Assessment of the genetic diversity of Cameroon indigenous chickens by the use of microsatellites

J C Fotsa, D Poné Kamdem, A Bordas*, M Tixier-Boichard* and X Rognon*

Mankon Specialized Station of Agricultural Research for Development (SSRAD)
Box 4099 Bamenda, Cameroon.
fotsajc2002@yahoo.fr
* INRA and AgroParisTech, UMR1313 GABI, 78350 Jouy-en-Josas, France

Abstract

The genetic diversity of Cameroon indigenous chickens was assessed on 141 DNA samples extracted from blood samples collected from 153 parent stocks bred on-station. Twenty-two microsatellite markers from AVIANDIV European’s project were used to characterize local chicken ecotypes collected from Centre (CE), South (SU), East (ES) and North-West/West (NW/OU) regions and five commercial lines (three broilers and two layers).
 

Results showed a total of 156 alleles for an average of 7.09 alleles per locus, though the number of alleles was greater for the local ecotypes. Furthermore, in local ecotypes the expected (He) and observed (Ho) heterozygosity varied from 0.617 to 0.634 and from 0.628 to 0.664, respectively. Genetic distances were very small within local ecotypes and varied from 0.015 (between CE and SU) to 0.042 (between NW/OU and ES (East)). Hence, birds from NW/OU were much more homogenous than those from CE and SU, which in turn were genetically closer to the commercial lines. These results would be considered as useful genetic information for Cameroon indigenous chickens conservation in view of their genetic improvement tailored to their historical, socio cultural, and environmental values.

Key words: Local fowls, molecular characterization, molecular markers

Evaluation de la diversité génétique des poules locales du Cameroun par l’usage des marqueurs microsatellites

Résumé

La diversité génétique a été évaluée sur 141 échantillons d’ADN extraits du sang de 153 poules parentales de souche locale élevées en station expérimentale au Cameroun. Le typage des échantillons d’ADN a été fait à l’aide des 22 marqueurs microsatellites issus du projet Européen AVIANDIV. Les populations des poules locales du Cameroun provenaient des régions du Centre (CE), du Sud (SU), de l’Est (ES) et du Nord-ouest/Ouest (NW/OU). Cinq lignées commerciales dont trois lignées chair et deux lignées ponte étaient associées à l’étude.

Les principaux résultats montrent un total de 156 allèles pour une moyenne de 7,09 allèles par locus. Toutefois, les populations locales présentent une bien plus grande richesse allélique que les lignées commerciales. Chez les populations locales, l’hétérozygotie attendue (He) et observée (Ho) varient respectivement de 0,617 à 0,634 et de 0,628 à 0,664. Les distances génétiques sont les plus faibles chez les populations locales et varient de 0,015 (entre CE et SU) à 0,042 (entre NW/OU et ES). Toutes ces populations locales apparaissent très peu différentiées entre elles. Cependant, celle du NW/OU semble plus homogène que celles du CE et du SU. Ces deux dernières semblent génétiquement plus proches des lignées commerciales. Ces résultats moléculaires seraient des informations génétiques utiles dans la conservation des races locales de poules en vue de leur amélioration génétique en liaison avec les valeurs historiques, socioculturelles et environnementales.

Mots clés: Poules locales, caractérisation moléculaire, marqueurs moléculaires

Introduction

Little is known about the genetic structure of Cameroon local chickens. Yet, such information could be useful in describing the phylogeny of these chickens in order to better manage their intra- and inter- genetic variability (Fotsa 2008). Some preliminary work by Mafeni et al (1997) on genetic fingerprints showed that the local chickens in North-West Cameroon were genetically close to Mediterranean and Bolivian breeds. On the phenotypic aspect, recent studies (Fotsa and Poné 2001; Keambou 2006; Fotsa et al 2010) showed that the great diversity observed in local chickens from both the western highlands and the dense forest regions of Cameroon were due to natural selection. In previous reports, Ngou Ngoupayou (1990) linked phenotype to production parameters as well as some sociological factors whereby  white plumage color was associated to meat production, golden and black plumage colours to egg production and black plumage color to rituals and magic practices.

