J Ornithol (2010) 151:797–803 DOI 10.1007/s10336-010-0515-y
ORIGINAL ARTICLE
Genetic diversity and differentiation of three Brazilian populations of Scarlet ibis (Eudocimus ruber) Evonnildo C. Gonc¸alves • Stephen F. Ferrari • Tibe´rio Ce´sar T. Burlamaqui • Leonardo Miranda • Marcelo S. Santos • Artur Silva • Maria Paula C. Schneider
Received: 13 January 2009 / Revised: 15 December 2009 / Accepted: 10 March 2010 / Published online: 27 March 2010 Ó Dt. Ornithologen-Gesellschaft e.V. 2010
Abstract The possible origin of the Scarlet ibis population of Cubata˜o in southern Brazil, and its levels of genetic diversity and differentiation in relation to populations from the country’s northern coast were investigated through the sequences of 980 base pairs of b-fibrinogen intron 7 from a sample of 37 specimens. A total of 19 haplotypes were recorded in the three populations. Despite observed discrepancies in the levels of genetic diversity (p = 0.0017– 0.0033; h = 0.60–0.95), AMOVA, K*st and Fst values all indicated that genetic differentiation among the populations was relatively low. This suggests that the Cubata˜o population was isolated recently from the panmictic population that was once distributed all along the Brazilian coast, although it does not totally refute its possible derivation from a specific population on the north coast. Given our results, genetic management should focus on the minimization of inbreeding, especially in the smaller populations, such as Cubata˜o. However, a more definitive study, including markers with higher evolutionary rates (e.g. microsatellites) and a much larger sample, would be required before any such actions can be taken.
Communicated by M. Wink. E. C. Gonc¸alves (&) T. C. T. Burlamaqui L. Miranda M. S. Santos A. Silva M. P. C. Schneider Laborato´rio de Polimorfismo de DNA, Departamento de Gene´tica, Universidade Federal do Para´, Caixa Postal 8607, 66075-970 Bele´m, Para´, Brazil e-mail:
[email protected] S. F. Ferrari Department of Biology, Universidade Federal de Sergipe, Av. Marechal Rondon, s/n Jardim Rosa Elze, 49100-000 Sa˜o Cristo´va˜o, Sergipe, Brazil
Keywords Scarlet ibis Eudocimus ruber Population genetic structure Conservation Brazil
Introduction The predatory exploitation of mangrove forests worldwide has had devastating effects on these unique ecosystems, with particularly serious consequences for the populations of vertebrates that inhabit them. One example is the Scarlet ibis (Eudocimus ruber), which was originally distributed along almost the whole of the Brazilian coast, between the border with French Guiana in the north and Santa Catarina in the south (Goeldi 1894). During the twentieth century, however, the species appeared to have disappeared from virtually the whole of the area east and south of the state of Ceara´, more than two-thirds of its original distribution, although a population was discovered in Cubata˜o, Sa˜o Paulo, in 1986 (Olmos 2003), some three thousand kilometres due south of Ceara´. While Scarlet ibis is considered to be of ‘‘least concern’’ by IUCN (BirdLife International 2008), given the presence of large populations along the northern coast of South America and the Caribbean, totalling an estimated 100,000–150,000 individuals (Wetlands International 2002), the apparently significant reduction in the distribution of Brazilian populations is obviously a source of concern. Antas et al. (1990) estimated the total Brazilian population at around 24,000 individuals, more than half of which are found at two locations, Canelas Island in the state of Para´ (10,000 individuals—Roma et al. 1996) and Cajual Island in Maranha˜o (3,000 individuals—Rodrigues and Martinez 1996). Most populations nevertheless suffer considerable anthropogenic pressure from local communities, not only
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indirectly in the form of the degradation of mangrove habitats, but also directly, through the predation of both eggs and adult animals. Nestlings may also be traded as pets. There are no figures on the impact of such activities in Brazil, although Spaans (1982) reported that such predation may have been the principal cause of a 50% decline in Scarlet ibis stocks in Suriname during the 1970s. While some data are available on demographic parameters, little is known of the current or historic levels of genetic diversity in Scarlet ibis populations. Given their recent history and highly dispersed distribution, information on the genetic characteristics of the Brazilian populations of Scarlet ibis may be essential for their successful conservation and management. Given this, the present study investigated patterns of genetic diversity and differentiation in three Brazilian populations, with the primary objective of identifying the possible origin of the Cubata˜o population.
