Skip to main content
NCBI home page
Annals of Botany logoLink to Annals of Botany
. 2003 Aug;92(2):223–230. doi: 10.1093/aob/mcg126

Allozyme Variation in Endangered Castanea pumila var. pumila

YUQING FU 1, FENNY DANE
PMCID: PMC4243649  PMID: 12829445

Abstract

Allozyme genetic variation in 12 populations of the endangered Castanea pumila var. pumila (Allegheny chinkapin), sampled across the natural range of the species in the United States, was evaluated using 11 loci from seven enzyme systems. At the species level, the percentage of polymorphic loci (Ps) was 72·7 %, the mean number of alleles per locus (As) was 1·9, the mean number of alleles per polymorphic locus (APs) was 2·3, the effective number of alleles per locus (Aes) was 1·5 and the genetic diversity (Hes) was 0·296. At the population level, Pp = 49·2 %, Ap = 1·5, Aep = 1·4, APp = 2·1 and Hep = 0·21. Most of the allozyme variation (70 %) in C. pumila var. pumila occurred within populations. Wright’s gene flow rate [Nm(W)] was as low as 0·57. Population differentiation along the species range was not detected. Populations of C. pumila var. pumila in Florida had the most variable levels of genetic diversity, but populations in Virginia and Mississippi also showed high levels. Based on the results of this study, conservation management strategies are discussed.

Key words: Allozyme, Allegheny chinkapin, Castanea pumila var. pumila, gene flow, genetic diversity, population structure, conservation

INTRODUCTION

Castanea pumila Mill. var. pumila (Allegheny chinkapin) is endemic to the south‐eastern United States and is widely distributed along the Appalachian mountain range south to Florida and Texas (Johnson, 1988). Chestnut blight, caused by Cryphonectria parasitica (Murrill) M.E.Barr, has dramatically destroyed the American chestnut [C. dentata (Marsh.) Borkh.] and severely affected the Ozark chinkapin [C. pumila var. ozarkensis (Ashe) G.E.Tucker], but C. pumila var. pumila exhibits slight resistance, although heavily cankered trees have been found in Florida and Alabama (Paillet, 1993; Fulbright, 1999). Unlike other Castanea taxa, C. pumila var. pumila is of little economic importance for nut and timber production because of its small nut and tree size. Its main use has been as rootstock for chestnut breeding and it provides a food source for wildlife. This species has long been neglected and conservation strategies need to be developed (Johnson, 1987; Payne et al., 1994).

Taxonomic studies by Johnson (1988) indicated that C. pumila var. pumila and var. ozarkensis can be considered as varieties of American chinkapin, characterized by one nut per two‐valved cupule. The varieties have similar vegetative and reproductive characters, including leaf, bud, twig and fruit characteristics, but differ in morphological traits and distribution as well as in their level of resistance to chestnut blight (Johnson, 1988). Castanea pumila var. pumila is a stoloniferous or non‐stoloniferous shrub or tree up to 15 m tall with one to five cupules at the base of an androgynous spike, variable leaf shapes, leaf blades of 4·1–21·7 × 1·5–8·3 cm, and with a seldom acuminate or long‐acuminate apex. In contrast, C. pumila var. ozarkensis is a large, multi‐stemmed shrub or tree up to 20 m tall with five to eight cupules at the base of an androgynous spike, leaf blades of 4·3–26·6 × 2·0–9·3 cm and an acuminate or long‐acuminate apex (Johnson, 1988; Payne et al., 1994). Owing to their differing levels of resistance to chestnut blight, C. pumila var. pumila is more widely distributed, while C. pumila var. ozarkensis is limited to the Ozark Plateau and most trees have been reduced to small stump sprouts (Johnson, 1987; Payne et al., 1994; Dane, pers. obs.).

Genetic variability of species is determined by geographical conditions, ecological factors, mating systems and also by historical events such as glacial periods (Hamrick et al., 1992; Dumolin‐Lapegue et al., 1997; Booy et al., 2000). In forest species, genetic variability is usually lower in marginal populations than in populations located more centrally or in the vicinity of ancient glacial refuges. Such a decrease in genetic variability towards species margins has been reported for Fagus (Demesure et al., 1996) and Quercus species (Dumolin‐Lapegue et al., 1997).

Allozyme markers have been used extensively since the mid‐1960s to study genetic diversity and genetic structure (McDonald and Hamrick, 1996; Godt and Hamrick, 1998), gene flow and genetic drift (Campbell, 1991), and hybridization and phylogenetics (Krutovskii and Bergmann, 1995). In chestnut, the mode of inheritance of allozymes has been studied in European sweet chestnut (C. sativa Mill.; Fineschi et al., 1990, 1991), and in Chinese chestnut (C. mollissima Bl.) and C. dentata (Huang et al., 1994a). Villani et al. (1991a, b) evaluated allozyme diversity and genetic structure in C. sativa populations and hypothesized that eastern Turkey is one of the centres of origin of this species. Research on allozyme diversity in chestnut species by Huang et al. (1994b) revealed high levels of expected heterozygosity (He = 0·305) for C. mollissima, and lower levels for Seguin chestnut (C. seguinii Dode; He = 0·203) and C. dentata (He = 0·183). These results were confirmed in later studies (Huang et al., 1998; Lang and Huang, 1999), and showed that the Changjiang river region of P.R. China, especially the Shennongjia area, was the origin of C. mollissima. In recent research on C. pumila var. ozarkensis, Dane et al. (1999) detected genetic diversity (He) of 0·272, higher than that previously reported for C. dentata.