Cameroon national poultry flock was estimated at 35 million, 70% being of local chickens (INS-Cameroon 2001; Fotsa et al 2007b). The 30% remaining were made of foreign breeds such as Arbor Acres, Hubbard, ISA, Rhode Island Red, Cornish, White Plymouth Rock, White Leghorn and many others introduced commercial lines. These introduced chickens have certainly contributed to the dilution of the genetic diversity of local chickens (Fotsa et al 2007a) and consequently could constitute a serious threat to local genetic erosion. On the molecular aspect, no study has ever been reported using microsatellite markers for polymorphism at the level of DNA other than the utilization of genetic fingerprints to show the genetic relationship between local chickens of the North West of Cameroon and the German Red Dahlem (Mafeni 1995; Mafeni et al 1997). On the other hand, it is not known if the ecotypes described in the humid forest zone and in the West and North West highlands of Cameroon represent identical or genetically distinct populations. Whatever the case, the genetic potentials of local breeds and their contribution to the future strategies for sustainable management require a prior knowledge of the prevailing genetic diversiy (Bordas et al 2004). Hence, the characterization of the genetic structure and the inbuilt variability constitute an important step towards identifying valuable genetic resources.
 

Globally, the uses of genetic marker polymorphisms such as microsatellites are reliable ways of assessing the differences within and between chicken populations (Nei 1972; Reynolds et al 1983; Caballero and Toro 2002). The aim of this study was to assess the genetic diversity of Cameroon local chickens collected from the highlands and dense humid forest zones through the use of microsatellite markers in order to clarify their phylogenetic relationships and to possibly identify genetically distinct ones for preservation for a better future use.

Materials and Methods

Characteristics of the studied areas

The Western Highlands Zone:

The Western Highlands covers the administrative Regions of West and North West of Cameroon. Situated at an average height of 1240 m above the sea level (a.s.l), the zone enjoys an annual average temperature of 19°C with a relative humidity above 80% and 2000 mm annual rainfall.

The humid forest zone with a biannual rainfall pattern:

The humid forest covers the majority of the southern part of Cameroon situated between 500 and 1000 masl (Figure 1). It has two rainy seasons and two dry seasons with an average annual temperature of 25°C, while relative humidity varies from 99.9 to 100%. The zone receives an annual average of 2000 mm of rain.  

Figure 1.  Map showing agro ecological zones of Cameroon (Source: Ambassa Kiki 2000)

Chicken samples and blood collection

From 2004 to 2005, 1679 fertilized eggs collected from 104 households within 30 divisions distributed all over the two agro ecological regions were jointly incubated to constitute the reference foundation stocks (Table 1) in this study. For each division per ecozone, 50 to 55 fertilized eggs were collected from at least three to four households sampled by the extension agents. Chosen households had at least a flock size of ten birds. Thus, 153 made of 131 hens and 22 cocks were hatched and bred at Mankon Experimental Station of the Agricultural Research for Development (IRAD) located at Bamenda – Cameroon from day old to maturity. These 153 native reference chickens were made of 4 populations from two ecological zones of Cameroon namely humid forest zones with three ecotypes from Centre, South and East regions and high land zone with one ecotype from North-West/West regions.

All ecotypes were fed on same commercial diet containing 21,5% crude protein, 5,6% ether extracts, 3,6% fiber and 6,7% minerals for 8 weeks then 19% crude protein, 6,97% ether extracts, 3,6% fiber, 0,45% methionine, 1% lysine and 0,7% phosphorus from 9 weeks to point-of-lay and 16% crude protein, 7,0% ether extracts, 4,0% fiber, 0,41% methionine, and 0,67% phosphorus from point of lay till end of trial. 

Reference populations

Genotypes of chickens belonging to five commercial lines (two broiler male lines, one broiler dam line, one White Leghorn layer and one Brown egg layer lines) from AvianDiv project were included in this study in order to highlight potential impacts of these lines in the local gene pools (distribution of these animals as a result of government sponsored projects and/or projects initiated by the farmers themselves). The commercial samples were obtained from the DNA bank established at the AvianDiv project (Hillel et al 2003) and managed by UMR INRA / AgroParisTech, Génétique et diversité animales. These characteristics made these reference commercial lines suited to be used in comparison to Cameroon indigenous chickens.