Materials and methods Samples In the present study, three Brazilian populations of Scarlet ibis were sampled, two from northern Brazil—Canelas Island in the state of Para´ (00°470 S, 46°430 W) and Caju´ Island in the Rio Parnaı´ba delta, between Maranha˜o and Piauı´ (02°410 S, 42°070 W)—and the southernmost known population from Cubata˜o (23°540 S, 46°240 W) in Sa˜o Paulo (Fig. 1). Blood samples of approximately 0.1 ml were obtained from the brachial vein of nestlings of 2–3 weeks of age. To minimize possible relatedness between specimens, only one nestling was selected per nest at each site. The genomic DNA of 37 samples (14 from Canelas, 16 from Caju´ and 7 from Cubata˜o) was isolated by enzymatic digestion, using proteinase K, extracted with phenol– chloroform, and precipitated with ethanol, following standard procedures (Sambrook et al. 1989). Amplification and sequencing Amplification via polymerase chain reaction (PCR) of b-fibrinogen intron 7 was carried out in a final volume of 50 ll containing 5–10 ng of DNA, 50 mM KCL, 2 mM MgCl2, 10 mM Tris–HCL, 50 lM of each DNTP, 0.5 lM of each oligonucleotide (FIB-17L/FIB-17U, Prychitko and Moore 1997), and one unit of Taq DNA polymerase (Invitrogen). The following amplification profile was used: 4 min at 94°C for initial denaturation; 30 cycles of 1 min at 94°C, 1 min at 53°C, 1 min at 72°C, and 10 min at 72°C to ensure complete extension of the PCR products.
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Fig. 1 Locations of the three Brazilian populations of Scarlet ibis sampled in the present study. Brazilian states: PA, Para´; MA, Maranha˜o; PI, Piauı´; CE, Ceara´; SP, Sa˜o Paulo
Amplification products were purified with the Qiaex II gel extraction kit (Qiagen) and cloned in Escherichia coli DH5a (Gibco) using the pGEM-T vector System I (Promega). The plasmid DNA of each clone was obtained using the QIAprep Spin plasmid Miniprep Kit (Qiagen) and sequenced automatically in a 3130 genetic analyzer (Applied Biosystems), according to the maker’s specifications. Both forward and reverse primers were used for sequencing reactions to confirm sequences. BioEdit software (Hall 2007) was used to align sequences. Data analyses Polymorphism levels within populations were evaluated through nucleotide (p) and haplotype (h) diversity, estimated in Arlequin 3.11 (Schneider et al. 2000), using the ‘‘open unphase data file’’ option. In order to test the null hypothesis of no genetic differentiation among populations at different localities we used three tests: K*st as implemented in DnaSP 4.10.3 (Rozas et al. 2003), analysis of molecular variance (AMOVA), and Wright’s (1931) Fst
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for pairwise comparisons, available in Arlequin 3.11. The K*st takes into account the number of nucleotide differences between different haplotypes, but does not give as much weight to large numbers of differences (Hudson et al. 1992). AMOVA uses genotype frequencies and the number of mutations between different haplotypes to test the significance of the components of the variance associated with three hierarchical levels of genetic population structuring: (a) intra-population; (b) between populations of the same geographic group; (c) between populations of different groups (Excoffier et al. 1992). DnaSP 4.10.3 (Rozas et al. 2003) was also used to test the hypothesis of neutral evolution of the sequences using the R2 statistic (Ramos-Onsins and Rozas 2002), which is also a powerful tool for the detection of demographic expansion. Significance was determined based on 10,000 coalescent simulations under a model of constant population size using empirical sample sizes, estimates of theta (h), and intermediate levels of the recombination parameter—R (Hudson 1987). Most nuclear genes appear to be intermediate for this parameter, including the b-fibrinogen intron 7 sequences from our data file, in which recombination has been detected at four nucleotide sites in previous analyses using DNAsp. Theta (h) = 4Nel for an autosomal gene of a diploid organism (Ne and l are the effective population size and the mutation rate per nucleotide site per generation, respectively). PAUP 4.0b8 (Swofford 2003) was used to assess the pattern of molecular evolution of the sequences among the different haplotypes observed, based on the estimates of the frequencies of nucleotide bases, substitution rates and the P distance between pairs of haplotypes. Phylogenetic relationships were characterized through Bayesian inference (BI) via Markov chain Monte Carlo (MCMC) tree searches using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), following the most appropriate model of evolution under the Akaike information criterion (AIC; Posada and Buckley 2004) selected by Modeltest 3.06 (Posada and Crandall 1998). b-Fibrinogen intron 7 sequences of Ciconia ciconia (EU739354) and Fregata magnificens (EU739415) were used as the outgroup. We also included sequences of Balaeniceps rex (EU739371), Ardea herodias (EU739368), Cochlearius cochlearius (EU739391), Harpiprion caerulescens (EF552768), Theristicus caudatus (EF552787), and Eudocimus albus (EU739409). We performed two parallel runs of four simultaneous MCMC searches for 5,000,000 generations each, sampling one tree every 1,000 generations, and discarding the results of the first 1250 trees (25% of the sample) as ‘‘burn in’’. The remaining 3,750 trees were used by MrBayes to estimate the posterior probability of each node in our phylogenetic reconstruction. Relationships among haplotypes were also analyzed through the
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construction of a haplotype network, using the Network program, version 4.2.0.1 (Bandelt et al. 1999). As the algorithm employed by the Network program was developed for nonrecombining biological molecules, a preliminary step that consisted of the reconstruction of haplotypes from population genotype data was performed using the Bayesian approach implemented in PHASE (Stephens et al. 2001). The input file for PHASE was obtained from SEQPHASE (Flot 2009).
