BACKGROUND
all models of speciation implicitly recognize two steps, whereby initial divergence is followed by evolution of reproductive isolation.
in the standard allopatric model , geographical isolation is viewed as a prerequisite for divergence  <cit> .
while geographical isolation may be due to dispersal or vicariance , the refuge theory argues that climate change is generally responsible for the fragmentation of a species range  <cit> .
for populations that remain isolated a long time, climate-driven vicariance events may be sufficient for reproductive isolation to evolve by drift alone  <cit> .
nevertheless, adaptation to local ecological conditions in different refuges can also accelerate speciation when divergence affects phenotypic traits involved in mate choice  <cit> .
accordingly, climatic fluctuations of the plio-pleistocene  may be expected to produce a stasis-punctuation model of evolution, where bursts of speciation are caused by major climatic changes  <cit>  .
however, while there is a consensus around the idea of past climatic oscillations having been determinant in shaping the current distribution range and genetic structure of many modern animal and plant species  <cit> , their importance in driving speciation remains a matter of controversies  <cit> , a situation that results from several factors.
firstly, the timing of speciation events is often inaccurate or too imprecise  <cit> .
secondly, despite an expanding literature on the role of ecology in driving the patterns of phenotypic differentiation and the levels of gene flow between sympatric or parapatric populations, the relationship between ecology and allopatric speciation remains poorly understood  <cit> .
thirdly, recent analyses suggested that the importance of the pleistocene for speciation events might actually vary among regions  <cit>  and habitats  <cit> .
however, the majority of studies focused on european and north american species, even if climatic cycles were as pronounced elsewhere  <cit> .
in africa for instance, frequent climatic shifts of moderate amplitude before  <dig>  million years ago  were followed by three major steplike shifts towards more arid conditions near  <dig>  ,  <dig>   and  <dig>   mya.
then, the climate changed quite abruptly around  <dig>  mya with an accentuation of amplitude and duration of climatic cycles .
periods of increased aridity were characterized by a contraction of forests and an expansion of savannas and deserts  <cit> , and this have affected the biotas that occupy these biomes.
fossil african bovid and rodent assemblages shifted towards arid-adapted species between  <dig>  and  <dig>  mya, and a further increase in the relative abundance of arid-adapted bovid species was noticed in africa near both  <dig>  and  <dig>  mya  <cit> .
also, these periods correspond to key junctures in early hominid evolution, including the emergence of the genus homo  <cit> .
in addition, some recent phylogeographical studies in africa revealed a good congruence between divergence times among species or phylogroups and the peak of aridification that occurred  <dig> mya .
furthermore, several intraspecific radiations appear to have occurred within the past 200– <dig> ky, corresponding to the last two or three major pleistocene glaciations .
however, generality of these results might be affected by biases in the current sampling of groups and biomes, with savanna-dwelling mammals  <cit>  and forest avian species  <cit>  being over represented in comparison with arid regions which are somewhat neglected.
as a consequence the role of the sahara in driving speciation events is still poorly understood  <cit> , while we may expect that periods of increased aridity, that actually started more than  <dig> million years ago  <cit>  and corresponded to an expansion of the sahara, promoted north-south vicariance events  <cit> .
since climatic fluctuations appeared to affect arid regions in the arabian peninsula and south-east asia  in a similar fashion , it is further possible that plio-pleistocene climatic fluctuations were responsible for additional east-west  vicariance events.
in this paper, we investigated the applicability of the climate-driven speciation hypothesis to avian populations distributed around the desert belt in north africa and eurasia.
in particular, we tested whether  vicariance events were concomitant with plio-pleistocene climatic changes and  current ecological factors  contribute to phenotypic divergence between allopatric populations.
we choose the crested and thekla lark species complexes as model because:  they have a very large old world distribution north and south of the sahara in africa, but also to the east within and beyond the arid belt of asia;  they show a high level of phenotypic diversification, resulting in a large number of described subspecies  <cit> ;  the crested and the thekla lark species group have diverged ~ <dig>  mya  <cit> , and thus represent two independent evolutionary trajectories through plio-pleistocene climatic fluctuations.
in addition, they are species of open grounds , for which relevant phylogeographic data in africa are scarce, particularly in comparison with mammals and forest birds .
prior analyses in the crested lark complex revealed the existence of two reproductively isolated and essentially allopatric species .
galerida randonii is an endemic of central maghreb while g. cristata has a much wider distribution  <cit>  .
limited geographic sampling further revealed the existence of two distinct genetic groups in g. cristata: cristata has the largest distribution from western morocco to the middle east and through eurasia, and possibly also in eastern sahel.
the senegallensis group was found on both sides of the saharan desert, in the western sahel and in the eastern maghreb  <cit> .
