Comparative cytogenetic and allozyme analysis of Mugil rubrioculus and M. curema (Teleostei: mugilidae) from Venezuela/Analisis comparativo citogenetico y aloenzimatico de Mugil rubrioculus and M. curema (Teleostei: Mugilidae) de Venezuela/Analise comparativa citogenetica e aloenzimatica de mugil rubrioculus and M. curema (Teleostei: Mugilidae) de Venezuela.
A comparative cytogenetic and aUozyme analysis of sympatric specimens of Mugil rubrioculus and M. curema from Venezuela is reported. Specimens of M. rubrioculus exhibit a 2n=48 karyotype with exclusively acrocentric (NF=48) chromosomes, one pair of NORs interstitially located on chromosome pair number 8 and constitutive heterochromatin distributed in pericentromeric position of all chromosomes. Specimens of M. curema show cytogenetic features significantly different in comparison to M. rubrioculus in terms of chromosome number and morphology (2n=24 biarmed chromosomes, NF=48) and NORs location (telomeric region of the largest metacentric pair). Starch gel electrophoresis analysis at 20 presumptive loci reveals a reduced genetic differentiation between the two species. In fact, though a total of ten private alleles are identified; all loci share alleles between the two species and the obtained Nei's genetic distance (D= 0.060) is lower than the values obtained between other congeneric mullet species. Thus, the cytogenetic and allozyme data sets indicate quite different degrees of genetic divergence between M. rubrioculus and M. curema. This could either reflect an underestimate of molecular divergence owing to cryptic variation or different rates of molecular/chromosomal evolution. Whatever the explanation, this study confirms the power of karyological data in discriminating species of Mugilidae.
KEYWORDS / C-banding / Chromosome / Fluorescent in situ Hybridization / Gene-enzyme / Karyotype / Mullets / NORs /
Se presentan los resultados del analisis comparativo citogenetico y aloenzimatico entre las especies simpatricas Mugil rubrioculus y M. curema de Venezuela. Los especimenes de M. rubrioculus presentan un cariotipo con 2n=48 cromosomas exclusivamente acrocentricos (NF=48), NORs intersticiales localizados en el par cromosomico numero 8 y heterocromatina constitutiva distribuida en posicion pericentromerica en todos los cromosomas. Los especimenes de M. curema presentan caracteristicas citogeneticas significativamente diferentes de M. rubrioculus en terminos de numero cromosomico y morfologia (2n=24 cromosomas de dos brazos y NF=48) y localizacion de las NORs (region terminal del par metacentrico mas grande). El analisis electroforetico en gel de almidon de 20 loci presuntivos revelo una diferenciacion genetica reducida entre las dos especies. De hecho, aun cuando un total de diez alelos especificos hayan sido identificados, no hay loci que no compartan alelos entre las dos especies y el valor de distancia genetica (Nei) obtenido (D= 0,060) es mas bajo que el obtenido entre otras especies congenericas de mugulidos. Asi los datos citogeneticos y los alozimicos indican diversos grados de divergencia entre el M. rubrioculus y M. curema. Esto podria reflejar una subestimacion de la divergencia molecular por variacion criptica o diferentes tasas de evolucion molecular y cromosomica. De cualquier manera, este estudio confirma el poder de los datos cariott'picos para discriminar especies de Mugilidae.
Apresentam-se os resultados de uma analise citogenetica e alozimica comparativa entre as especies simpatricas Mugil rubrioculus e M. curema da Venezuela. Especimes de M. rubrioculus apresentam cariotipo com 2n=48 e exclusivamente cromossomos acrocentricos (NF=48), NORs intersticiais localizados no par cromossomico numero 8 e heterocromatina constitutiva distribuida em posicao pericentromerica de todos os cromossomos. Especimes de M. curema apresentam caracteristicas citogeneticas significativamente diferentes de M. rubrioculus em termos de numero cromossomico e morfologia (2n=24, cromossomos de dois bracos e NF=48) e localizacao das NORs (regiao terminal do maior par metacentrico). A analise de eletroforeses em gel de amido de 20 presumiveis loci revelou uma diferenciacao genetica reduzida entre as duas especies. De fato, apesar de que um total de dez alelos especificos tenham sido identificados, nao ha loci que nao compartilhem alelos entre as duas especies e a distancia genetica de Nei (D = 0,060) e menor do que os valores obtidos entre outras especies de tainhas. Assim, os dados citogeneticos e alozimicos indicam graus de divergencia diferentes entre M. rubrioculus e M. curema. Isto pode ser reflexo de uma divergencia molecular subestimada por variacao criptica ou a uma taxa de evolucao molecular e cromossomica diferente. De qualquer maneira, esse estudo confirma o poder dos dados cariotipicos na discriminacao de especies de Mugilidae.
