-EDTA (TBE)buffer for 3–4 h (depending upon the locus) and thenexposure to BioMax MR X-ray film (Kodak) for 1–3 days asnecessary. Development of the microsatellites followedAldrich

et al

. (1998). Variability of all markers employed issummarized in Table 2.

Analysis of parentage

Analysis of parentage within the plot was conducted usingthe software package

cervus

and is described in detail byMarshall

et al

. (1998). The program assigns parentage toa most likely parent based upon Log of the Odds (LOD)

LocusNo. of alleles HE PIC

First parent exclusion

Second parent exclusion

MicrosatellitesD2 8 0.812 0.786 0.455 0.631L6 8 0.829 0.805 0.481 0.655E8 8 0.823 0.797 0.468 0.643Totals (means) 24 (0.825) (0.794) 0.850 0.955AllozymesPGM1 2 0.156 0.144 0.012 0.072PGM2 2 0.170 0.155 0.014 0.078PGI 2 0.041 0.040 0.001 0.020DIA 2 0.067 0.060 0.002 0.032SKDH 2 0.049 0.047 0.001 0.024Totals (means) 10 (0.095) (0.057) 0.030 0.208Overall totals 34 0.855 0.964

Expected heterozygosity (HE), polymorphic information content (PIC), and exclusion probabilities for single-parent analysis (maternity analysis), and second parent analysis when one parent is known (paternity analysis) are included. Total values represent pooled values for HE, PIC, first and second parent exclusion probability. The number of alleles is a sum of all alleles present at the three microsatellite and five allozyme loci, respectively.

Table 2 Summary of genetic diversity at eachlocus

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scores following the methods of Meagher (1986). Theprogram assesses parentage in three steps. The first stepin the analysis is a summary of the allele frequency datafrom the parents and progeny to be analysed. The secondstep is a simulation of parentage based upon the observedallele frequency data that are used to define a confidencelimit in assignment of parentage. At this second step, parentalexclusion probabilities are calculated [with the specificformulae in Appendix 2 of Marshall

et al

. (1998)] and are basedon the distribution of allele frequencies in the population.Different exclusion probabilities are calculated based uponwhether the analysis seeks to identify a single parent withonly the offspring genotype to work from, as well aspaternity exclusion where maternal and offspring genotypesare known. Both the maternity (single parent) and paternity(second parent) exclusion probabilities calculated from ourallele frequency data are given in Table 2. The third step isthe estimation of LOD scores for the set of parents andoffspring specified and assignment of parentage. The methodof assignment of a most likely parent has been criticized(Devlin & Ellstrand 1990) because of the increased likelihoodof assignment to homozygous individuals. This problemis largely circumvented by the relatively high number ofalleles (three loci each with eight alleles) and high levels ofheterozygosity (82.5%) of the microsatellite loci resultingin a majority of the parentage assignment coming fromcomplete exclusion of alternate parents. Since the specieswas dioecious, the number of possible parents was effectivelyhalved relative to a hermaphroditic plant species.

The analysis of parentage in

cervus

may be appliedwhere neither parent is known (single-parent analysis ora maternity analysis) or where one parent of the offspringis known (as in a paternity analysis). It was decided toanalyse all candidate mothers within the colonizing plotfirst and then to link identified mother–offspring pairs withmales through paternity analysis. The high exclusion prob-ability (which exceeded 86% for single-parent analysis andover 96% for paternity analysis) should readily distinguishamong the limited set of candidate maternal parents. Byidentifying maternal parents first any problems associatedwith parent-pair analysis, which generates ambiguity inthe assignment of parentage as a result of the large numberof parent pairs that must be tested (Schnabel

et al

. 1998),were avoided. Thus maternal plants were identified withinthe plots and then both maternal and progeny genotypeswere used to test paternity among males.