Blood collection

A total of 141 blood samples were obtained from adult chickens in Cameroon as indicated in Table 1. For each chicken, about 1.5 ml of blood was drawn from the wing vein using Sarstedt syringes with needles of 22G x 1¼ bore size containing 5% of 0.5 M EDTA as an anti-coagulant agent (to prevent clotting in the needle or syringe). The contents of the syringes (blood + anticoagulant) were slowly dispensed into tubes and conserved at -20°C in 20% (final volume) sterile glycerol until use.

Extraction and purification of genomic DNA from blood samples

The 141 frozen blood samples were transported to the Biotechnology Laboratory of the College of Agriculture and Consumer Sciences, University of Ghana, Legon, where genomic DNA was extracted using the QIAGEN^® DNeasy Tissue Kit (QIAGEN, Valencia, CA, USA).

Genotyping

Molecular genotyping of the samples was performed by the GIE LABOGENA with 22 microsatellite loci (AvianDiv panel, Hillel et al 2003), after amplification by PCR with fluorescently labelled primers and migration on a capillary sequencer (ABI PRISM 3100 Genetic Analyzer, Applied Biosystems). Polymerase chain reaction (PCR) was carried out with a multiplex of two to five primer-pairs, with each reaction comprising 20 ng of DNA template, 10 pmol each of forward and reverse primers, and 1 mM tetramethylammonium chloride.  The amplification protocol involved initial denaturation of DNA and enzyme activation at 95^oC (15min) followed by 35 cycles of denaturation at 95^oC (1min), primer annealing at a temperature varying between 58^oC and 63^oC (1 min), extension at 72^oC (1min), and final extension at 72^oC (10 min) using an automated thermal cycler (Mastercycler, Eppendorf, Hamburg, Germany).  Subsequently, the PCR products were electrophoresed on an ABI 3100 DNA Sequencer (Applied Biosystems Division, Foster City, CA, USA) and the sizes of the fragments were estimated based on fluorescently labelled forward primers (FAM, NED and HEX) using the GENESCAN and GENOTYPER software (Applied Biosystems)

Data analyses

The observed (Ho) and expected (He) heterozygosities, the average number of alleles (A) per locus and the average number of effective alleles (Ae) per locus were calculated using the software GENETIX 4.4 (Belkhir et al 2000). These parameters provide information on within population diversity. The F_ST (coefficient of genetic differentiation) between pairs of populations was calculated using the method of Weir and Cockerham (1984), as implemented in FSTAT 2.9.3.2 (Goudet 2001) to estimate the level of differentiation between populations.  To determine phylogenetic relationships, a neighbour-joining tree was drawn based on Nei’s genetic distances (DA) calculated between individuals (Nei et al 1983), using POPULATION 1.2.28 and TREEPLOT 0.7 by Olivier Langella (http://www.pge.cnrs-gif.fr/bioinfo/populations/).

Results

Characteristics of markers

A total of 156 alleles were detected for 22 microsatellite markers (table 2), corresponding to an average of 7.09 alleles per marker. The highest allele numbers (16, 12 and 11) were obtained from LEI234, LEI094 and MC034 loci, respectively while the smallest allele numbers came from MCW098 with 2 loci. The majority of alleles found in the commercial lines (108/117) were also found in Cameroon indigenous chickens, which total alleles numbered 147. Local chickens’ populations showed 94.2% of total allele number, compared with 75% for commercial lines. For both commercial lines and local chicken’s populations, the total common alleles represented 69.2% of total count. The rate of the share alleles between a given local chicken population group and the commercial line as a whole were similar and varied from 78.6% for Centre population to 80.6% for South population and 82% for North West/west population.

Intra population genetic diversity

Table 3 shows an important number of alleles per marker and per ecotype, averaging 5.474, but for the East region (Cn-E) where this number was only 3.36 alleles. Commercial population showed ellelic diversity with 3.26 alleles on average for broiler commercial lines and 2.55 for layer commercial lines. For Cameroon local chickens population, observed heterozygosity rate (Ho) varied from 0.626 (Cn-NO) to 0.664 (Cn-E). For broiler chicken lines, these rates varied from 0.475 (Br-m-49) to 0.512 (Br-m-47) while for layer lines, these rates varied from 0.295 (WEL-37) to 0.360 (BEL-44). Generally, observed heterozygouzities rate were always greater for Cameroon chickens than for commercial lines (broiler and layer). Similar observations were evident for Cameroonian populations (0,622-0,634) when compared with commercial lines (0,297-0,499) concerning the expected values of heterozygosity (He).

