Impacts of forest fragmentation on thereproductive success of white spruce (Piceaglauca)

Lisa M. O’Connell, Alex Mosseler, and Om P. Rajora

Abstract: The fragmentation of forests into small, isolated remnants may reduce pollen quantity and quality in naturalplant populations. The reproductive success of white spruce (Picea glauca (Moench) Voss) was assessed in a landscapefragmented by agriculture in northern Ontario, Canada. We sampled a total of 23 stands and 104 white spruce trees fromthree different stand size classes. Each sampled stand was separated by 250–3000 m from the nearest neighbouring stand.Reproductive success, measured as the number of filled seeds per cone, increased with stand size. The total number ofseeds per cone, a measure that includes both filled and aborted seeds, also increased with stand size, suggesting that pollenreceipt limits the number of seeds in a cone. The proportion of empty seeds (postzygotic abortions) was highest in the twosmallest stand size classes, suggesting that inbreeding levels were also highest in these stands. We detected no differencein germination success, seedling growth, and growth of trees up to 10 years from seeds produced by trees from differentstand size classes. These results suggest that inbred individuals are largely eliminated during the seed development stage.We estimated that a threshold population size of 180 trees is needed to reduce the negative effects of pollen limitation andinbreeding and maintain seed yields observed in large contiguous stands.

Key words: conifer, forest fragmentation, inbreeding, Picea glauca, pollen limitation, wind pollination.

Resume : La fragmentation des forets diminue la dimension et augmente l’isolement des populations naturelles de plantes,limitant ainsi la quantite et qualite du pollen recu. Nous avons evalue le succes reproductif de l’epinette blanche (Piceaglauca (Moench) Voss) dans des populations fragmentees par l’agriculture, dans le nord de l’Ontario, Canada. Nous avonsechantillones 23 peuplements et 104 arbres, regroupes en trois differentes classes de dimension de peuplement. Chaquepeuplement echantillone etait separe d’au moins 250–3000 m du peuplement le plus proche. Nous avons observe que lesucces reproductif (le nombre de graines pleines par cone) augmente avec le nombre d’arbres par peuplement. Le nombretotal de graines par cone, qui inclus les graines pleines et les graines avortees, augmente aussi avec la dimension du peu-plement, suggerant que le nombre de graines est limite par le pollen. Le pourcentage de graines vides (avortements post-zygotique) etait plus eleve dans les deux plus petites classes de dimension de peuplement, ce qui suggere que les croise-ments consanguins etaient plus eleves dans ces classes. Nous n’avons pas decele de difference dans le pourcentage de ger-mination des graines et la croissance des semis provenant des peuplements de differentes classes. Ceci suggere que lesindividus provenant de croisement consanguin sont elimines au stade du developpement des graines. Nous avons estimequ’une population minimum de 180 arbres est requise pour reduire la perte de graines causee par la limitation par le pollenet la consanguinite, et pour maintenir le niveau de production de graines observe dans les plus grands peuplements.

Mots cles : conifere, consanguinite, fragmentation des forets, limitation par le pollen, Picea glauca, pollinisation anemo-phile.

Introduction

Forested landscapes have been fragmented by naturalevents such as fires, insects, and diseases and by human ac-tivities such as agriculture, urbanization, and forest harvest-ing. Tree populations have become more isolated withdecreasing population sizes, numbers, and densities and in-creasing distances to other populations. The impact of frag-mentation on natural populations is a common researchtopic. Fahrig (2003) found over 1600 scientific articles thatused the term ‘‘habitat fragmentation’’. In natural plant pop-ulations, habitat fragmentation can potentially reduce repro-ductive success by decreasing the amount of pollen availableand increasing inbreeding through self-pollination or pollina-tion by closely related neighbours. In a recent review, Reed(2005) showed that reproductive success (measured through

Received 4 January 2006. Published on the NRC Research PressWeb site at http://canjbot.nrc.ca on 31 July 2006.

L.M. O’Connell1,2 and A. Mosseler. Natural ResourcesCanada, Canadian Forest Service, Atlantic Forestry Centre, P.O.Box 4000, Fredericton, NB E3B 5P7, Canada.O.P. Rajora.2 Forest Genetics and Biotechnology Group,Department of Biology, Life Sciences Centre, DalhousieUniversity, Halifax, NS B3H 4J1, Canada.

1Corresponding author (e-mail: oconnell@unb.ca).2Present address: Canada Research Chair in Forest andConservation Genomics and Biotechnology, Faculty of Forestryand Environmental Management, University of NewBrunswick, P.O. Box 44555, 28 Dineen Drive, Fredericton, NBE3B 6C2, Canada.