Results Molecular evolution of b-fibrinogen intron 7 Alignment of the nucleotides of the 37 Scarlet ibis specimens returned sequences of 980 base pairs (excluding primers), which presented 15 polymorphic sites, all but one of which had only two variants (Fig. 2). Twelve of these sites were informative for parsimony analysis. Of the 14 sites with just two variants, nine corresponded to transitions (6 A $ G, 3 T $ C), and five to transversions (1 A $ T, 2 A $ C, 1 G $ T, 1 G $ C). Base frequencies (31.72% for adenines, 31.5% for thymines, 18.79% for guanines and 17.99% for cytosines) varied little among haplotypes (v2 = 0.296, df = 54, P = 1.000). Mean sequence divergence (P distance) among the haplotypes recorded here was 0.0041 (±0.002), with a range of 0.0010–0.0092.
233333444555566 Absolute 903779369455802 Frequency 338282221627963 Can Caj Cub Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap Hap
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19
ATACTCAAATTTAAT ...AC.......G.. .......G....... .............G. GCC..G......... .CC..G......... ...........A... ...A.......A... ..........CA..G GCC..G...A..... ..............G ........GGCA... ......G.GG....G .........A..... .CC..G.G......G ...A........... ...........A..G ......G....A..G ..........C....
9 1 1 1 1 1 -
5 1 2 1 1 1 1 1 1 1 1 -
2 1 1 1 1 1
Fig. 2 Map of the polymorphic sites for the b-fibrinogen intron 7 sequences (GenBank accession GQ331113–GQ331131) from three Brazilian populations of Scarlet ibis. Noninformative sites for parsimony analysis are shaded in grey. Hap, Haplotype; Can, Canelas; Caj, Caju´; Cub, Cubata˜o
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Diversity and differentiation
clustered 15 haplotypes with high statistical support (Fig. 3). The low phylogenetic resolution reflects the reduced P distances among the haplotypes. Clusters derived from BI are common in the network analysis (Fig. 4), which provides some evidence of phylogenetic structuring and also indicates that haplotype 6 is the most likely ancestral one.
The 37 sequences resulted in 19 haplotypes (GenBank accession GQ331113–GQ331131, Figs. 2 and 4). Mean pairwise distance (nucleotide diversity, p) between all individuals was 0.0026, while the probability that two individuals selected at random from the sample population as a whole had different haplotypes (h) was 0.82. Values for these parameters were lowest in the Canelas population, while the highest p value was recorded at Caju´ and the highest h at Cubata˜o (Table 1). The AMOVA revealed considerably more genetic variation within populations than among them. Additionally, it is interesting to note that P values for these analysis were highly nonsignificant (Table 2). This is further emphasized by the lack of significant differences (P [ 0.05) for the K*st and Fst values recorded between pairs of populations (Table 3). The P values of R2 for Caju´ and Cubata˜o and all the populations together were as expected according the hypothesis of b-fibrinogen intron 7 neutrality. In this case, the statistical significance of the R2 value for Canelas (Table 1) may be evidence of demographic expansion. Modeltest selected the TVM ? G substitution model, with empirical base frequencies (A = 0.3051, C = 0.1768, G = 0.1952, T = 0.3229), rate matrix ([A-C] = 1.0000, [A-G] = 3.1597, [A-T] = 0.6778, [C-G] = 1.6356, [C-T] = 3.1597, [G-T] = 1.0000) and gamma distribution shape parameter = 0.9657. The Bayesian consensus tree
