Allozyme Variation of the Egyptian Rousette (Rousettus
egyptiacus; Chiroptera, Pteropodidae) in the Gulf of Guinea
(West-Central Africa)
JAVIER JUSTE B.,*1: ANNIE MACHORDOMt and CARLOS IBAÑEZ*
Estación Biologica de Doñana (CSIC), Aptdo 1056,41080, Sevilla, Spain;
tMuseo Nacional de Ciencias Naturales (CSIC), Cl José Gutiérrez Abascal 2, 28006, Madrid, Spain;
Present Address: Department of Biological Sciences. Texas Tech University,Lubbock, Texas 79409-3131,
U.S.A.
Key Word lndex—Rousettus egyptiacus; Pteropodidae; African Rousette; fruit bats; allozyme; population
variation; island genetics.
Abstract—Genetic variation among the populations of Rousettus egyptiacus from the islands of the Gulf
of Guinea (Bioko, Principe and São Tome) and two other continental populations of R. egyptiacus, (Rio
Muni and Guinea Conakry) was studied in order to evaluate their systematic and evolutionary relationships. Insular and mainland populations had similar levels of genetic variation. The data support the subspecific assignment of the São Tome and Prlncipe populations of R. egyptiacus. Copyright C 1996
Published by Elsevier Science Ltd
Introduction
The Egyptian Rousette (Rousettus egyptiacus Geoffroy, 1810), ranges throughout
the sub-Saharan continent and reaches the Mediterranean along the Nile basin,
northward to Cyprus and Southern Turkey, and eastward into Pakistan (Hayman
and Hill, 1971; Koopman, 1993; Bergmans, 1994). Andersen (1912) first studied
the geographic variation of R. egyptiacus and distinguished three subspecies (R. e.
egyptiacus, R. e. leachil and R. e. arabicus) based on variation in wing, cranial and
dental traits. Eisentraut (1959) described a fourth form (R. e. occidentalis), later
shown by Koopman (1966) to be synonymous with R. e. unicolor (Gray, 1870),
which had been disregarded by Andersen. The four subspecies have disjunct distributions (Bergmans, 1979, 1994), the nominate species being found in Eastern
Mediterranean Africa and southward to 20°N; R. e. arabicus occurring along the
coast of the Southern Arabian Peninsula northeast to Pakistan; R. e. leachll ranging
across East Africa from Ethiopia to a narrow coastal strip of the southern African
coast; and R. e. un/color distributed throughout Western Africa from Senegal to
central Angola (Bergmans, 1994).
Recently, two more subspecies have been described from West Africa, R. e.
tomensis and R. e. princeps, respectively, found on the islands of São Tome and
Principe in the Gulf of Guinea (Juste B. and lbáñez, 1993). Despite noticeable
morphological differences between these insular subspecies, they share wing,
cranial and dental characteristics that suggest a close phylogenetic affinity, as well
as an inter-island colonization process rather than independent colonization
events from the mainland.
Allozyme electrophoresis was used to analyze genetic variation among the
populations of Rousettus egyptiacus from the islands of the Gulf of Guinea.
Genetic differentiation was compared between these populations and two
500
J.JUSTEB.ETAL.
continental populations, and their taxonomic status and evolutionary relationships
examined. In addition, the systematic value of the allozymic approach is assessed
within the African fruit bats, as well as the congruence between morphological
and biochemical systematic approaches.
Material and Methods
Samples. Rousettus egyptiacus was collected from 1987 to 1992 along the Gulf of Guinea from three
insular populations: Bioko island (Republic of Equatorial Guinea: 3’30’N, 840’E, N=9); PrIncipe island
(Democratic Republic of São Tome and Principe: 1 ‘37’N, 7’25’E, N = 19); São Tome island (Democratic
Republic of São Tome and Principe: 0°1 2’N, 6°39’E, N 20); and one continental population, Rio Muni
(Republic of Equatorial Guinea: 1 °30’N, 1 0°30’E, N 10) (Fig. 1). Two additional continental specimens
from Dubreka (Guinea Conakry: 9°48’N, 1 3°31‘W, N = 2), thought to belong to the same mainland subspecies, were used to compare genetic divergence among continental populations. Specimens were preserved as standard museum vouchers and deposited in the Estación Biológica de Doñana collections.
