Polymorphic heterologous microsatellite loci for population genetics studies of the white-faced ibis Plegadis chihi (Vieillot, 1817) (Pelecaniformes, Threskiornithidae).

We screened 44 heterologous microsatellites isolated in species of the families Threskiornithidae, Ciconiidae and Ardeidae for their use in a migratory waterbird, the white-faced ibis Plegadis chihi (Vieillot, 1817) (Threskiornithidae). Of the screened loci, 57% amplified successfully and 24% were polymorphic. In two breeding colonies from southern Brazil (N = 131) we detected 32 alleles (2–10 alleles/locus). Average He over all loci and colonies was 0.55, and the combined probability of excluding false parents, 98%. There was no departure from HWE in any loci or population. Eru6 and Eru4 loci were in non-random association in the Alvorada colony, and NnNF5 and Eru5 in both populations. AMOVA analysis indicated that most of the genetic diversity was contained within populations. Structure analysis suggested a single population, and FST value showed weak genetic structuring (FST = 0.009, p = 0.05). The two populations are apparently connected through gene-flow. The panel of six microsatellites optimized here was sufficiently informative for characterizing the genetic diversity and structure in these natural populations of the white-faced ibis. The information generated could be useful in future studies of genetic diversity, relatedness and the mating system in Plegadis chihi and related species.


Introduction
The white-faced ibis Plegadis chihi (Vieillot, 1817) (Pelecaniformes, Threskiornithidae) is a migratory longlegged wading bird, with a long, downward-curved bill and attractive metallic bronze plumage (Dark-Smiley and Keinath, 2003). Occurring from North to South America, it primarily inhabits freshwater wetlands and marshes, and forages in flooded areas and agricultural fields (Ryder and Manry, 1994). Populations in the U.S.A. nest in marshes in the western region, and winter in large flocks in the western parts of the country and Mexico (Dark-Smiley and Keinath, 2003). In South America, it occurs in Brazil, Bolivia, Paraguay, Uruguay, Argentina and Chile. In Brazil, breeding takes place in colonies in coastal lagoons in Rio Grande do Sul State, in the southernmost part of the country (Petry and Fonseca, 2005). Although flocks engage in regional movements between the breeding and wintering periods, their specific migration routes remain unknown (Ryder and Manry, 1994).
So far, populations of the white-faced ibis have not been investigated as to genetic variability, an important property of populations. Population-genetic parameters can be determined with the aid of molecular markers, such as microsatellites, also called simple sequence repeats (SSR). DNA sequences, two to six nucleotides long, repeated in tandem and widely dispersed in all eukaryotic genomes (Tautz, 1989). These codominant and highly polymorphic markers (Zane et al., 2002) have high mutation rates (10 -2 -10 -6 per meiotic event) (Li et al., 2002), the majority being selectively neutral (Selkoe and Toonen, 2006). Accordingly, SSRs have become powerful tools for use in forensic studies, kinship investigation, gene mapping, conservation biology and population genetics (Zane et al., 2002).
In spite of their utility and wide application, there is still a major drawback in the use of microsatellites, since any study aimed at employing such markers requires the isolation of new loci and the development of new primers. However, the isolation of loci for a given species, besides being exceedingly labour-intensive, requires considerable funds, time and technical experience (Mukesh et al., 2011). Moreover, in avian species, the process of microsatellite isolation can pose an additional challenge, since microsatellites are less abundant in avian genomes than in any other vertebrate group (Primmer et al., 1997). An alternative cost-effective approach is the use of primers developed for closely related species, denominated cross-species microsatellite amplification or heterologous amplification, commonly applied in bird studies (Barbará et al., 2007;Mukesh et al., 2011). The success of this process depends on the degree of conservation of primer annealing sites among related species. High evolutionary divergence between the species from which the microsatellite loci were originally isolated (the source species) and the species to which they will be applied (the target species) results in a low number of amplified loci (Primmer et al., 2005). A necessary first step in any study involving the use of heterologous markers is the screening and evaluation of their level of polymorphism in the target species.
Microsatellite loci constitute an adequate tool for estimating such important properties of populations, as genetic diversity, population structure, effective population size and gene flow, all useful in defining units for conservation (Haig et al., 2011). This lack of species-specific microsatellite markers is one of the reasons why, in populations of the white-faced ibis, nuclear neutral genetic variability remains neglected. Thus, the goal in the present study was to identify and optimize heterologous microsatellite loci for population studies in this bird-species. Further information is provided on population genetic parameters and patterns of genetic structuring in samples of two breeding colonies located in the extreme geographic distribution of this species in south Brazil.
