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The population genetic structure of the Greater Rhea (Rhea americana) in an agricultural landscape
Biological Conservation 99 (2001) 277±284
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The population genetic structure of the Greater Rhea (Rhea americana) in an agricultural landscape Juan L. Bouzat * Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403-0212, USA
Abstract Although population genetic structure and geographic dierentiation have been reported for many bird species with insular distributions, high vagility typically results in high levels of gene ¯ow and thus little local dierentiation among continental populations. This study provides the ®rst survey of population genetic variation in a South American ratite, suggesting that, in an agricultural landscape, recent fragmentation and isolation may aect the genetic structure of ¯ightless bird species. Using randomly ampli®ed polymorphic DNA (RAPD) analysis, I evaluated the population genetic structure of the greater rhea, Rhea americana albescens, by quantifying genetic variability within and among four isolated wild populations located in a highly fragmented region of the Argentinean Pampas. Levels of genetic diversity of the natural populations were compared to those of a captive population used as an inbred ``control.'' An analysis of molecular variance (AMOVA) based on a phenotypic analysis of 54 RAPD markers showed that 94.38% of the total observed variance was explained by dierences within populations (P<0.001) whereas 6.37% was due to dierences among populations (P=0.006). In addition, measures of genetic diversity (D: mean genetic diversity, and P: percentage of polymorphic bands) estimated for the wild populations were similar to those of the inbred ``control.'' The pattern of genetic variation reported combined with data on the sizes of eight wild populations and the reproductive success of a focal population are consistent with the idea that relatively recent fragmentation and isolation may have increased local genetic dierentiation and decreased within-population levels of genetic variation as a result of genetic drift and inbreeding. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Genetic diversity; Fragmentation; RAPD; AMOVA; Rhea americana
1. Introduction As a result of human activities, signi®cant declines in the distribution and abundance of many species have increased their risk of global extinction (SouleÂ, 1986; Mee and Carroll, 1994). Habitat destruction has led to the fragmentation of natural populations and today, many species that were originally distributed in continuous habitats exist as metapopulations, i.e. groups of interacting populations, often with limited gene ¯ow (Gilpin and Hanski, 1991). The genetic eects of fragmentation are directly related to the stochastic processes associated with small population size (Gilpin, 1987, 1991). As populations become isolated with small eective sizes, genetic bottlenecks and founder events through extinction and recolonization processes may reduce the genetic variability within each island population. Such * Tel.: +1-419-372-9240; fax: +1-419-372-2024. E-mail address: [email protected] (J.L. Bouzat).
reduction in genetic diversity may result directly from a founder event as well as from the combined eects of genetic drift and inbreeding (Wright, 1978). The signi®cance of genetic variation in conservation and extinction of natural populations has been widely addressed (Gilpin and SouleÂ, 1986; Soule and Wilcox, 1980; SouleÂ, 1987; Schonewald-Cox et al., 1983). Two aspects of genetic diversity are of importance. First, loss of genetic diversity can result in reduced evolutionary potential for future adaptation through evolutionary change (Frankel, 1983). Second, there is evidence that reduced genetic variability at both the individual and population levels can result in a reduction in ®tness (e.g. Quattro and Vrijenhoek, 1989; Bouzat et al., 1998a,b; Westemeier et al., 1998). Thus, populations with depauperate levels of genetic diversity generally face increased risk of extinction (Gilpin and SouleÂ, 1986). The greater rhea, Rhea americana, represents a critical model organism to study the potential eects of fragmentation, isolation and small eective size on the
0006-3207/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0006-3207(00)00193-2
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genetic diversity of natural populations that have been highly aected by human activities. The ¯ightless condition of rheas, a characteristic that contrasts with the majority of continental bird species, and the increasing isolation of their populations as a result of habitat loss and fragmentation have the potential for profoundly aecting patterns of genetic variation. In addition, the rheas' mating system of harem polygyny (Bruning, 1974), in which few males contribute to the formation of the following generations, may decrease eective population sizes intensifying stochastic events such as genetic drift (Lande and Barrowclough, 1987). Although population genetic structure has been reported for many bird species with insular geographic distributions (e.g. Gibbs and Grant, 1989; Baker et al., 1990; Grant and Grant, 1992), many continental bird species fail to show any genetic structuring because of their high vagility, which typically results in high levels of gene ¯ow (Barrowclough, 1980; Rockwell and Barrowclough, 1980; Evans, 1987). In addition, the lack of control populations in most studies intended to assess the genetic eects of fragmentation and small population size (e.g. bottlenecks) prevents one from inferring a direct causal link between population size, isolation and genetic diversity in natural populations (Mee and Carroll, 1994; Bouzat et al., 1998a,b). This study provides the ®rst survey of population genetic variation in a South American ratite suggesting that relatively recent fragmentation and isolation may have increased local genetic dierentiation and decreased within-population levels of genetic variation. The population genetic structure of R. a. albescens was evaluated using randomly ampli®ed polymorphic DNA (RAPD) analysis. Although there have been both methodological and analytical controversies surrounding the use of RAPDs to estimate genetic variability (Grosberg et al., 1996), several studies have demonstrated their usefulness in addressing issues of ecological and evolutionary importance. RAPD studies included the analysis of genetic variation within and among vertebrate populations (e.g. Kimberling et al., 1996; Nusser et al., 1996), levels of genetic variability in plant populations (e.g. Hsiao and Lee, 1999; Maki and Horie, 1999), measures of genetic distance associated with ®tness (Trame et al., 1995), and parental analysis (Gronemeyer et al., 1997). In this study, 54 polymorphic RAPD markers were used for quantifying genetic variability within and among four isolated wild populations of the greater rhea located in a region highly aected by agricultural development and cattle breeding. The within-population component of genetic diversity from the natural populations studied was compared to that of a captive population with a small number of founders used as an inbred ``control.'' Speci®cally, I addressed the following questions: (1) what patterns of genetic diversity exist
within and among fragmented populations of the greater rhea? (2) Are estimates of within-population genetic variation observed in nature similar to those of a captive population with a small founder size? Complementary data on the sizes of eight natural populations of R. a. albescens and on the reproductive success of a focal wild population (measured as number of successful nests produced during the studied reproductive season) are also reported to evaluate potential factors that might have led to the observed genetic patterns in the wild. Implications for the conservation of the greater rhea are discussed in light of the current status of the studied populations as well as their observed population genetic structure. 2. Materials and methods 2.1. Studied populations and sampling Population genetic structure of R. a. albescens was assessed from the analysis of 50 samples collected from ®ve populations (Fig. 1). These included four wild populations (Trebolar, OlavarrõÂa, Rastros I and Rastros II) and a captive population with a small number of founders (Captive). The four natural populations (38 samples) were grouped according to their geographic location. The Trebolar±OlavarrõÂa group was separated from the Rastros I±Rastros II group by more than 400 km. Populations within the groups were located within 50 km from each other. Population Trebolar (a focal
Fig. 1. Geographic location of wild populations of subspecies R.a. albescens used in this study. Stars indicate populations used in the RAPD analysis. Filled circles indicate populations censussed.