Relationship between local chicken populations and commercial lines

The differentiation between four Cameroon local chickens populations, three broiler commercial lines and two layer commercial lines was evaluated using F_ST calculation (table 4). The general F_ST mean for pair of local populations and commercial lines was 0.22 ± 0.13. Furthermore, highest values were obtained for commercial lines (Mean F_ST = 0.37 ± 0.10), and between these lines and Cameroon local populations (0.21 ± 0.07). On the contrary, Cameroon local chickens appeared slightly differentiated (0.03 ± 0.01) as smaller F_ST values corresponded to significant differentiation and varied from 0,015 (between Cn-C and Cn-S) to 0.042 (between Cn-NO and Cn-E). Only two pairs of Cameroon ecotypes did not differentiate (CnE/CnS and CnE/CnC), this could be explained by the smaller number of birds that came from East region.

The genetic relation tree between all individuals (Figure 2) was constructed from Nei genetic distances DA (Nei 1983). A code was used to attribute a given color to each population. Within the local Cameroon chickens and contrary to commercial lines, each individual was identified by a number. This approach helped to quickly assess the magnitude of populations’ heterogeneity. Results showed that commercial lines appeared highly homogenous as all individuals gathered in one whole group per line. The local populations presented were very disparate. Indeed, 60% of them (88/141) were clearly gathered in a single group, separately from commercial lines, while 54 others were scattered.

Globally,

• In the Centre  Cn-C, 62 % of birds were in the ‘local’ group (32/52) versus 38% (20/52) between different ‘commercial’ groups;
• In the South population Cn-S, 53 % were in this local group (25/47) while 47% (22/47) were scattered within "commercial" groups;
• In the North West/West population Cn-NO, almost all individuals (84 %) belonged to the local group while the smallest percentage of animals (16 %) or 6/37 were in the "commercial" groups;
• In the East population Cn-E, all individuals belonged to the "commercial" group but the number of birds was too limited and did not permit firm conclusions.

Figure 2.  Genetic relationship tree between all the individuals on the basis of their genotype for 22 microsatellite markers (Each breed is represented by a different color. The commercial chicken lines are not individually labeled. For Cameroonian local chicken population, each individual is identified by its population code, ID number, as well as color).

Discussion
 

Reliability of results

Results obtained per tested microsatellite marker were consistent with Barker’s (1994) recommendations that at least four alleles are needed to reduce the standard errors in their distances estimation. Therefore, the use of 21 microsatellite markers in this study was sufficient; although microsatellite (MCW098) marker registered only two alleles. Out of the 22 markers used, 21 were previously used (Qu et al 2004; Muchadeyi et al 2005; Granevitze et al 2007). Results further showed mean number of alleles per locus to be inferior to other results obtained with a large population size. In fact, Granevitze et al (2007) reported, on 64 populations sampled from all continents, a mean alleles per locus of 11.4 with extreme values varying from 2 for MCW0103 locus to 28 for LEI0234 locus. Similarly, studying 78 breeds of Chinese chickens with 27 markers, Qu et al (2004) observed 18.6 alleles per marker with the number of alleles varying from 6 to 51.

Although the number of markers used in the present study was less than FAO recommendation, it appeared that the set of 22 markers helped to clearly differentiate commercial populations from their local counterparts. The high frequency level of common alleles found between Cameroon local chickens’ populations and commercial lines would suggest the introgression of commercial lines into the Cameroon local populations. This is consistent with earlier reports wherein many exotic lines were being introduced in Cameroon starting from the 50’s (Belot et Hardouin 1981; Teleu Ngandeu and Ngatchou 2006), and could also stem from common ancestors relationship of the two genetic types that accumulated during commercial exchanges between Africa and Asia (Carter 1971). Besides, 77.3% of markers (17/22) carried proper alleles to Cameroon local populations versus 31.82% for commercial lines (7 markers / 22). The presence of private alleles in commercial lines suggested also the absence of important and recent mixing with Cameroon local populations. The existence of many specific alleles in Cameroon local populations suggests that the sampled populations were quite genetically distant from commercial lines, despite the intensive used of commercial lines in Cameroon’s poultry industry. These specific alleles to Cameroon local chicken populations could be used as “diagnostic markers” to attest the local origin of chicken meat and to detect fraudulent importation in the territory (Osman et al 2006). The use of private alleles for the traceability of poultry products depends on their frequency in the local population; one single test can detect alleles from many loci. The small population size sampled in the East Region was due to the remoteness and transport difficulties of fertile eggs that resulted in lower hatchability rates. This constrained any scientific discussion on the molecular characterization of this ecotype.