Allozyme variation at the species and population level has been neglected in C. pumila var. pumila compared with other Castanea species. The present study addressed the following questions: (1) what is the genetic variability of C. pumila var. pumila in comparison with other Castanea species; (2) do populations have significantly different levels of genetic variation; (3) can migration patterns along the natural geographical distribution be detected; and (4) can this information be used to develop appropriate conservation strategies for C. pumila var. pumila?

MATERIALS AND METHODS

Plant material

In autumn 1998 and 1999, 12 C. pumila var. pumila populations were sampled from along its natural distribution in Alabama (populations from Uchee, AL‐U, and Mobile, AL‐M), Mississippi (population MS from Saucier), Ohio (population OH from a naturalized stand in east Ohio), Florida (six populations: FL‐B, FL‐D, FL‐E, FL‐G, FL‐I and FL‐L from Eglin Air Force Base) and Virginia (two populations VA and VB from Iron Mountain) (Fig. 1). Some of the natural populations were small, consisting of several trees only. Two to 30 trees were sampled in each population, and open‐pollinated nuts were randomly collected from each individual tree. If nuts were not available, several twigs with dormant buds were collected because nut and dormant bud tissues give the best and most reproducible allozyme definition (Huang, 1993). When possible, leaf, nut and cupule characteristics of individual trees were recorded.

graphic file with name mcg126f1.jpg

Open in a new tab

Fig. 1. Collection sites (stars) of Castanea pumila var. pumila populations used in this study.

Allozyme analysis

Allozymes were extracted from individual nut or dormant bud tissue from each tree. Four to 12 randomly selected open‐pollinated nuts or one sample of dormant bud tissue were analysed per tree. Samples were ground with 0·5 µl 2 % glycine extraction buffer (pH 8·6) per mg of fresh nut tissue or with 3 µl 0·1 m phosphate extraction buffer (pH 7·2) per mg of fresh bud tissue. Extracts were incubated at 4 °C for at least 60 min and centrifuged for 3 min at 10 000 g (Eppendorf Centrifuge 5415C; Brinkman Instruments, Westbury, NY, USA). Electrophoresis was performed on precast isoelectric focusing agarose gels (Hypure Gel FS‐5080, pH 4–6; EG&G WALLAC, Akron, OH, USA) at 40 W for about 60 min at 12–15 °C after loading 8·5 µl supernatant per well. Gels were assayed for nine enzyme systems: acid phosphatase (ACP), alcohol dehydrogenase (ADH), aconitase (ACO), diaphorase (DIA), esterase (EST), malic enzyme (ME), peroxidase (PRX), shikimate dehydrogenase (SKD), superoxide dismutase (SOD) and phosphoglucoisomerase (PGI). The staining procedures followed were those of Wendel and Weeden (1989) with minor modifications according to staining methods provided by Hypure. The following loci were scored where possible: Acp‐2, Acp‐4, Adh, Aco, Dia‐1, Dia‐6, Est‐3, Est‐4, Me, Prx‐4, Skd‐1, Skd‐2, Pgi‐2 and Sod‐1.

Genotype data analysis

A series of genetic parameters was used to calculate genetic diversity within populations and species (Berg and Hamrick, 1997) using BIOSYS‐1 developed by Swofford and Selander (1981). These parameters were: percentage of polymorphic loci (Pp, Ps), mean number of alleles per locus (Ap, As), mean number of alleles per polymorphic locus (APp, APs), effective number of alleles per locus (Aep, Aes), observed heterozygosity within population (Ho) and Nei’s (1978) unbiased estimate of expected heterozygosity under Hardy–Weinberg equilibrium (Hep, Hes), where subscripts ‘s’ and ‘p’ indicate species and population level, respectively.

Partitioning of genetic diversity was estimated using Nei’s genetic diversity statistics (Nei, 1973, 1977, 1978). Total genetic diversity (HT), within‐population genetic diversity (HS), among‐population genetic diversity (DST), and proportion of total genetic diversity occurring among populations (GST) were calculated for each polymorphic locus. Based on the fixation index among populations (FST) (or GST) values, indirect estimates of gene flow (Nm, number of migrants per generation) were computed using Wright’s formula: FST = 1/(1 + 4Nm) (Wright, 1951).

Populations were analysed for deviation from Hardy–Weinberg equilibria at each variable locus using Chi‐square tests performed using BIOSYS‐1 (Swofford and Selander, 1981), as well as Wright’s fixation index (FIS) for all loci and populations and a Chi‐square test to detect deviation of the fixation indices from zero. Expected genotype frequencies were calculated using Levene’s correction for small sample size (Levene, 1949). For loci consisting of more than two alleles at a locus, Chi‐square tests with pooling were used (Swofford and Selander, 1981).

For small numbers of individuals in populations, Nei’s (1978) unbiased genetic identities, I, and unbiased genetic distances, D, were estimated. A dendrogram based on unbiased genetic distance was constructed using the unweighted pair‐group method using arithmetic averages (UPGMA) (BIOSYS‐1; Swofford and Selander, 1981).