J.L. Bouzat / Biological Conservation 99 (2001) 277±284
population used for behavioral studies) had an estimated size of 283 individuals. The other three populations sampled were known to be smaller than 100 individuals each (J. Flores and G. Wendor, personal communications). In addition, 12 samples were obtained from a captive population of 57 rheas from Cargill's farm near Pilar city, CoÂrdoba province, in 1993. This population was founded in 1974±1975 by seven rheas (two adult individuals and ®ve yearlings from the same brood) and remained isolated over more than 20 years (A. Rimada, personal communication). This captive population was used as an experimental ``control'' population for comparison to evaluate if similar founding processes might be occurring in the wild. Because this population was unmanaged, and most founders were ®rst-degree relatives, it is likely highly inbred. Although environmental eects were not ruled out, morphological asymmetries observed in two of the 57 rheas from this population may be the result of the high levels of inbreeding. Small sample sizes resulted from logistic diculties in capturing adult birds. Samples from multiple nests, however, were included, thereby increasing representation of the reproductive population. Communal nests in the greater rhea include egg clutches from multiple females (Bruning, 1974). To increase the probability of sampling genotypes from dierent maternal lines two individual chicks were sampled from 14 dierent nests. Sampling from the same nest may, however, bias the estimate of population genetic diversity due to the relatedness between some of the individuals sampled. Sampling variance results from both the number of individuals sampled from a given population and the number of polymorphic markers sampled in every individual. Given the logistic problems of reducing the sampling variance among individuals due to small sample size, I maximized the number of RAPD markers analyzed (54) to reduce the variance heterogeneity among RAPD markers. To evaluate the relative importance of both population size and the eective number of individuals contributing to reproduction on the genetic structure of wild populations of R. a. albescens, data on the sizes of several populations as well as on the reproductive success of a focal population were collected. Census data were collected from eight wild populations located in Buenos Aires province, Argentina, a region known to have historically large continuous populations of the greater rhea, during the breeding season of 1993 (July± December; Fig. 1). Population sizes (adult and juveniles) were estimated by direct censusing using a 2060-mm spotting scope. Ospring hatched during the studied reproductive season were not included. Repeated censusing (n=7) in an area of 900 ha resulted in a mean estimate of 179 individuals with a standard error of 2.4. Although one cannot disregard the possibility of an underestimation of actual population sizes, the prominent size of rheas and the characteristic open landscape of the
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Argentinean Pampas make these estimates relatively precise. On the basis of the repeated censusing, population estimates probably represent more than 95% of actual population sizes. A focal study was carried out in population Trebolar (General Lamadrid county) to evaluate the rhea's mating system (J. L. Bouzat, unpublished data) and to estimate the total numbers of nests and successful nests produced throughout the breeding season (July±December 1993). Average clutch size was estimated from seven nests assumed to have a complete clutch (males were incubating and no females were observed in close proximity). Field observations were made monthly for a period of about 5 days for a total of 27 days (179 h of total observation time). The area in which the population was located (1990 ha) was searched intensively to detect all possible nests. In addition, this area was surveyed by airplane to detect nest sites that may have been overlooked during ground surveys (three nests were detected by air surveys). 2.2. DNA isolation and RAPD methods Blood samples (100±200 ml) for DNA analyses were collected by puncture of the brachial vein and stored in 2 ml of Queen's lysis buer (0.01 M Tris, 0.01 M Na2EDTA, 0.01 M NaCl and 1% n-lauroylsarcosine) at 4 C (Seutin et al., 1991) until further analysis at the University of Illinois. Samples were collected with permits from the National Division of Fauna of Argentina. CITES permits were obtained for sample transportation. DNA from blood samples was extracted following Seutin et al. (1991). Fifty to seventy-®ve microliters of blood stored in Queen's lysis buer were digested overnight at 65 C with Proteinase K (3U in 500 ml of lysis buer). Samples were subjected to two phenol-chloroform extractions and DNA was then precipitated by adding 10% volume of 7.5 M ammonium acetate and one volume of isopropanol. DNA was ®nally resuspended in 50 ml of TE buer and stored at 4 C. Following extraction, DNA was quanti®ed using a ¯uorometer and degradation was assessed by electrophoresis on a 1% agarose gel. Non-degraded DNA samples were diluted to a concentration of 30 ng/ml for further PCR analysis. RAPD ampli®cation reactions (Welsh and McClelland, 1990; Williams et al., 1990) were performed in 25 ml ®nal volumes containing 30 ng of DNA template in 10 mM Tris (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 100 uM of each dNTP, 5 pmol of a 10-base pair random primer, and 0.5 U of Taq polymerase. DNA, PCR buffer, primer, MgCl2, and water were combined to a volume of 15 ml, capped with 10 ml of liquid wax, and subjected to a ``hot-start'' for 5 min at 85 C (Chou et al., 1992). Then, Taq polymerase, dNTPs and water were added to each reaction to a ®nal volume of 25 ml. Ampli®cations were carried out in an MJ Research
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J.L. Bouzat / Biological Conservation 99 (2001) 277±284
PTC-100 thermocycler (MJ Research, Waltham, MA) with a 3-min denaturation step at 94 C followed by 44 cycles of 30 s denaturation at 94 C, 30 s annealing at 36 C, and 1.5 min extension at 72 C, and then a ®nal 10-min extension step at 75 C. Ampli®ed products were then mixed with 0.5 volumes of a loading dye solution and run on a 1% agarose gel electrophoresis at 70 V for 1±2 h. RAPD products were stained with ethidium bromide and visualized under UV light (Fig. 2). Based on the presence of clear and distinct banding patterns, 18 of 30 random primers screened were selected to analyze 12 samples from Trebolar, 7 from OlavarrõÂa, 11 from Rastros I, 8 from Rastros II and 12 from the Captive population. Selected primers included A01, A02, A04, A08, A09, A10, A14, A16, A18, C01, C02, C05, C06, C09, C10, C11, C18 and C19 from Operon Technologies Primer Kits A and C (Operon Technologies, Alameda, CA). From these 18 RAPD primers, 54 polymorphic bands were scored. For each primer, a master mix with all reaction components, except DNA template, was used to minimize artifact bands that might result from dierent PCR starting conditions. In addition, all RAPD reactions for a given primer were performed at the same time using the same thermocycler. For each RAPD primer, a sub-sample of ®ve individuals was replicated to ensure repeatability of banding patterns (Grosberg et al., 1996). 2.3. Genetic variability and statistical analysis To estimate measures of genetic diversity with RAPD markers, a phenotypic analysis of the ampli®ed bands (conventional band frequency scoring) was used (Fritsch and Rieseberg, 1996). Bands that co-migrated were assumed to be homologous. Although this analysis is likely to underestimate levels of genetic variation, it does not assume Hardy±Weinberg equilibrium, a required condition for the genetic analysis of allele frequencies (Fritsch and Rieseberg, 1996). RAPD variation within and among natural populations of the greater rhea was estimated by an analysis of molecular variance (AMOVA; Excoer et al., 1992)
Fig. 2. Example of RAPD markers ampli®ed using Operon primer C06 in the greater rhea. Arrows indicate polymorphic bands scored and used in the analysis of molecular variance. Lanes 1Kb are standard molecular weight markers.
using the AMOVA 1.55 program provided by Laurent Excoer (Department of Anthropology and Ecology, University of Geneva, Switzerland). The AMOVA allows one to estimate variance components for RAPD phenotypes, partitioning the variation among individuals/ within populations, and among populations/within geographic regions (Excoer et al., 1992). Variance components were inferred from metric distances among RAPD fragment patterns estimated as E N1
2Nxy =2N;
where Nxy is the number of bands shared by individuals x and y, and N is the total number of polymorphic markers (Hu et al., 1993). Signi®cance levels for variance component estimates were computed by non-parametric permutation procedures, determining the probability of obtaining a more extreme variance component than the observed values by chance alone (1000 permutations). Genetic variability was also estimated using mean genetic diversity (D) and the percentage of polymorphic bands (P) within populations. Mean genetic diversity (D) was calculated as the average of pair-wise relative distances (d) within populations. Pair-wise relative distances between any two given individuals were estimated using the following equation: d1
Nxy =N;
where Nxy is the number of bands shared by individuals x and y, and N is the total number of polymorphic sites. The proportion of polymorphic bands (P) was estimated for each population as the number of polymorphic bands divided by the total number of markers analyzed. Levels of within-population genetic diversity for the four studied natural populations were compared to those of the captive population founded in 1974±1975. Dierences in D among populations were tested using a permutation analysis. Pair-wise comparisons among populations were based on 40,000 permutations, computing a distribution for the p studentized range, R max fi; jgfmean i mean j= var i var jg; where (i) and (j) represent the d values for two given populations, and var (i) and var (j) their variances, respectively. Signi®cant dierences between populations (P<0.05) were determined by estimating con®dence interp vals of the form mean i mean j RCRIT var i var j; where RCRIT is the 95% quantile of the studentized range distribution generated through 40,000 permutations. 3. Results A total of 54 polymorphic markers were ampli®ed by PCR using 18 RAPD primers. PCR replicates showed
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repeatable banding patterns. Bands that were not consistently ampli®ed in the replicates were not considered. The average number of bands per RAPD primer was three (S.E.=0.42, range=1±7). The four wild populations studied showed that, on average, 35% of their RAPD markers were monomorphic, the same proportion of monomorphic markers observed in the captive ``control'' population. Each population group (Trebolar±OlavarrõÂa and Rastros I±Rastros II) showed ®ve monomorphic markers (9% of the total number of markers analyzed). The AMOVA results were based on the metric distances among multiple RAPD band patterns as the dependent variable. Estimated phenotypic distances among RAPD patterns showed considerable variation within each population. Every individual sampled had a genetically unique RAPD pattern (i.e. the distance matrix used in the AMOVA did not show any zero values). Results of the hierarchical partitioning of the variance among the four wild populations are shown in Table 1. The variance component between the two geographic groups considered (Trebolar±OlavarrõÂa and Rastros I±Rastros II) was not statistically dierent from zero (P=0.6653). The proportion of the ``among populations'' variance component within each geographic group was small (6.37%) relative to the ``within population'' variance component (94.38%). These were highly signi®cant, indicating that the observed dierences among and within populations were larger than that expected by chance. Comparisons of levels of genetic variation (mean genetic diversity within populations (D) and the proportion of polymorphic RAPD markers (P)) for each of the four wild populations and the captive population with small founder size are shown in Table 2. The permutation test showed no signi®cant dierences between the mean genetic diversity of the wild populations and that of the captive control. However, analysis of D for the wild populations revealed that Rastros I and Trebolar had signi®cantly higher genetic diversity than OlavarrõÂa (Table 2; studentized range test based on 40,000 permutations; P<0.05). The proportion of
Table 1 Hierarchical analysis of molecular variance (AMOVA) based on metric distances among randomly ampli®ed polymorphic DNA (RAPD) haplotypes for four wild populations of the greater rheaa Variance component
d.f. Variance % Total
Between groups 1 Among populations/within groups 2 Within populations 34
0.056 0.476 7.046
0.75 6.37 94.38
P 0.6653 0.0060 <0.0010
a In an AMOVA, small negative values may occur for the higher hierarchical levels because these statistics are not computed as sum of squares (Excoer et al., 1992).