Discussion Genetic diversity and differentiation Perhaps surprisingly, given the geographic distances involved, genetic differentiation was more pronounced within than between the Scarlet ibis populations studied here, and at least one b-fibrinogen haplotype was shared by all three populations. The lack of significant differentiation between populations suggests that they are not structured genetically. Two principal factors may account for the low levels of genetic divergence observed. One is ongoing gene flow or a relatively recent process of isolation. In theory, migration of only one individual per generation would be sufficient to uphold genetic homogenization between geographically distinct populations (Hartl and Clark 1989). While the Scarlet ibis is a colonial species (Sick 1997), which may limit migrations between populations, Palmer (1962) noted that subadults often disperse over substantial distances. In the present day, significant anthropogenic translocations may also occur. Despite its reduced capacity for dispersal, the original, continuous distribution of the species along the coast of Brazil would likely have permitted panmictic breeding, a hypothesis supported by the low levels of genetic differentiation reported here. In this case, the lack of distinct genetic lineages in present-day populations may reflect a relatively recent process of fragmentation. Alternatively, reduced differentiation may reflect recent colonization and founder effects related to processes such as strong selective pressures on certain haplotypes, and extinction and recolonization on a local scale (Stamatis et al. 2004), although no evidence of such selective
Table 1 Genetic diversity (±SD) and neutrality tests for the bfibrinogen intron 7 sequences of three Brazilian populations of Scarlet ibis Population
Diversity p
Neutrality test R2 (P)
h
All (n = 37)
0.0026 ± 0.002
0.82 ± 0.07
0.08 (0.15)
Canelas (n = 14)
0.0017 ± 0.001
0.60 ± 0.15
0.10 (0.02)
Caju (n = 16) Cubata˜o (n = 7)
0.0033 ± 0.002
0.91 ± 0.06
0.12 (0.23)
0.0030 ± 0.002
0.95 ± 0.10
0.16 (0.17)
In the neutrality tests, P is the probability of an R2 value being less than the observed value based on 10,000 coalescent simulations
Table 2 Analysis of molecular variance (AMOVA), based on the b-fibrinogen intron 7 sequences of three Brazilian populations of Scarlet ibis Groups
Fst
Fsc
Fct
Percentage of variation Among groups
P
Among populations within groups
Within populations
-0.25
100.25
0.44
{Canelas, Caju, Cubata˜o} {Canelas, Caju}, {Cubata˜o}
-0.002 -0.045
0.034
-0.082
-8.16
3.69
104.47
0.45
{Canelas, Cubata˜o}, {Caju} {Caju, Cubata˜o}, {Canelas}
0.002 0.019
-0.016 -0.071
0.018 0.084
1.76 8.42
-1.61 -6.49
99.85 98.07
0.45 0.44
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–
–
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Table 3 Estimates of Fst (below the diagonal) and Kst* (above the diagonal) for pairwise comparisons of three Brazilian populations of Scarlet ibis Canelas
Caju´
Cubata˜o
Canelas Caju´
0.03719
–
0.01945
-0.02491
Cubata˜o
0.00673
-0.07857
–
–
0.02514
P values are all greater than 0.05, i.e. not significant
pressures was recorded. While human activities have been responsible for the decline of Scarlet ibis in many areas, they may also have played a role in its introduction into areas such as the southern United States (American Ornithologists’ Union 1998), where it is known to hybridize with the White ibis, Eudocimus albus. This close relationship is also evident in our Bayesian consensus tree. There is in fact some evidence that the Cubata˜o population represents a recent reintroduction, as suggested by Olmos (2003), based on the lack of records from the nineteen century onwards, and reports of the release into this area of 18 breeding pairs from Maranha˜o in 1967– 1969. As Maranha˜o State is located between Canelas and Caju´ (Fig. 1), it is likely that the genetic characteristics of these pairs would have reflected those of the panmictic population of this region. Given the relatively short period of time since these events, current levels of diversity may still reflect the original scenario.