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seed set, seeds per plant, seed mass, or percent germination)was positively correlated with population size in 11 speciesof herbaceous plants. In trees, most research on the impactof forest fragmentation on the reproductive success hasbeen carried out on tropical species, and there is some evi-dence that fragmentation does reduce reproductive success(e.g., Aizen and Feinsinger 1994; Nason and Hamrick 1997;Nason et al. 1998; Fuchs et al. 2003). These species usuallyoccur at naturally low densities and are generally animalpollinated. Habitat fragmentation can restrict pollinatormovement between plants and thus decrease pollination suc-cess and increase inbreeding. In contrast, temperate zoneforest trees often grow in large stands and at high densitiesand are usually wind pollinated. Because wind-pollinatedplants do not rely on a mutualistic relationship with animalpollinators, they are not expected to be as sensitive to theimpact of forest fragmentation (Totland and Sottocornola2001; Knight et al. 2005). Little is known, however, aboutthe impact of population fragmentation in wind-pollinatedtrees or wind-pollinated plants in general (Koenig and Ash-ley 2003; Davis et al. 2004).

Pollen limitation is common in plants, and in two reviewsof studies using controlled supplemental pollination experi-ments, pollen-limited reproductive success was detected in62% and 63% of plant species (Burd 1994; Knight et al.2005, respectively). Knight et al. (2005) failed to find a sig-nificant difference in pollen limitation between animal- andwind-pollinated plants but pointed out that there were toofew data available on pollen limitation in wind-pollinatedplants. Nevertheless, Owens (1995) stated that the lack ofpollination is the major cause of ovule abortion in temperateconifers. By counting the proportion of ovules without pol-len grains, Owens and colleagues estimated that more than

50% of aborted ovules in some species of conifers are dueto a lack of pollen (Colangeli and Owens 1990; Owens etal. 1991). Other causes for the loss of ovules in conifers in-clude low spring temperatures, diseases, and insect infesta-tions (Owens 1995).

There is evidence of long-distance pollination events inconifers (e.g., Schuster and Mitton 2000), but there are fewdata on the frequency of successful pollinations over longdistances. It is not clear how habitat fragmentation affectsgene flow in wind-pollinated trees. Wind pollination is ex-pected to increase gene flow between fragments comparedwith insect-pollinated plants, therefore minimizing matingwith neighbours in small populations (Young et al. 1996;Weidema et al. 2000). Dyer and Sork (2001) found a reduc-tion in pollen dispersal distances in Pinus echinata Mill.when surrounded by denser vegetation compared with moreopen, less dense stands. On the other hand, forest fragmenta-tion could potentially increase inbreeding in isolated frag-ments if a reduction in population size increases the levelof relatedness among plants and thus increases mating be-tween relatives. Gapare and Aitken (2005) found higher lev-els of relatedness among trees in peripheral populations ofPicea sitchensis (Bong.) Carriere with lower adult densitythan in denser core populations. Conifers generally showlow levels of self-fertility, expressed by a dramatic decreasein seed set following self-pollination (reviewed in Husbandand Schemske 1996; Kormu’tak and Lindgren 1996). Small,isolated populations of these trees are likely to be suscepti-ble to the effects of inbreeding depression if they are polli-nated by related neighbours. There does not need to be anincrease in family structure in small stands, however, to in-crease mating between relatives. Large stands may alsoshow similar levels of family structure, but the higher den-

Fig. 1. Map of study site near the western arm of Lake Nipissing, Ontario, showing the sampled white spruce (Picea glauca) stands. Smallstands are represented by triangles, medium stands by circles, and large stands by squares. Only sampled stands are indicated on the map.The location of the study site is indicated on the inset map by an open square. Descriptions of stands are given in Table 1.

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sity of the surrounding pollen cloud should decrease the pro-portion of pollen contributed by local trees.

In our study, we use seed set, seed viability, and seedlingand tree height growth data to assess the impact of forestfragmentation on white spruce (Picea glauca (Moench)Voss) reproductive success. Specifically, we examinewhether seed yield and quality and seedling growth and sur-vival increase with stand size. We then estimate the mini-mum population size needed to maintain the reproductivesuccess attained in large, continuous stands of white spruce.

Materials and methods

Study speciesWhite spruce is an ecologically and commercially impor-

tant conifer species, with a transcontinental range in thenorthern part of North America. In eastern forests, whitespruce is found in either pure stands or mixed-species stands

associated with black spruce (Picea mariana (Mill.) BSP),white birch (Betula papyrifera Marsh.), trembling aspen(Populus tremuloides Michx.), red spruce (Picea rubensSarg.), and balsam fir (Abies balsamea (L.) Mill.) (Nien-staedt and Zasada 1990). Female buds are concentrated intop whorls, and male buds are generally located in the mid-dle to lower crown (Eis and Inkster 1972; Nienstaedt andZasada 1990). Female receptivity and pollen shedding coin-cide and usually last from 3 to 5 d (Owens and Molder1979). Following self-pollination, seed set in P. glauca isgreatly reduced; relative self-fertility (the proportion offilled seeds following self-pollination compared with cross-pollination) in New Brunswick populations is less than 20%(Coles and Fowler 1976; Fowler and Park 1983). In a con-trolled pollination experiment in natural white spruce popu-lations, Coles and Fowler (1976) found that trees pollinatedby neighbours within a 100 m radius produced 28% fewerfilled seeds than when they were pollinated by more distant

Table 1. Description of 23 white spruce (Picea glauca) stands sampled near Lake Nipissing, Ontario, and stand means ofsampled tree measurements (ranges are in parentheses).