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polymorphic markers (P) for each individual population showed similar trends. Both Rastros I and Trebolar had the highest P compared to the other populations (Table 2). In addition, population OlavarrõÂa revealed the lowest estimated values for both D and P. Data on the sizes of the eight wild populations surveyed are shown in Table 3. Estimated population sizes ranged from eight to 283 individuals. Five of the eight populations were smaller than 100 individuals in size. In addition, there were considerable dierences in the size of the areas in which these populations were located (Table 3). Population Trebolar, the largest wild population studied, produced at least 26 nests during the reproductive season of 1993. Average clutch size was 20.9 (S.E.=2.0). Of the 26 nests produced, only six (23%) were successful. Unsuccessful nests resulted mainly from ¯ooding (4 nests) and direct or indirect disturbances [from cattle or other animals (14 nests), and humans (2 nests)]. At the population level, there were at least 260 eggs produced (including both successful and unsuccessful eggs) during the breeding season. Of these, 42 (16%) were isolated eggs laid on the ground in areas with no nest in close proximity.
Table 2 Estimates of genetic variability for four wild greater rhea populations and a captive population with small founder size (D, mean genetic diversity; P, proportion of polymorphic bands)a Population Captive OlavarrõÂa Rastros I Rastros II Trebolar Wild populations mean (S.E.)
D 0.22 0.18 0.27 0.24 0.32
P AB A B AB B
0.25 (0.03)
0.65 0.41 0.78 0.55 0.87 0.65 (0.10)
a
Values with dierent letters indicate signi®cant dierences (studentized range test based on 40,000 permutations, P<0.05).
Table 3 Estimated population sizes (N) and area in hectares (A) of eight wild populations of the greater rhea located in Buenos Aires province, Argentina Population Trebolar Las Cortaderas La DivisioÂn 17 Agosto La MarõÂa Pringles FernaÂndez El OmbuÂ
N
A
283 88 118 39 85 8 99 165
1990 5545 4229 300 1000 300 856 3000
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4. Discussion During the last few years, there has been considerable debate concerning the possible eects of fragmentation, isolation and small population size on the genetic variability of bird populations. The high vagility of many bird species typically results in high levels of gene ¯ow and thus little local genetic dierentiation (Barrowclough, 1980; Rockwell and Barrowclough, 1980; Evans, 1987). Only a limited number of studies have demonstrated genetic eects as a result of fragmentation of continental bird populations. For example, high levels of genetic dierentiation have been reported for the endangered red-cockaded woodpecker, Picoides borealis, a non-migratory bird species with high site ®delity (Stangel et al., 1992; Haig et al., 1993). Most of these studies, however, lack control populations (e.g. unfragmented populations or populations with a known demographic history), thus preventing direct testing for the potential genetic eects of fragmentation and small population size. In this study, I evaluated the population genetic structure of the greater rhea in an agricultural landscape, and compared levels of withinpopulation genetic diversity to that of an isolated captive population used as a highly inbred control. The pattern of genetic variation observed within and among the four natural populations of R. a. albescens is consistent with the idea that recent fragmentation and isolation may have increased local genetic dierentiation and decreased within-population genetic variability as a result of stochastic events associated with small population size and inbreeding. The high proportion of polymorphic markers that showed ®xed phenotypes in one or more of the wild populations suggests that stochastic events such as genetic drift may be leading to the ®xation of alternative genetic variants in dierent populations. The AMOVA showed a statistically signi®cant genetic divergence among the four populations nested within the two geographic groups (6.37% of the total observed variance, Table 1). This was also true for all populations independent of the grouping. Alternatively, dierences in P among populations may be overestimated due to limitations of small sample sizes. This was apparent by the fact that the AMOVA showed no signi®cant variation between the two geographic groups. However, the proportion of ®xed markers remained relatively high (9%) when samples from different populations were pooled into two geographically distinct groups, suggesting some levels of genetic dierentiation due to the presence of population-speci®c RAPD bands. The large ``within population'' component of the variance detected by the AMOVA (94.38%) is the result of using metric distances among multiple RAPD banding patterns as the dependent variable for the analysis of variance. In this study, every individual sampled showed
a unique RAPD pattern as a result of the large number of RAPD markers analyzed. Although the ``within population'' component of variance has been shown to be small for RAPD markers in highly inbred species with many generations of arti®cial selection, such as in highly inbred cultivars (Hu and Quiros, 1991; Kazan et al., 1992, Wilde et al., 1992), this does not appear to be true for natural populations of outcrossing species (Hu et al., 1993). In this study, measures of genetic diversity from the captive population were used as an inbred ``control'' population to evaluate the relative levels of within population genetic variation observed in the wild. Although the eects of genetic drift in a single isolated population cannot be predicted (i.e. drift may result in loss of a little, a lot, or no genetic variation), inbreeding will consistently lead to a decrease in genetic variability over time. Comparisons of mean genetic diversity estimates showed that, overall, wild populations had similar levels of genetic variability as the captive ``control'' population, which was probably subjected to more than 10 generations of inbreeding (Table 2). This suggests that similar processes to those operating in an isolated population with a small number of founders might be occurring in the wild. The levels of polymorphism reported in this study can be compared to those observed in other endangered species that have substantially decline in numbers from historic levels. For example, a RAPD study of ®ve populations of the endangered clapper rail (Rallus longirostris), a waterbird inhabiting isolated marshes, showed that 54% of 16 polymorphic RAPD bands analyzed were ®xed in one or more populations (Nusser et al., 1996). The lower proportion of ®xed markers observed in wild populations of the greater rhea (35%) may be the result of relatively recent fragmentation. In contrast to the greater rhea, the historical range of clapper rails was probably restricted to small fragmented populations long time before the impacts of human development (Nusser et al., 1996). The ®xation of alternative genetic variants in dierent populations and geographic groups of the greater rhea cautions against making the simple assumption that avian vagility, which typically results in high levels of gene ¯ow, decreases by default the genetic structuring of continental bird species. The population genetic structure reported in this study is consistent with the idea that fragmentation, isolation and small population size may be aecting the genetic variability of the greater rhea. The data on population sizes and reproductive success are indicative of a population structure of isolated populations with small eective sizes in which only a few reproductive males contribute to the formation of the following generations. Relative abundances reported here indicate that, in Buenos Aires province, most populations are small (e.g. less than 120 individuals; Table 3). The two
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largest populations studied (Trebolar and El OmbuÂ) are exceptions because the landowners of the farms in which rheas are present actively protect them. Estimates of reproductive success suggest that extremely low eective population sizes may be a common feature of most rhea populations inhabiting agricultural landscapes. With an estimated size of 283 rheas, population Trebolar produced only six successful nests during the breeding season of 1993. Assuming the occurrence of no extra-pair fertilizations and no loss of broods due to predation, the above data indicates that only six males would be successfully contributing to reproduction in this population. If we assume that the genetic pattern reported here is the result of stochastic events due to small population size and inbreeding, then we can conclude that fragmentation and isolation may be aecting greater rhea's genetic diversity. The greater rhea is another example of the potential threats of habitat loss and direct human impacts on a wild species. Once an abundant and continuously distributed species throughout the Argentinean Pampas, greater rheas have become rare in areas of intensive agriculture and cattle breeding. Prior to 1991 commercial hunting probably contributed greatly to the decline of rhea populations. For example, during the four-year period, 1976±1979, the number of rheas (including both Rhea and Pterocnemia) exported from Buenos Aires harbor was 102,543 (Mares and Ojeda, 1984). Therefore, hunting pressure probably acted as an important factor in reducing population sizes of rheas, in conjunction with habitat loss and fragmentation as a result of human activities. However, further studies would be needed to determine whether or not recent fragmentation, isolation and small population size have an eect on the genetic structure of rheas that could lead to an increase in their risk of extinction. Acknowledgements I would like to thank Ken N. Paige, Scott Robinson, Je Brawn, Chris Phillips, Harris Lewin, Marcel Amills, Mark W. Schwartz, and two anonymous reviewers for their constructive criticisms on earlier drafts of the manuscript, which originally appeared as part of my PhD dissertation (Bouzat, 1998). I would also like to acknowledge Professor Stephen Portnoy and Maria Bidart for statistical advice, and Brian Dilger for helping in the lab during the DNA preparations. I am particularly grateful to the Rivera family from ``Estancia El Trebolar'' who provided a warm home during my ®eld season observing rheas in Argentina, and to MoÂnica Martella and Gerardo Wendor who facilitated sampling of the captive and Rastros populations, respectively. Financial support for this study was provided by grants from the Chicago Zoological Society, the Frank
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Chapman Fund of the American Museum of Natural History, the Phillip Smith Fund from the Illinois Natural History Survey, and the Center for Latin American Studies and the Graduate College of the University of Illinois. Autolatina S. A. supplied a vehicle for the ®eld studies in Argentina.