Electrophoresis. Samples of liver, heart, kidney and muscle were dissected from netted specimens and
immediately stored in liquid nitrogen. After transportation from the field, samples were stored at —70°C
until analyzed. Horizontal starch gel electrophoresis was performed using standard techniques (Pasteur et
al., 1987) to assess genetic variation at 31 presumptive loci encoding 22 enzymatic systems (Table 1).
Alleles at each locus were distinguished by decreasing mobility using side-by-side comparisons, and
designated alphabetically with the most common allele designated as ‘a’.
FIG. 1. STUDY AREA AND COLLECTING POINTS: CONTINENTAL POPULATIONS FROM RIO MUNI (a) AND GUINEA
CONAKRY (e); LAND-BRIDGE ISLAND OF BIOKO (b); AND THE OCEANIC ISLANDS OF PRINCIPE (c) AND SÃO
TOME (d).
ALLOZYME VARIATION OF THE EGYPTIAN ROUSETTE
501
TABLE 1. ALLOZYMES EXAMINED AND STAINING PROCEDURES USED IN THE STUDY. Enzyme Commission Number
follows the Nomenclature Committee of the International Union of Biochemistry (1984)
Protein
E.C.N.
Organ
Buffer
Aspartate aminotransferase (Aat)
Aconitate hydratase (Acon)
Acid phosphatate (Acp)
Adenylate kinase (Ak)
Creatine kinase (Ck)
Fumarate hydratase (Fum)
Glycerol-3-phosphate dehydrogenase (Gpd-1)
(Gpd-2)
Glucose-6-phosphate isomerase (Gpi)
lsocitrate dehydrogenase (Idh)
Leucinamide peptidase (Lap)
Lactate dehydrogenase (Ldh)
Malate dehydrogenase (Mdh)
Malic enzime (Me)
Mannose.6-phosphate isomerase (Mpi)
Purine nucleoside phosphorylaae (Np)
Leucyl-tyrosine peptidase (Pep A)
Leucyl-glycyl-glycinepeptidase (Pep B)
Phenyl-proline peptidase (Pep D)
6-Phosphogluconic acid dehydrogenase (6Pgd)
Phosphoglucomutase (Pgm)
Sorbitol dehydrogenase (Sdh)
Superoxide dismutaae (Sod)
Xanthine dehydrogenase (Xdh)
2.6.1.1
4.2.1.3
3.1.3.2
2.7.4.3
2.7.3.2
4.2.1.2
1.1.1.8
H/L
H
L
H
H
H
TME 6.9
TC 6.7
TME 6.9/TC 6.4
TC 6.4
TC 6.4
TME 6.9
TC 8/TBE 8.6
TC 8/TBE 8.6
TME 6.9
TC 6.7
TBE 8.6
TC 6.7
TC 6.7
TC 6.7
TC 6.7
L1OH 8.3/TBE 8.6
TC 8
TC8
TC 8
TC 6.4
TC 6.4/TME 6.9
TC 6.4
TC 6.4
TBE 8.6
5.3.1.9
1.1.1.42
3.4.11.1
1.1.1.27
1.1.1.37
1.1.1.40
5.3.1.8
2.4.2.1
3.4.11.
3.4.11.
3.4.11.
1.1.1.44
2.7.5.1
1.1.1.14
1.15.1.1
1.2.1.37
H/L
L
H
H
L
H
H
H
H/L
H/L
H
H
H
H
H/L
L
L
H
H = heart L= liver; TC=tris/cistrate, TME =tris/maleate/EDTA, TBE =tris/borate/EDTA, according to Pasteur eta!. (1987),
except for TC 8, TME 6.9 and LiOH 8.3, in which a half dilution of buffer was used for the gels.
Data analysis. Data were analyzed using the BIOSYS-1 computer package (Swofford and Selander,
1989). Genetic variability was calculated for populations represented by three or more specimens, and was
estimated as the mean number of alleles per locus, percentage of polymorphic loci (P), observed (directcount) and expected heterozygosities per locus (H), and Wright’s F-statistics (Wright, 1965). Since no
comparisions yielded observed values of heterozygosity deviating significantly from expected HardyWeinberg equilibrium (Nei, 1975), only values for observed heterozygosity are presented. Genetic relatedness among populations was evaluated using Nei’s (1978) unbiased distances (DN) and Rogers’
(1972) distance (Op). Correlations between the matrix of Rogers’ (1972) distances and the matrices of
both geographical distances and the Euclidean morphological distances were assessed using Mantel tests
(Sokal, 1979) for all the populations except Guinea Conakry, utilizing the procedure MXCOMP in the
NTSYS-pc package (Rohlf, 1988). Based on results of a previous morphological comparison (Juste B.
and lbáñez. 1993), morphological distances were obtained after averaging the factor scores for each
population on the first two components of a PCA for 54 characters.