RESULTS

Morphological variation

Several populations of C. pumila var. pumila were located in the open‐canopied pine forests of north‐west Florida to Mississippi. Leaves collected from trees from the Florida sandhills were consistently smaller (mean 2·3 × 7·9 cm) than those collected from other C. pumila var. pumila populations (VA and VB: 4·3 × 12·0 cm; AL‐U: 4·0 × 11·3 cm). Burs with multiple nuts were detected at low frequency in all populations.

Allozyme variation

An overall pattern of widespread sharing of allozyme alleles was found in C. pumila var. pumila populations (Table 1). A rare allele, Dia‐1c, previously identified in C. pumila var. ozarkensis (Dane et al., 1999), was detected in samples from populations AL‐M, MS, FL‐B, FL‐D and FL‐E. Alleles Dia‐1b and Dia‐1c were found in six of the 12 populations and five of the 12 populations, respectively. Allele Prx‐4a was detected in all populations except OH, whereas Prx‐4b was found in eight populations. Acp‐4c was found in only two populations, AL‐M and FL‐D. Eight loci (Dia‐1, Aco, Acp‐2, Acp‐4, Prx‐4, Est‐4, Est‐5, Skd‐1 and Skd‐2) were polymorphic in at least some of the populations, and three loci, Sod‐1, Dia‐6 and Pgi‐1, were monomorphic (Table 1).

Table 1.

Frequencies of allozyme alleles at polymorphic loci in 12 Castanea pumila var. pumila populations, and Chi‐square tests for deviation from Hardy–Weinberg equilibrium

Population
Locus and alleles AL‐U AL‐M MS FL‐B FL‐D FL‐E FL‐G FL‐I FL‐L OH VA VB
Dia‐1
 a 1·000 0·500 0·650 0·684 0·706 0·500 1·000 1·000 1·000 1·000 0·841 1·000
 b 0·000 0·222 0·033 0·158 0·088 0·150 0·000 0·000 0·000 0·000 0·159 0·000
 c 0·000 0·278 0·317 0·158 0·206 0·350 0·000 0·000 0·000 0·000 0·000 0·000
Deviation ** ** n.s. n.s. * n.s.
Prx‐4
 a 0·571 1·000 1·000 0·816 1·000 1·000 0·375 0·750 0·625 0·000 0·659 0·531
 b 0·429 0·000 0·000 0·184 0·000 0·000 0·625 0·250 0·375 1·000 0·341 0·469
Deviation n.s. n.s. n.s. n.s. n.s. * n.s.
Acp‐2
 a 0·286 0·500 0·667 0·632 0·441 0·750 0·250 0·750 0·375 0·500 0·750 0·750
 b 0·714 0·500 0·333 0·368 0·559 0·250 0·750 0·250 0·625 0·500 0·250 0·250
Deviation n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. * n.s.
Acp‐4
 a 0·429 0·000 0·567 0·395 0·382 0·525 0·500 0·750 0·438 0·643 0·614 0·562
 b 0·571 0·500 0·433 0·605 0·500 0·475 0·500 0·250 0·562 0·357 0·386 0·438
 c 0·000 0·500 0·000 0·000 0·118 0·000 0·000 0·000 0·000 0·000 0·000 0·000
Deviation n.s. ** n.s. ** n.s. * n.s. n.s. n.s. n.s. n.s. n.s.
Est‐3
 a 0·286 0·500 0·333 0·158 0·000 0·350 1·000 1·000 0·938 1·000 0·841 0·969
 b 0·714 0·500 0·667 0·842 1·000 0·650 0·000 0·000 0·062 0·000 0·159 0·031
Deviation n.s. ** ** n.s. * n.s. *** n.s.
Est‐4
a 0·000 0·000 0·150 0·000 0·000 0·000 0·000 0·000 0·000 0·143 0·159 0·250
b 1·000 1·000 0·850 1·000 1·000 1·000 1·000 1·000 1·000 0·857 0·841 0·750
Deviation *** *** n.s. n.s.
Skd‐1
a 1·000 0·000 0·383 0·658 0·706 0·950 1·000 1·000 0·938 1·000 0·704 0·969
b 0·000 1·000 0·617 0·342 0·294 0·050 0·000 0·000 0·062 0·000 0·296 0·031
Deviation *** n.s. n.s. n.s. n.s. * n.s.
Skd‐2
a 0·857 0·000 0·483 0·500 0·735 0·925 0·875 1·000 0·750 1·000 0·818 0·781
b 0·143 1·000 0·517 0·500 0·265 0·075 0·125 0·000 0·250 0·000 0·182 0·219
Deviation n.s. *** n.s. n.s. *** n.s. n.s. * *
Open in a new tab

*** P < 0·001, ** P < 0·01, * P < 0·05, n.s., non‐significant.

Among the 12 populations, no population harboured all 18 alleles. Population VA exhibited 88·9 % of the alleles (16), and populations FL‐I and OH each maintained the fewest alleles (11). Most of the alleles were common and widespread (allele frequency >5 %, found in >10 % of the populations; Marshall and Brown, 1981).