An earlier cytogenetic analysis, based on Giemsastained karyotypes, revealed that specimens identified as Mugil gaimardianus (sensu Cervigon, 1993), from Panama and Margarita Island, Venezuela, possess a 2n=48 uniarmed chromosome karyotype (Nirchio et al., 2003). In contrast, specimens of M. curema (sensu Cervigon, 1993), also collected from Margarita Island, have a 2n=24, entirely biarmed chromosome karyotype (Nirchio et al., 2003). Thus, cytogenetic data provided important support to the hypothesis that nominal M. gaimardianus, although very similar in appearance to M. curema, was a distinct species, as previously noted by some authors (Menezes, 1983; Cervigon, 1993).
Subsequent morphological comparisons of the specimens identified by Nirchio et al. (2003) as M. gaimardianus with type specimens or representative material from other nominal species of Mugil occurring in the Western Central Atlantic, revealed that the specimens identified as M. gaimardianus were not conspecific with any other nominal species. However, as noted by Harrison et al. (2007), the original description of M. gaimardianus by Desmarest in Bory de Saint-Vincent (1831) was uninformative and the name had been suppressed (Alvarez-Lajonchere et al., 1992; ICZN, 1994). Therefore, a new species description and name were required for Nirchio et al.'s (2003) specimens; this description has been made by Harrison et al. (2007), with provision of the new name Mugil rubrioculus.
This study reports a comparative cytogenetic and allozyme analysis of sympatric specimens of M. rubrioculus and M. curema from La Restinga lagoon, Margarita Island, Venezuela, carried out in order to provide genetic data on the specimens used for the taxonomic description (Harrison et al., 2007) and to expand the knowledge on the genetic divergence between these two, very similar species. The cytogenetic characterization of M. rubrioculus includes data on constitutive heterochromatin by C-banding and on nucleolus organizer regions (NORs) by silver staining (Ag-NORs) and fluorescent in situ hybridization (FISH). The chromosomal constitution for the same cytogenetic features has also been monitored in specimens of M. curema, in consideration of the important chromosome variauon described among M. curema from Venezuela and from Brazil (Nirchio et al., 2005a). Additionally, comparative analyses of specimens of M. rubrioculus and M. curema are made using starch gel electrophoresis of 11 gene-enzyme systems.
Materials and Methods
Specimens of M. rubrioculus and M. curema were collected from La Restinga lagoon, Margarita Island, Venezuela (10[degrees]57'46.6"N, 064[degrees]10'44.9"W) between April 4, 2004 and March 2, 2005. The voucher specimens deposited in the collections of the Escuela de Ciencias Aplicadas del Mar (ECAM) used for the karyological (K) and alloyzyme (A) analyses are reported in Table I. Additional specimens were used in electrophoretic studies for M. rubrioculus (21) and M. curema (25). The meristics and morphometrics of some voucher specimens are provided in Harrison et al. (2007).
A total of 14 specimens of M. rubrioculus and 12 specimens of M. curema were cytogenetically analyzed. Each fish was injected with 0.0125% colchicine solution (1ml/100g body weight) and maintained in a well-aerated aquarium for 50min, prior to being killed by a sharp blow to the head. The mitotic chromosome preparations followed the technique described by Bertollo et al. (1978). C-bands were obtained according to Sumner (1972). Nucleolus Organizer Regions (NORs) were localized by chromosome impregnation with silver nitrate (Howell and Black, 1980) and by Fluorescent in situ Hybridization (FISH). FISH was performed according to the method described by Pinkel et al. (1986) with some modifications (Martins and Galetti, 2001). The 18S rDNA probe was obtained from Oreochromis niloticus and was labeled by nick translation with biotin-14-dATP according to manufacturer (Bionick labeling system-Invitrogen) instructions. The metaphase chromosome slides were incubated with RNase (40pg/ml) for 1.5h at 37[degrees]C. After denaturation of chromosomal DNA in 70% formamide/2xSSC for 4min at 70[degrees]C, hybridization mixtures containing 200ng of denatured probe, 10mg/ mi Dextran sulfate, 2xSSC and 50% of Formamide were dropped on the slides and the hybridization was performed overnight at 37[degrees]C. Hybridization washes included 2xSSC and 50% Formamide at 37[degrees]C and 2xSSC and 4xSSC at room temperature.