The general pattern of gene flow among populations was estimated quantitatively by means of Nm, the
number of effective migrants among populations per generation. Given the physical isolation of the
populations, this value was calculated by Slatkin’s (1985) private alleles formula [lnp(1) — 0.505
ln(Nm)—2.44, where p(1) is the average frequency of private alleles]. To adjust the constants of this
formula, Nm value was obtained after correcting for sample sizes following Slatkin (1985). Rates of
values between populations.
interpopulation gene flow were estimated from
Mid-point rooted phenograms were constructed from the matrix of Rogers’ (1 972) genetic distances
using both the unweighted-pair group method (UPGMA, Sneath and Sokal. 1973) and the distanceWagner procedure, which allows the detection of different rates of genetic evolution. Times of evolutionary divergence among taxa were estimated from Nei’s (1978) distance by using Nei’s (1 975) formula,
assuming a linear relationship between time and genetic distance for small genetic distances.
Results
Patterns of variability
Of the 31 loci surveyed, 18 (58.1%) were monomorphic in all the populations
studied (Aat-2, Acp, Ak, Ck, Fum, Idh-2, Ldh- 1, Ldh-2, Mdh-2, Me, Np, Pep-B, Pgi
Sdh-1, Sdh-2, Sod-i, Sod-2, Xdh). Allele frequencies at the remaining 13 poly
502
J.JUSTE B.EFAL.
morphic loci are presented in Table 2. All populations showed at least one unique
allele (Table 2). Bioko’s land-bridge island population differed from the Rio Muni
mainland population mainly in the frequency of the alleles, although the former
exhibited different alleles for three loci (Gpd-1, Pep-D, 6Pgd). São Tome’s
population showed unique alleles for six loci (Acon, Gpd-2, Lap, Mdh-1, Mp1
Pgm- 1), 46.1% of all polymorphic loci; PrIncipe’s showed two unique alleles (PepA, Pgm-1), and both populations shared one allele atAat-1. Bioko and Rio Muni
were monomorphic for the same allele at Pgm-/, whereas all remaining populations each showed unique alleles at this locus (Table 2).
Genetic variability
Polymorphism among populations ranged from P =0.097 in the Rio Muni’s
sample to P=0.29 at São Tome population (Table 3). Mean heterozygosity
(direct-count) within populations ranged from H = 0.031 on PrIricipe island to
H =0.056 on Bioko island (Table 3). There was no apparent correlation among
polymorphism and heterozygosity values. The mean number of alleles per locus
was very similar in all the populations and averaged 1.22 (up to 4 per locus). The
Principe and São Tome populations, with the highest values of polymorphism,
showed low heterozygosity as did the Rio Muni’s population. The mean value of
Wright’s F-statistics among the populations was very high (F=0.493). It was
TABLE 2. ALLELE FREQUENCIES AT 13 POLYMORPHIC LOCI FOR FIVE POPULATIONS OF EGYPTIAN ROUSETTES.