Genetic diversity analysis

Three measures of genetic diversity were used, expectedheterozygosity (

H

E

), mean number of alleles per locus(

A

) and polymorphic information content (PIC). Expectedheterozygosity was calculated as the proportion of indi-viduals that will be heterozygous given the assumptions of

Hardy–Weinberg:

H

E

= 1

, where

p

i

is the frequencyof the

i

th allele at a locus. A single value of

H

E

was used todescribe the mean expected level of heterozygosity forthe entire population at all loci scored. In this analysiscolonizing cohorts of the same age from each plot weregrouped together for analysis, thus all the individuals thatarrived in 1997 in all three plots were grouped togetherand were compared to all individuals from 1996 and 1995.It was therefore possible to compare estimates of geneticdiversity among cohorts and among sample sites. Micro-satellite and allozyme data were grouped separately becauseof their inherently different levels of allelic variability. Thesecond diversity measure employed is the sum of the allelespresent in a given cohort at all eight loci scored. PIC is ameasure of informativeness for one or more loci and isanalogous to paternity exclusion probability where the greaterthe number of alleles at a locus and the more even theirfrequency, the greater the PIC value (Botstein

et al

. 1980;Hearne

et al

. 1992). PIC was calculated as the arithmeticaverage of the PIC values at each locus. PIC was calculatedin a similar manner to

H

E

and

A

, where individuals from agiven year’s cohort were bulked across plots for comparisonand microsatellite and allozyme data were treated separately.

Results

Demography

Three distinct recruiting cohorts were identified for thisstudy: 1995 and earlier (referred to as

1995 since thiscohort included individuals that were present when thestudy began and thus arrived prior to 1995), 1996 and 1997(Fig. 2). A total of 377 individuals from the

1995 cohort(137 from the core plots and 240 from the buffer zones), 61individuals from the 1996 cohort, and 232 individuals fromthe 1997 cohort were mapped and sampled over the courseof the study (Table 1). Recruitment in plots T1, T2 and T3for all 3 years and in both the core plot and buffer zone isshown in Fig. 2. For the purpose of measuring geneticdiversity, all individuals in the core plot were genotypedand included in the analyses. Individuals from the bufferzones were not included in the genetic diversity analysessince it would exaggerate the sample size of the

1995cohort relative to the 1996 or 1997 cohorts, which were onlysampled from the core plots. Only flowering individualsfrom the core and buffer zones were included in theparentage analyses. Thus in 1996 there were 20 floweringindividuals in plot T1, 41 in T2, and 25 in T3. In the bufferzones of T1, T2 and T3 there were 33, 157 and 34 floweringindividuals in 1996. The number of flowering males andfemales was determined in the spring of 1996, where thenumber of females and males roughly followed theexpected 50 : 50 (Table 1). It was assumed that individualsflowering in 1996 had also flowered in 1995, therefore the

pi2

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same group of potential parents was used to analyse par-entage for the 1996 and 1997 cohorts. Individuals floweringin the spring of 1996 would have produced seed thatmatured in the autumn of 1996 and become establishedin late spring–early summer of 1997. The number of indi-viduals in plots T1 and T3 more than doubled during the1997 recruitment season, whereas, very few recruits (3% oftotal recruitment of 1997) arrived in the T2 site despite itshigh number of flowering individuals. The T2 site hadnearly four times the number of flowering individuals aseither T1 or T3, yet it is the latter two that shared 97% of the1997 colonizing cohort.

Genetic diversity

There was little difference in

H

E

and PIC among cohortsand no difference in the total number of alleles presentamong cohorts for the eight loci analysed (Table 2). Fur-thermore, there was little difference in the frequency ofthe most common allele or three most common alleles ateach locus among cohorts (Table 3), suggesting a randommixing of the propagule pool from which the colonistswere derived. Essentially all of the genetic diversity presentin the population appears to have arrived by the time ofsampling the 1995 cohort. Doubling the number of indi-viduals present within the T1 and T3 sites between 1995 and1997 had no effect on the levels of genetic diversity present.Likewise, there was little or no difference in heterozygosity,PIC, or number of alleles present among the three colonizingsites. The three microsatellites had a maximum of eightalleles each, which is a low level of diversity for the markertype, and no allozyme marker had more than two alleles.Yet the low level of genetic diversity observed at the markerloci did not affect the spatial distribution of genetic diversityamong the colonizing sites on the island, with recruitmentdrawing upon a homogeneous propagule pool.