Estimates of genetic diversity

Genetic parameters estimated using one randomly selected nut per tree or bud samples are shown in Table 2. At the species level, the percentage of polymorphic loci (Ps) was 72·7 %, the mean number of alleles per polymorphic locus (APs) was 2·3 and the effective number of alleles per locus (Aes) was 1·5. The expected genetic diversity was 0·296. At the population level, values of Pp, APp and Aep were 49·2 %, 2·1 and 1·4, respectively. The percentage of polymorphic loci ranged from 27·3 % for populations OH and FL‐I to 72·7 % for population VA. The mean number of alleles per polymorphic locus varied from 2·0 to 2·4, and the effective number of alleles per locus from 1·2 to 1·5. The mean genetic diversity at the population level (Hep) was 0·205 ± 0·052 (mean ± s.d.), ranging from a low of 0·114 ± 0·062 detected in the naturalized population OH to a high of 0·285 ± 0·071 in population MS. Similarly high values of genetic diversity were detected in populations FL‐B and VA (0·275 and 0·262, respectively). Genetic diversity values were higher when the number of allozyme loci was increased from 11 in population VA and VB [He(VA) = 0·262, He(VB) = 0·207) to 16 [He(VA) = 0·283, He(VB) = 0·226]. North‐west Florida was the most variable resource for Castanea pumila var. pumila. The percentage of polymorphic loci (Pp) for the six Florida populations investigated ranged from 27·3 to 63·6 %, APp ranged from 2·0 to 2·4, Aep from 1·2 to 1·5 and Hep from 0·136 to 0·275.

Table 2.

Allozyme genetic variation for 12 populations of Castanea pumila var. pumila

Population Tree (No.) Tissue analysed P AP A A e Ho (s.d.) He (s.d.)
AL‐U 7 Nut 45·5 2·0 1·5 1·3 0·286 (0·109) 0·200 (0·072)
AL‐M 9 Bud 36·4 2·3 1·5 1·4 0·323 (0·140) 0·204 (0·086)
FL‐B 19 Nut 63·6 2·1 1·7 1·5 0·258 (0·074) 0·275 (0·070)
FL‐D 17 Nut 45·5 2·4 1·6 1·4 0·209 (0·075) 0·219 (0·077)
FL‐E 20 Nut, Bud 54·5 2·2 1·6 1·4 0·255 (0·102) 0·202 (0·073)
FL‐G 4 Nut 36·4 2·0 1·4 1·3 0·136 (0·062) 0·162 (0·072)
FL‐I 2 Nut 27·3 2·0 1·3 1·2 0·136 (0·070) 0·136 (0·070)
FL‐L 8 Nut 54·5 2·0 1·5 1·3 0·193 (0·072) 0·198 (0·070)
MS 30 Bud 63·6 2·1 1·7 1·5 0·218 (0·090) 0·285 (0·071)
OH 14 Nut 27·3 2·0 1·3 1·2 0·117 (0·079) 0·114 (0·062)
VA 22 Nut 72·7 2·0 1·7 1·4 0·169 (0·047) 0·262 (0·055)
VB 16 Nut 63·6 2·0 1.6 1.4 0.170 (0·061) 0·207 (0·066)
Mean 49·2 2·1 1·5 1·4 0·206 0·205
s.d. 15·2 0·1 0·2 0·1 0·065 0·052
Species 72·7 2·3 1·9 1·5 0·296
Open in a new tab

P, Percentage of polymorphic loci; AP, mean number of alleles per polymorphic locus; A, mean number of alleles per locus; Ae, effective number of alleles per locus; Ho, observed heterozygosity; He, unbiased estimate of expected heterozygosity (Nei, 1978).

Significant heterogeneity in allele frequency was found among populations. Chi‐square tests for heterogeneity of allele frequency showed that allele frequencies among populations were significantly different for all eight polymorphic loci. Populations were tested for conformity to the Hardy–Weinberg equilibrium at each variable locus, and results indicated that loci Dia‐1 and Acp‐4 deviated significantly from the Hardy–Weinberg equilibrium in three populations, loci Prx‐4 and Acp‐2 in one population each, Est‐3 and Skd‐2 in four populations each, and Est‐4 and Skd‐1 in two populations each (Table 3).

Table 3.

Fixation index (FIS) values at eight polymorphic allozyme loci for each population of Castanea pumila var. pumila

Locus
Population Dia‐1 Prx‐4 Acp‐2a Acp‐4 Est‐3 Est‐4 Skd‐1 Skd‐2
AL‐U 0 –0·750 –0·400 –0·750 –0·400 0 0 –0·167
AL‐M –0·604** 0 –0·111 –1·000** –1·000** 0 0 0
MS –0·470** 0 0·250 –0·222 –0·500** 0·869** 0·929** 0·933**
FL‐B 0·126 0·124 0·321 –0·652** –0·188 0 0·182 0·263
FL‐D –0·042 0 0·165 –0·097 0·000 0 0·150 –0·058
FL‐E –0·322* 0 –0·067 –0·504* –0·538* 0 –0·053 0·640***
FL‐G 0 0·467 –0·333 0 0 0 0 –0·143
FL‐I 0 –0·333 –0·333 –0·333 0 0 0 0
FL‐L 0 –0·067 –0·067 –0·270 –0·067 0 –0·067 0·333
OH 0 0 –0·143 –0·556 0 1·000*** 0 0
VA 0·151 0·494* 0·394* –0·054 0·830*** 0·189 0·672* 0·389*
VB 0 0·373 0·333 –0·270 –0·032 0 –0·032 0·451*
Open in a new tab

*** P < 0·001, ** P < 0·01 and * P < 0·05.

Genotype analysis of six populations was also conducted using several nuts per tree to study the occurrence of non‐random mating and to estimate outcrossing rates. While the percentage of polymorphic loci per population increased, observed heterozygosity decreased in the smaller populations (AL‐U and OH), but increased in the larger Virginia populations (Table 4). Allele frequencies and expected heterozygosity values were not affected by an increase in the number of nut samples per tree. Estimates of outcrossing rates varied from 0·61 for population VA to 1·13 in FL‐D (Table 4).