the distribution of the thekla lark is disjoint, with one set of populations in the western mediterranean region, and the other one in the horn of africa .
in addition, g. malabarica of coastal india might form a superspecies with the thekla lark  <cit> .
since these three sets of populations are currently separated by arabo-saharan deserts, we may speculate that they became isolated following one or several episodes of increased aridity in these areas .
however, a single study has examined genetic variation in g. theklae, but it was limited to morocco and did not detect any phylogeographical structure among five described subspecies  <cit> .
in this study, important new sampling  and new analyses  were used to investigate the evolutionary history of modern taxa belonging to the crested and thekla lark species complexes and its links with past climatic events.
in addition to examining historical patterns of diversification events in these two partially co-distributed species complexes, phylogenetic and population genetics data were used to understand the processes which underlie complex patterns of phenotypic differentiation in these taxa.
in particular, we tested the hypothesis that recurrent adaptation to similar living environments provides a tenet for long-standing systematic controversies  <cit> .
by studying traits  that are prominent in mate choice in passerine birds  <cit> , this study also brought insights into speciation patterns in these taxa and biogeographic zones.
firstly, previous analyses in the crested lark complex revealed a striking syndrome of body size increase in the maghreb for two different lineages  <cit> .
however, previous studies did not make further attempts to explain this pattern, which could be explained either by climatic effect , and/or by interspecific competition .
the design of the present study allowed us to address the climatic component of the variation  while simultaneously controlling for the possible influence of interspecific competition  and phylogenetic effects .
secondly, subspecies that live in desertic areas tend to have a very pale sandy plumage, seemingly mainly as a result of drastic reduction in the amount of melanin deposition in feathers centre of the head, back, wings and breast  <cit>  .
however, such phenotypic similarity might reflect close phylogenetic relationships rather than independent adaptation to desert conditions.
for instance, no mtdna divergence was found between two desert subspecies of the thekla lark  in a previous study in morocco  <cit> .
in this study, phylogenetic differentiation of desert subspecies was investigated for geographically remote populations, both in the thekla lark complex  and in the crested complex .
in addition, a coalescence-based method was used to investigate the possible influence of gene flow in establishing similarity between desert subspecies.
in summary, this paper addresses the influence of the climate on speciation and phenotypic variation in two widespread species complexes of larks.
incidentally, this study should also bring new insights into the biogeography of poorly-known regions such as the sahara, and to the phylogenetic status of currently isolated populations in india or the horn of africa.
RESULTS
phylogeny
as expected, two major groups broadly corresponding to the thekla lark complex on one hand, and to the crested lark complex on the other hand were identified: for nuclear data, 6– <dig> mutations were fixed between the two species complexes, as compared to 0– <dig> mutation among sequences within groups, and for mtdna, both groups were found to be monophyletic with strong bootstrap support .
however, despite its thekla-like morphology, g. malabarica turned out to be a member of the crested lark complex.
this result is supported by identical mtdna sequences of three different specimens of g. malabarica , and by the  <dig> bp of the β-fibrinogen gene obtained for four different g. malabarica : the four specimens shared three mutations that are diagnostic of crested larks , but they were not fixed for any mutation diagnostic of the thekla lark complex .
bootstrap support  and estimates of divergence times in million years ago  derived for various nodes of the mitochondrial phylogeny  using the standard molecular clock of 2% per million year .
possible coincidence with some climatic events in mya  is indicated, as well as modified estimates derived from slightly different molecular clocks .
<dig> haplotypes for the short  cytochrome b fragment .
<dig> alleles  for intron  <dig> of the β-fibrinogen gene ; s = c and g; y = c and t. the geographic origin of samples is indicated as follows: mo = morocco; tu = tunisia; ch = chad; se = senegal; ke = kenya; ir = iran.
further resolution is revealed by mtdna data.
within the crested lark complex, the phylogenetic tree revealed three monophyletic groups corresponding to three previously recognized species: g. randonii  <cit> , g. malabarica and g. cristata .
lack of resolution for the basal nodes is suggestive of a fairly simultaneous vicariant event for these three species, coincident with the second aridification event at ~ <dig>  mya .
g. randonii and g. malabarica show little intraspecific genetic variation, which might be due to their narrow geographic distribution and/or small sample size .
within the more widely distributed g. cristata, three haplotypes cluster with allopatric distribution could be identified   the cristata group  is by far the most widespread, being present throughout eurasia , but also in ne africa and nw morocco;  the senegallensis group  is located in the western sahel and eastern maghreb; the type specimen of the subspecies helenae in the central sahara clearly belongs to that group ;  the somaliensis group  is only found in kenya.
geographic distribution of haplotypes for the short - <dig> bp- cytochrome b fragment.