Stand Stand size Neighbour (m) Height (m) DBH (cm) Age (years) Density (m)

SmallA 3 400 6.7 (6–7) 14.0 (13–16) 24.3 (23–25) 5B 1 2000 11 23 29 naC 3 3000 7.0 (4–11) 11.0 (5–20) 31.7 (19–52) 14.7D 3 1500 17.3 (16–19) 28.7 (25–33) 49.3 (44–57) 9.3E 1 300 10 20 35 naF 4 300 14.0 (12–15) 36.8 (31–44) 41.8 (36–46) 43.7G 1 700 14 31 48 naH 3 500 15.3 (13–18) 31.3 (28–36) 60.7 (52–66) 16.8I 1 250 15 38 58 naJ 8 300 10.9 (9–15) 25.6 (15–42) 35.6 (28–58) 18.6K 4 250 16.5 (15–18) 44.8 (41–53) 64.0 (56–72) 54

MediumL 21 300 12.3 (8–17) 26.2 (19–34) 41.3 (26–58) 27.9M 25 250 10.8 (10–12) 29.5 (22–40) 37.0 (34–40) 29.5N 56 450 12.5 (10–15) 24.5 (18–35) 42.6 (33–53) 27.3O 43 300 13.2 (10–22) 28.7 (21–44) 48.8 (36–73) 14.7P 56 300 14.7 (9–21) 32.3 (24–42) 50.7 (31–72) 22.5Q 20 300 12.7 (12–16) 26.2 (18–41) 40.3 (30–65) 20.4

LargeR 100 400 15.2 (12–18) 29.3 (21–43) 46.7 (33–72) 13.9S 180 400 13.2 (11–17) 26.3 (21–33) 42.5 (35–58) 14.8T 180 300 12.3 (10–14) 27.3 (14–38) 46.7 (32–60) 21U >500 na 13.3 (11–17) 29.7 (23–39) 42.3 (33–55) 15.4V >500 na 15.5 (10–19) 36.2 (21–55) 48.5 (30–66) 14.5W >500 na 12.8 (10–16) 28.3 (21–40) 40.3 (31–52) 17

Tree means (SE)Small (N = 32) 12.4 (0.7) 27.9 (2.0) 42.9 (2.7) 19.6Medium (N = 36) 12.6 (0.5) 27.8 (1.2) 43.3 (2.1) 23.7Large (N = 36) 13.8 (0.4) 29.7 (1.3) 44.8 (1.7) 16

ANOVA F ratio (p)SSC (df 2) 0.58 (0.57) 0.22 (0.81) 0.07 (0.93) 1.87 (0.19)a

Stand (SSC) (df 16–20) 3.71 (<0.001) 4.03 (<0.001) 2.79 (<0.001) 4.4 (<0.001)

Note: The difference in stand traits among stand size classes (SSC) are tested using a nested ANOVA. Cones were collected from eachreproductive tree in small stands and from six trees in each of the medium and large stands. Stand size, number of reproductive trees perstand; neighbour, distance to the closest neighbouring stand; height, tree height; DBH, diameter at breast height; density, mean distance to thefive closest trees; na, not applicable.

aSingle trees are excluded (N = 100).

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neighbours. This suggests that there is a family structure inwhite spruce, which can potentially contribute to biparentalinbreeding and an increase in the proportion of empty seeds.

Study siteThe area around the western arm of Lake Nipissing in

central Ontario (46820’N, 80810’W) is an ideal site to studythe impacts of fragmentation on temperate forest tree species(Fig. 1). This area is an old glacial lakebed interspersed witha number of rocky outcrops of varying sizes containing re-sidual forest cover composed of, for the most part, whitespruce, eastern white pine (Pinus strobus L.), trembling as-pen, and white birch. These isolated patches of trees occurwithin an agricultural landscape used largely for corn anddairy production. A number of small islands in Lake Nipiss-ing also contain stands of isolated trees. All white sprucestands in this area grow on ecologically similar rocky out-crops. The surrounding tracts of continuous forest are com-posed of less than 5% white spruce and generally occur tothe northwest of the sampled populations (G. Thauvette, On-tario Ministry of Natural Resources, personal communica-tion), and the prevailing winds are from the southwest.

Sampling and cone collectionsCones were collected between 7 August and 3 September