SAMPLE SIZES WITHIN PARENTHESES
Locus
Allele
Aat-7
a
b
a
b
a
b
a
b
a
b
cxGpd-1
IGpd-2
Idh-1
Lap
Md/i-i
Mp/
Pep-A
Pep-D
6Pgd
Pgm- 1
ci
a
b
a
b
a
b
a
b
a
b
a
b
a
b
Population
Rio Muni
(10)
Bioko
(9)
Principe
(19)
São Tome
(20)
Guinea-Conakry
(2)
1.000
1.000
1.000
1.000
0.889
0.111
1.000
1.000
0.100
0.900
0.950
0.050
0.275
0.725
0.969
0.031
0.975
1.000
1.000
0,211
0.789
1.000
0.500
0.025
0.964
0.500
1.000
0.036
0,975
1.000
1.000
0,300
1.000
0.250
0.050
0.400
0.944
0056
0.556
0.056
0.806
0.028
0389
0875
0.125
0.167
0.875
0.125
1.000
1 000
1.000
1.000
1 000
1.000
1.000
1 000
0.969
0.031
1.000
1.000
1.000
1.000
0.250
0.750
0.944
0.056
1 000
0.025
0.972
0.028
1.000
1.000
1.000
1.000
1.000
1.000
1 000
1.000
1.000
1.000
1.000
0.921
0.975
0.025
0.500
0079
Pgm-2
d
a
b
0.889
0.111
1 000
1.000
1.000
0.500
1.000
ALLOZYME VARIATION OF THE EGYPTIAN ROUSE1TE
503
TABLE 3. GENETIC VARIABILITY MEASURES FOR FOUR POPULATIONS OF EGYPTIAN ROUSETTE
Population
Mean sample size
per locus
Mean number of
allelles per locus
Percent polymorphic loci
(P)
Average heterozygosity
(H)
Rio Muni
Bioko
Prlncipe
São Tome
Mean
8.8
7.8
16.0
16.5
12.3
1.16
1.19
1.19
1.29
1.21
9.7
16.1
16.1
29.0
17.7
0.033
0.056
0.031
0.032
0.038
based on a low negative inbreeding coefficient within populations (F5= —0.041),
and a high positive inbreeding coefficient relative to the whole population
(F1= 0.472). The corresponding Nm value was 0.248, suggesting a restricted gene
flow among all the populations. Estimated gene flow is highest between RIo Muni
and Bioko populations and lowest among Rio Muni and the oceanic islands
(Table 4). The populations from RIo Muni and Bioko island showed the lowest
levels of genetic divergence, and RIo Muni and São Tome the highest values
(Table 5). Mantel tests showed similar congruence between the matrix of Rogers’
genetic distances and both the matrix of geographic distances (r = 0.974, t = 1.737,
P0.9588), and that of morphological distances (r0.979, t=1.715,
P = 0.9568).
The UPGMA phenogram (Fig. 2) was a fair representation of the original distances (cophenetic correlation r=0.869), although the tree produced by the
Wagner-distance method (Fig. 3) provided a better fit to the genetic distance
matrix (r=0.994). Both analyses suggested similar relationships among populations, producing trees with two main clusters; one included Bioko and the two
continental populations (Rio Muni and Guinea Conakry), with the second comprised of the two oceanic islands populations (São Tome and PrIncipe). These
latter populations were also distinctly separated (Fig. 3).
TABLE 4. MATRIX OF FST VALUES (ABOVE THE DIAGONAL) AND NMVALUES (BELOWTHE DIAGONAL) AMONG FIVE
POPULATIONS OF EGYPTIAN ROUSETES
Guinea Conakry
Guinea Conakry
Rio Muni
Bioko
Prlncipe
São Tome
0.134
0.498
0.252
0.265
Rio Muni
Bioko
Principe
São Tome
0.400
0.260
0.058
0.316
0.469
0.335
0.474
0.574
0.441
0.219
—
1.504
0.071
0.138
—
0.152
0.371
—
0.846
—
TABLE 5. GENETIC DISTANCES BETWEEN FIVE POPULATIONS OF EGYPTIAN ROUSETTES. Neis (1978) distance values
are above the diagonal (DN), and Rogers (1972) distance values are below the diagonal (DR)
Guinea Conakry
Guinea Conakry
Rio Muni
Bioko
Prfncipe
São Tome
0.061
0.053
0.056
0.089
Rio Muni
Bioko
Prlncipe
São Tome
0.039
0.020
0.001
—
0.026
0.062
0.040
0.066
0.096
0.056
0.092
0.063
0.019
0.043
—
0.027
0.081
0.114
504
J. JUSTE B. ETAL.
0.05
0.04
0.03
0.02
0.01
0
RIo Muni
Bioko
G. Conakry
PrIncipe
São Tome
•
0.05
-- -
-
---
0.04
-
0.03
I
I
I
0.02
0.01
0
DISTANCE
FIG. 2. UPGMA CLUSTER ANALYSIS OF ROGERS (1972) GENETIC DISTANCES AMONG FIVE POPULATIONS OF
ROUSETTUS EGYPTIACUS. Cophenetic correlation coefficient is 0.869.