Parentage

Seedlings from the 1996 and 1997 cohorts were subjected toanalyses of parentage (Table 4) to determine their source.Through genealogical reconstruction and the exclusionof parentage for recruiting seedlings it was possible todetermine that seed immigration occurred when a maternalparent for a seedling could not be identified; and pollengene flow was inferred when a maternal parent withinthe site was identified but a male parent was not. Lastly,individuals that were derived from mating among flowering

Fig. 2 Scatter plot of the three sites, T1, T2 and T3, showing thenumber and distribution of individuals at three stages. Eachcolumn represents the sites at three stages of sampling. The firstrow shows individuals collected in the < 1995 (�) and 1996 (�)cohorts. The second row shows the number and distributionof individuals sampled from the buffer zone that were collectedin 1996. The last row shows the number and distribution ofindividuals collected from the 1997 cohort.

Table 3 Comparison of genetic diversity among the three colonizing cohorts, combined (pooled) across all three colonizing sites. Alsopresented, are data from the established population for comparison with the colonizing sites

Cohort HE (SD) PICNo. of alleles

Three most common alleles

≤ 1995 microsatellites 0.796 (0.012) 0.756 23 0.667(n = 124) allozymes 0.061 (0.016) 0.057 10 na1996 microsatellites 0.823 (0.018) 0.785 20 0.688(n = 61) allozymes 0.051 (0.019) 0.041 8 na1997 microsatellites 0.823 (0.009) 0.797 21 0.667(n = 232) allozymes 0.082 (0.014) 0.077 10 naIsland microsatellites 0.833 0.801 21 0.667(n = 51) allozymes 0.088 0.083 10 na

Data on microsatellites and allozymes are summarized separately. HE is expected heterozygosity, PIC is polymorphic information content averaged for all loci, and no. of alleles is the total number of alleles present in that cohort for all loci. Standard deviations of HE, calculated via bootstrapping, are shown in parentheses with the mean. The three most common alleles is the combined frequency of the three most common alleles at the locus with the highest heterozygosity; this value is not calculated for allozymes since only two alleles were present at any locus.

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colonists within the study sites were not considered asgene flow events, but were analysed for their effect oninbreeding or kin structure depending on the frequency ofsuch mating as reflected in the population of the recruitingcohorts. With an 80% confidence applied to parentage, 10seedlings (6%) from T1, nine seedlings (7.7%) from T3 andno seedlings from T2 were assigned to flowering femaleplants within the colonizing sites. Parent pairs within thecolonizing sites were identified for seedling recruits onlytwice, once in T1 and once in T3. Thus a majority of theseedlings with maternal plants in the colonizing plots wasderived from pollen gene flow into the sites. Calculationof total per cent gene flow was based upon the haploidcontribution, such that seed gene flow counted twice thatof pollen gene flow. Thus the total rate of gene flow into thethree sites was very high (Table 4).

There was an even distribution of maternity amongmaternal plants that were identified at the 80% confid-ence level for parentage. Of the 19 seedlings that wereassigned to maternal plants within all three sites combined,15 (79%) had unique mothers and only one maternalplant was assigned parentage to more than two seedlings.Thus there was little variance in reproductive successamong individuals contributing to recruitment withinthe colonizing sites. This eliminates the possibility of agenetic bottleneck caused by over-representation of asingle parent in the 1996 or 1997 recruiting cohorts. Overall,the population exhibited low levels of genetic diversityrelative to plant species with similar breeding and life-history characters, yet this paucity of genetic variation couldnot be attributed to a genetic bottleneck within the studysites, nor was evidence uncovered for inbreeding or variancein mating success.