1994 from a total of 104 trees from 23 natural white sprucestands varying in size from single, isolated trees to contigu-ous stands with hundreds of trees from a sample area cover-ing 378 km2 (Fig. 1; Table 1). Cones were collected fromone branch from each of the four cardinal directions at thetop of each tree and then pooled. To study the effect ofstand size on reproductive success, we sampled trees fromthree different stand size classes (SSCs) that differed by anorder of magnitude: 11 small stands (1–<10 trees, mean 2.9trees), six medium stands (10–<100 trees, mean 36.8 trees),and six large stands (‡100 trees, mean 326.7 trees). Standswere chosen so that a similar number of trees from eachSSC was sampled. In small stands, every reproductive tree(one to eight trees) was sampled (mean 2.9 trees per stand,32 trees in total); in medium and large stands, six trees perstand were sampled (36 trees from each SSC) (Table 1). Inlarge and medium stands, sampled trees were selected sothat they were at least 30 m from any other sampled tree tominimize the sampling of related trees. While pollen candisperse over several kilometres in wind-pollinated trees,the majority of pollen will disperse within 50–100 m (Ri-chards 1997). Each sampled stand was located between 250and 3000 m from the closest neighbouring other whitespruce stand, except for three stands that were part of a con-

tiguous forest (i.e., stands with more than 500 trees) (Fig. 1;Table 1). Stand size and isolation distance were highly cor-related so that we could not test for the effect of isolationdistance. All highly isolated stands ( >700 m from the near-est neighbour) were small, and all large stands were eitherpart of a contiguous forest or separated by <400 m fromother neighbouring stands (Table 1). Nevertheless, withinsmall stands, we found no correlation between isolation dis-tance and reproductive fitness (results not shown). For eachtree, height and diameter at breast height (DBH) were meas-ured, and age was determined from a core sample. The aver-age distance to the five closest trees (or all other trees instands with less than five trees) was measured. The height,DBH, age, and mean distance to the five closest trees foreach maternal tree were compared among SSC using anested ANOVA (Table 1). SSC was a fixed effect and stand,nested within SSC, a random effect. We used the softwareJMPTM version 3.2.1 (SAS Institute Inc. 1997) for this stat-istical analysis as well as the others reported below. Therewas no significant difference in height, DBH, age, or meandistance to other trees among SSCs (Table 1).

Reproductive successTo measure reproductive success in each tree, up to 25

cones per tree (mean 23.6) were sampled from the pooledcones. For the most isolated stand (stand C, located 3 kmfrom the nearest other stand), very few cones were availablefor sampling, so that seed counts were obtained from onlythree cones from one of the three trees. This is in contrastwith all other stands, where 25–200 cones were sampled perstand. Thus, reproductive success data were available foronly 102 of the 104 trees sampled. To obtain seeds percone measures, the total number of seeds sampled from atree was divided by the total number of cones sampled.Seven measures of reproductive success were calculated foreach tree as follows. (i) The total number of seeds per cone.In white spruce, seed development requires pollination andfertilization (Owens and Molder 1979). The total number ofseeds with enlarged seed coats, whether filled or empty, isequal to the number of ovules fertilized and thus is an esti-mate of the amount of pollen received. (ii) The number offilled seeds per cone. Filled seeds have viable embryos. (iii)The number of empty seeds per cone. Empty seeds are fer-tilized but have aborted embryos. (iv) The percentage ofempty seeds per cone. Some embryos will abort owing toenvironmental effects, such as low temperatures and dis-eases, and produce empty seeds. The remaining proportionof the empty seeds will be due largely to the deleterious ef-fects of inbreeding depression. In conifers, the percentage of

Table 2. Pearson’s correlation coefficients between seven reproductive success measures in 102 white spruce (Picea glauca) trees.

No. of total seedsper cone

No. of filledseeds per cone

No. of emptyseeds per cone % empty seeds Seed mass

Conemass

No. of filled seeds per cone 0.766*No. of empty seeds per cone 0.523* –0.148ns% empty seeds –0.086ns –0.689* 0.781*Seed mass 0.166ns 0.107ns 0.113ns 0.031nsCone mass 0.421* 0.462* 0.036ns –0.235ns 0.597*Reproductive efficiency 0.610* 0.819* –0.148ns –0.596* 0.081ns –0.003ns

Note: *, statistical significance at p < 0.001; ns, p > 0.0045, nonsignificant after sequential Bonferroni correction.

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empty seeds can be used to approximate the amount of nat-ural inbreeding (Mosseler et al. 2000; Rajora et al. 2000,2002). We assume that significant differences in the percent-age of empty seeds among trees were largely due to differ-ences in the amount of inbreeding, either through self-pollination or through mating with relatives. (v) The massof 1000 filled seeds. To obtain a per seed mass, the mass ofall the filled seeds sampled in a tree was divided by the totalnumber of filled seeds sampled and then the per seed masswas multiplied by 1000. The mass of filled seeds was usedto calculate reproductive efficiency. (vi) Cone mass, meas-ured as the dry cone mass without seeds. Cone mass wasused to correct for environmental differences among moth-

ers and stands, such as resource limitations. (vii) Reproduc-tive efficiency, calculated as the total mass of filled seeds ina cone, divided by cone mass. Reproductive efficiency is theproportion of the total cone mass, including seeds, allocatedto reproductive output.