within the thekla lark complex, populations currently separated by the sahara form two highly divergent monophyletic assemblages that diverged concomitantly with the first aridification event at ~ <dig>  mya  and table 5).
within g. theklae sensu stricto , we only found a shallow genetic division among the whole sample .
by contrast, in the horn of africa, we found two old mitochondrial lineages, namely praetermissa and ellioti whose split coincided with the second aridification event at ~ <dig>  mya .
they correspond to two previously unrecognized evolutionary units.
the sequence obtained for a single specimen of the race hueii  turned out to be most closely related to g. praetermissa, even if it is quite divergent .
geographic distribution of haplotypes for the short - <dig> bp- cytochrome b fragment.
population genetics
population subdivision
in both g. cristata and g. theklae, none pairwise Φst value between populations within lineages was significant , while as expected, all pairwise Φst among lineages were significant .
population pairwise Φst using kimura two parameter distance.
p-values, without bonferonni correction: ns p >  <dig> ; * p <  <dig> ; ** p <  <dig> ; *** p <  <dig> .
population pairwise Φst using kimura two parameter distance.
p-values, without bonferonni correction: ns p >  <dig> ; * p <  <dig> ; ** p <  <dig> ; *** p <  <dig> .
test of demographic stability
taken separately, each geographic population of g. cristata or g. theklae showed non-significant fu's fs value , with the exception of moroccan populations of g. theklae .
evidence of expansion was found in the theklae lineage of g. theklae  and in the cristata lineage of g. cristata .
for the senegallensis lineage of g. cristata, evidence of expansion was weaker, with nearly significant result .
mismatch-distribution analyses were performed for the theklae, cristata and senegallensis lineages.
all three distributions fitted the sudden expansion model, as indicated by non-significant tests of goodness of fit , supporting the hypothesis of a recent expansion in senegallensis also.
the time to expansion event t estimated from mismatch-distribution analyses  was t ~  <dig> kya  for senegallensis, t ~  <dig> kya  for theklae and t ~  <dig> kya  for cristata.
because expansions are detected in three distinct lineages that are also the most widespread , and because three independent and fairly simultaneous selective sweeps appear unlikely, we feel confident that these patterns reflect demography and not selection  <cit> .
test of recurrent gene flow across the sahara  – eight independent mdiv runs were conducted and yielded highly consistent results.
the data are most consistent with a non-null period of divergence , but with complete isolation .
the latter result is indicated by a strictly decreasing curve for proba as long as the prior for tmax is set <  <dig>  which seems reasonable given the estimated mode of t .
phenotypic evolution
morphometry
the topology of the tree obtained from the six morphological traits is very different from the genetic one .
such a discrepancy can be explained by two factors:  taxa do not always cluster according to the species complex they belong to.
for instance, g. malabarica cluster with members of the thekla lark complex and not with the more closely related g. cristata or g. randonii; and  within species complexes, populations tend to cluster on the basis of geographic -and not phylogenetic-affinities, suggesting morphological convergence.
a multiple regression analysis revealed the role of ecological competition and current climate in driving the patterns of body size variation .
within both species complexes, populations tended to evolve a larger body size in the presence of a competitor and in more arid areas.
however, this latter effect was eclipsed by a significant interaction between aridity and competition .
generalized estimating equations  were used to control for phylogenetic relationships between populations .
they yielded significant results consistent with ordinary regression .
in addition, the significant interaction between species complex and competition factors  suggested that body size shift in the presence of competition affects mainly the crested larks, thus supporting the character displacement hypothesis.
however, this latter effect was not found again when the analysis was limited to males .
in contrast with these results for body size, neither competition nor climate explained a significant part of the variation in body shape, as no factor was retained in the multiple regression procedure .
anova table presenting factors included in the best regression model for body size : n =  <dig> observations; r <dig> =  <dig> ; f <dig>  =  <dig> , p <  <dig> .
for the sc and sc × comp effects, the sign of student's t ) is given here with the thekla lark complex as a standpoint.
for body shape , no factor was included in the best model.
color variation
in g. cristata, the three chadian light-plumaged isabellina specimens shared the most common haplotype of the cristata lineage , while the algerian light-plumaged type specimen of helenae had the most common senegallensis haplotype .
the hypothesis of reciprocal monophyly of cristata and senegallensis is weakly supported .
nevertheless, mdiv results suggested that they diverged around  <dig> kya , seemingly without any subsequent gene flow , while the origin of helenae and isabellina within each lineage is probably much more recent.
the cristata lineage mostly includes dark-plumaged populations, e.g.
in s europe or turkey  and the same is true for the senegallensis lineage .
hence, each subspecies holds widespread haplotypes of two genetic groups that are mainly made of typical/dark populations.
our molecular results are thus consistent with the hypothesis that several convergence events explain the evolution of plumage color patterns in g. cristata.