Polymerase chain reactions (PCR) were carried out in 10-12 mL reaction mixtures containing by 1 x PCR buffer (1 mM Tris-HCL, pH 8.4, 50 mM KCl), 1.5-2.5 mM MgCl 2 , 0.2 mM dNTPs (Amersham Biosciences), 0.5 U of Taq DNA polymerase (Biotools B&M Labs) and 10-30 ng of template DNA. Except for loci isolated in P. ajaja and M. americana (HEX-labeled), all the primers were dynami-cally labeled with HEX, FAM or TET, using M13 methodology (Schuelke, 2000). Reactions were carried out in an Eppendorf Mastercycler Gradient® (Eppendorf AG, Hamburg, Germany) thermal cycler. The annealing temperatures described for the source species were initially tested, and then modified accordingly, in order to increase the probability of amplifying heterologous loci (Primmer et al., 2005). Successful amplification conditions are subsequently described. For Aaju3 and Aaju5 loci (HEXlabeled), the PCR cycling parameters were: 1 min of initial denaturation at 94°C, followed by 29 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 35 s and extension at 72°C for 1 min, and a final extension at 72°C for 20 min. For the loci NnNF5, NnCE11, NnDD9 and NnHB12 (M13-labeled), conditions were: 4 min of initial denaturation at 94°C, followed by 38 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 1 min and extension at 72°C for 30 s, 8 cycles of 94°C for 30 s, 45 s at 53°C and extension at 72°C for 45 s with a final extension at 72°C for 20 min. Eru3, Eru4, Eru6 and Eru9 loci (M-13 labeled) were amplified with the following PCR parameters: 4 min of initial denaturation at 94°C, followed by 35 cycles of 94°C for 45 s, annealing at specific temperature for 35 s and extension at 72°C for 1 min, 8 cycles of 94°C for 30 s, 45 s at 53°C and extension at 72°C for 1 min with a final extension at 72°C for 20 min. Eru2, Eru5, Eru7, Eru8 and Eru11 loci (M-13 labeled) were amplified as follows: initial denaturation at 94°C for 3 min, followed by 19 cycles at 94°C for 45 s, annealing for 1 min at locus-specific temperatures and 72°C for 1 min, 8 cycles of 94°C for 30 s, 45 s at 53°C and extension at 72°C for 1 min with a final extension at 72°C for 20 min. For WS03, WS04 and WS09 loci (HEX-labeled), amplification conditions were: 5 min of initial denaturation at 94°C, followed by 21 cycles of 94°C for 30 s, 35 s at 55°C and extension at 72°C for 1 min, 8 cycles of 94°C for 30 s, 45 s at 53°C and extension at 72°C for 1 min with a final extension at 72°C for 20 min. For Bi1, Bi5, Bi7, Bi9, Bi10, Bi11 and Bi12 loci (M-13 labeled), PCR parameters were: initial denaturation at 5 min for 94°C, followed by 20 cycles of 94°C for 30 s, annealing for 1 min at locus-specific temperatures and extension at 72°C for 45 s, 15 cycles of 94°C for 30 s, annealing for 1 min at locus-specific temperatures and extension at 72°C for 45 s, 12 cycles of 94°C for 30 s, 45 s at 53°C and extension at 72°C for 45 s with a final extension at 72°C for 20 min.
In order to detect amplification, PCR products underwent horizontal electrophoresis in 1% agarose gels stained with ethidium bromide. Genotyping was carried out in a MegaBACE 1000 (Amersham Biosciences) automatic sequencer. PCR products were diluted with Tween 0.1% and added to ET550-R Size Standard (GE Healthcare). Alleles were sized after analyzing electropherograms in MegaBace Fragment Profiler software (GE Healthcare). Souza et al. 75 The Micro-Checker (Van Oosterhout et al., 2004) program was applied for assessing genotyping quality, the occurrence of null alleles, allelic dropout and stuttering. The number of alleles (A), expected (He) and observed (Ho) heterozygosity, exclusion probability (PE), identify probability (PI), Hardy-Weinberg equilibrium (HWE) per locus, and analysis of molecular variance (AMOVA) were estimated using GenAlex version 6.4 (Peakall and Smouse, 2006). Polymorphic information content (PIC) was computed in Cervus version 3.0 (Kalinowski et al., 2007). Allelic richness (Ra), genic diversity (DG, Nei, 1987), inbreeding coefficient (F IS ) and population differentiation (F ST ) were computed using Fstat version 2.9.3.2 (Goudet, 1995). Linkage disequilibrium (LD) between loci was computed in Arlequin version 3.5.1.2 (Excoffier and Lischer, 2010). P-values for all tests were corrected for multiple comparisons using the sequential Bonferroni procedure (Rice, 1989). The Genepop version 4.0.10 program (Rousset, 2008) was applied for computing a multilocus estimate of the effective number of migrants (Nm) (Slatkin, 1985;Slatkin and Barton, 1989). Signals of reduction in population size were assayed with the Wilcoxon test (Luikart et al., 1998), as implemented in Bottleneck version 1.2.02 software (Cornuet and Luikart, 1996). Population structure for microsatellite variation was also examined using the Structure version 2.1 program (Pritchard et al., 2000). By this Bayesian clustering method, estimates are made of the number of genetic clusters (subpopulations; k) that best explain the data, and thus, based on their genotypes and disregarding sampling locations, assign individuals to subpopulations. Ten replicates of each run for k = 1 and k = 2 were carried out using the admixture model with a burn-in period of 100,000, and 10,000 MCMC reps after burn-in.
