Heat induced male sterility in Drosophila melanogaster: adaptive genetic variations among geographic populations and role of the Y chromosome
CNRSUPR 9034, Avenue de la Terrasse, Laboratoire Populations, Génétique et Evolution, F-91 198 Gif sur Yvette Cedex, France
* Author for correspondence (e-mail: joly{at}pge.cnrs-gif.fr)
Accepted 12 May 2004
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Summary |
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Key words: heat stress, geographic race, heat tolerance, spermatogenesis, climatic adaptation, Drosophila
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Introduction |
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The drosophilid family, with diverse species living under different
climatic regimes, has been a superb model for ecological, ecophysiological,
genetical and evolutionary analyses, especially because several hundred
species tolerate laboratory culture and are amenable to experimentation. Here
we focus on a poorly investigated trait, the induction of male sterility by
chronic exposure to high temperature, and its genetic variability in
Drosophila melanogaster. Most strains are continuously fertile at a
29°C but not at 30°C (Parsons,
1973). David et al.
(1971
) established that this
upper limit was due to the sterilization of males, and that sterile males
could recover fertility after a few days at a lower, non-stressful
temperature. This phenomenon, although mentioned in reviews
(David et al., 1983
), has
attracted little attention and was considered mainly as a physiological
curiosity analogous to the male sterility in mammals with undescended testes.
Surprisingly, a strain collected from a very hot locale, N'Djamena (Chad
Republic), by L. Tsacas, was fertile at 30°C and could tolerate permanent
culture at that temperature (J. R. David, unpublished). Although this
observation was not published, the strain was provided to various Russian
laboratories where it was further selected for increased heat tolerance, so
that permanent culture could be kept at 3132°C (see
Zatsepina et al., 2001
).
Here we characterize the geographic pattern of male sterility thresholds and show that most tropical populations from different continents produce fertile males when grown at 30°C, while temperate populations do not. We investigate this variation further in selected strains by characterizing the frequency of male sterility and cytological abnormalities of spermatogenesis after development at high temperatures, and the recovery process after a return at a mild temperature. The results establish that male sterility thresholds are genetically variable and are consistent with adaptation, as populations living under hot climates are more tolerant to heat stress. Unexpectedly, much of the divergence between tropical and temperate strains appears due to the Y chromosome.
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Materials and methods |
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From data in Table 1, two thermosensitive (Draveil and Prunay) and two thermotolerant (Niamey and Delhi) populations were chosen for more detailed investigation in which we measured the fertility of males grown at various temperatures in 0.5°C steps. Two of these populations (Prunay and Delhi) were then chosen for a genetic analysis involving crosses (F1 and F2 males) and repeated backcrosses towards the female parent. The aim of backcrosses was to introduce the Y chromosome of each strain into the genetic background (X chromosome and autosomes) of the other strain.
Fertility recovery (time needed for males to produce progeny at 21°C) was investigated in two populations, Bordeaux (from France) and Delhi (from India). Recovery was also investigated by counting the proportion of fertile males after a return at 21°C. Finally the cytological abnormalities of spermiogenesis in males grown at high temperature were investigated in the populations of Prunay and Delhi.
Fertility/sterility after development at 30°C: progeny production
Male fertility/sterility at 30°C was analysed using the following
procedure. Several sets of 10 pairs from a mass culture were isolated as
parents of the experimental flies. These parents oviposited at
2021°C in culture vials containing a killed yeast, high nutrient
medium (David and Clavel,
1965). Oviposition lasted 6 h and then the culture vials were
transferred at 30°C until emergence of adults. These adults, grown at
30°C, were kept at the same temperature and mass-transferred to fresh food
every 24 days, for at least 2 weeks. Males were considered as fertile
when progeny larvae appeared in vials so that a permanent culture could be
established at 30°C. When no progeny appeared in a sample of several
hundreds of flies, males were classified as fully sterile. In all cases,
females laid numerous eggs and, when mated with males grown at 25°C, and
kept at 30°C always produced numerous progeny. In some cases, fertility
after development at 30°C was not clearcut. A few larvae could appear in
one vial but not in others, and this low fertility was insufficient to
establish a permanent culture. For such populations, the experiment was
repeated, generally with the same conclusion. The threshold for absolute
(100%) sterility in these cases was presumably slightly greater than 30°C
(e.g. 30.1°C or 30.2°C). Such strains are classified as `uncertain' in
Table 1.