Discussion

The acquisition of genetic diversity within populations isrelevant to their ecology and evolution. An investigationwas made into how genetic diversity arrives within colo-nizing segments of a growing population of the shrub,Myrica cerfera, as it invaded new habitat on an islandenvironment. A description was sought of the process bywhich genetic variation is introduced to the new colonizingsites, in addition to describing the patterns of acquisition.Thus genealogical reconstruction was used to deduce therelative contribution made by seed and pollen dispersalto the recruiting colonists, as well as the contribution ofmating patterns such as inbreeding and variance in matingsuccess to the acquisition of genetic diversity.

Genetic diversity in colonizing sites

A rapid acquisition of genetic diversity was seen within thethree study sites. All three study sites were established in1995, with varying numbers of pre-1995 colonists alreadypresent. One site was chosen that had a large number ofearly colonists (T2) many of whom were flowering, as ahedge against the possibility that little recruitment wouldoccur without a large number of flowering individualswithin the plots. However, recruitment was most active inthose sites with low numbers of individuals present inthe 1995 cohort (sites T1 and T3, Table 1). This increasein recruitment coincided with the onset of flowering amongearly colonists of the 1995 cohort in site T1 and T3. Thenumber of individuals flowering was observed to almostdouble in the T1 and T3 sites between 1995 and 1996, andcould therefore provide a mechanism that accounted for

Table 4 Parentage and gene flow for the 1996 and 1997 seedling cohorts from the three study plots T1, T2 and T3 analysed at a significanceof 80%

Plot Cohort RecruitsSeed dispersal

Female parents

Pollen dispersal

Male parents

Seed gene flow

Pollen gene flow

Total gene flow

T1 1996 39 38 1 1 0 97.4% 1.3% 98.7%1997 118 109 9 8 1 92.4% 3.4% 95.7%

T2 1996 13 13 0 0 0 100% 0% 100%1997 6 6 0 0 0 100% 0% 100%

T3 1996 9 8 1 1 0 88.8% 5.6% 84.4%1997 108 100 8 7 1 92.6% 3.2% 95.8%

Total 293 274 19 17 2 93.5% 2.9% 96.4%

Recruits represent the number of seedling recruits collected for each cohort in each study plot. Seed dispersal is the number of seedling recruits with no female parent within the plots, including the buffer zone. Female parent(s) is the number of female plants that have a seedling recruit assigned to it through maternity analysis. Pollen dispersal is the number of seedlings identified to have a maternal parent within the study site that was derived from pollen gene flow into the study site. Male parents are the number of male parents that were identified through paternity analysis based on mother offspring genotype. Seed gene flow is the number of seeds derived through seed dispersal divided by the number of seedling recruits. Pollen gene flow is the number of seedlings derived from pollen dispersal divided by twice the number of seedling recruits (to account for the haploid genome contribution from pollen).

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the increased recruitment in the 1996 and 1997 cohorts. Itwas anticipated that mating among flowering colonists, aswell as pollen gene flow into the colonizing sites, wouldresult in an increased number of seeds produced withinthe sites, that would in turn contribute to in situ recruit-ment. With active recruitment, it was also anticipated thatthere would be an increase in the genetic diversity withinthe sites, as well as a reduction of genetic differentiationamong colonizing sites. However, the results differed fromexpectations. The earliest cohort examined, the 1995 cohort,had equivalent levels of genetic diversity to subsequentcohorts, as well as to the extant population on the island.Furthermore, there was little differentiation among 1995cohorts from the three sites, with similar numbers of allelesand levels of heterozygosity in each 1995 cohort (Table 3).Thus although the 1995 cohorts were small in two sites, T1and T3, they did not exhibit evidence of a founder effect.This suggests that the seed pool from which they werederived is relatively uniform, and that there is littlevariance in the genetic structure of the propagule pool.Had colonists for a given cohort or site all shared aparent (maternal or paternal) this would result in significantgenetic structuring among cohorts or colonizing sites,which was not observed. Thus although there are examplesof increased kin-structured dispersal for plants withbird-dispersed seed (Furnier et al. 1987; Bruederle et al.1998), there is no evidence of this in our experiment,suggesting that dispersal of seed is random throughoutthe population.