Germination, survival, and growthIn January 1995, seed germination and survival percent-

age differences were tested using a randomized completeblock design with four replicates (i.e., blocks) of 32 seedsfrom each of 100 families (i.e., seeds from the same mater-nal tree) for a total of 12 800 sown seeds. Seeds were from23 stands (11 small, six medium, and six large) and therewere one to eight sampled trees per stand (Table 1). Theseed from two missing trees in the reproductive successdata (from population C) are included in these 100 families,but seed from four other trees, all from small populations,were not available to be tested for germination and survival.Seeds were germinated and seedlings grown under opera-tional greenhouse conditions at ambient light and at temper-atures between 20 and 25 8C. Germination and survivalpercentages were recorded at 39 d. From these seedlings,three replicates of 32 seedlings from each of the 100 fami-lies were tested for survival, and seedling height was meas-ured at 120 d. A subset of these seedlings were randomlyselected and established at the Petawawa Research Forest,Chalk River, Ontario, Canada (45856’N, 77824’W) in arandomized complete block design consisting of threeblocks of nine-tree plot squares from each family of 100families, with trees spaced at 1 m � 1 m. Heights weremeasured at 4 years (August 1998) and again at 10 years(October 2004) after establishment.

Data analysesWe used Pearson’s correlation coefficients to test for pair-

wise correlations between all pairs of reproductive successmeasures as well as between reproductive success measuresand maternal tree traits (height, DBH, age, and mean dis-tance to the five closest trees). A tablewide sequential Bon-ferroni correction was applied to adjust the significancelevel for multiple tests over all correlation coefficients(Rice 1989).

To test for a difference in the seven measures of repro-

Table 3. Effects of stand size class (SSC) and stand (nested within SSC) on seed and cone traits in 102 white spruce (Picea glauca)trees from small, medium, and large stands.

ANOVA SSC

SSC Stand (SSC) Small (N = 30) Medium (N = 36) Large (N = 36)

F[2,101] p F[20,101] p Mean (SE) Mean (SE) Mean (SE)

No. of total seeds per cone 5.702 0.011 2.23 0.006 47.7b (3.2) 58.0a (2.6) 64.7a (2.4)No. of filled seeds per cone 8.037 0.003 2.02 0.015 26.2b (2.2) 29.6b (2) 42.6a (2.4)No. of empty seeds per cone 4.56 0.023 1.63 0.06 21.5b (1.9) 28.5a (2) 22.0b (1.5)% empty seedsa 5.28 0.01 2.47 0.002 0.44ab (0.03) 0.49a (0.02) 0.35b (0.02)1000 seed mass (g) 1.68 0.21 1.95 0.02 2.50a (0.06) 2.47a (0.06) 2.30a (0.07)Cone mass (g) 0.91 0.42 1.25 0.24 1.02a (0.04) 1.01a (0.05) 0.102a (0.005)Reproductive efficiencya 10.69 0.0007 2.14 0.0094 0.064b (0.005) 0.073b (0.004) 0.97a (0.05)

Note: N, total number of trees sampled per SSC. Within a row, means with different letters are significantly different.aData were arcsine transformed before statistical analyses. SSC with p > 0.017 and stands with p > 0.0125 are not significantly different after a

sequential Bonferroni correction.

Fig. 2. Regression of the mean number of filled seeds per cone onstand size (number of trees) in 23 white spruce (Picea glauca)stands. The solid line represents the nonlinear regression showing athreshold stand size of 180 trees. The equation for the nonlinear re-gression is number of filled seeds per cone = 26.2 + 0.111N –0.132N � (N – 180) � (N ‡ 180), where N is the number trees perstand. The broken line represents the simple linear regression.

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ductive success among the three SSCs, we used a nestedANOVA. The effects were SSC (fixed effect) and standnested within SSC (random effect). A post hoc contrast ofleast square means was used to test for differences amongSSCs. Data for the proportion of filled seeds and reproduc-tive efficiency were arcsine transformed. A sequential Bon-ferroni correction was applied to the significance levels ofeach of the effects of the ANOVA.

To identify the minimum stand size needed to attain themaximum reproductive success, we first regressed the meannumber of filled seeds per cone in each stand on stand size(n = 23). To identify the threshold population size to attainthe maximum reproductive success, we conducted a nonlin-ear split-line regression using following formula:

y ¼ b0 þ b1 � N þ b2 � ðN � TÞ � ðN � TÞ

where y is the number of filled seeds per cone, b0 is the y-intercept, b1 and b2 are the slopes below and above thethreshold stand size, respectively, N is the stand size, and Tis the threshold stand size. We used the nonlinear fit plat-form in JMP to estimate the different parameters in the re-gression with � = 0.05 (SAS Institute Inc. 1997). Weincrease T by increments of 10 until we identified thethreshold stand size that gave the lowest sum of squares er-ror (SSE). We tested if the nonlinear regression model(model 2) was a better fit to the data than a simple linearregression model (model 1) with a sum of squares reductiontest. The F ratio was calculated using the following formula:

F ¼ ½ðSSE1 � SSE2Þ=ðdf1 � df2Þ�=MSE2

where SSE is the sum of squares error and df is the degreesof freedom for model 1 and 2, respectively, and MSE2 is themean squares error for model 2. The F ratio degrees of free-dom for the numerator is df = df1 – df2 and for the denomi-nator is df = df2.