Genetic diversity of Cameroon chicken’s populations

The number of alleles for the three Cameroon chicken populations (Cn-C, Cn-S and Cn-NO) was higher than those observed in the commercial ‘broiler’ and ‘layer’ lines; in agreement with Muchadeyi et al (2005) in a test involving 5 Malawian local chickens’ populations collected from three different agro ecological zones. Crooijmans et al (1997) and Tabahaski et al (1998) reported that alleles’ mean values per locus varied from 4 (4 to 9) to 5.6 (2 to 10), respectively. Granevitze et al (2007) studied 64 chicken populations from different continents and reported average number of alleles to vary from 5 to 6 for non selected local populations and an overall allele’s mean number of 3.6. It is found that local populations had a mean allele’s number higher than that of standardized commercial lines. Higher numbers were found in Japan on 34 populations from 28 local breeds (allele average number = 10.85; Osman et al 2006) and in Zimbabwe on 13 populations of local chickens (average allele number = 9.7 ± 5.1; Muchadeyi et al 2007). Average alleles’ number found in this study was higher than that obtained in Japanese local chickens (1.75 – 4.70; Osman et al 2006) but was intermediate to french local chickens (2.68 – 4.73) and Vietnamese (5.2-7) reported by Berthouly et al (2007) and Berthouly et al (2008) with same microsatellite markers.

The higher rate of heterozygosity in the present study for Cameroon chickens revealed an important polymorphism at the molecular level, which was coherent with the greater phenotypic variability observed earlier (Fotsa and Poné, 2001; Keambou et al 2007). Expected heterozygosity (He) and observed heterozygosity (Ho) values were higher in Cameroon local populations than those obtained in the commercial lines, respectively. Similar observations were reported by Wimmers et al (2000) when comparing African local breeds with two commercial breeds (Dahlem Red and Rhode Island Red).

Ho values between Cameroon local populations and commercial lines were higher than those reported by Kayang et al (2002) with 0.205 (2 to 4 alleles per locus) and 0.127 (2 to 5 alleles per locus), respectively. For both He and Ho, the highest values were observed in the East population Cn-E followed closely by those of other Cameroon ecotypes. The values of He and Ho obtained in the present study were higher than those reported by Granevitze  et al (2007), but were closer to the Ho=0,622 value reported by Qu et al (2004) and He = 0.21 to 0.67 reported by Osman et al (2006). In the local Cameroon chickens, the obtained Ho value was higher than the average Ho=0.55 obtained on the 13 ancient local French breeds (Tixier-Boichard et al 2006) studied with the same markers used in the AvianDiv European Project. Moreover, it was not observed deficit of heterozygosity in the local Cameroon chicken’s populations compared to the expected rates under Hardy-Weinberg’s hypothesis. In the commercial lines in contrary, the deficit were observed in BEL-44, WEL-37 and Br-49 to be –0.046 ; -0.002) and -0.006 respectively for the unknown reasons which would probably be due to the exchange breeding stocks with these lines during the selection programs.

However, average heterozygosity rates obtained in the Cameroon local chicken’s populations are lower than the values observed in the other species such as fish (0,65, Rutten et al 2004) and pigs (0,74, Behl et al 2006).

In general, high values of heterozygosity rates in the Cameroon local chicken’s populations were in coherent with the absence of organized selection for a given production type. Small values obtained with the commercial lines could be explained by the intensive selection of each line for a given production type. Cameroon local chicken’s population have the highest heterozygosity rate indicating a great genetic diversity as it had been always shown in the populations reared without selection diagram contrary to what had always been observed in the commercial lines or in the population with breed’s standard (Wimmers et al 2000; Berthouly et al 2007; Granevitze et al 2007; Muchadeyi et al 2007). These results bring in the best available objective information on genetic variability of local populations prior to the genetic improvement program or the conservation of local breeds of chickens (Ruane 1999) to be developed; they also confirmed the use of microsatellite markers for genetic diversity characterization in Cameroon local populations of chickens.