Patterns of paternity and maternity

Studies that have employed genealogical reconstructionhave often demonstrated a high degree of variability inmating success among individual plants. It is not un-common for a small set of reproductive individuals tocontribute disproportionately to the recruitment of newindividuals and for mating to be more common amongclosely spaced individuals (Aldrich & Hamrick 1998; Dow& Ashley 1996). The consequence of such mating patternscan be high levels of genetic structure, with significantdifferentiation within populations or among sites wheredifferent sets of parents contribute to recruitment. Thisgenetic structuring might also be seen among cohorts thatare temporally separated, as a result of a shift in the set ofcontributing adults. Such differentiation may break downover time as a result of continued gene flow, but shouldresult in the fine-scale genetic structuring that is oftenobserved in natural populations.

Examination of patterns of reproduction through theuse of genealogical reconstruction allowed for the identi-fication of the ecological mechanisms that produced theobserved patterns of genetic diversity. In the sites showingthe greatest recruitment (T1 and T3), parentage analyses for

newly established seedlings demonstrated that a majorityof the seedlings had neither a maternal nor paternalparent present within the site (Table 4). Of the 293 recruitsin the 1996 and 1997 cohorts, less than 1% was derived frommating among colonists within the study plots, and withonly two seedling recruits having male parents presentwithin the colonizing sites. Thus nearly all seedling recruit-ment was derived from seed dispersal. Only 3% of all geneflow into the colonizing sites was attributable to pollengene flow, where pollen from outside the study sites polli-nated a female plant within the site. Had the species notbeen dioecious, the contribution of males from within thecolonizing site might have been higher because of self-pollination, but wind pollination produced a large, andapparently uniform, pollen pool that masked the smalleramount of pollen produced by resident males in the colon-izing plots. Overall, variability in mating success amongearly colonists was of limited importance since only 6.5%of the seedling recruits were derived from female parentswithin the study plots. However, when maternal plantswithin the study site were identified for seedling colonists,the majority of the successful pollen originated from out-side the site. Thus only 21 (3.6%) of the 586 gametes repre-sented in the 1996 and 1997 seedling cohorts originatedwithin the study sites (Table 4).

While studies of paternity and maternity routinelyidentify significant variance in reproductive success, noevidence of this was observed, either among the colonistsor among the seed pool from outside the colonizing sites,which would manifest itself through genetic differenti-ation among colonizing sites or among cohorts. Two factorsmay affect this result, one is methodological and relatedto analysis by the program cervus and the other is relatedto the natural history of the species. cervus is based uponmethods of Meagher (1986) although methods of genealo-gical reconstruction are employed (Meagher & Thompson1987; Prodohl et al. 1998; see Oddou-Muratorio et al. 2003for a review of methods and issues). In our analysis, therewas a high power of resolution to identify potential par-ents of seedlings, with a maternal exclusion probability ofover 86% and a paternity exclusion (when consideringmaternal and offspring genotypes jointly) of over 96%. Byanalysing the male and female parents separately therewas greater power to identify parents than had a parent-pair analysis been conducted, which have a lower power ofresolution when the number of offspring is large relative tothe number of parents (Schnabel et al. 1998). cervus alsodetermines a threshold LOD score as a confidence estimatethat the most likely parent is the true parent. While this canlead to exclusion of all parents when the number of parentsis large and the difference in LOD scores between them issmall, when the number of parents is relatively small, thismethod provides greater certainty that the most likely par-ent is the true parent. Both a strict (95%, not reported) and

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a relaxed (80%, Table 4) confidence was used for parentageand it was found that at high confidence very few femaleand no male plants within the study sites contributed torecruitment. The 80% estimate of confidence was chosen,which identified more female and male parents as a way ofshowing the distribution of male and female contributionto recruitment.