0
0.01
0.02
0.03
0.04
0.05
0.06
Muni
PrIncipe
São Tome
0
0.01
0.02
0.03
0.04
0.05
0.06
DISTANCE FROM ROOT
FIG. 3. WAGNER PHENOGRAM (ROOTED AT MIDPOINT OF THE LONGEST PATH) AMONG FIVE POPULATIONS OF
ROUSETTUS EGYPTIACUS BASED ON ANALYSIS OF ROGERS (1972) GENETIC DISTANCES. Cophenetic correlation
coefficient is 0.994.
ALLOZYME VARIATION OF THE EGYPTIAN ROUSETE
505
Discussion
Genetic variability
The unweighted average polymorphism among the Egyptian Rousette populations
(P = 0.177) was comparable to that typically given for mammals (P = 0.147; Nevo,
1978) and less than the values that Peterson and Heaney (1993) reported for the
Asian pteropodids Cynopterus brachyotis (P = 0.381) and Haplonycteris fischeri
(P=0.228). Rousette polymorphism is similar to the average values (P=O.196)
obtained for the bat family Phyllostomidae (Koop and Baker, 1983), and slightly
more than the average value reported for the phyllostomid genus Sturnira
(P=0.142, Pacheco and Patterson, 1991). Average heterozygosity among populations (H =0.038) was similar to the values reported for mammals (H =0.036;
Nevo, 1978) and Neotropical bats (H = 0.032; Straney eta!., 1979). The average
heterozygosity of the Egyptian Rouesette is similar to that reported for H. fisheri
(H = 0.034; Peterson and Heaney, 1993), and the Asian pteropodids Cynopterus
sphinx (H = 0.028) and C. horsfieldi (H 0.032; Schmitt et a!., 1995). However,
heterozygosity is lower than the values reported for other species of Cynopterus
(Peterson and Heaney, 1993; Schmitt et a!., 1995) and the fruit bat Aetha/ops
alecto (H = 0.055; Kitchener eta!., 1993). Direct comparisons are limited, however,
because different enzymatic loci are involved (Simon and Archie, 1985).
Reduced genetic variability is characteristic of island or isolated populations
(Lewontin, 1974). The highest average heterozygosity of Bioko’s island population of R. egyptiacus may be due to its recent vicariant origin (Juste B. and
lbáñez, 1994) and gene flow from the nearby mainland (Table 4). Surprisingly, the
highest polymorphism is shown by the São Tome population, although its heterozygosity is similar to that found in the other populations. This results from the
occurrence of rare alleles at some loci. However, comparisons to the mainland
must be made with caution because of differences in sample size, and some polymorphism may be missed from the continental population of Rio Muni. Some
previous reports for other bats (Greenbaum and Baker, 1976), show no reduction
of genic variability in insular versus mainland populations; this is also true for some
insular populations (Azores) of chaffinches (Baker eta!., 1990). Finally, in our
study increasing genic variability is not correlated with increasing island size as
was suggested among insular fruit bat populations from the Philippines (Peterson
and Heaney, 1993).
Genetic divergence
The fixation indices indicate considerable genetic differentiation among the R.
egyptiacus populations. The mean value (F=0.493) is relatively high, although
varies widely among insular populations of the few fruit bats studied,
this
ranging from F0.066 forAetha!opsa!ecto (Kitchener eta!., 1993) to F=0.606
for Haplonycteris fischeri (Peterson and Heaney, 1993). When calculated separately between populations, the values of
and Nm point to sufficient gene flow
to overcome genetic differentiation (Nm> 1) only between continental Rio Muni
and the land-bridge island of Bioko. The values between São Tome and PrIncipe
populations suggest some flow between them of about one migrant per generation. The fixation index between the two continental populations (Guinea
Conakry and Rio Muni) is similar to those calculated among islands. The Nm value
suggests one migrant per five generations.
High values of DN and DR for the two oceanic island populations point to isolation, resulting in genetic differentiation. The differences between PrIncipe and
São Tome are low (DN=0.0l9), but still higher than the mean value among (a)
conspecific populations of the phyllostomid Macrotus (DNO.005; Straney eta!.,
1979) or (b) conspecific subspecies of the vespertilionid Myotis lucifugus
506
J. JUSTE B. ETAL.
(DN=O.Oll; Herd, 1987), or the average inter-island genetic distance for Cynopterus from Indonesia (DNO.0l36 Schmitt eta!., 1995). Peterson and Heaney
(1993) found genetic distances up to DN = 0.113 among inter-island populations
of Haplonycteris fischer/. This indicates that at least some bats show relatively low
genetic distances, even among congeneric species (Schmitt eta!., 1995).