To test the effect of SSC on the percent seed germinationand percent survival to 120 d, we used a mixed-model AN-OVA. The factors included replicate (i.e., block), SSC, andstand nested within SSC. Replicates and stands were randomeffects and SSC a fixed effect. Percent seed germination andpercent survival were arcsine transformed before the analy-ses. There were a total of 23 stands (11 small, six medium,and and large) and 100 families (one to eight families perstand) and 32 seeds or seedlings per family. There werefour complete replicates for germination and survival to39 d (N = 400) and three replicates for survival to 120 d (N= 300). There was no difference in survival between 39 and120 d; thus only the results for survival to 39 d are pre-sented. To test for the effect of SSC on height at 120 d,

4 years, and 10 years, we used a mixed-model ANOVAwith the factors including replicate, SSC, stand (nestedwithin SSC), and maternal family (nested within stand).Replicate, stand, and family were random effects and SSC afixed effect. Pearson’s correlation coefficients were used totest for correlations in mean family heights between years.

Results

Correlations among measure of reproductive successMost reproductive success measures were significantly

correlated (Table 2). The total number of seeds per conewas positively and significantly correlated with the numberof filled seeds per cone, the number of empty seeds percone, cone mass, and reproductive efficiency. The numberof filled seeds per cone was positively and significantly cor-related with cone mass and reproductive efficiency and neg-atively correlated with the proportion of empty seeds percone. In contrast, the total number of seeds per cone wasnot significantly correlated with the percentage of emptyseeds per cone. Reproductive efficiency was not correlatedwith either seed mass or cone mass. Only p values <0.0045were statistically significant after a tablewide sequentialBonferroni correction. None of the reproductive successmeasures were significantly correlated with maternal treeheight, DBH, age, or the mean distance to the five closesttrees (all p > 0.06, results not shown).

Reproductive success and stand sizeThe reproductive success measures in white spruce dif-

fered significantly among stands and SSCs (Table 3). Thetotal number of seeds per cone increased with increasingstand size and was significantly lower in trees from smallstands compared with medium and large stands. The numberof filled seeds per cone was significantly higher in treesfrom large stands than in trees from small and mediumstands. In contrast, there were more empty seeds per conein medium stands than in either small or large stands butdid not differ significantly among SSC. The percentage ofempty seeds was lower in small and medium stands butonly differed significantly between medium and large SSCs.There was no significant difference in seed or cone massamong the three SSCs. Reproductive efficiency, however,was significantly higher in large stands than in either smallor medium stands.

Minimum stand sizeThe linear regression of the mean number of seeds per

cone on stand size was positive and statistically significant(r2 = 0.19, F = 5.016, p = 0.036, n = 23). We estimated

Table 4. ANOVA of the effect of replicate, stand size class (SSC), and stand (nested withinSSC) on germination and survival of white spruce (Picea glauca) to 39 d.

% germination % survival

Source of variation df (N = 398) MS F ratio MS F ratio

Replicate 3 0.0071 1.455ns 0.00998 1.870nsSSC 2 0.01093 0.689ns 0.01108 0.717nsStand (SSC) 20 0.01564 3.201* 0.01522 2.853*Error 372 0.004877 0.005334

Note: *, statistical significance at p < 0.0001; ns, not statistically significant (p > 0.05).

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that the threshold stand size that minimized the sum ofsquares error in a nonlinear regression was T = 180 (Fig. 2).The sum of squares error did not decrease at higher thresh-old sizes. The sum of squares reduction test showed that thenonlinear regression was a significantly better fit than a sim-ple linear regression: F1,20 = 5.09, p < 0.05.

Seedling survival and height growthThe overall percent germination (mean = 94.2%) and per-

cent survival of seedlings to 39 and 120 d (mean = 93.9%)were high. Only the individual stand had a significant effecton the percent germination and percent survival (Table 4).SSC or individual stand did not show a significant effect onseedling or tree height at any age, but both replicate and in-dividual family had a significant effect on height at 120 dand 10 years, and only family had an effect on height at4 years (Table 5). The mean heights of seedlings from afamily were positively correlated between years. The meanfamily heights of seedlings measured in 1995 (at 120 d)were significantly and positively correlated with the meanfamily heights in 1998 (at 4 years: Pearson’s r = 0.32, p =0.0013, N = 100) and 2004 (at 10 years: r = 0.35, p =0.0003). The mean family heights of trees at 4 years werealso strongly and positively correlated with the heights at10 years (r = 0.68, p < 0.0001).

Discussion

Pollen limitationAlthough reproductive success in wind-pollinated trees is

not expected to be very susceptible to habitat fragmentation,we found a strong effect of stand size on the reproductiveoutput of white spruce trees in fragmented stands. We de-tected a significant increase in the total number of seeds percone (fertilized ovules) and number of filled seeds per cone(viable seeds) with stand size. Trees in small and mediumstands produced 38% and 30% fewer filled seeds per cone,respectively, than trees in large stands. While some seedloss was due to postzygotic abortions, pollen limitationseems to be the main factor limiting reproductive success insmall isolated stands of white spruce in this study.