Detection of hybridized probes was performed with Avidin-FITC conjugate (Sigma) followed by two rounds of signal-amplification. After each amplification step, the slides were washed in the blocking buffer (1.26% NaHC[O.sub.3], 0.018% sodium citrate, 0.0386% triton, 1% nonfat dried milk) at 42[degrees]C. Chromosomes were counterstained with propidium iodide, and the slides were mounted with Antifade (Vector). Metaphases were examined in a Zeiss Axiophot photomicroscope and photographed with Kodak Gold Ultra 400 ASA film. The images were digitalized and processed with Adobe Photoshop v. 7.0. This software was also used for constructing the karyogram, with chromosomes organized in decreasing size order. Chromosome morphology followed Levan et al. (1964).
[FIGURE 1 OMITTED]
A total of 35 specimens of each M. rubrioculus and M. curema were used for electrophoretic comparisons of 11 enzyme systems. Samples of liver, muscle and eye lens tissue were obtained from each fish. Each tissue sample was homogenized in a cold buffer solution of Tris-HCl 0.05M pH 7.00 in a 1:3 proportion (weight: volume), centrifuged at 9000rpm for ~10min and the supernatant was electrophoresed in horizontal 12% starch gels employing several buffer systems (Table II). Histochemical staining was performed according to Harris and Hopkinson (1976). Allelic frequencies, effective number of alleles, observed and expected heterozygosity, probability for goodness of fit to Hardy-Weinberg equilibrium, and Nei's (1978) genetic distance index (D) were computed using the software Genes in Populations v. 2 (May and Krueger, 1995).
Results and Discussion
Specimens of Mugil rubrioculus exhibit a 2n=48 karyotype (Figure 1a) with exclusively acrocentric chromosomes (Fundamental Number of arms, NF=48). Chromosomes gradually decrease in size, not allowing a clear distinction of the homologues, with the exception of chromosome pair number 8, which is clearly identifiable by the presence of an interstitial secondary constriction. Silver staining (Figure 1b), as well as FISH with the 18S ribosomal genes (Figure 1c), produce signals located interstitially, close to the centromere, on one pair of chromosomes (apparently chromosome pair number 8), allowing the identification of one single pair of NOR-bearing chromosomes in the chromosome complement. C-band positive segments (Figure 1d), more pronounced on chromosome pair number 8, were observed in pericentromeric position in all chromosomes.
The chromosome complement and the cytogenetic features of the analyzed specimens of Mugil curema from Margarita correspond to those, previously described (Nirchio et al., 2001, 2005a; Rossi et al., 2005) in conspecific specimens from the same area. These specimens have a 2n=24 (NF=48) karyotype (Figure 1e), with 22 metacentric and two submetacentric chromosomes, and NORs, as detected by Ag-staining (Figure 1f) or FISH with the 18S rDNA probe (Figure 1g), located on the telomeric region of the long arms of the largest metacentric pair. C-banding (Figure 1h) produce strong positive signals in the pericentromeric position of all chromosome pairs and at the terminal region of pair number 1, corresponding to NORs.