References Barrowclough, G.F., 1980. Gene ¯ow, eective population sizes and genetic variance components in birds. Evolution 34, 789±798. Baker, A.J., Dennison, M.D., Lynch, A., Le Grand, G., 1990. Genetic divergence in peripherally isolated populations of chanches in the Atlantic islands. Evolution 44, 981±999. Bouzat, J.L., 1998. The genetic consequences of fragmentation and small population size in two grassland bird species: the greater rhea and the greater prairie chicken. PhD thesis, University of Illinois. Bouzat, J.L., Cheng, H.H., Lewin, H.A., Westemeier, R.L., Brawn, J.D., Paige, K.N., 1998a. Genetic evaluation of a demographic bottleneck in the greater prairie chicken. Conservation Biology 12, 836±843. Bouzat, J.L., Lewin, H.A., Paige, K.N., 1998b. The ghost of genetic diversity past: historical DNA analysis of the greater prairie chicken. The American Naturalist 152, 1±6. Bruning, D.F., 1974. Social structure and reproductive behaviour in the greater rhea. Living Bird 13, 251±294. Chou, Q., Russell, M., Birch, D., Raymond, J., Block, W., 1992. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number ampli®cations. Nucleic Acids Research 20, 1717± 1723. Evans, P.G.H., 1987. Electrophoretic variability of gene products. In: Cook, F., Buckley, P.A. (Eds.), Avian Genetics. Academic Press, London, pp. 105±168. Excoer, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479±491. Frankel, O.H., 1983. The place of management in conservation. In: Schonewald-Cox, C.M., Chambers, S.M., MacBryde, F., Thomas, L. (Eds.), Genetics and Conservation: A Reference for Managing Wild Animal and Plant Populations. Benjamin/Cummings, Menlo Park, California, pp. 1±14. Fritsch, P., Rieseberg, L., 1996. The use of random ampli®ed polymorphic DNA (RAPD) in conservation genetics. In: Smith, T.B., Wayne, R.K. (Eds.), Molecular Genetic Approaches in Conservation. Oxford University Press, NY, pp. 54±73. Gibbs, H.L., Grant, P.R., 1989. Inbreeding in Darwin's medium ground ®nches (Geospiza fortis). Evolution 43, 1273±1284. Gilpin, M., 1987. Spatial structure and population vulnerability. In: SouleÂ, M.E. (Ed.), Viable Populations for Conservation. Cambridge University Press, NY, pp. 125±139. Gilpin, M., 1991. The genetic eective size of a metapopulation. Biological Journal of the Linnean Society 42, 165±175. Gilpin, M., Hanski, I. (Eds.), 1991. Metapopulation Dynamics: Empirical and Theoretical Investigations. Academic Press, NY. Gilpin, M., SouleÂ, M.E., 1986. Minimum viable populations: processes of species extinction. In: SouleÂ, M.E. (Ed.), Conservation Biology: the Science of Scarcity and Diversity. Sinauer, Sunderland, MA, pp. 19±34. Grant, P.R., Grant, B.R., 1992. Hybridization of bird species. Science 256, 193±197. Gronemeyer, P.A., Dilger, B.J., Bouzat, J.L., Paige, K.N., 1997. The eects of herbivory on paternal ®tness in scarlet gilia: better moms also make better pops. The American Naturalist 150, 592±602.
284
J.L. Bouzat / Biological Conservation 99 (2001) 277±284
Grosberg, R.K., Levitan, D.R., Cameron, B.B., 1996. Characterization of genetic structure and genealogies using RAPD-PCR markers: a random primer for the novice and nervous. In: Ferraris, J.D., Palumbi, S.R. (Eds.), Molecular Zoology. Wiley-Liss, NY, pp. 67±95. Haig, S.M., Beltho, J.R., Allen, D.H., 1993. Examination of population structure in red-cockaded woodpeckers using DNA pro®les. Evolution 47, 185±194. Hsiao, J.Y., Lee, S.M., 1999. Genetic diversity and microgeographic dierentiation of Yushan cane (Yushania niitakayamensis; Poaceae) in Taiwan. Molecular Ecology 8, 263±270. Hu, J., Quiros, C.F., 1991. Identi®cation of broccoli and cauli¯ower cultivars with RAPD markers. Plant Cell Rep. 10, 505±511. Hu, D.R., Peakall, R., Smouse, P.E., 1993. RAPD variation within and among natural populations of outcrossing bualograss [Buchloe dactyloides (Nutt.) Engelm.]. Theoretical Applications in Genetics 86, 927±934. Kazan, K., Manners, J.M., Cameron, D.F., 1992. Genetic variation in agronomically important species of Stylosanthes determined using random ampli®ed polymorphic DNA markers. Theoretical Applications in Genetics 85, 882±888. Kimberling, D.N., Ferreira, A.R., Shuster, S.M., Keim, P., 1996. RAPD marker estimation of genetic structure among isolated northern leopard frog populations in the south-western USA. Molecular Ecology 5, 521±529. Lande, R., Barrowclough, G.F., 1987. Eective population size, genetic variation, and their use in population management. In: SouleÂ, M.E. (Ed.), Viable Populations for Conservation. Cambridge University Press, NY, pp. 87±123. Maki, M., Horie, S., 1999. Random ampli®ed polymorphic DNA (RAPD) markers reveal less genetic variation in the endangered Cerastium ®scherianum var. molle than in the widespread conspeci®c C. ®scherianum var. ®scherianum (Caryophyllaceae). Molecular Ecology 8, 145±150. Mares, M.A., Ojeda, R.A., 1984. Faunal commercialization and conservation in South America. Bioscience 34, 580±584. Mee, G.K., Carroll, C.R., 1994. Principles of Conservation Biology. Sinauer, Sunderland, MA. Nusser, J.A., Goto, R.M., Ledig, D.B., Fleischer, R.C., Miller, M.M., 1996. RAPD analysis reveals low genetic variability in the endangered light-footed clapper rail. Molecular Ecology 5, 463±472.
Quattro, J.M., Vrijenhoek, R.C., 1989. Fitness dierences among remnant populations of the endangered Sonoran topminnow. Science 245, 976±978. Rockwell, R.F., Barrowclough, G.F., 1980. Gene ¯ow and the genetic structure of populations. In: Cook, F., Buckley, P.A. (Eds.), Avian Genetics. Academic Press, London, pp. 223±255. Schonewald-Cox, C.M., Chambers, S.M., MacBryde, F., Thomas, L. (Eds.), 1983. Genetics and Conservation: A Reference for Managing Wild Animal and Plant Populations. Benjamin/Cummings, Menlo Park, California. Seutin, G., White, B.N., Boag, P.T., 1991. Preservation of avian blood and tissue samples for DNA analyses. Canadian Journal of Zoology 69, 82±90. SouleÂ, M.E. (Ed.), 1986. Conservation Biology: The Science of Scarcity and Diversity. Sinauer, Sunderland, MA. SouleÂ, M.E., 1987. Viable Populations for Conservation. Cambridge University Press, Cambridge. SouleÂ, M.E., Wilcox, B.A., 1980. Conservation Biology: An Evolutionary-Ecological Perspective. Sinauer Associates, Sunderland, MA. Stangel, P.W., Lennartz, M.R., Smith, M.H., 1992. Genetic variation and population structure of red-cockaded woodpeckers. Conservation Biology 6, 283±292. Trame, A.M., Coddington, A.J., Paige, K.N., 1995. Field and genetic studies testing optimal outcrossing in Agave schottii, a long-lived clonal plant. Oecologia 104, 93±100. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213±7218. Westemeier, R.L., Brawn, J.D., Simpson, S.A., Esker, T.L., Jansen, R.W., Walk, J.W., Kreshner, E.L., Bouzat, J.L., Paige, K.N., 1998. Tracking the long-term decline and recovery of an isolated population. Science 282, 1695±1698. Wilde, J., Waugh, R., Powell, W., 1992. Genetic ®ngerprinting of Theobroma clones using randomly ampli®ed polymorphic DNA markers. Theoretical Applications in Genetics 83, 871±877. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymorphism ampli®ed by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531±6535. Wright, S., 1978. Evolution and the Genetics of Populations: Variability Within and Among Natural Populations. University of Chicago Press, Chicago.