Proportion of fertile males observed after dissection
To estimate the proportion of sterile males, we followed the procedure of
Chakir et al. (2002). Flies
were cultured in incubators regulated at 0.5°C intervals
(±0.1°C) between 28.5° and 31.5°C. After emergence, males
were separated from females and kept at their developmental temperature on
cornmealsugar medium seeded with live yeast. After 56 days,
males were dissected in Drosophila Ringer solution, seminal vesicles
were opened with tiny needles and their content examined for the presence of
motile sperm. A complete absence of sperm, or the presence of a few non-motile
sperm in seminal vesicles, was the criterion for male sterility. At a critical
temperature, when for example 50% of males were scored as sterile, the amount
of sperm among fertile ones appeared variable. We did no try to quantify this
variability.
At each temperature, 50 males grown in several vials were scored for motile
sperm. Percentage of sterile males increases with temperature and the response
curve has a sigmoid shape. These response curves were adjusted to a logistic
function (STATISTICA software) as described in Chakir et al.
(2002). This adjustment
estimates two parameters: the temperature at the inflection point (TIP), which
gives the value at which 50% of males are sterile (median threshold), and a
slope coefficient at the inflection point (SIP), which reflects the steepness
of the curve.
Genetic analysis: male sterility in crosses and backcrosses
A thermotolerant (Delhi) and a thermosensitive (Prunay) strain were
selected. Preliminary observations on crosses between Indian and French
strains revealed that reciprocal F1 males were clearly different
and resembled the male parent, suggesting a role of the Y chromosome. We
precisely investigated reciprocal F1 and F2 crosses
between Delhi and Prunay. F1 males were also backcrossed to the
females of their maternal parent, and the same kind of backcross was repeated
for six successive generations to introduce the Y chromosome of a given strain
into the genetic background (X chromosome, autosomes and cytoplasm) of the
other strain. All crosses and backcrosses were done with flies grown at
25°C. Eggs of each investigated generation were transferred at the various
experimental temperatures, and male fertility was analyzed by dissection as
described above.
Recovery after a return at a mild temperature
Sterile males may recover when returned to a mild, permissive temperature.
Ten young males aged 0 to 1-day posteclosion, grown at various high
temperatures, were isolated in culture vials each with three normal virgin
females grown at 21°C. These vials were kept at 21°C, changed daily
and then examined for the appearance of progeny. A male was classified as
fertile when at least one larva appeared in a vial. Progeny number was not
counted and males were discarded after recovery. This procedure was
simultaneously applied to Bordeaux and Delhi populations.
We also analyzed male recovery by dissecting the testis and observing the presence of motile sperm in the seminal vesicles. This experiment was done on two strains, Prunay and Delhi, grown at their sterility temperatures of 30°C and 31°C, respectively. Young males of both strain were distributed into several culture vials and transferred to 21°C. These males were dissected regularly up to the age of 9 days. For a given age, 50 males were analyzed.
Cytological observations
Developmental factors leading to male sterility were investigated by
analyzing spermiogenesis in males reared at the threshold of absolute
sterility, i.e. 31°C for the thermotolerant (Delhi) and 30°C for the
thermosensitive (Prunay) strain. Testes were dissected and opened in a drop of
Ringer solution, and their content dispersed by gentle movements. Spermatozoa
are produced within cysts of 64 spermatocytes arising from four mitotic and
two meiotic divisions of a primary gonial cell. These 64 spermatocytes develop
synchronously, due to incomplete cytokinesis, through eight postmeiotic stages
of spermatogenesis (Lindsley and Tokuyasu,
1980). Four traits were investigated to evaluate the pre- or
post-meiotic abnormalities involved in sterility: (1) the number of
spermatocytes per cyst, after DAPI staining, to confirm normal cell division;
(2) the length of the cyst, which is species specific
(Joly et al., 1989
) and
slightly greater than that of the sperm; (3) the localisation of spermatocyte
nuclei along the cyst, as an indicator of the elongation process of each
spermatid; (4) the chromatin condensation within sperm nuclei, as abnormal
condensation is expected to produce a non-functional, sterile gamete.