Cytogenetic studies in Mugilidae, reviewed in Sola et al. (2007), show that a karyotype composed of 2n=48 uniarmed chromosomes, NF=48, is conserved through several species of mugilids. Thus, the karyotypes with 2n=24, or 2n=28, NF=48, observed in M. curema appear to be an apomorphic condition. More extensive taxonomic sampling within Mugil and basal mugilid lineages such as Agonostomus and Joturus (Harrison and Howes, 1991) are required in order to make any substantial phylogenetic conclusions about these karyological characters. Nevertheless, the karyological information available for Mugilidae (references in Sola et al., 2007) shows that NORs are generally born by a single chromosome pair, and two groups of species can be identified according to the location of their NORs. In the first group, which includes M. cephalus, M. platanus (=M. cephalus according to Thomson, 1997), M. liza, and M. curema from Venezuela (Nirchio et al., 2005a), NORs are located in a terminal position on the long arm of the largest chromosome pair. In the second group, which includes Liza aurata, L. ramado, L. saliens, Chelon labrosus and Oedalechilus labeo, NORs are located on the short arm of a unique subtelocentric chromosome pair. Also in M. curema from Brazil, NORs have been recently localized (Nirchio et al., 2005a) on the short arms of one of the two subtelocentric chromosome pairs of the chromosome complement. Thus, M. rubrioculus, though sharing with the other studied mugilid species the presence of a single NOR-bearing chromosome pair, shows a completely different location of NORs, compared to most other species. The interstitial position of NORs in M. rubrioculus has been seen among other mugilids only in M. trichodon (Nirchio et al., 2005b).
As far as the analysis of the allozymes is concerned, seven (GLUDH*, LDH-1*, LDH-2*, LDH-3*, MDH-1*, PROT-1*, PROT-2*) out of the 20 presumptive loci investigated were monomorphic in both species. The allele frequencies at the remaining 13 polymorphic loci are reported in Table III. Among these loci, four showed more than one allele only in one of the two species: MDH-2* in M. rubrioculus; ADH-2*, EST3* and SOD* in M. curema. Mean observed heterozygosity (Ho) among all loci is 0.153 [+ or -] 0.055 in M. rubrioculus and 0.129 [+ or -] 0.047 in M. curema. Allele distribution shows the presence of five private alleles at four loci in M. rubrioculus (ACP*, DIA*, EST-1*, MDH-2*), and five, scattered on five loci in M. curema (ACP*, ADH-2*, EST-3* EST-4* and SOD*). The presence of 10 privates alleles among the 37 alleles observed does provide indication of divergence between the two taxa; nevertheless, the absence of loci with no shared alleles between the two species represents a result inconsistent with the number of diagnostic loci detected in other congeneric comparisons for mugilids. Between 5 and 12 diagnostic loci have been identified for species comparisons within Mugil (Rossi et al., 1998b; Turan et al., 2005), and between 2 and 16 have been identified within Liza (Lee et al., 1995; Papasotiropoulos et al., 2001; Rossi et al., 2004; Turan et al., 2005).
Nei's (1978) genetic distance (D) between M. rubrioculus and M. curema is 0.060. This value is one order of magnitude lower than the values of genetic distances reported in other congeneric comparisons, including Mugil. Within Liza, D= 0.237 has been obtained between L. affinis and L. macrolepis (Lee et al., 1995), and D values ranging from 0.249 to 0.530 (Papasotiropoulos et al., 2001), from 0.466 to 0.659 (Rossi et al., 2004) and from 0.319 to 0.714 (Turan et al., 2005) have been observed in multispecies comparisons. Within Mugil, the lowest genetic distances are as high as D= 0.620 between M. cephalus and M. curema from East Pacific (Rosenblatt and Waples, 1986), rising to a maximum of D= 0.898 between M. gyrans (probably = M. trichodon; Thomson, 1997; Harrison, 2002) and M. curema from Florida (Rossi et al., 1998b). Turan et al. (2005) reported D= 0.606 between M. cephalus and M. soiuy (from the Black Sea); however, the latter species is not validly accepted and most reports of M. soiuy from the Black Sea are misidentifications of Liza haematocheilus (Bogutskaya and Naseka, 2004; Harrison, 2004). Therefore, it is unclear what material Turan et al. (2005) had before them. The D value obtained between M. rubrioculus and M. curema falls within the wide range of distance values obtained by Rossi et al. (1998a) for global populations of M. cephalus, from 0 to 0.242 (mean= 0.117). The most differentiated of the populations of M. cephalus examined by Rossi et al. (1998a) have, however, been proposed to be at a stage of incipient speciation.
Thus, the cytogenetic and allozyme data sets suggest quite different degrees of genetic divergence between M. rubrioculus and M. curema. The cytogenetic features, that are conservative in most species of fishes and particularly in Mugilidae (Sola et al., 2007), reveal striking differences between the two species, both in the chromosome complement and in the position of ribosomal genes. On the other hand, allozyme data, which are generally quite variable at intraspecific and interspecific levels, reveal an unsubstantial genetic differentiation between M. rubrioculus and M. curema which, in the absence of chromosomal and morphological data, would suggest an ongoing gene flow between them.