The high levels of seed gene flow into the colonizingsites are not entirely surprising given that birds disperseseed from Myrica cerifera. The normal pattern of seed dis-persal, where the density of seed dispersed decreases withdistance from the parent, may be altered as a result of thebehaviour of the dispersal agent. Thus the yellow-rumpedwarbler Dendroica coronata, the primary dispersal vector,may truncate the dispersal curve and essentially reduce thesignificance of spatial separation between sources and sinks.

Seed and pollen dispersal

In plants, two dispersal agents are responsible for the dis-tribution of genetic diversity, seed and pollen. The relativecontribution of these two is often unknown in studies ofgenetic variation and population genetic structure. Therehave been a number of studies, which directly investigatethe distribution of genetic variation at nuclear and cyto-plasmic (usually chloroplast) markers in plants (Ennos1994; El Mousadik & Petit 1996). The distribution of thesemarker types is often a function of the natural history of thesystem under study, with low estimates of genetic structureat nuclear markers relative to cytoplasmic markers in thegenus Quercus (Ennos 1994; Hu & Ennos 1997), which sharesthe dioecous, wind pollination characteristics of M. cerifera.These results are interpreted to mean that pollen dispersalis more common, and of greater importance to the dis-tribution of genetic diversity. However, these conclusionsare strictly inferential, and do not document the relativecontribution of each. It was possible to observe very highrates of seed dispersal (> 94% of gene flow), and relativelylow rates of pollen gene flow (∼3%) into the colonizingsites. While seed dispersal is exclusive early in colonization,upon flowering, colonists become receptive to pollendispersal, and the relative contribution of pollen gene flowmay increase with time. However, few of the female plantswithin the study site contributed seed to recruitment(Table 4), and even fewer males within the site contributedpollen to the formation of observed seedlings. It wasdemonstrated that although the onset of flowering withinthe colonizing sites coincided with increased recruitment,it was not driven by pollen gene flow to maternal plantsin the colonizing sites. Rather, seed dispersal from outsidethe sites was the dominant mechanism that accountedfor the acquisition of genetic variation. The large sourcepopulation on the island largely drives this result, as doesthe small size of our sample plots.

Conclusions

Questions concerning genetic structure and diversityare of increasing relevance to the study of ecology as bothecologists and evolutionary biologists further integratetheir fields of study. The use of genetics in ecology is ex-panding because of its power to elucidate the outcome ofecological events such as mating patterns and dispersal, aswell as a recognition that the genetics of populations willaffect their ecology. This is particularly true in areas suchas invasive species biology and metapopulation biology.In metapopulations, the distribution of genetic variationis largely determined by patterns of extinction and re-colonization, as opposed to more traditional determinantssuch as gene flow among populations (Hanski & Gilpin1997). It has been observed that subpopulations withina metapopulation that carry increased genetic diversityare more stable and that metapopulations that containgreater variation in sum are more stable and less prone toextinction (Saccheri et al. 1998). Patterns of genetic diversityin newly established populations of invasive species mayalso offer insights into the origins and sources of invadingpopulations, thus helping us understand how invasive speciesare introduced and where to look for biological controlagents (Fonseca et al. 2000; Downie 2002; although see Tsutsuiet al. 2000 for an exception). The ability of biologists toreconstruct the ecological processes that affect geneticvariability within populations, particularly within new orexpanding populations, may provide further insightsinto the mechanisms promoting stability and sustainabilityof populations in increasingly fragmented and disturbedlandscapes. Our study demonstrates the relevance ofapplying methods of genealogical reconstruction to notonly document patterns of genetic change in recruitingsegments of an expanding population, but also to determinethe contribution of different ecological factors to theacquisition and distribution of genetic variation withinand among newly colonized sites of M. cerifera.

Acknowledgements

We would like to thank, C. Fenster, D. Fonseca, R. Malmberg,C. Murren and R. Wyatt for critical reviews of the study and themanuscript. We would also like to thank C. Ivey, S. Jorgensen,J. Spitler and J. Williams for assistance in the field. In addition,P. Aldrich, C. Chavarriaga, B. Lance and W. Smart provided invalu-able technical assistance. This research was funded by a NationalScience Foundation training grant award (BIR-9220329 and DBI-9602223) to D.L.E.