Genetics and conservation While genetic and ecological processes may exert a strong synergetic influence on the probability of extinction of fragmented populations (Lande 1988; Gaines et al. 1997), ecological factors may be more important over the short term. This is due primarily to fluctuations in population size, which influence genetic variables through processes such as genetic drift and inbreeding. Whatever the exact origin of the Cubata˜o population, ecological processes do appear to be the most important in this case. The first census in 1986 recorded a total of 82 individuals, which rose to around 400 in 1994, and 500 in 1997, and has remained at this level ever since. Although rapid population growth tends to favour the retention of new mutations (Avise et al. 1984; Watterson 1984), this does not appear to be the case at Cubata˜o, considering the lack of significant P values in the R2 analysis. While no census data are available for the Canelas and Caju´ populations, the evidence from the present study indicates that only Canelas has been expanding. Expansion is important for the maintenance of diversity levels, which generally reduces the risk of extinction. The role of genetic factors in the extinction of wild populations is poorly understood, but may be very important in many cases (Frankham 2003). Effective population size is the principal factor influencing the evolutionary potential of a species over the medium to long term, and populations of fewer than 500 individuals—as was the case for the original Cubata˜o population—may be
Fig. 3 Bayesian inference tree for the 19 haplotypes of bfibrinogen intron 7 observed in the present study using the TVM ? gamma model of DNA substitution. Bayesian posterior probabilities appear above each node. The scale bar represents the number of substitutions per nucleotide site
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Hap 13 Hap 12 Hap 18 Hap 10
Hap 09 Hap 17
Hap 05 Hap 07 Hap 08
Hap 06
Hap 11 Hap 19
Hap 16
Hap 01
Hap 15
Hap 03 Hap 04
Hap 14
Hap 02
Fig. 4 Haplotype network of b-fibrinogen intron 7 for three Brazilian populations of Scarlet ibis. Each circle represents a haplotype (Fig. 2). Circle size is proportional to haplotype frequency, and arm length to the number of nucleotide changes. Circles in grey, Canelas; black, Caju´; white, Cubata˜o
especially vulnerable to extinction (Frankham et al. 2002). As the population size remains at this threshold, there is a clear need for the implementation of a conservation program to ensure its long-term survival. As the populations analyzed here present similar levels of diversity and reduced differentiation, genetic management should focus on the minimization of inbreeding, especially in the smaller populations, such as Cubata˜o. A putative strategy in this case would be the translocation of individuals between populations, preferably involving the pairing of appropriately screened individuals. However, any such intervention would require more conclusive evidence on genetic variability—including markers with higher evolutionary rates, such as microsatellites—and a larger sample of individuals. Among other questions, this would probably help to decipher the marked exclusivity of haplotypes observed in this study, and whether this reflects an incipient process of differentiation which would eventually elevate these populations to the level of evolutionarily significant (management) units. This would favour a situation in which the flow of maladaptive alleles could result in outbreeding depression, with deleterious consequences for the fitness of the populations.
Zusammenfassung Genetische Vielfalt und Differenzierung von drei Populationen des brasilianischen Scharlachsichler(Eudocimus ruber) Die mo¨gliche Herkunft der Scharlachsichler-Population in Cubata˜o im Su¨den Brasiliens und ihre genetische Vielfalt
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und Differenzierung im Vergleich zu Populationen der no¨rdlichen Ku¨ste Brasiliens wurde anhand der Sequenzen von 980 Basenpaaren des Introns 7 von b-Fibrinogen in Proben von 37 Individuen untersucht. Insgesamt wurden in den drei Populationen 19 verschiedene Haplotypen gefunden. Obwohl Unterschiede im Grad der genetischen Vielfalt festgestellt wurden (p = 0.0017–0.0033; h = 0.60– 0.95), deuten AMOVA, K*st- und Fst-Werte auf eine relativ geringe genetische Differenzierung zwischen den Populationen hin. Diese Ergebnisse fu¨hren zu der Annahme, dass die Population aus Cubata˜o erst ku¨rzlich von der panmiktischen Population isoliert wurde, die einst entlang der gesamten brasilianischen Ku¨ste verbreitet war. Sie widerlegen aber nicht vollsta¨ndig die Theorie, dass die Cubata˜o-Population von einer bestimmten Population an der Nordku¨ste abstammen ko¨nnte. In Anbetracht unserer Ergebnisse, sollte sich genetisches Management besonders in kleinen Populationen, wie Cubata˜o, darauf konzentrieren, Inzucht zu vermeiden. Doch bevor solche Maßnahmen ergriffen werden ko¨nnen, ist eine erga¨nzende Studie vonno¨ten, mit gro¨ßerer Stichprobenzahl und Markern, die eine ho¨here Evolutionsrate haben (z. B. Mikrosatelliten). Acknowledgments Financial support was provided by the National Council for Technological and Scientific Development (CNPq) and the Brazilian Ministry of the Environment (MMA) PROBIO initiative. CNPq also supported individual authors through grants 152757/ 2007-4 (ECG) and 302747/2008-7 (SFF). Specimen collection was authorized by the Brazilian Environment Institute (license number 105/2003-DIFAS/IBAMA). We are grateful to Antoˆnio Augusto Ferreira Rodrigues for the collection of specimens on Caju Island, and Silvanira Barbosa for help with data collection. We also thank an anonymous reviewer for helpful comments on an earlier version of the manuscript.
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