Genetic structuring of local populations and commercial lines

No genetic structure was clearly observed between different local populations in the studied areas even though low values of F_ST showed, as a whole, a significant differentiation. The absence of this type of structuring was also observed in other non selected local populations as in Vietnam (Berthouly et al 2007) or in Zimbabwe (Muchayedi et al 2007), even if other scientists seemed to find some geographic grouping as it has been the case within Kenyan poultry population (Mwacharo et al 2007). Local population distribution on both sides of the tree could be explained by the movements of Cameroonian human populations in these areas alongside with their animals. Local chickens have been often used as natural gifts to a relative or to an important guest (Ekue et al 2002; Fotsa et al 2007a, c) living in different region of Cameroon; this leads to a movement of birds from one region to another. These movements can easily explain why mixture of human population can bring in a mixture of chicken’s populations by crossbreeding.

Moreover, large industrial farms were found around the city capital of Yaoundé (Centre Region) and the proximity of this Centre Region to the south region partly explained the connection of local chicken populations to commercial improved lines. Although during the egg collection that hatched chickens for the present study, questionnaires were used to avoid as much as possible obtaining samples from populations of chickens which could have been mixed with commercial lines, it appeared that some birds had nevertheless preserved the mark of hybridization with commercial strains. One could see the past or recent introduction of commercial animals in the villages in the last decades and influence of proximity of industrial poultry farms which were taking place around urban centers. In fact, many improved poultry breeds, lines and strains were introduced in Cameroon for industrial production of chickens (Téleu Ngandeu and Ngatchou, 2006). However, local chicken populations of Centre Cn-C and south Cn-S were too close to commercial lines and less homogenous because these two regions are very close geographically. Moreover, large industrial farms were found around the city capital of Yaoundé (Centre Region) and the proximity of this central region to the south region partly explained the connection of local chicken populations to commercial improved lines. Local chicken population of highland plateaus of North west and west (Cn-NO) were too homogenous showing that, even though industrial farms were strongly present in these two regions of West and North West, it did not seem to have mixing of birds in these regions, because breeding of improved birds constituted revenue activity for semi industrial and industrial poultry farmers situated in the areas allowing them to sell in the large consumption cities (Douala with about 3 million inhabitants and Yaoundé with about 2.5 millions of inhabitants) while local chickens were really used for family consumption and for ritual, traditional and socio-cultural uses as reported by Ekue  et al (2002) and Fotsa  et al (2007c).

Conclusion and perspectives

• This study gave the opportunity to confirm the molecular genetic diversity often observed phenotypically on local Cameroon chickens. However, the geographical location of each of the studied populations does not allowed the identification of any specific breed amongst local ecotypes.  A close comparison with commercial lines revealed a high rate of introgression among some local chicken populations varying from 16% for High lands ecotypes to 47% for South ecotype in the forest zone. This introgression is likely to reduce the stability of local chicken populations.

• Hence, a conservation and improvement programme for local chicken breeds could be launched with those animals showing little similarity with commercial strains as reported in Kenya and other east African Countries (Mwacharo et al 2007).

Acknowledgements

The authors hereby express their gratitude to all those who contributed in the management of experimental animals and collection of biological samples. Specifically, our acknowledgements go to Ms. Annah Takieh Neh, Mrs. Ngu Suzanne, Mrs. Abudu Felicia, Mr. Romanus Bache, Mr. Valentine Evi, Mr. Francis Ayumdi, Mr. Asana Joseph Atacho, Mr. Biassi Silvère, Mr. Thimoty Ndoum Mbobe, Mr. Teko George Tah, Ms. Kaham Divine, Mrs. Poné Kamdem Malanie, Dr. Tih Shefe Joseph, Mrs. Bi Henrietta Amabo, and Mr. Tah Joseph Wanye. We are also thankful to IRAD, BAD, REPARAC, the French Foreign Ministry through its Embassy in Cameroon, Dr. Boniface Kayang, the Head of Department of Animal Productions of the Food and Consummers College of the University of Leghorn in Ghana for a familiar assistance (ADN extraction and translation of part of the manuscript), INRA/AgroParisTech, Unité Mixte de Recherche 1236 de Jouy-en-Josas en France, for providing the exotic strains from the European Project AVIANDIV.

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Received 11 October 2010; Accepted 13 November 2010; Published 1 May 2011

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