Cyst length was measured in parental males (N=50) and in males (N=25) from three successive odd generations (i.e. F1, G3 and G5, see Table 2) reared at 21°C and at the sterility temperature. The numbers, positions and condensations of spermatocyte nuclei within the cyst were counted in 20 cysts in males of each generation investigated. Two categories were distinguished for sperm nuclei position along the cyst: `apical', when all nuclei were found in the apical part of the cyst as usual in fertile males, and `distal', when sperm nuclei were dispersed along the cyst or even found only in the distal part of the cyst. Chromatin condensation within cysts was classified as `maximal' when nuclei were needle-shaped with intense fluorescence, `minimal' when nuclei were roundshaped with low fluorescence, and `variable' when the shape of nuclei and their respective fluorescence varied from one spermatocyte to another within the same cyst.
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Results |
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As seen in Table 1, fertile strains were from the Afrotropical region, the Caribbean and India. No geographic pattern is evident other than a strong relationship with latitude. The average latitude of origin for the three kinds of strains is: fertile at 30°C: latitude=12.93±3.89° (N=10; mean ± S.E.M.); sterile at 30°C: latitude=33.25±3.86° (N=10); uncertain: latitude= 8.92±6.11° (N=4).
A statistical analysis of variance (ANOVA; not shown) revealed a strong latitudinal effect (F2,21=9.133, P<0.01) and a post-hoc test suggested two groups: sterile strains are from high latitudes and fertile and uncertain strains from low latitudes. This pattern shows that populations from temperate places are more thermosensitive than those from tropical climates.
Male sterility as a function of growth temperature in two temperate and two tropical populations
The response curves of two temperate (Draveil and Prunay, France) and two
tropical (Niamey, Africa and Delhi, India) populations are shown in
Fig. 1 and the parameters
obtained after logistic adjustments are given in
Table 2. In all four cases, the
experimental points are close to the adjusted curves with very high
r2 values, indicating the validity of the model. For TIP
(median threshold), which estimates the temperature that produces 50% of
sterile males, the mean of the two tropical populations is
30.39±0.15°C (mean ± S.E.M.), while it is
1.04°C less (29.35±0.13°C) for the two temperate populations.
The slopes at inflection point (SIP) are quite variable among populations and
not related to the geographic origin. Slopes are steeper for the Draveil and
the Delhi populations. A smoother slope, as observed with Prunay and Niamey
(see Fig. 1),indicates a higher
phenotypic variability among males (Sokal
and Rohlf, 1995) and might reflect a genetic heterogeneity.
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Genetic analysis between a temperate and a tropical population
Preliminary experiments revealed a difference between reciprocal
F1 males, suggesting an effect of the Y chromosome. This problem
was investigated in more detail, using a larger set of growth temperatures, in
crosses between the French Prunay and the Indian Delhi strains. The design was
to introduce the Y chromosome of Delhi into the Prunay background, and
vice versa. All these crosses were at 25°C, and progeny
transferred at various experimental temperatures, to estimate the median
sterility threshold (TIP).
The results are given in Table 2. The validity of logistic adjustments is, as previously, shown by very high r2 values. SIPs were quite variable among generations with no regular pattern.
The TIP values are more interesting. In the first series (Y chromosome of Delhi introgressed into the Prunay background), the three values of parents F1 and F2 are almost identical (30.53±0.02, mean ± S.E.M.) suggesting a complete dominance of thermotolerance (Fig. 2A). This stability is followed by a decrease of 0.44°C in G2. The decrease does not proceed further in the following generations, however, and indeed the values increase slightly. The average for backcross generations 36 is 30.23±0.01°C. This corresponds to a slight but highly significant decrease of 0.31°C with respect to the three parental values (t=18.5, d.f.=5, P=<0.001), which is due to the replacement of the Delhi background by the Prunay background. Most interesting, however, is that, although we almost completely replaced the Delhi by the Prunay background in G6, the TIP remained much higher than in the pure French strain. The difference, which is 0.76°C, must be explained by the Indian Y chromosome.