Table 4.

Genetic variation for populations of Castanea pumila var. pumila using allozyme variation of several nuts per tree

Population Tree (No.) Nuts/tree P A A e Ho (s.d.) He (s.d.) t
AL‐U 7 10·3 66·7 1·75 1·33 0·197 (0·034) 0·199 (0·058) 0·98
FL‐B 19 3·4 63·6 1·73 1·47 0·262 (0·045) 0·264 (0·067) 0·99
FL‐D 17 3·5 54·6 1·73 1·39 0·226 (0·043) 0·213 (0·070) 1·13
OH 14 5·0 50·0 1·50 1·18 0·107 (0·028) 0·108 (0·049) 0·98
VA 22 9·4 75·0 2·00 1·53 0·223 (0·025) 0·295 (0·059) 0·61
VB 16 7·9 75·0 1·83 1·36 0·202 (0·030) 0·210 (0·061) 0·92
Mean 64·2 1·76 1·38 0·203 0·215
Open in a new tab

P, Percentage of polymorphic loci; A, mean number of alleles per locus; Ae, effective number of alleles per locus; Ho, observed heterozygosity; He, unbiased estimate of expected heterozygosity (Nei, 1978); t, outcrossing rate, estimated using the equation Fe = (1 – t)/(1 + t) and Fe = (He – Ho)/He (Berg and Hamrick, 1992).

Population structure and gene flow

Differentiation among populations was estimated using Nei’s statistics (Nei, 1973, 1978). Seventy per cent of the diversity resided within populations (GST = 0·30 calculated only for polymorphic loci). Among the eight polymorphic loci, GST ranged from 0·111 for locus Acp‐4 to 0·557 for locus Est‐3 (Table 5). The high GST value of 0·30 indicated that C. pumila var. pumila populations are highly differentiated and isolated. A low level of gene flow (Nm) was detected, 0·57 migrant per generation.

Table 5.

Gene diversity statistics for eight polymorphic allozyme loci in 12 Castanea pumila var. pumila populations†

Locus H T H S D ST G ST F IS χ2
Dia‐1 0·385 0·305 0·080 0·208 –0·245 82·52 (22)**
Prx‐4 0·372 0·223 0·150 0·401 0·046 156·60 (11)**
Acp‐2 0·477 0·415 0·062 0·130 0·004 31·35 (11)**
Acp‐4 0·536 0·477 0·059 0·110 –0·388 134·57 (22)**
Est‐3 0·499 0·221 0·278 0·557 –0·384 165·07 (11)**
Est‐4 0·153 0·132 0·021 0·137 0·364 35·37 (11)**
Skd‐1 0·406 0·197 0·209 0·515 0·419 125·15 (11)**
Skd‐2 0·423 0·267 0·156 0·369 0·328 92·50 (11)**
Mean (Nei) 0·296 0·203 0·092 0·221 0·013
Mean (Poly) 0·406 0·279 0·127 0·303 0·018
Open in a new tab

† Three loci, Dia‐6, Pgi‐1, and Sod‐1 were monomorphic.

HT, Total genetic diversity; HS, within‐population genetic diversity; DST, among‐population genetic diversity; GST, among‐population differentiation; FIS, fixation index within populations.    ** P < 0·01.

In the UPGMA cluster analysis (Fig. 2), large variation in genetic identity (I = 0·696–1·000) and distance (D = 0·000–0·512) were observed (data not shown). Population AL‐M had the lowest identity to the other populations. The 12 populations were separated into two distinct groups. Group 1 consisted of population AL‐M only. Group 2 was separated into two main branches, the first consisted of the Alabama population AL‐U, Florida populations FL‐G, FL‐I and FL‐L, and Virginia populations VA and VB, and the second of Florida populations FL‐B, FL‐D and FL‐E, and Mississippi population MS. The naturalized OH population, which originated from nuts collected in Virginia (G. Miller, pers. comm.), showed high identity to the Virginia populations.

graphic file with name mcg126f2.jpg

Open in a new tab

Fig. 2. UPGMA dendrogram based on Nei’s (1978) unbiased genetic distance for 12 Castanea pumila var. pumila populations using allozymes.

Migration patterns

Population divergence in allele frequency was studied at two levels: divergence in allele frequency between populations and divergence in allele occurrence (Bradshaw, 1975). Because several of the natural C. pumila var. pumila populations investigated in this study are small and are threatened with extinction from chestnut blight, as well as being located far from each other, population divergence in allele occurrence was easily observed (Table 1). Divergences in allele frequency were detected only for alleles Acp‐2a and Acp‐2b; however, no trends in allele frequency distribution along the natural range were observed.

DISCUSSION

At the species level, the genetic parameters (P, AP, A and Ae) calculated using allozyme data from all populations were consistent with the estimates obtained using only the large populations (MS, FL‐B, FL‐D, FL‐E, VA, VB and OH, with 14–30 trees per population). The expected genetic diversity (He) was 0·292 using allozyme data from large populations only vs. 0·296 using all populations.