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The population genetic structure of the Greater Rhea (Rhea americana) in an agricultural landscape Juan L. Bouzat * Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403-0212, USA
Abstract Although population genetic structure and geographic dierentiation have been reported for many bird species with insular distributions, high vagility typically results in high levels of gene ¯ow and thus little local dierentiation among continental populations. This study provides the ®rst survey of population genetic variation in a South American ratite, suggesting that, in an agricultural landscape, recent fragmentation and isolation may aect the genetic structure of ¯ightless bird species. Using randomly ampli®ed polymorphic DNA (RAPD) analysis, I evaluated the population genetic structure of the greater rhea, Rhea americana albescens, by quantifying genetic variability within and among four isolated wild populations located in a highly fragmented region of the Argentinean Pampas. Levels of genetic diversity of the natural populations were compared to those of a captive population used as an inbred ``control.'' An analysis of molecular variance (AMOVA) based on a phenotypic analysis of 54 RAPD markers showed that 94.38% of the total observed variance was explained by dierences within populations (P<0.001) whereas 6.37% was due to dierences among populations (P=0.006). In addition, measures of genetic diversity (D: mean genetic diversity, and P: percentage of polymorphic bands) estimated for the wild populations were similar to those of the inbred ``control.'' The pattern of genetic variation reported combined with data on the sizes of eight wild populations and the reproductive success of a focal population are consistent with the idea that relatively recent fragmentation and isolation may have increased local genetic dierentiation and decreased within-population levels of genetic variation as a result of genetic drift and inbreeding. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Genetic diversity; Fragmentation; RAPD; AMOVA; Rhea americana
1. Introduction As a result of human activities, signi®cant declines in the distribution and abundance of many species have increased their risk of global extinction (SouleÂ, 1986; Mee and Carroll, 1994). Habitat destruction has led to the fragmentation of natural populations and today, many species that were originally distributed in continuous habitats exist as metapopulations, i.e. groups of interacting populations, often with limited gene ¯ow (Gilpin and Hanski, 1991). The genetic eects of fragmentation are directly related to the stochastic processes associated with small population size (Gilpin, 1987, 1991). As populations become isolated with small eective sizes, genetic bottlenecks and founder events through extinction and recolonization processes may reduce the genetic variability within each island population. Such * Tel.: +1-419-372-9240; fax: +1-419-372-2024. E-mail address: [email protected] (J.L. Bouzat).
reduction in genetic diversity may result directly from a founder event as well as from the combined eects of genetic drift and inbreeding (Wright, 1978). The signi®cance of genetic variation in conservation and extinction of natural populations has been widely addressed (Gilpin and SouleÂ, 1986; Soule and Wilcox, 1980; SouleÂ, 1987; Schonewald-Cox et al., 1983). Two aspects of genetic diversity are of importance. First, loss of genetic diversity can result in reduced evolutionary potential for future adaptation through evolutionary change (Frankel, 1983). Second, there is evidence that reduced genetic variability at both the individual and population levels can result in a reduction in ®tness (e.g. Quattro and Vrijenhoek, 1989; Bouzat et al., 1998a,b; Westemeier et al., 1998). Thus, populations with depauperate levels of genetic diversity generally face increased risk of extinction (Gilpin and SouleÂ, 1986). The greater rhea, Rhea americana, represents a critical model organism to study the potential eects of fragmentation, isolation and small eective size on the
0006-3207/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0006-3207(00)00193-2
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genetic diversity of natural populations that have been highly aected by human activities. The ¯ightless condition of rheas, a characteristic that contrasts with the majority of continental bird species, and the increasing isolation of their populations as a result of habitat loss and fragmentation have the potential for profoundly aecting patterns of genetic variation. In addition, the rheas' mating system of harem polygyny (Bruning, 1974), in which few males contribute to the formation of the following generations, may decrease eective population sizes intensifying stochastic events such as genetic drift (Lande and Barrowclough, 1987). Although population genetic structure has been reported for many bird species with insular geographic distributions (e.g. Gibbs and Grant, 1989; Baker et al., 1990; Grant and Grant, 1992), many continental bird species fail to show any genetic structuring because of their high vagility, which typically results in high levels of gene ¯ow (Barrowclough, 1980; Rockwell and Barrowclough, 1980; Evans, 1987). In addition, the lack of control populations in most studies intended to assess the genetic eects of fragmentation and small population size (e.g. bottlenecks) prevents one from inferring a direct causal link between population size, isolation and genetic diversity in natural populations (Mee and Carroll, 1994; Bouzat et al., 1998a,b). This study provides the ®rst survey of population genetic variation in a South American ratite suggesting that relatively recent fragmentation and isolation may have increased local genetic dierentiation and decreased within-population levels of genetic variation. The population genetic structure of R. a. albescens was evaluated using randomly ampli®ed polymorphic DNA (RAPD) analysis. Although there have been both methodological and analytical controversies surrounding the use of RAPDs to estimate genetic variability (Grosberg et al., 1996), several studies have demonstrated their usefulness in addressing issues of ecological and evolutionary importance. RAPD studies included the analysis of genetic variation within and among vertebrate populations (e.g. Kimberling et al., 1996; Nusser et al., 1996), levels of genetic variability in plant populations (e.g. Hsiao and Lee, 1999; Maki and Horie, 1999), measures of genetic distance associated with ®tness (Trame et al., 1995), and parental analysis (Gronemeyer et al., 1997). In this study, 54 polymorphic RAPD markers were used for quantifying genetic variability within and among four isolated wild populations of the greater rhea located in a region highly aected by agricultural development and cattle breeding. The within-population component of genetic diversity from the natural populations studied was compared to that of a captive population with a small number of founders used as an inbred ``control.'' Speci®cally, I addressed the following questions: (1) what patterns of genetic diversity exist
within and among fragmented populations of the greater rhea? (2) Are estimates of within-population genetic variation observed in nature similar to those of a captive population with a small founder size? Complementary data on the sizes of eight natural populations of R. a. albescens and on the reproductive success of a focal wild population (measured as number of successful nests produced during the studied reproductive season) are also reported to evaluate potential factors that might have led to the observed genetic patterns in the wild. Implications for the conservation of the greater rhea are discussed in light of the current status of the studied populations as well as their observed population genetic structure. 2. Materials and methods 2.1. Studied populations and sampling Population genetic structure of R. a. albescens was assessed from the analysis of 50 samples collected from ®ve populations (Fig. 1). These included four wild populations (Trebolar, OlavarrõÂa, Rastros I and Rastros II) and a captive population with a small number of founders (Captive). The four natural populations (38 samples) were grouped according to their geographic location. The Trebolar±OlavarrõÂa group was separated from the Rastros I±Rastros II group by more than 400 km. Populations within the groups were located within 50 km from each other. Population Trebolar (a focal
Fig. 1. Geographic location of wild populations of subspecies R.a. albescens used in this study. Stars indicate populations used in the RAPD analysis. Filled circles indicate populations censussed.