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In the second series of backcrosses, the Y chromosome of Prunay was introduced into the Delhi background (Table 2 and Fig. 2B). The first generations (F1 and F2) were intermediate between the parental values. TIP decreased slightly in G2, matching a similar phenomenon in G2 of the reciprocal crosses. This parallelism suggests some accidental phenomenon, which might be a slight perturbation in growth temperatures. Starting from G3 onward the values are very similar and stable, with a mean of 30.12±0.02°C (N=4). This value is close to that obtained in the reciprocal series (30.23±0.01). The difference is, however, significant (paired data; d=0.105±0.022, P<0.05). Again the interpretation of the results is straightforward: introducing a temperate Y chromosome in the Indian background has decreased the TIP of 0.42°C. The difference of 0.65°C above that of the temperate parental strain corresponds to an increase of the thermotolerance due to the Indian genetic background.
Progeny production after a return at a permissive temperature
Males of two different populations (Bordeaux and Delhi) were grown at nine
different constant temperatures from 28 to 32°C. Upon emergence, males
were isolated with three normal virgin females at 21°C, and vials were
changed daily. The first appearance of a larva in a vial defines the age at
recovery. For each temperature, ten males were investigated.
Results for Bordeaux and Delhi are shown in Fig. 3. For the three lowest pre-adult temperatures (28, 28.5 and 29°C), males were all normally fertile after emergence in both populations. Such was also the case for the Indian males up to 30°C, and these results match the data shown in Fig.1. For higher growth temperatures, the recovery time is greater in each population, from 1 up to 910 days. At very high temperatures, only a fraction of the males recovered. In all cases, the recovery time was longer in the temperate than in the tropical population. The difference between the two strains is significant for three growth temperatures, 29.5, 30 and 30.5°C (Student t test, P<0.001). In other words, males of the heat-sensitive, temperate population always required a longer time to recover and produce their first viable spermatozoa.
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Proportion of fertile males after a return at a permissive temperature
Fig. 3 shows that French
males grown at 30°C recovered on average in 6 days, while a quite similar
duration (8 days) was evident for Indian males grown at 31°C. In other
words, we expect that French males grown at their absolute sterility threshold
(30°C) will show the same functional alterations as Indian males grown at
31°C. This expectation was further tested by comparing Prunay and Delhi
populations. Males, grown at 30 and 31°C, respectively, were transferred
to 21°C after emergence, and sets of 50 males were dissected regularly up
to 9 days post-eclosion. Initially all males were sterile (no sperm visible)
but at the end almost all were fertile, with highly motile sperm
(Fig. 4). For young males,
there was a slight difference between the two populations: fertile males
appeared after 3 days in the Delhi strain but after 5 days in Prunay. The
overall curves have similar shapes, however, which were adjusted to a logistic
model. The ages at the inflection point were similar: 4.97±0.13 and
5.32±0.37 days for Prunay and Delhi, respectively. These values, which
indicate a mean recovery time, are slightly less than those obtained by direct
progeny observation (Fig. 3).
Such a small difference is not surprising and we already know that the
presence of sperm, even motile, in the seminal vesicles is not a certitude for
offspring production (Araripe et al.,
2004).
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Spermatogenesis defects due to high temperature
Cyst length
In control males grown at 21°C, neither strain nor generation affected
cyst length (ANOVA, not shown), which averaged 1.915±0.010 mm (mean
± S.E.M., N=250)
(Table 3). Cysts produced at a
sterilizing temperature (30°C for Prunay, 31°C for all other cases)
were shorter than in controls, the reduction varying between 24 and 44%. Cyst
length varied among generations (ANOVA, not shown), but in no regular
pattern.