At the species level, C. pumila var. pumila showed a high level of genetic variation (Ps = 72·7, APs = 2·3, As = 1·9, Aes = 1·5 and Hes = 0·296) when compared with levels of allozyme variation in other woody species (Ps = 64·7 ± 2·7, Aes = 1·24 ± 0·02 and Hes = 0·177 ± 0·010), other species with a regional distribution (Ps = 52·9 ± 2·1, Aes = 1·20 ± 0·01 and Hes = 0·150 ± 0·008), with wind‐pollinated species (Ps = 66·1 ± 2·7, Aes = 1·21 ± 0·02 and Hes = 0·162 ± 0·009), and with species whose seeds are dispersed by gravity or animals (Ps = 69·3 ± 3·5, Aes = 1·23 ± 0·07 and Hes = 1·66 ± 0·037) (Hamrick and Godt, 1989). The results for C. pumila var. pumila are consistent with previous reports of allozyme diversity in other Castanea species (Villani et al., 1991a, b; Huang et al., 1998; Dane et al., 1999; Lang and Huang, 1999). The genetic diversity (Hes = 0·296) was much higher than that observed for C. dentata (Hes = 0·167), which is geographically sympatric with C. pumila var. pumila (Johnson, 1988; Paillet, 1993), and was similar to that of the closely related C. pumila var. ozarkensis (Hes = 0·272). Paillet (1993) studied the distribution, growth form and stem histories of C. dentata and C. pumila var. ozarkensis and pumila, and found that the C. pumila var. ozarkensis was intermediate between C. pumila var. pumila and C. dentata in life cycle and stem forms as well as in resistance to chestnut blight, although only a few signs of possible introgression were found and recognizable F1 hybrids between C. pumila and C. dentata were relatively rare (Johnson, 1988).

Levels of genetic variation in C. pumila var. pumila were lower than those in C. mollissima (Hes = 0·311), but higher than those in C. seguinii (Hes = 0·186), and similar to those in C. henryi (Hes = 0·261) (Huang et al., 1994; Lang and Huang, 1999). At the population level, C. pumila var. pumila was less variable than C. sativa (Hep = 0·210–0·27) (Villani et al., 1991a, b).

Based on allozyme variation, studies of intra‐ and intercontinental genetic divergence in Castanea in eastern Asia and eastern North America have indicated that C. dentata has similar genetic distances to C. pumila var. pumila (0·328) and C. pumila var. ozarkensis (0·316) (Dane et al., 2003). However, American Castanea spp. do differ from Asian Castanea spp. Whereas all American Castanea spp. showed the highest distance from C. seguinii (0·709 with C. dentata, 0·749 with C. pumila var. pumila, and 0·833 with C. pumila var. ozarkensis), C. pumila var. pumila showed the lowest distance from C. mollissima (0·674), and C. pumila var. ozarkensis showed the lowest distance from C. henryi (0·654). The evolutionary pathway and phylogeographic structure should be more clearly revealed by studying patterns of plastid DNA polymorphism. Such exploration has been done in other closely related genera and in C. sativa (Dumolin‐Lapegue et al., 1997; Fineschi et al., 2000).

In this study, higher levels of deviation from Hardy–Weinberg equilibrium were detected than in other Castanea species and other woody species (Villani et al., 1991a, b; McDonald and Hamrick, 1996; Kremer and Zanetto, 1997; Huang et al., 1998). Twenty of the 65 Chi‐square tests were significant, indicating drift and non‐random mating. This conclusion was verified by our observations that Castanea pumila var. pumila produced few nuts although the trees are primarily wind‐pollinated and should cross freely. This was especially evident in several populations at Eglin Air Force Base in Florida, where most trees showed limited nut production. A decrease in population size as a result of chestnut blight and low gene flow, as well as non‐random mating, are possible causes of such high levels of disequilibrium.

Unlike the strong association between genetic distance and geographic distance observed for C. dentata (r = – 0·708, P < 0·01; Huang et al., 1998), a geographic pattern of genetic differentiation in the C. pumila var. pumila populations could not be detected. However, the Florida sandhills showed the most variable genetic resources for C. pumila var. pumila.

The proportion of genetic diversity among populations (30·4 %, GST = 0·304) was higher than that of temperate species (24·6 %), regional plant species (21·6 %), woody species (7·6 %), wind‐pollinated, outcrossing species (9·9 %), species whose seeds are dispersed by gravity or animals (12·4 %), and late‐successional species (10·1 %) (Hamrick and Godt, 1989). It is also higher than that found for C. pumila var. ozarkensis (14·7 %) and other Castanea species, such as C. dentata (11 %), C. sativa (10 %), C. mollissima (7·5 %), C. seguinii (10·9 %) and C. henryi (22·1 %) (Villani et al., 1991a, b; Huang et al., 1998; Dane et al., 1999; Lang and Huang, 1999). Gene flow, estimated in C. pumila var. pumila as less then one migrant per generation [Nm(W) = 0·57], was lower than that for C. mollissima [Nm(W) = 3·20], C. seguinii [Nm(W) = 2·05], C. dentata [Nm(W) = 2·62] and C. sativa [Nm(W) = 1·72] (Lang and Huang, 1999). When only large populations (consisting of more than 20 trees) were used, the proportion of genetic diversity among populations was lower than that estimated using all populations (GST = 0·217), and gene flow was estimated to be higher than the value obtained using all populations (Nm = 0·90), although it was still low. Estimates of outcrossing rates of 0·62 for VA to 1·13 for FL‐D (Table 4) were similar to those reported for other wind‐pollinated species (Vogler and Kalisz, 2001).