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population used for behavioral studies) had an estimated size of 283 individuals. The other three populations sampled were known to be smaller than 100 individuals each (J. Flores and G. Wendor, personal communications). In addition, 12 samples were obtained from a captive population of 57 rheas from Cargill's farm near Pilar city, CoÂrdoba province, in 1993. This population was founded in 1974±1975 by seven rheas (two adult individuals and ®ve yearlings from the same brood) and remained isolated over more than 20 years (A. Rimada, personal communication). This captive population was used as an experimental ``control'' population for comparison to evaluate if similar founding processes might be occurring in the wild. Because this population was unmanaged, and most founders were ®rst-degree relatives, it is likely highly inbred. Although environmental eects were not ruled out, morphological asymmetries observed in two of the 57 rheas from this population may be the result of the high levels of inbreeding. Small sample sizes resulted from logistic diculties in capturing adult birds. Samples from multiple nests, however, were included, thereby increasing representation of the reproductive population. Communal nests in the greater rhea include egg clutches from multiple females (Bruning, 1974). To increase the probability of sampling genotypes from dierent maternal lines two individual chicks were sampled from 14 dierent nests. Sampling from the same nest may, however, bias the estimate of population genetic diversity due to the relatedness between some of the individuals sampled. Sampling variance results from both the number of individuals sampled from a given population and the number of polymorphic markers sampled in every individual. Given the logistic problems of reducing the sampling variance among individuals due to small sample size, I maximized the number of RAPD markers analyzed (54) to reduce the variance heterogeneity among RAPD markers. To evaluate the relative importance of both population size and the eective number of individuals contributing to reproduction on the genetic structure of wild populations of R. a. albescens, data on the sizes of several populations as well as on the reproductive success of a focal population were collected. Census data were collected from eight wild populations located in Buenos Aires province, Argentina, a region known to have historically large continuous populations of the greater rhea, during the breeding season of 1993 (July± December; Fig. 1). Population sizes (adult and juveniles) were estimated by direct censusing using a 2060-mm spotting scope. Ospring hatched during the studied reproductive season were not included. Repeated censusing (n=7) in an area of 900 ha resulted in a mean estimate of 179 individuals with a standard error of 2.4. Although one cannot disregard the possibility of an underestimation of actual population sizes, the prominent size of rheas and the characteristic open landscape of the
279
Argentinean Pampas make these estimates relatively precise. On the basis of the repeated censusing, population estimates probably represent more than 95% of actual population sizes. A focal study was carried out in population Trebolar (General Lamadrid county) to evaluate the rhea's mating system (J. L. Bouzat, unpublished data) and to estimate the total numbers of nests and successful nests produced throughout the breeding season (July±December 1993). Average clutch size was estimated from seven nests assumed to have a complete clutch (males were incubating and no females were observed in close proximity). Field observations were made monthly for a period of about 5 days for a total of 27 days (179 h of total observation time). The area in which the population was located (1990 ha) was searched intensively to detect all possible nests. In addition, this area was surveyed by airplane to detect nest sites that may have been overlooked during ground surveys (three nests were detected by air surveys). 2.2. DNA isolation and RAPD methods Blood samples (100±200 ml) for DNA analyses were collected by puncture of the brachial vein and stored in 2 ml of Queen's lysis buer (0.01 M Tris, 0.01 M Na2EDTA, 0.01 M NaCl and 1% n-lauroylsarcosine) at 4 C (Seutin et al., 1991) until further analysis at the University of Illinois. Samples were collected with permits from the National Division of Fauna of Argentina. CITES permits were obtained for sample transportation. DNA from blood samples was extracted following Seutin et al. (1991). Fifty to seventy-®ve microliters of blood stored in Queen's lysis buer were digested overnight at 65 C with Proteinase K (3U in 500 ml of lysis buer). Samples were subjected to two phenol-chloroform extractions and DNA was then precipitated by adding 10% volume of 7.5 M ammonium acetate and one volume of isopropanol. DNA was ®nally resuspended in 50 ml of TE buer and stored at 4 C. Following extraction, DNA was quanti®ed using a ¯uorometer and degradation was assessed by electrophoresis on a 1% agarose gel. Non-degraded DNA samples were diluted to a concentration of 30 ng/ml for further PCR analysis. RAPD ampli®cation reactions (Welsh and McClelland, 1990; Williams et al., 1990) were performed in 25 ml ®nal volumes containing 30 ng of DNA template in 10 mM Tris (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 100 uM of each dNTP, 5 pmol of a 10-base pair random primer, and 0.5 U of Taq polymerase. DNA, PCR buffer, primer, MgCl2, and water were combined to a volume of 15 ml, capped with 10 ml of liquid wax, and subjected to a ``hot-start'' for 5 min at 85 C (Chou et al., 1992). Then, Taq polymerase, dNTPs and water were added to each reaction to a ®nal volume of 25 ml. Ampli®cations were carried out in an MJ Research
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PTC-100 thermocycler (MJ Research, Waltham, MA) with a 3-min denaturation step at 94 C followed by 44 cycles of 30 s denaturation at 94 C, 30 s annealing at 36 C, and 1.5 min extension at 72 C, and then a ®nal 10-min extension step at 75 C. Ampli®ed products were then mixed with 0.5 volumes of a loading dye solution and run on a 1% agarose gel electrophoresis at 70 V for 1±2 h. RAPD products were stained with ethidium bromide and visualized under UV light (Fig. 2). Based on the presence of clear and distinct banding patterns, 18 of 30 random primers screened were selected to analyze 12 samples from Trebolar, 7 from OlavarrõÂa, 11 from Rastros I, 8 from Rastros II and 12 from the Captive population. Selected primers included A01, A02, A04, A08, A09, A10, A14, A16, A18, C01, C02, C05, C06, C09, C10, C11, C18 and C19 from Operon Technologies Primer Kits A and C (Operon Technologies, Alameda, CA). From these 18 RAPD primers, 54 polymorphic bands were scored. For each primer, a master mix with all reaction components, except DNA template, was used to minimize artifact bands that might result from dierent PCR starting conditions. In addition, all RAPD reactions for a given primer were performed at the same time using the same thermocycler. For each RAPD primer, a sub-sample of ®ve individuals was replicated to ensure repeatability of banding patterns (Grosberg et al., 1996). 2.3. Genetic variability and statistical analysis To estimate measures of genetic diversity with RAPD markers, a phenotypic analysis of the ampli®ed bands (conventional band frequency scoring) was used (Fritsch and Rieseberg, 1996). Bands that co-migrated were assumed to be homologous. Although this analysis is likely to underestimate levels of genetic variation, it does not assume Hardy±Weinberg equilibrium, a required condition for the genetic analysis of allele frequencies (Fritsch and Rieseberg, 1996). RAPD variation within and among natural populations of the greater rhea was estimated by an analysis of molecular variance (AMOVA; Excoer et al., 1992)
Fig. 2. Example of RAPD markers ampli®ed using Operon primer C06 in the greater rhea. Arrows indicate polymorphic bands scored and used in the analysis of molecular variance. Lanes 1Kb are standard molecular weight markers.
using the AMOVA 1.55 program provided by Laurent Excoer (Department of Anthropology and Ecology, University of Geneva, Switzerland). The AMOVA allows one to estimate variance components for RAPD phenotypes, partitioning the variation among individuals/ within populations, and among populations/within geographic regions (Excoer et al., 1992). Variance components were inferred from metric distances among RAPD fragment patterns estimated as E N1
2Nxy =2N;
where Nxy is the number of bands shared by individuals x and y, and N is the total number of polymorphic markers (Hu et al., 1993). Signi®cance levels for variance component estimates were computed by non-parametric permutation procedures, determining the probability of obtaining a more extreme variance component than the observed values by chance alone (1000 permutations). Genetic variability was also estimated using mean genetic diversity (D) and the percentage of polymorphic bands (P) within populations. Mean genetic diversity (D) was calculated as the average of pair-wise relative distances (d) within populations. Pair-wise relative distances between any two given individuals were estimated using the following equation: d1
Nxy =N;
where Nxy is the number of bands shared by individuals x and y, and N is the total number of polymorphic sites. The proportion of polymorphic bands (P) was estimated for each population as the number of polymorphic bands divided by the total number of markers analyzed. Levels of within-population genetic diversity for the four studied natural populations were compared to those of the captive population founded in 1974±1975. Dierences in D among populations were tested using a permutation analysis. Pair-wise comparisons among populations were based on 40,000 permutations, computing a distribution for the p studentized range, R max fi; jgfmean i mean j= var i var jg; where (i) and (j) represent the d values for two given populations, and var (i) and var (j) their variances, respectively. Signi®cant dierences between populations (P<0.05) were determined by estimating con®dence interp vals of the form mean i mean j RCRIT var i var j; where RCRIT is the 95% quantile of the studentized range distribution generated through 40,000 permutations. 3. Results A total of 54 polymorphic markers were ampli®ed by PCR using 18 RAPD primers. PCR replicates showed
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repeatable banding patterns. Bands that were not consistently ampli®ed in the replicates were not considered. The average number of bands per RAPD primer was three (S.E.=0.42, range=1±7). The four wild populations studied showed that, on average, 35% of their RAPD markers were monomorphic, the same proportion of monomorphic markers observed in the captive ``control'' population. Each population group (Trebolar±OlavarrõÂa and Rastros I±Rastros II) showed ®ve monomorphic markers (9% of the total number of markers analyzed). The AMOVA results were based on the metric distances among multiple RAPD band patterns as the dependent variable. Estimated phenotypic distances among RAPD patterns showed considerable variation within each population. Every individual sampled had a genetically unique RAPD pattern (i.e. the distance matrix used in the AMOVA did not show any zero values). Results of the hierarchical partitioning of the variance among the four wild populations are shown in Table 1. The variance component between the two geographic groups considered (Trebolar±OlavarrõÂa and Rastros I±Rastros II) was not statistically dierent from zero (P=0.6653). The proportion of the ``among populations'' variance component within each geographic group was small (6.37%) relative to the ``within population'' variance component (94.38%). These were highly signi®cant, indicating that the observed dierences among and within populations were larger than that expected by chance. Comparisons of levels of genetic variation (mean genetic diversity within populations (D) and the proportion of polymorphic RAPD markers (P)) for each of the four wild populations and the captive population with small founder size are shown in Table 2. The permutation test showed no signi®cant dierences between the mean genetic diversity of the wild populations and that of the captive control. However, analysis of D for the wild populations revealed that Rastros I and Trebolar had signi®cantly higher genetic diversity than OlavarrõÂa (Table 2; studentized range test based on 40,000 permutations; P<0.05). The proportion of
Table 1 Hierarchical analysis of molecular variance (AMOVA) based on metric distances among randomly ampli®ed polymorphic DNA (RAPD) haplotypes for four wild populations of the greater rheaa Variance component
d.f. Variance % Total
Between groups 1 Among populations/within groups 2 Within populations 34
0.056 0.476 7.046
0.75 6.37 94.38
P 0.6653 0.0060 <0.0010
a In an AMOVA, small negative values may occur for the higher hierarchical levels because these statistics are not computed as sum of squares (Excoer et al., 1992).