Both mainland populations (Rio Muni and Guinea Conakry) were more
divergent than anticipated. The Guinea Conakry specimens are from the western
range of the distribution of R. e. un/co/or, where these fruit bats are slightly smaller
on average than those of the central population (Bergmans, 1994). Even if R.
egyptiacus is not closely linked to the rainforest, it is possible that isolation of the
western and central rain forest blocks could have restricted gene flow between
populations, as occurred with other mammal species (Robbins, 1978). The São
Tome and PrIncipe populations of R. egyptiacus would have changed faster morphologically than this western continental population. Further genetic and ecological studies are necessary to better understand these patterns of genetic
divergence. It is especially critical to determine if some of the rare alleles of the São
Tome population are shared with its closest mainland population from Port Gentil,
Gabon.
The UPGMA and Wagner dendrograms and the phenetic tree based on morphological distances among populations (Juste B. and lbáñez, 1993) are highly
concordant as shown by the Mantel test, making the inferred systematic
arrangement more reliable (Hillis, 1987). The diverse character suites support the
taxonomic consideration of São Tome and PrIncipe populations as subspecies of
R. egypt/acus. These data also verify the adequacy of the allozyme electrophoresis
for discriminating among subspecies of fruit bats.
Evolutionary relationships
The correlation between geographic distances and genetic distances suggests
that geographic isolation has played a role in allozymic differentiation among the
populations. Peterson and Heaney (1993) found significant correlation between
genetic and historical geographic distances for two species of Philippine fruit bats
and Schmitt et a/. (1995) found significant correlations between genetic distances
and both actual and historical geographic distances among the Indonesian island
populations of C. nusatenggara. Nevertheless, in the Gulf of Guinea islands, the
evolutionary scenario seems to have been very different. In this case, Pleistocene
events probably had similar effects on all the island populations (except for the
land-bridge island of Bioko). Both the distances to the mainland and the small
sizes of the oceanic islands have hampered colonization and genetic interchanges.
On the Philippine archipelago, a large minimum island size (12,500 km2) is
required for the presence of a single endemic fruit bat (Heaney, 1991); São Tome
island supports an endemic species of Myonycteris with only an area of 836 km2.
Since the distance between São Tome and PrIncipe islands (146 km) is of the
same order of magnitude as each island to the mainland (280 and 220 km.
respectively), independent colonization events from the mainland may be more
likely. Our results suggest that both island subspecies share a common evolutionary history. Their phylogenetic affinity would be supported by shared alleles,
although allele frequencies were phylogenetically uninformative. The higher
genetic differentiation shown by R. e. tomensis may reflect a more complex colonization history for São Tome, possibly with multiple colonization events.
The divergence between subspecies is more than an order of magnitude greater
than between the Bioko and Rio Muni populations. Using genetic distances to
estimate timing of divergence (Nei, 1975), the Bioko and RIo Muni populations
diverged approximately 5000 years BP. This date agrees with the estimated time of
ALLOZYME VARIATION OF THE EGYPTIAN ROUSETE
507
the complete separation of Bioko from the Cameroon coast at the end of the last
glacial period (Thys van den Adenauerde, 1967). Distances between, and sizes of,
the Gulf of Guinea islands have changed dramatically during the series of climatic
cycles since (at least) the early Pleistocene (Juste B. and lbáñez, 1994). The
changes have primarily affected Principe Island, which could have increased in
area up to tenfold and reduced (by more than 25%) its distance from São Tome
during the drops in sea !evet. These conditions would favor contacts between the
two insular populations of Rousettus. During the last 100,000 years (their
approximate time of divergence), R. e. tomensis and R. e. prInceps would have had
several opportunities to experience such contacts, which would explain the relatively high value of Nm between them. Nevertheless, a wider study, or a more
detailed approach with other techniques, seems necessary to fully understand this
colonization event.
Acknowledgements—Support for field work was financed by the governmental Agencia Espanola de
Cooperaciôn Internacional and Consejeria de Educación y Ciencia of the Junta de Andalucia. We thank
Anton Ayong and Carlos Ruiz for help in the field and Celia Lôpez Gonzalez, Meredith Hamilton, ira
Greenbaum and especially Lou Densmore, Mark Engstrom and Stephen Kasper for their comments and
suggestions on earlier drafts.
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