The total number of ovules per cone, which would affectthe number of seeds per cone, was not available for thisstudy. It is unlikely, however, that environmental differencesamong SSCs could have caused an increase in the numberof ovules per cone with stand size and consequently the ob-served increase in the number of total and filled seeds percone with stand size. First, all stands grew under similar en-

vironmental conditions. They occurred on shallow soils onsimilar rocky outcrops and in open stands with similar treedensities. Cone production in white spruce is also fairly uni-form over extensive areas and a wide range of habitats (Eisand Inkster 1972). Second, dry cone mass and seed mass didnot differ among SSCs or stands, suggesting that resourcelimitation differences among stands probably did not affectthe number of ovules per cone either. Furthermore, repro-ductive efficiency, the total filled seed mass divided bycone mass (without seeds), also showed a significant in-crease with SSC. Reproductive efficiency is a measure ofthe reproductive output per cone and should correct for ma-ternal effects and most environmental differences amongstands.

Similar data from other wind-pollinated tree species arelacking, but the increase in reproductive success (i.e., filledseeds per cone) with stand size is found in many herbaceousplant species (Reed 2005). Aizen and Feinsinger (1994)found lower seed set (number of filled seeds per fruit) infragmented forest stands than in continuous stands in 10 outof 16 insect pollinated tropical tree species. They found thatthe number of pollen grains per ovule and the number offilled seeds per fruit were both about 20% lower in frag-mented versus continuous stands, suggesting pollen-limitedseed set in fragmented stands. Studies on pollen limitationand reproductive success in wind-pollinated trees are scarce.Reproductive success in a few species has been associatedwith local population density. Allison (1990) found that pol-lination success in the coniferous shrub Taxus canadensisMarsh. was strongly correlated with the mean closest neigh-bour distance. Work on wind-pollinated oaks (Quercus dou-glasii Hook. & Arn.and Quercus lobata Nee) shows pollenlimitation and an average pollen dispersal distance limitedto 60–80 m (Knapp et al. 2001; Sork et al. 2002; Koenigand Ashley 2003). Although wind-pollinated plants areknown for their capability of dispersing pollen over longdistances, the isolation distances between stands in our study(250–3000 m) were sufficient to significantly reduce seedset in small stands of white spruce.

Seed production varies greatly from year to year in amasting species such as white spruce, and the degree of pol-len limitation may be different in a mast year. Knapp et al.(2001) only found a significant relationship between the dis-tance of pollen-producing neighbours and acorn productionin blue oak (Quercus macrocarpa Michx.) in two out of thefour years of their study. This relationship was strongest in ahigh acorn production (mast) year, as trees produced almostno acorns during the other years of the study. The year that

Table 5. ANOVA for the effect of replicate, stand size class (SSC), stand (nested within SSC), and family (nested within stand)on the height of white spruce (Picea glauca) offspring from 100 white spruce trees (i.e., family).

Height at 120 d (N = 8891) Height at 4 years (N = 2553) Height at 10 years (N = 2540)

Source of variation df MS F ratio df MS F ratio df MS F ratio

Replicate 2 3031547 3295.1** 2 213.44 1.098ns 2 20936.1 7.089*SSC 2 9809.65 0.530ns 2 1789.3 5.275ns 2 19793.1 1.277nsStand (SSC) 20 28640.6 0.967ns 20 679.24 0.649ns 20 19851 1.291nsFamily (stand) 77 20878.9 22.694** 77 721.98 3.715** 77 11581.9 3.922**Error 8789 920 2451 194.36 2433 2953.3

Note: *, statistical significance at p < 0.001; **, statistical significance at p < 0.0001; ns, not statistically significant (p > 0.05).

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we sampled for this study, 1994, was not a mast year forwhite spruce. In a mast year, we would expect greater pollenproduction and an increase in the pollen cloud density. Wemight therefore expect to see less of a difference in seedproduction among different-sized stands in white spruce dur-ing a mast year.

InbreedingSeed abortion as a result of inbreeding will depend on lo-

cal family structure, pollen dispersal distances, and differen-ces in inbreeding depression among individual trees.Nevertheless, we observed some patterns in seed abortionrates associated with stand size. The overall percentage ofempty seeds (mean = 42.5%) observed in this study waslow compared with the percentage of empty seeds observedin other white spruce populations. In previous studies, evenin controlled, completely outcrossed pollinations, high levelsof empty seeds have been observed ranging from 40% to68% (Coles and Fowler 1976; Fowler and Park 1983). Wefound that the percentage of empty seeds was lowest in thelarge SSC, suggesting that inbreeding levels are lower inthese stands. The small and medium SSCs showed increasesin the proportion of empty seeds of 14% and 22%, respec-tively, compared with the large stand class. These resultssuggest that trees in small and medium stands receive alarger proportion of self-pollen, or pollen from related trees,than do trees in larger stands. Inbreeding may be mainly dueto mating with relatives, as opposed to self-fertilization. Allsingle isolated trees, which have no neighbours, showed thelowest proportion of empty seeds per cone (23%–39%, n = 4trees), even though they also showed the lowest number offilled seeds per cone. In this study, the range in age amongtrees within small and medium stands is large enough to in-clude overlapping generations (Table 1). The younger repro-ductive trees could be offspring of the older ones. Over 90%of seeds in white spruce disperse within 50 m of the mother,and less than 4% disperse beyond 100 m (Nienstaedt andZasada 1990). The small and medium populations that wesampled were isolated by more than 250 m from any otherspruce stands and thus were likely to show some familystructure. Gapare and Aitken (2005) attributed the higher de-gree of family structure that they observed in a disjunct pop-ulation of P. sitchensis to offspring establishment nearmaternal plants and fewer overlapping seed shadows in pop-ulations with low tree density. Family structure may still bepresent in large stands leading to some mating between rela-tives; however, a pollen cloud containing a higher propor-tion of unrelated pollen may be contributing to the lowerlevels of empty seeds observed in larger stands.