References

Aldrich PR, Hamrick JL (1998) Reproductive dominance ofpasture trees in a fragmented tropical forest mosaic. Science, 281,103–105.

Page 10: Ecological determinants of genetic diversity in an expanding population of the shrub Myrica cerifera

1664 D . L . E R I C K S O N , J . L . H A M R I C K and G . D . K O C H E R T

© 2004 Blackwell Publishing Ltd, Molecular Ecology, 13, 1655–1664

Aldrich PR, Hamrick JL, Chavarriaga P, Kochert G (1998) Micro-satellite analysis of demographic genetic structure in fragmentedpopulations of the tropical tree Symphonia globulifera. MolecularEcology, 7, 933–944.

Botstein D, White RL, Skolnick M, Davis RW (1980) Construction ofa genetic linkage map in man using restriction fragment lengthpolymorphisms. American Journal of Human Genetics, 32, 314–331.

Broyles SB, Schnabel A, Wyatt R (1994) Evidence for long-distancepollen dispersal in milkweeds (Asclepias exaltata). Evolution, 48,1032–1040.

Bruederle LP, Tomback DF, Kelly KK, Hardwick RC (1998) Popu-lation genetic structure in a bird-dispersed pine, Pinus albicaulis(Pinaceae). Canadian Journal of Botany, 76, 83–90.

Devlin B, Ellstrand NC (1990) The development and application ofa refined method for estimating gene flow from angiospermpaternity analysis. Evolution, 44, 248–259.

Dow BD, Ashley MV (1996) Microsatellite analysis of seed dis-persal and parentage of saplings in bur oak, Quercus macrocarpa.Molecular Ecology, 5, 615–227.

Downie DA (2002) Locating the sources of an invasive pest, grapephylloxera, using a mitochondrial DNA gene genealogy. Mole-cular Ecology, 11, 2013–2026.

Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue.Focus, 12, 13–15.

El Mousadik A, Petit RA (1996) Chloroplast DNA phylogeo-graphy of the argan tree of Morocco. Molecular Ecology, 5, 547–555.

Ennos RA (1994) Estimating the relative rates of pollen and seedmigration among plant populations. Heredity, 72, 250–259.

Fonseca DM, LaPointe DA, Fleisher RC (2000) Bottlenecks andmultiple introductions: population genetics of the vector ofavian malaria in Hawaii. Molecular Ecology, 9, 1803–1814.

Frankham R (1995) Conservation genetics. Annual Review of Genetics,29, 305–327.

Furnier GR, Knowles P, Clyde MA, Danick BP (1987) Effects ofavian seed dispersal on the genetic structure of whitebark pinepopulations. Evolution, 41, 607–612.

Hamrick JL, Nason D (1996) Consequences of dispersal in plants.In: Population Dynamics in Ecological Space and Time (eds RhodesOR, Chesser RK, Smith MH), pp. 203–236. University of ChicagoPress, Chicago.

Hanski I, Gilpin ME (1997) Metapopulation Dynamics: Ecology,Genetics and Evolution. Academic Press, San Diego, CA, USA.

Hayden BP, Santos MCFV, Shao G, Kochel RC (1995) Geomor-phologic controls on coastal vegetation at the Virginia CoastReserve. Geomorphology, 13, 283–300.

Hearne CM, Ghosh C, Todd JA (1992) Microsatellites for linkageanalysis of genetic traits. Trends in Genetics, 8, 288–294.

Hu XS, Ennos RA (1997) On estimation of the ratio of pollen toseed flow among plant populations. Heredity, 79, 541–552.

Ingvarsson PK (1997) The effect of delayed population growthon the genetic differentiation of local populations subject tofrequent extinctions and recolonizations. Evolution, 51, 29–35.