Considering the two data sets, two possible scenarios can be hypothesized. On the one hand, an underestimate of molecular divergence between M. curema and M. rubrioculus could have been obtained, due to the presence of cryptic variation and alleles with identical electrophoretic mobility, but different nucleotide sequences (Aquadro and Avise, 1982). On the other hand, a significant structural intrachromosomal reorganization might have taken place at the time of speciation and the low divergence of molecular data would mark the recentness of the separation between the two taxa. The application of other molecular markers to M. curema, M. rubrioculus and other Western Atlantic mugilids is required to test these hypotheses further. Such studies will provide additional characters to ascertain the phylogenetic relationships of these species and to test the assumption that M. rubrioculus and M. curema are closely related (based on their similar appearance).
Nevertheless, there is little doubt that chromosomal rearrangements play an important role in establishing reproductive barriers. Thus, karyological data constitute a useful diagnostic tool for examining species divergence within Mugilidae, and this has applications to studying their biology and ecology. The data presented confirm the power of karyological data in discriminating sympatric specimens of M. rubrioculus and M curema. Moreover, by comparing the data reported with that reported for global populations of Mugil cephalus (Rossi et al., 1998a), for which genetically differentiated populations share the chromosome complement and NOR location (Rossi et al., 1996), it is suggested that different rates of molecular/chromosomal evolution might act within the genus Mugil.
The authors thank Jose Marcano for his help with specimen collection and acknowledge financial support provided by the Consejo de Investigacion, Universidad de Oriente, Venezuela, by the Italian Ministry of Instruction, University and Research (MIUR), DM n. 219, 22-12-05, National Relevant Interest Research Projects (PRIN), 2005, and by the Brazilian Conselho Nacional de Desenvolvimento Cientifico e Tecnologico and Fundacao de Amparo a Pesquisa de Sao Paulo. Ian Harrison is grateful to staff and students at the Universidad de Oriente, Venezuela.
Received: 03/26/2007. Accepted: 09/21/2007.
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Mauro Nirchio. M.Sc. in Marine Sciences, Universidad de Oriente (UDO), Venezuela. Professor, Escuela de Ciencias Aplicadas del Mar (ECAMUDO), Venezuela. Address: Apartado Postal 147-Porlamar, Margarita, Venezuela. e-mail: firstname.lastname@example.org
Claudio Oliveira. PhD. in Genetics, Universidade de Sao Paulo, Brazil. Professor, Instituto de Biociencias, Universidade Estadual Paulista (UNESP) Botucatu, SP, Brazil.
Irani A. Ferreira. M.Sc. in Genetics and Ph.D. student, UNESP, Botucatu, SP, Brazil.
Julio E. Perez. Ph.D. in Biology, University of Southampton, UK. Profesor, Instituto Oceanografico de Venezuela, UDO, Venezuela.
Juan I. Gaviria. M. Aq. Auburn University, USA. Professor, ECAM-UDO, Venezuela.
Ian Harrison. Ph.D. in Zoology, University of Bristol, U.K. Bio diversity Scientist. American Museum of Natural History, New York, USA.
Anna Rita Rossi. PhD in Evolutionary Biology and Researcher, Universita de Roma "La Sapienza", (URLS) Italy.
Luciana Sola. Doctor in Biology and Professor, URLS, Italy.