A greater percentage of empty seeds in smaller standssuggests higher inbreeding levels at the early stage of seeddevelopment. In contrast, there was no difference in germi-nation, seedling survival, and tree height growth among thedifferent SSCs. This suggests that inbred seeds are largelyeliminated early in the process of embryo development andseed formation rather than at later stages in the life cycle ofindividuals. In other studies, molecular markers alone havebeen used to assess inbreeding levels in some fragmentedtree populations (e.g., Hall et al. 1996). Because only filledseeds are usually assayed with molecular markers, the levelof inbreeding at the fertilization stage is underestimated

(Mosseler et al. 2000; Rajora et al. 2000, 2002). By measur-ing the percentage of empty seeds, we have been able to de-tect possible inbreeding in embryos that would not beassayed with molecular markers.

Minimum population sizeFrom our data, we can estimate that a population size of

‡180 reproductive trees should maintain levels of reproduc-tive success similar to large contiguous stands of trees (i.e.,stands with >500 trees). Eis and Inkster (1972) observed thatcone production in trees of the same size, regardless of hab-itat, produced a similar number of cones, and thus an in-crease in the number of viable seeds per cone shouldtranslate into an increase in the number of viable seeds atthe tree level for trees of the same size. Based on our non-linear regression formula, an increase in stand size from 10to 100 trees leads to a 36% increase in viable seeds percone, while an increase from 10 to 180 trees leads to a 68%increase in filled seeds per cone. Above the threshold standsize of 180 trees, the regression is slightly negative. In her-baceous plants, Reed (2005) observed that reproductive suc-cess increased linearly with a logarithmic increase inpopulation size, and reproductive success did not show anasymptote, even at very large population size, for any of thespecies that he studied and thus no threshold populationsize. This method leads to the prediction of biologically un-reasonable minimum viable population sizes in the thou-sands. By plotting our data on a logarithmic scale, we alsodid not observed an asymptote in the number of filled seedsper cone, even at the largest stand sizes, and using Reed’s(2005) method predicted a minimum viable population size>10 000 trees for white spruce (results not shown). By usinga nonlinear split-line regression to identify the minimumpopulation size, we obtained a more biologically reasonablethreshold estimate of 180 trees.

Conclusions and conservation implications

Widespread, wind-pollinated tree species produce prodi-gious amounts of pollen and are not expected to be sensitiveto habitat fragmentation. We have found, however, that for-est fragmentation negatively impacts the reproductive suc-cess of white spruce. The results of our study suggest that areduction in stand size can reduce the number of seeds percone in white spruce, first by restricting the amount of pol-len that reaches and successfully fertilizes ovules and secondby increasing inbreeding and thus embryo abortion insmaller populations. We found that reproductive success inwhite spruce trees increased with population size and at astand size of at least 180 trees reaches levels of reproductivesuccess comparable with that of large contiguous stands.The most important guideline to maintain high pollinationsuccess and to minimize inbreeding in white spruce, andother conifers with similar mating systems, is to maintainlarge stands of several hundred reproductive trees. To fur-ther explore the impacts of fragmentation on the geneticcomposition of the pollen received, levels of inbreeding inwhite spruce, and diversity of the resultant seeds, we haveconducted a study using allozyme markers(L.M. O’Connell, A. Mosseler, and O.P. Rajora, unpublisheddata).

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AcknowledgmentsWe thank L. Dobrich for locating stands and collecting

seed, J. Lavereau for processing seed, I. DeMerchant andR. Simpson for producing the map of the study site,G. Thauvette, Ontario Ministry of Natural Resources, for in-formation on forest composition, and G. Oostermeijer,R. Cox, and Y.-S. Park for helpful suggestions on earlierversions of the manuscript. L.M.O. was supported by a Nat-ural Sciences and Engineering Research Council of Canada(NSERC) Postdoctoral Visiting Fellowship while at NaturalResources Canada (NRCAN) and an NSERC PostdoctoralFellowship while at the University of New Brunswick(UNB). O.P.R. held the Stora Enso Senior Chair in ForestGenetics and Biotechnology at Dalhousie University, whichwas supported by Stora Enso Port Hawkesbury Ltd., andholds the Senior Canada Research Chair in Forest and Con-servation Genomics and Biotechnology at UNB, which issupported by NSERC. The research was funded by anNSERC discovery grant to O.P.R. and NRCAN operatingfunds to A.M.

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