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polymorphic markers (P) for each individual population showed similar trends. Both Rastros I and Trebolar had the highest P compared to the other populations (Table 2). In addition, population OlavarrõÂa revealed the lowest estimated values for both D and P. Data on the sizes of the eight wild populations surveyed are shown in Table 3. Estimated population sizes ranged from eight to 283 individuals. Five of the eight populations were smaller than 100 individuals in size. In addition, there were considerable dierences in the size of the areas in which these populations were located (Table 3). Population Trebolar, the largest wild population studied, produced at least 26 nests during the reproductive season of 1993. Average clutch size was 20.9 (S.E.=2.0). Of the 26 nests produced, only six (23%) were successful. Unsuccessful nests resulted mainly from ¯ooding (4 nests) and direct or indirect disturbances [from cattle or other animals (14 nests), and humans (2 nests)]. At the population level, there were at least 260 eggs produced (including both successful and unsuccessful eggs) during the breeding season. Of these, 42 (16%) were isolated eggs laid on the ground in areas with no nest in close proximity.
Table 2 Estimates of genetic variability for four wild greater rhea populations and a captive population with small founder size (D, mean genetic diversity; P, proportion of polymorphic bands)a Population Captive OlavarrõÂa Rastros I Rastros II Trebolar Wild populations mean (S.E.)
D 0.22 0.18 0.27 0.24 0.32
P AB A B AB B
0.25 (0.03)
0.65 0.41 0.78 0.55 0.87 0.65 (0.10)
a
Values with dierent letters indicate signi®cant dierences (studentized range test based on 40,000 permutations, P<0.05).
Table 3 Estimated population sizes (N) and area in hectares (A) of eight wild populations of the greater rhea located in Buenos Aires province, Argentina Population Trebolar Las Cortaderas La DivisioÂn 17 Agosto La MarõÂa Pringles FernaÂndez El OmbuÂ
N
A
283 88 118 39 85 8 99 165
1990 5545 4229 300 1000 300 856 3000
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4. Discussion During the last few years, there has been considerable debate concerning the possible eects of fragmentation, isolation and small population size on the genetic variability of bird populations. The high vagility of many bird species typically results in high levels of gene ¯ow and thus little local genetic dierentiation (Barrowclough, 1980; Rockwell and Barrowclough, 1980; Evans, 1987). Only a limited number of studies have demonstrated genetic eects as a result of fragmentation of continental bird populations. For example, high levels of genetic dierentiation have been reported for the endangered red-cockaded woodpecker, Picoides borealis, a non-migratory bird species with high site ®delity (Stangel et al., 1992; Haig et al., 1993). Most of these studies, however, lack control populations (e.g. unfragmented populations or populations with a known demographic history), thus preventing direct testing for the potential genetic eects of fragmentation and small population size. In this study, I evaluated the population genetic structure of the greater rhea in an agricultural landscape, and compared levels of withinpopulation genetic diversity to that of an isolated captive population used as a highly inbred control. The pattern of genetic variation observed within and among the four natural populations of R. a. albescens is consistent with the idea that recent fragmentation and isolation may have increased local genetic dierentiation and decreased within-population genetic variability as a result of stochastic events associated with small population size and inbreeding. The high proportion of polymorphic markers that showed ®xed phenotypes in one or more of the wild populations suggests that stochastic events such as genetic drift may be leading to the ®xation of alternative genetic variants in dierent populations. The AMOVA showed a statistically signi®cant genetic divergence among the four populations nested within the two geographic groups (6.37% of the total observed variance, Table 1). This was also true for all populations independent of the grouping. Alternatively, dierences in P among populations may be overestimated due to limitations of small sample sizes. This was apparent by the fact that the AMOVA showed no signi®cant variation between the two geographic groups. However, the proportion of ®xed markers remained relatively high (9%) when samples from different populations were pooled into two geographically distinct groups, suggesting some levels of genetic dierentiation due to the presence of population-speci®c RAPD bands. The large ``within population'' component of the variance detected by the AMOVA (94.38%) is the result of using metric distances among multiple RAPD banding patterns as the dependent variable for the analysis of variance. In this study, every individual sampled showed
a unique RAPD pattern as a result of the large number of RAPD markers analyzed. Although the ``within population'' component of variance has been shown to be small for RAPD markers in highly inbred species with many generations of arti®cial selection, such as in highly inbred cultivars (Hu and Quiros, 1991; Kazan et al., 1992, Wilde et al., 1992), this does not appear to be true for natural populations of outcrossing species (Hu et al., 1993). In this study, measures of genetic diversity from the captive population were used as an inbred ``control'' population to evaluate the relative levels of within population genetic variation observed in the wild. Although the eects of genetic drift in a single isolated population cannot be predicted (i.e. drift may result in loss of a little, a lot, or no genetic variation), inbreeding will consistently lead to a decrease in genetic variability over time. Comparisons of mean genetic diversity estimates showed that, overall, wild populations had similar levels of genetic variability as the captive ``control'' population, which was probably subjected to more than 10 generations of inbreeding (Table 2). This suggests that similar processes to those operating in an isolated population with a small number of founders might be occurring in the wild. The levels of polymorphism reported in this study can be compared to those observed in other endangered species that have substantially decline in numbers from historic levels. For example, a RAPD study of ®ve populations of the endangered clapper rail (Rallus longirostris), a waterbird inhabiting isolated marshes, showed that 54% of 16 polymorphic RAPD bands analyzed were ®xed in one or more populations (Nusser et al., 1996). The lower proportion of ®xed markers observed in wild populations of the greater rhea (35%) may be the result of relatively recent fragmentation. In contrast to the greater rhea, the historical range of clapper rails was probably restricted to small fragmented populations long time before the impacts of human development (Nusser et al., 1996). The ®xation of alternative genetic variants in dierent populations and geographic groups of the greater rhea cautions against making the simple assumption that avian vagility, which typically results in high levels of gene ¯ow, decreases by default the genetic structuring of continental bird species. The population genetic structure reported in this study is consistent with the idea that fragmentation, isolation and small population size may be aecting the genetic variability of the greater rhea. The data on population sizes and reproductive success are indicative of a population structure of isolated populations with small eective sizes in which only a few reproductive males contribute to the formation of the following generations. Relative abundances reported here indicate that, in Buenos Aires province, most populations are small (e.g. less than 120 individuals; Table 3). The two
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largest populations studied (Trebolar and El OmbuÂ) are exceptions because the landowners of the farms in which rheas are present actively protect them. Estimates of reproductive success suggest that extremely low eective population sizes may be a common feature of most rhea populations inhabiting agricultural landscapes. With an estimated size of 283 rheas, population Trebolar produced only six successful nests during the breeding season of 1993. Assuming the occurrence of no extra-pair fertilizations and no loss of broods due to predation, the above data indicates that only six males would be successfully contributing to reproduction in this population. If we assume that the genetic pattern reported here is the result of stochastic events due to small population size and inbreeding, then we can conclude that fragmentation and isolation may be aecting greater rhea's genetic diversity. The greater rhea is another example of the potential threats of habitat loss and direct human impacts on a wild species. Once an abundant and continuously distributed species throughout the Argentinean Pampas, greater rheas have become rare in areas of intensive agriculture and cattle breeding. Prior to 1991 commercial hunting probably contributed greatly to the decline of rhea populations. For example, during the four-year period, 1976±1979, the number of rheas (including both Rhea and Pterocnemia) exported from Buenos Aires harbor was 102,543 (Mares and Ojeda, 1984). Therefore, hunting pressure probably acted as an important factor in reducing population sizes of rheas, in conjunction with habitat loss and fragmentation as a result of human activities. However, further studies would be needed to determine whether or not recent fragmentation, isolation and small population size have an eect on the genetic structure of rheas that could lead to an increase in their risk of extinction. Acknowledgements I would like to thank Ken N. Paige, Scott Robinson, Je Brawn, Chris Phillips, Harris Lewin, Marcel Amills, Mark W. Schwartz, and two anonymous reviewers for their constructive criticisms on earlier drafts of the manuscript, which originally appeared as part of my PhD dissertation (Bouzat, 1998). I would also like to acknowledge Professor Stephen Portnoy and Maria Bidart for statistical advice, and Brian Dilger for helping in the lab during the DNA preparations. I am particularly grateful to the Rivera family from ``Estancia El Trebolar'' who provided a warm home during my ®eld season observing rheas in Argentina, and to MoÂnica Martella and Gerardo Wendor who facilitated sampling of the captive and Rastros populations, respectively. Financial support for this study was provided by grants from the Chicago Zoological Society, the Frank
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Chapman Fund of the American Museum of Natural History, the Phillip Smith Fund from the Illinois Natural History Survey, and the Center for Latin American Studies and the Graduate College of the University of Illinois. Autolatina S. A. supplied a vehicle for the ®eld studies in Argentina.