At the level of families and orders, the percentages of successful amplification and polymorphism were higher than, or of the same magnitude as, those reported in the literature for the group`birds'. Herein, when considering loci isolated in other genera of the same family as P. chihi, the 84% of successful heterologous amplification was of the same magnitude as that obtained by Chang et al. The PIC index, an efficient indicator of loci adequacy in population genetics studies, ranged from 0.35 to 0.76, with a mean of 0.50 in our panel of microsatellites (Table 1). It is commonly accepted that PIC values > 0.50 are characteristic of the most useful markers, 0.25 < PIC > 0.50 of those moderately informative, and PIC < 0.25 of the less informative (Andres and Kapkowska, 2011). Thus, the loci optimized in this study can thus be considered moderate to highly informative. The potential of our set of markers for identifying individuals within the population was quite high, i.e., across loci PI = 0.0002 in Alvorada and PI = 0.0001 in Tapes. Exclusion probability (PE) across loci of 98% falls within the range observed in other waterbirds (Miño et al., 2009). This relatively high probability of excluding a false parent indicates that this panel of markers is useful not only in the estimation of the overall level of genetic variability in white-faced ibis populations, but also in the diagnosis of relationships between individuals.
Population genetic parameters were estimated using the entire sample (N = 44 for Alvorada; N = 87 for Tapes). Analysis with the Micro-Checker program indicated null alleles in Eru6 loci due to homozygote excess in both populations, probably caused by stuttering in the electropherograms. According to Chi-square tests, with sequential Bonferroni correction, there was no departure from HWE in any of the analyzed loci after multiple comparisons (Table 2). Across population samples, the mean number of alleles per locus was 4.83, average He 0.55 and average Ho 0.57. Average Ra per population was 4.70 in Alvorada and 4.42 in Tapes, whereas mean DG was 0.56 in both populations (Table 2). Inbreeding coefficients (F IS ) did not deviate significantly from random mating (p = 0.004), neither per locus nor population. The exact test of LD indicated association (p < 0.05) between Eru6 and Eru4 loci in the Alvorada colony, and between NnNF5 and Eru5 loci in both populations. Thus, NnNF5 and Eru6 loci were excluded when subsequently estimating population genetic parameters.
Overall expected heterozygosity observed in the white-faced ibis (He: 0.55) was similar to that observed in 17 species of aquatic birds (He: 0.62 ± 0.15). This He value was higher than the range of most common heterozygosity values (He: 0.40-0.50) observed in 17 aquatic bird species (Eo et al., 2011), besides being higher than that observed in the wood stork, of the same magnitude as that noted in the jabiru stork (Jabiru mycteria), and lower than in the roseate spoonbill and great egret (Table 3). Furthermore, on considering only the aquatic species phylogenetically more related to the white-faced ibis (the great cormorant, Phalacrocorax carbo, and the great blue heron, Ardea herodias), mentioned in the Eo et al. (2011) study, it can be seen that in the white-faced ibis, He values were lower (Table 3). Moreover, the mean number of alleles per locus observed in the white-faced ibis (A: 4.83) was lower than the average for the 17 former mentioned species examined by Eo et al. (2011) (A: 6.59 ± 6.94).
dered as a single population cluster. This is in agreement with the estimated number of migrants per generation (Nm = 1.7) and indicates that gene flow may be occurring between the studied populations. In theory, the migration of one individual per generation is sufficient to homogenize the genetic pools of two different populations (Hartl and Clark, 1989). Thus, the low significant differentiation de-tected between these two white-faced ibis colonies is probably a consequence of minor genetic divergence. It must be stressed that a study with more representative samples, collected throughout the entire species-distribution range, using additional microsatellite loci, would help to clarify this issue.
In conclusion, the panel of heterologous loci optimized in this study is suitable for future investigations on genetic structure and relatedness in both natural and captive populations of the white-faced ibis. Future studies including additional loci and populations, should be carried out in order to characterize overall genetic structuring of breeding colonies distributed throughout Brazil. We believe that the data herein would be useful for studies on important questions regarding population genetics, mating systems and kinship in the white-faced ibis and related species. More specifically, the information provided in the present study can benefit studies of population genetics in other species within the genus Plegadis, viz., P. falcinellus and P. ridgwayi.  Non-significant after sequential Bonferroni correction (p > 0.05). The study by Miño et al. (2011) refers to nine microsatellite loci, whereas that by Van Den Bussche et al. (1999) refers to four loci, also mentioned in Eo et al. (2010). Original references: Goostrey et al. (1998) and Piertney et al. (1998).