TABLE I LIST OF VOUCHER SPECIMENS * USED FOR THE KARYOLOGICAL (K) AND/OR ALLOZYME (A) ANALYSES M. rubrioculus Mugil curema ECAM-00038 (K) ** ECAM-00034 (K) ECAM-00039 (K) ** ECAM-00035 (K) ECAM-00040 (A) ECAM-00036 (K) ECAM-00043 (K/A) ECAM-00037 (K) ECAM-00045 (K/A) ECAM-00062 (K) ECAM-00045 (K/A) ECAM-00063 (K) ECAM-00047 (K/A) ECAM-00064 (K) ECAM-00052 (K/A) ECAM-00065 (K) ECAM-00067 (K) ECAM-00073 (K) ECAM-00068 (K) ECAM-00149 (K) ECAM-00087 (K) ECAM-00150 (A) ECAM-00088 (K) ECAM-00153 (K/A) ECAM-00151 (K) ECAM-00158 (K/A) ECAM-00152 (K) ECAM-00159 (A) ECAM-00154 (K) ECAM-00160 (A) ECAM-00167 (A) ECAM-00161 (A) ECAM-00168 (A) ECAM-00162 (A) ECAM-00169 (A) ECAM-00164 (A) ECAM-00170 (A) ECAM-00165 (A) ECAM-00171 (A) ECAM-00166 (A) ECAM-00172 (A) ECAM-00176 (A) ECAM-00177 (A) * Deposited in the collections of the Escuela de Ciencias Aplicadas del Mar (ECAM), Universidad de Oriente, Venezuela. ** Transferred to collections of American Museum of Natural History (see Harrison et al., 2007) TABLE II ENZYME SYSTEMS ASSAYED BUFFERS AND TISSUES Enzyme system Abbreviation E.C. No. N Of loci Acid phosphatase ACP 18.104.22.168 1 Alcohol dehydrogenase ADH 22.214.171.124 2 Catalase CAT 126.96.36.199 1 Diaphorase DIA 1.8.14 1 Esterase EST 3.1.1.- 4 Glutamate dehydrogenase GLUDH 188.8.131.52 1 Glucose dehydrogenase GDH 184.108.40.206 1 Lactate dehydrogenase LDH 220.127.116.11 3 Malate dehydrogenase MDH 18.104.22.168 2 Total protein Prot 3 Superoxide dismutase SOD 22.214.171.124 1 Enzyme system Buffer Tissue Acid phosphatase TC Liver Alcohol dehydrogenase TC Liver Catalase TC Liver Diaphorase LB Liver Esterase LB Liver Glutamate dehydrogenase TEB Liver Glucose dehydrogenase TC Liver Lactate dehydrogenase TC Eye Malate dehydrogenase TC Muscle Total protein TC Muscle Superoxide dismutase TEB Liver TC: buffer gel 0.97g/l Tris, 0.63g/l citrate, 0,11g/l NaOH, pH 6.7; buffer tray 27g/l Tris, 18.07g/l citrate, 2g/l NaOH, pH 6.3. LB; buffer gel 3.63g/l Tris, 0.96g/l citrate, 10ml buffer Tray, pH 8.5; buffer tray: 2.52g/l LiOH, 18,55g/1 boric acid, pH 8.1. TEB: buffer gel 1 tray: 3H20, pH 8.7; buffer tray 21,9g/l Tris, 6,18g/l boric acid, 1,17g/l EDTA, pH 8,70. TABLE III ALLELE FREQUENCIES AT THE 13 POLYMORPHIC LOCI IN Mugil rubrioculus AND M. curema Locus Allele M. rubrioculus M. curema (n) 34 34 98 0 0.162 ACP* 100 0.309 0.838 102 0.529 0 104 0.162 0 (n) 30 35 ADH-1 * 102 0.900 0.829 102 0.100 0.171 (n) 22 28 ADH-2 * 100 1.000 0.750 102 0 0.250 (n) 35 35 CAT * 100 0.700 0.700 102 0.300 0.300 (n) 34 35 98 0.015 0 100 0.044 0.743 DIA * 102 0.618 0.171 104 0.221 0.029 106 0.103 0.057 (n) 30 35 98 0.067 0.068 EST-1 * 100 0.917 0.914 102 0.017 0 (n) 35 35 EST-2 * 100 0.971 0.686 102 0.029 0.314 (n) 35 35 EST-3 * 98 0 0.014 100 1.000 0.986 (n) 33 35 96 0.167 0.057 EST-4 * 98 0.030 0.057 100 0.803 0.857 102 0 0.029 (n) 34 34 96 0.059 0.147 GDH-2 * 98 0.397 0.103 100 0.412 0.588 102 0.188 0.147 104 0.015 0.015 (n) 35 35 MDH-2 * 98 0.029 0 100 0.971 1.000 (n) 35 35 PROT-3 * 100 0.586 0.886 102 0.414 0.114 (n) 35 35 SOD * 98 0 0.014 100 1.000 0.986
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|Author:||Nirchio, Mauro; Oliveira, Claudio; Ferreira, Irani A.; Perez, Julio E.; Gaviria, Juan I.; Harrison,|
|Date:||Nov 1, 2007|
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