References Barrowclough, G.F., 1980. Gene ¯ow, eective population sizes and genetic variance components in birds. Evolution 34, 789±798. Baker, A.J., Dennison, M.D., Lynch, A., Le Grand, G., 1990. Genetic divergence in peripherally isolated populations of chanches in the Atlantic islands. Evolution 44, 981±999. Bouzat, J.L., 1998. The genetic consequences of fragmentation and small population size in two grassland bird species: the greater rhea and the greater prairie chicken. PhD thesis, University of Illinois. Bouzat, J.L., Cheng, H.H., Lewin, H.A., Westemeier, R.L., Brawn, J.D., Paige, K.N., 1998a. Genetic evaluation of a demographic bottleneck in the greater prairie chicken. Conservation Biology 12, 836±843. Bouzat, J.L., Lewin, H.A., Paige, K.N., 1998b. The ghost of genetic diversity past: historical DNA analysis of the greater prairie chicken. The American Naturalist 152, 1±6. Bruning, D.F., 1974. Social structure and reproductive behaviour in the greater rhea. Living Bird 13, 251±294. Chou, Q., Russell, M., Birch, D., Raymond, J., Block, W., 1992. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number ampli®cations. Nucleic Acids Research 20, 1717± 1723. Evans, P.G.H., 1987. Electrophoretic variability of gene products. In: Cook, F., Buckley, P.A. (Eds.), Avian Genetics. Academic Press, London, pp. 105±168. Excoer, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479±491. Frankel, O.H., 1983. The place of management in conservation. In: Schonewald-Cox, C.M., Chambers, S.M., MacBryde, F., Thomas, L. (Eds.), Genetics and Conservation: A Reference for Managing Wild Animal and Plant Populations. Benjamin/Cummings, Menlo Park, California, pp. 1±14. Fritsch, P., Rieseberg, L., 1996. The use of random ampli®ed polymorphic DNA (RAPD) in conservation genetics. In: Smith, T.B., Wayne, R.K. (Eds.), Molecular Genetic Approaches in Conservation. Oxford University Press, NY, pp. 54±73. Gibbs, H.L., Grant, P.R., 1989. Inbreeding in Darwin's medium ground ®nches (Geospiza fortis). Evolution 43, 1273±1284. Gilpin, M., 1987. Spatial structure and population vulnerability. In: SouleÂ, M.E. (Ed.), Viable Populations for Conservation. Cambridge University Press, NY, pp. 125±139. Gilpin, M., 1991. The genetic eective size of a metapopulation. Biological Journal of the Linnean Society 42, 165±175. Gilpin, M., Hanski, I. (Eds.), 1991. Metapopulation Dynamics: Empirical and Theoretical Investigations. Academic Press, NY. Gilpin, M., SouleÂ, M.E., 1986. Minimum viable populations: processes of species extinction. In: SouleÂ, M.E. (Ed.), Conservation Biology: the Science of Scarcity and Diversity. Sinauer, Sunderland, MA, pp. 19±34. Grant, P.R., Grant, B.R., 1992. Hybridization of bird species. Science 256, 193±197. Gronemeyer, P.A., Dilger, B.J., Bouzat, J.L., Paige, K.N., 1997. The eects of herbivory on paternal ®tness in scarlet gilia: better moms also make better pops. The American Naturalist 150, 592±602.
284
J.L. Bouzat / Biological Conservation 99 (2001) 277±284
Grosberg, R.K., Levitan, D.R., Cameron, B.B., 1996. Characterization of genetic structure and genealogies using RAPD-PCR markers: a random primer for the novice and nervous. In: Ferraris, J.D., Palumbi, S.R. (Eds.), Molecular Zoology. Wiley-Liss, NY, pp. 67±95. Haig, S.M., Beltho, J.R., Allen, D.H., 1993. Examination of population structure in red-cockaded woodpeckers using DNA pro®les. Evolution 47, 185±194. Hsiao, J.Y., Lee, S.M., 1999. Genetic diversity and microgeographic dierentiation of Yushan cane (Yushania niitakayamensis; Poaceae) in Taiwan. Molecular Ecology 8, 263±270. Hu, J., Quiros, C.F., 1991. Identi®cation of broccoli and cauli¯ower cultivars with RAPD markers. Plant Cell Rep. 10, 505±511. Hu, D.R., Peakall, R., Smouse, P.E., 1993. RAPD variation within and among natural populations of outcrossing bualograss [Buchloe dactyloides (Nutt.) Engelm.]. Theoretical Applications in Genetics 86, 927±934. Kazan, K., Manners, J.M., Cameron, D.F., 1992. Genetic variation in agronomically important species of Stylosanthes determined using random ampli®ed polymorphic DNA markers. Theoretical Applications in Genetics 85, 882±888. Kimberling, D.N., Ferreira, A.R., Shuster, S.M., Keim, P., 1996. RAPD marker estimation of genetic structure among isolated northern leopard frog populations in the south-western USA. Molecular Ecology 5, 521±529. Lande, R., Barrowclough, G.F., 1987. Eective population size, genetic variation, and their use in population management. In: SouleÂ, M.E. (Ed.), Viable Populations for Conservation. Cambridge University Press, NY, pp. 87±123. Maki, M., Horie, S., 1999. Random ampli®ed polymorphic DNA (RAPD) markers reveal less genetic variation in the endangered Cerastium ®scherianum var. molle than in the widespread conspeci®c C. ®scherianum var. ®scherianum (Caryophyllaceae). Molecular Ecology 8, 145±150. Mares, M.A., Ojeda, R.A., 1984. Faunal commercialization and conservation in South America. Bioscience 34, 580±584. Mee, G.K., Carroll, C.R., 1994. Principles of Conservation Biology. Sinauer, Sunderland, MA. Nusser, J.A., Goto, R.M., Ledig, D.B., Fleischer, R.C., Miller, M.M., 1996. RAPD analysis reveals low genetic variability in the endangered light-footed clapper rail. Molecular Ecology 5, 463±472.
Quattro, J.M., Vrijenhoek, R.C., 1989. Fitness dierences among remnant populations of the endangered Sonoran topminnow. Science 245, 976±978. Rockwell, R.F., Barrowclough, G.F., 1980. Gene ¯ow and the genetic structure of populations. In: Cook, F., Buckley, P.A. (Eds.), Avian Genetics. Academic Press, London, pp. 223±255. Schonewald-Cox, C.M., Chambers, S.M., MacBryde, F., Thomas, L. (Eds.), 1983. Genetics and Conservation: A Reference for Managing Wild Animal and Plant Populations. Benjamin/Cummings, Menlo Park, California. Seutin, G., White, B.N., Boag, P.T., 1991. Preservation of avian blood and tissue samples for DNA analyses. Canadian Journal of Zoology 69, 82±90. SouleÂ, M.E. (Ed.), 1986. Conservation Biology: The Science of Scarcity and Diversity. Sinauer, Sunderland, MA. SouleÂ, M.E., 1987. Viable Populations for Conservation. Cambridge University Press, Cambridge. SouleÂ, M.E., Wilcox, B.A., 1980. Conservation Biology: An Evolutionary-Ecological Perspective. Sinauer Associates, Sunderland, MA. Stangel, P.W., Lennartz, M.R., Smith, M.H., 1992. Genetic variation and population structure of red-cockaded woodpeckers. Conservation Biology 6, 283±292. Trame, A.M., Coddington, A.J., Paige, K.N., 1995. Field and genetic studies testing optimal outcrossing in Agave schottii, a long-lived clonal plant. Oecologia 104, 93±100. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213±7218. Westemeier, R.L., Brawn, J.D., Simpson, S.A., Esker, T.L., Jansen, R.W., Walk, J.W., Kreshner, E.L., Bouzat, J.L., Paige, K.N., 1998. Tracking the long-term decline and recovery of an isolated population. Science 282, 1695±1698. Wilde, J., Waugh, R., Powell, W., 1992. Genetic ®ngerprinting of Theobroma clones using randomly ampli®ed polymorphic DNA markers. Theoretical Applications in Genetics 83, 871±877. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymorphism ampli®ed by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531±6535. Wright, S., 1978. Evolution and the Genetics of Populations: Variability Within and Among Natural Populations. University of Chicago Press, Chicago.