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Sperm heads per cyst
Control flies (21°C) differed between generations in both the Prunay
and Delhi series (ANOVA, not shown; Table
3). However, the overall means in the two series were similar
(59.80±0.49 in Prunay and 58.04±0.6 in Delhi). These numbers are
less than the expected number (64), suggesting that during cyst maturation
some spermatids die and do no produce a visible nucleus. In the heat-grown
males, the number of sperm heads decreased in four of eight cases. In three
cases, however, the heated cysts had more nuclei than the controls. We suggest
that development at a high temperature tends to increase spermatid mortality,
but the effect is small.
Nuclear position
Cysts with at least one abnormal nucleus were more numerous in heat-grown
flies, from 1.25% (controls) to 8.75% in the Prunay series, and from 6.25% to
12.5% in the Delhi series (compare Fig.
5A,G and B,H). The difference between the two temperatures,
however, was not significant (2=0.163, d.f.=1,
P=0.685). Development at a high temperature does impair cyst
elongation, but the heterogeneity of nucleus position among spermatids in the
same cysts increases only slightly.
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Chromatin condensation of sperm heads
In normal cases, most sperm heads in a cyst are elongated with strong DAPI
staining (Fig. 5A,B). Some
abnormalities are evident, however, even after development at 21°C. These
abnormalities correspond to variation either in the strength of condensation
or in the proportion of non-condensed nuclei. At 21°C, the proportion of
cysts harboring such abnormalities was 26.25% and 32.50% in the Delhi and
Prunay series, respectively. But abnormalities were significantly more
frequent (2=1.285, d.f.=1, P=0.256) and often more
pronounced in males grown at 31°C (see
Fig. 5CH). The
frequencies of such cysts was 67.50% in Prunay and 77.50% in Delhi
(N=80 in each case).
All these cytological observations suggest that sterility arises mainly from incomplete elongation of the spermatids, and also from abnormalities in the chromatin condensation of the sperm heads. However, heat treatment has only a marginal effect on heterogeneity among spermatids in the same cyst and on their mortality.
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Discussion and conclusion |
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Populations from diverse geographic regions differed in progeny production
at the absolute threshold (30°C) in a direction consistent with climatic
adaptation: tropical populations are more heat tolerant than temperate ones.
Whether there is latitudinal clinal pattern awaits more precise measurement of
the median threshold. In D. melanogaster, ancestral populations occur
in the Afrotropical region (David and
Capy, 1988). In other words, a significant loss of heat tolerance
has accompanied the geographic expansion of the species into colder regions.
Previously the heat tolerance of the N'Djamena strain (see Introduction)
appeared unique, possibly related to some idiosyncratic mutations in a very
hot and arid place. We now show that the heat tolerance of spermatogenesis,
which permits permanent laboratory culture at 30°C, is widespread. To our
knowledge, the most tolerant populations occur in the African Sahel, south of
Sahara, and in Tropical India. Both regions experience extremely hot summers,
with daily maximum temperature above 38°C (see
Gibert et al., 1998
, for more
climatic information in the Delhi region). If heat tolerance is considered an
ancestral trait, we have to explain why this tolerance disappeared when D.
melanogaster extended its range toward colder, temperate countries. In
other words, why did thermotolerance disappear when it was no longer subjected
to selection? A general response is that thermotolerance implies a cost, due
to unknown pleiotropic effects. A possibility is that heat-tolerant flies
might be less cold-tolerant, but the occurrence of a functional trade-off
requires further investigation.
Laboratory selection seldom breaches the limit of 30°C for continuous
laboratory culture (Parsons,
1973; David et al.,
1983
; Zatsepina et al.,
2001
). We report here one more unsuccessful attempt (J. R. David,
unpublished). Two French mass populations were submitted to the following
selection procedure. Development proceeded at 31°C. After emergence,
adults were transferred at 20°C for recovery. About 2 weeks later,
numerous progeny were obtained and the larvae transferred back to 31°C.