Irvin SD, Wetterstrand KA, Hutter CM, Aquadro CF (1998)Genetic variation and differentiation at microsatellite loci inDrosophila simulans: evidence for founder effects in new worldpopulations. Genetics, 150, 777–790.

Leblois R, Rousset F, Tikel Det al. (2000) Absence of evidence forisolation by distance in an expanding cane toad (Bufo marinus)population: an individual-based analysis of microsatellitegenotypes. Molecular Ecology, 9, 1905–1909.

Lee CE (2002) Evolutionary genetics of invasive species. Trends inEcology and Evolution, 17, 386–391.

Loveless MD, Hamrick JL (1984) Ecological determinants ofgenetic structure in plant populations. Annual Review of Ecologyand Systematics, 15, 65–95.

Manos PS, Steele KP (1997) Phylogenetic analyses of ‘higher’Hamamelididae based on plastid sequence data. American Journalof Botany, 84, 1407–1419.

Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statisticalconfidence for likelihood-based paternity inference in naturalpopulations. Molecular Ecology, 7, 639–655.

Meagher TR (1986) Analysis of paternity within a natural popu-lation of Chamaelirium luteum I. Identification of most-likely maleparents. American Naturalist, 128, 199–215.

Meagher TR, Thompson E (1987) Analysis of parentage for natur-ally established seedlings of Chamaelirium luteum (Liliaceae).Ecology, 68, 803–812.

Mitton JB, Linhart YB, Sturgeon KB, Hamrick JL (1979) Allozymepolymorphisms detected in mature needle tissue of ponderosapine. Journal of Heredity, 70, 86–89.

Oddou-Muratorio S, Houot ML, Demesure-Musch B et al. (2003)Pollen flow in the wildservice tree, Sorbus torminalis (L.) Crantz.I. Evaluating the paternity analysis procedure in continuouspopulations. Molecular Ecology, 12, 3427–3439.

Prodohl PA, Loughry WJ, McDonough CM et al. (1998) Geneticmaternity and paternity in a local population of armadillosassessed by microsatellite DNA markers and field data. AmericanNaturalist, 151, 7–19.

Radford AE, Ahles HE, Bell CR (1968) Manual of the Vascular Floraof the Carolinas. University of North Carolina Press, Chapel HillNC.

Saccheri I, Kuussaari M, Kankare M et al. (1998) Inbreeding andextinction in a butterfly metapopulation. Nature, 392, 491–494.

Schnabel A, Nason JD, Hamrick JL (1998) Understanding the popu-lation genetic structure of Gleditsia triacanthos L. seed dispersaland variation in female reproductive success. Molecular Ecology,7, 819–832.

Tsutsui ND, Suarez AV, Holway DA et al. (2000) Reduced geneticvariation and the success of an invasive species. Proceedings ofthe National Academy of Science USA, 97, 5948–5953.

Wade MJ, McCauley DE (1988) Extinction and colonization: theireffects on the genetic differentiation of local populations. Evolu-tion, 42, 995–1005.

White GM, Boshier DH, Powell W (2002) Increased pollen flowcounteracts fragmentation in a tropical dry forest: an examplefrom Swieteia hamilis Zuccarini. Proceedings of the National Acad-emy of Science USA, 99, 2038–2042.

Young DR, Shao G, Porter JH (1995) Temporal and spatial growthdynamics of barrier island shrub thickets. American Journal ofBotany, 82, 638–645.

David Erickson received a PhD in Botany from the University ofGeorgia, and this manuscript represents a portion of that work. Heis now a Postdoctoral researcher at the Smithsonian Institute’sLaboratory of Analytical Biology where he is working on a QTLmapping project. J. L. Hamrick is a research professor in theDepartments of Plant Biology and Genetics at the University ofGeorgia where his laboratory conducts research into the popu-lation and ecological genetics of natural plant populations. GaryKochert is an emeritus professor at the department of Plant Biologyat the University of Georgia who has worked on genetic improve-ment of crop species.