Population outcrossing rate and gene flow estimates indicate that the present genetic diversity in the endangered C. pumila var. pumila populations is predominantly the result of plant history (e.g. glacial refuges, migration) (Geburek, 1997). Seventy per cent of all allozyme variation was within C. pumila var. pumila populations. In terms of conservation, optimal sampling strategies should focus on the collection of at least one copy of common populational alleles with a frequency greater than 0·05 (Marshall and Brown, 1981). Thus populations with a high number of alleles per locus should be given priority. For the C. pumila var. pumila populations investigated, populations MS, FL‐B and VA harboured a high number of alleles per locus (A = 1·7 for each population), and showed higher genetic diversity. These populations should receive priority for conservation management purposes. For the initial stage of a conservation programmes, sampling eight to 15 trees from population VA and sampling nine to 17 trees from population FL‐D would capture at least one to two copies of the common alleles. Since the naturalized OH population showed significantly lower genetic diversity, sampling nuts from a small number of trees at one location only should be avoided. Efforts should be made to promote the propagation of trees using artificial supplementary pollination within regions (Dane et al., 1999). In the long term, carrying out a backcross breeding programme to transfer chestnut blight resistance genes from Asian Castanea species into the American chinkapin will be most promising (Payne et al., 1994).

ACKNOWLEDGEMENTS

We thank USDA‐ARS for partial support of this work; Joe Norton, Fred Hebard, Paul Sisco, Scott Hassell, Neil Hoskins, Greg Miller, Tom Kubisiak and Bill Piercy for their help with field sampling; and Rasima Bakhtiyarova for technical assistance.

Supplementary Material

Content Snapshot
supp_92_2_223__index.html (994B, html)

Received: 14 February 2002; Returned for revision: 22 July 2002; Accepted: 6 May 2003    Published electronically: 26 June 2003