The selection was repeated for 30 generations but, at the end, tolerance of
heat-induced male sterilization had not increased: the recovery time at the
permissive temperature was not shortened. Against this background, the ability
of the N'Djamena strain to grow initially at 30°C and later at
3132°C (see Zatsepina et al.,
2001
) is remarkable. This occurrence suggests that the
thermotolerance of spermatogenesis was increased by selection, although the
thermal thresholds were, to our knowledge, never determined. Other positive
responses to laboratory selection have been obtained. Several strains from
Niamey and Delhi region have now been cultured permanently at 30°C. At the
beginning and when population size was too low, strains had to be put at
29°C for one generation to recover. These difficulties disappeared after
about 100 generations. Indeed, the Indian strains were eventually cultured
permanently at 30.5°C, and fertile males may occur at 31°C. Thus,
permanent culture at a high temperature increased thermotolerance of male
reproduction, at least in the already heat-tolerant populations of Sahel and
tropical India.
Our crosses between a French and an Indian population demonstrate that much
of the genetic difference is due to the Y chromosome. Whether the same
phenomenon exists for the Sahel populations is still unknown. Cytological
abnormalities at the absolute threshold appeared to result mainly from a
perturbation of the elongation process, which may explain why motile sperm are
not produced. Chromatin condensation abnormalities increase in sperm heads,
suggesting that heat may also affect several different physiological
processes. After transfer to a mild, permissive temperature, males may
eventually recover fertility. In several species, recovery time is
proportional to the sterilizing temperature
(Chakir et al., 2002;
Vollmer et al., 2004
;
Araripe et al., 2004
) and may
take up to 10 days, which is equivalent to the duration of spermatogenesis
(Lindsley and Tokuyasu, 1980
).
Possibly, therefore, heat-induced perturbations are expressed at the level of
germ cells themselves. Interestingly, cytological abnormalities related to
deletions of Y fertility genes were often observed in early stages of
spermatogenesis (Hardy et al.,
1981
). In this respect, the cytological abnormalities produced by
different temperatures above the absolute thermal threshold remain to be
investigated.
The importance of the Y chromosome was unexpected because this mostly
heterochromatic chromosome bears only a few genes which, however, are
generally necessary for male fertility
(Kennison, 1981; Carvalho et
al., 2000
,
2001
). Genes on the Y
chromosome generally have a low level of molecular polymorphism
(Zurovcova and Eanes, 1999
).
Within a population, all Y chromosomes might be identical, which would leave
little variation on which selection could act. On the other hand, we now have
evidence that Y chromosomes in distant geographic populations can differ, and
in ways that are relevant to spermatogenesis. Three out of nine genes
identified on the Y chromosome belong to the dynein family
(Carvalho et al., 2001
) and may
be related to sperm motility. Possibly heat inactivation of at least one such
protein is responsible of the elongation abnormality, and also this protein in
tropical populations is more tolerant of high temperature. This hypothesis is,
however, difficult to reconcile with the differing time courses of recovery.
Also unclear is how dynein inactivation might affect the function of germ
cells. Perhaps a complete explanation may lie in genes on the Y chromosome
whose function remains unknown (Carvalho et
al., 2001
).
The significant role of genetic background (genes on autosomes and X), as
suggested by backcrosses and presumably also by selection, is also difficult
to explain. A role for increased Hsp70 is unlikely because the N'Djamena
strain has comparatively low expression of this heat-shock protein
(Zatsepina et al., 2001). But
many other genes are involved in spermiogenesis and interact with temperature
(Yue et al., 1999
;
Rajendra et al., 2001
;
Rockett et al., 2001
).
Interestingly, male sterility at high temperature is evident in all species
investigated so far (Chakir et al.,
2002; Vollmer et al.,
2004
; Araripe et al.,
2004
). The sterility threshold, however, varies among species, and
most temperate species cannot be grown at 30°C. For example, for the
European D. subobscura the upper development limit is 26°C
(Moreteau et al., 1997
), but
males are sterile at 25°C (J. R. David, unpublished observation). Possibly
this sterility phenomenon is general among drosophilids and plays an important
role in determining the climatic distribution of species, as proposed by
Araripe et al. (2004
).
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Acknowledgments |
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