References

  1. BergEE, Hamrick JL.1997. Quantification of genetic diversity at allozyme loci. Canadian Journal of Forest Research 27: 415–424. [Google Scholar]
  2. BooyG, Hendriks RJJ, Smulders MJM, van Groenendael JM, Vosman B.2000. Genetic diversity and the survival of populations. Plant Biology 2: 379–395. [Google Scholar]
  3. BradshawAD.1975. Population structure and the effect of isolation and selection. In: Frankel OH, Hawkes JG, eds. International biological programme 2. Crop genetic resources for today and tomorrow. Cambridge: Cambridge University Press. [Google Scholar]
  4. CampbellDR.1991. Comparing pollen dispersal and gene flow in a natural population. Evolution 45: 1965–1968. [DOI] [PubMed] [Google Scholar]
  5. DaneF, Hawkins LK, Huang H.1999. Genetic variation and population structure of Castanea pumila var. ozarkensis Journal of the American Society for Horticultural Science 124: 666–670. [Google Scholar]
  6. DaneF, Lang P, Huang H, Fu Y.2003. Inter‐continental genetic divergence of Castanea (Fagaceae) in eastern Asia and eastern North America. Heredity 91 (in press). [DOI] [PubMed] [Google Scholar]
  7. DemesureB, Comps B, Petit RJ.1996. Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe. Evolution 50: 2515–2520. [DOI] [PubMed] [Google Scholar]
  8. Dumolin‐LapegueS, Demesure B, Fineschi S, Le Corre V, Petit RJ.1997. Phylogeographic structure of white oaks throughout the European continent. Genetics 146: 1475–1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. FineschiS, Gillet E, Malvolti ME.1990. Genetics of sweet chestnut (Castanea sativa Mill.) III. Genetic analysis of zymograms of single tree offspring. Silvae Genetica 39: 188–194. [Google Scholar]
  10. FineschiS, Gillet E, Malvolti ME.1991. Segregation among fruits from single burrs of sweet chestnut (Castanea sativa Mill.). In: Fineschi S, Malvolti ME, Cannata F, Hattemer HH, eds. Biochemical markers in the population genetics of forest trees. The Netherlands: SPB Academic Publishing BV. [Google Scholar]
  11. FineschiS, Taurchini D, Villani F, Vendramin GG.2000. Chloroplast DNA polymorphism reveals little geographical structure in Castanea sativa Mill. (Fagaceae) throughout southern European countries. Molecular Ecology 9: 1495–1503. [DOI] [PubMed] [Google Scholar]
  12. FulbrightDW.1999. Chestnut blight and hypovirulence. In: Stacey G, Keen NT, eds. Plant microbe interactions. St Paul, MN: APS Press. [Google Scholar]
  13. GeburekT.1997. Isozymes and DNA markers in gene conservation of forest trees. Biodiversity and Conservation 6: 1639–1654. [Google Scholar]
  14. GodtMJW, Hamrick JL.1998. Allozyme diversity in the endangered pitcher plant Sarracenia rubra ssp. alabamensis (Sarraceniaceae) and its close relative S. rubra ssp. rubra. American Journal of Botany 85: 802–810. [PubMed] [Google Scholar]
  15. HamrickJL, Godt MJW.1989. Allozyme diversity in plant species. In: Brown AHD, Clegg MT, Kahler AL, Weir BS, eds. Plant population genetics, breeding and genetic resources. Sunderland, MA: Sinauer Associates Inc. [Google Scholar]
  16. HamrickJL, Godt MJW, Sherman‐Broyles SL.1992. Factors influencing levels of genetic diversity in woody plant species. New Forests 6: 95–124. [Google Scholar]
  17. HuangH.1993.Inheritance, diversity of allozyme coding loci, and linkage relationships between allozyme and morphological markers in chestnut (Castanea). PhD Thesis, Auburn University, Auburn, USA. [Google Scholar]
  18. HuangH, Dane F, Kubisiak TL.1998. Allozyme and RAPD analysis of the genetic diversity and geographic variation in wild populations of the American chestnut (Fagaceae). American Journal of Botany 85: 1013–1021. [PubMed] [Google Scholar]
  19. HuangH, Dane F, Norton JD.1994a. Genetic analysis of 11 polymorphic isozyme loci in chestnut species and characterization of chestnut cultivars by multi‐locus allozyme genotypes. Journal of the American Society for Horticultural Science 119: 840–849. [Google Scholar]
  20. HuangH, Dane F, Norton JD.1994b. Allozyme diversity in Chinese, Seguin and American chestnut (Castanea spp.). Theoretical and Applied Genetics 88: 981–985. [DOI] [PubMed] [Google Scholar]
  21. JohnsonGP.1987. Chinquapins: taxonomy, distribution, ecology, and importance. Northern Nut Growers’ Association Annual Report 78: 58–62. [Google Scholar]
  22. JohnsonGP.1988. Review of Castanea sect. Balanocastanon (Fagaceae). Journal of the Arnold Arboretum 69: 25–49. [Google Scholar]
  23. KremerA, Zanetto A.1997. Geographical structure of gene diversity in Quercus petraea (Matt.) Liebl. II: Multilocus patterns of variation. Heredity 78: 476–489. [Google Scholar]
  24. KrutovskiiKV, Bergmann F.1995. Introgressive hybridization and phylogenetic relationships between Norway, Picea abies (L.) Karst. and Siberian, P. obovata Ledeb. spruce species studied by isozyme loci. Heredity 74: 464–480. [Google Scholar]
  25. LangP, Huang H.1999. Genetic variation and population structure of three endemic Castanea species in China. Acta Horticulturae 494: 269–276. [Google Scholar]
  26. LeveneH.1949. On a matching problem arising in genetics. Annals of Mathematical Statistics 20: 91–94. [Google Scholar]
  27. McDonaldDB, Hamrick JL.1996. Genetic variation in some plants of Florida scrub. American Journal of Botany 83: 21–27. [Google Scholar]
  28. MarshallDR, Brown AHD, 1981.Wheat genetic resources. In: Evans LT, Peacock WJ, eds. Cambridge: Cambridge University Press. [Google Scholar]
  29. NeiM.1973. Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences of the USA 70: 3321–3323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. NeiM.1977. F‐statistics and analysis of gene diversity in subdivided populations. Annals of Human Genetics 41: 225–233. [DOI] [PubMed] [Google Scholar]
  31. NeiM.1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89: 583–590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. PailletFL,1993. Growth form and life histories of American chestnut and Allegheny and Ozark chinkapin at various North American sites. Bulletin of the Torrey Botanical Club 120: 257–268. [Google Scholar]
  33. PayneJA, Miller G, Johnson GP, Senter SD.1994.Castanea pumila (L.) Mill.: an underused native nut tree. HortScience 29: 62, 130–131. [Google Scholar]
  34. SwoffordDL, Selander RB.1981. BIOSYS‐1: a FORTRAN program for the comprehensive analysis of electrophoretic data in population genetics and systematics. Journal of Heredity 72: 281–283. [Google Scholar]
  35. VillaniFS, Pigliucci M, Benedettelli S, Cherubini M.1991a. Genetic differentiation among Turkish chestnut (Castanea sativa Mill.) populations. Heredity 66: 131–136. [Google Scholar]
  36. VillaniFS, Benedettelli S, Paciucci M, Cherubini M, Pigliucci M.1991b. Genetic variation and differentiation between natural populations of chestnut (Castanea sativa Mill.) from Italy. In: Fineschi S, Malvolti ME, Cannata F, Hattemer HH, eds. Biochemical markers in the population genetics of forest trees. The Netherlands: SPB Academic Publishing BV Press. [Google Scholar]
  37. VoglerDW, Kalisz S.2001. Sex among flowers: the distribution of plant mating systems. Evolution 55: 202–204. [DOI] [PubMed] [Google Scholar]
  38. WendelJF, Weeden NF.1989.Genetics of plant isozymes In: Soltis ED, Soltis PS, eds. Isozymes in plant biology. Portland, Oregon: Dioscorides Press, 9–63. [Google Scholar]
  39. WrightS.1951. The genetical structure of populations. Annals of Eugenetics 15: 323–354. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Content Snapshot
supp_92_2_223__index.html (994B, html)
supp_92.2.223_mcg168f7.gif (9.9KB, gif)

Articles from Annals of Botany are provided here courtesy of Oxford University Press

RESOURCES