in g. theklae, all three light-plumaged carolinae from se morocco  had haplotypes from the theklae haplogroup .
by contrast, each light-plumaged deichleri of s tunisia had a distinct haplotype that belonged either to theklae  or to superflua haplogroups .
rather than incomplete lineage sorting, mdiv favours a model of recent divergence between populations in morocco and tunisia , associated with weak but non-null gene flow .
hence, it is not possible to exclude a single origin of the light colored plumage that characterizes desertic phenotypes in the thekla lark.
discussion
support for the climate-driven speciation hypothesis
vicariance diversification events coincide with past climatic events
as could be predicted on the basis of traditional systematic hypotheses and results of a previous study  <cit> , both mitochondrial and nuclear dna data revealed an old  division between members of two well-supported species groups corresponding to two traditionally recognised species: the thekla lark and the crested lark.
all subsequent diversification events were remarkably congruent with major late pliocene and pleistocene climatic events in africa, particularly steplike shifts towards more arid conditions near  <dig> ,  <dig>  and  <dig>  mya, and with radiations in co-distributed unrelated species groups  <cit>  , bringing support to the climate-driven speciation hypothesis.
periodic expansion of the sahara appears to have played a major role in cladogenesis, by repeatedly isolating vicariant populations respectively north and south of the main desert landmass , but periods of aridification also impacted populations of the thekla lark complex living in the horn of africa, with  a split between populations in highland steppes  and xeric grasslands  that occurred ~ <dig>  mya;  the separation of highland steppes populations by the rift valley dated at ~ <dig>  mya.
this scenario should of course be refined in the future and more accurate tests performed.
first, the general history of climate change in africa as described by demenocal  <cit>  shows local or even regional exceptions  <cit> , which could be taken into account with wider geographical sampling.
perhaps more importantly, the coincidence between divergence and climatic events rely on an assumed cytochrome b mutation rate of 2% per my, which has not been calibrated for these larks.
although the widespread use of this rate has been criticized  <cit> , there is no alternative to date when no fossil record is available  <cit> , and this rate seems to be largely consistent among avian groups  <cit> , being also relevant in the single songbird clade calibrated to date  <cit> .
in any case, if we except unlikely low values , all diversification events we report would still be consistent with a late pliocene or pleistocene influence, with the possible exception of the palearctic/ethiopian split in the thekla lark .
finally, confidence intervals around estimated values of divergence times are quite large .
in the future, a multilocus approach could be used to reduce these confidence limits  <cit> .
prior to this work, the role of past climatic fluctuations was essentially noticed for savanna-dwelling mammals and forest avian species .
the present study suggests that the same general pattern also applies for ground-dwelling birds adapted to open habitats .
interestingly, the diversification pattern we describe here for species of steppe-like and montane habitats in and near the tropics is in accordance with the standard model for temperate species  <cit> , but differs from what has been reported for lowland tropical forest species, where most speciation events seem to predate the late pliocene and pleistocene epochs  <cit>  .
overall, these results highlight the importance of comparative phylogeographic approaches to unravel the factors underlying diversification processes  <cit> .
current ecological factors shape phenotypic variation
in agreement with previous reports in these taxa , we found overall a strong discordance between phylogenetic relationships inferred from neutral molecular markers and phenetic similitude among groups based on phenotypic data sets.
this suggests a major role for natural selection in driving the patterns of phenotypic evolution in galerida larks, especially given that in birds a strong genetic component has been reported in both morphological traits  <cit>  and melanin-based plumage traits  <cit> .
however, the exact contribution of phenotypic plasticity cannot be addressed with present data.
morphology
the most striking example concerns morphological convergence in the maghreb.
for the crested lark, convergence is found between g. randonii and both senegallensis and cristata lineages of g. cristata .
for the thekla lark, convergence is found between theklae and superflua .
as the trend in both complexes is for increasing size , this suggested a common, possibly climatic, cause .
however, a multiple regression analysis indicated that variations in body size – such as the trends towards larger body size in the maghreb- are mainly driven by interspecific competitive interactions between sympatric members of each species complex.
nevertheless, this response to competition is stronger in more arid areas, which is consistent with the view that phenotypic response to natural selection should be strongest during periods or in areas where resources are most limiting  <cit> .
our analysis also provided some evidence that competition might affect primarily representatives of the crested lark complex, thereby supporting the character displacement hypothesis .
mitochondrial data suggest that in g. cristata, the maghreb was invaded twice : 1) by the cristata lineage, from southern europe  to the western maghreb , and 2) by the senegallensis lineage, from sub-saharan africa  to eastern maghreb .
in presumed source areas , g. cristata is entirely  or predominantly  allopatric with g. theklae.
interestingly, the body size of source g. cristata populations are strikingly similar to the body size of maghreban thekla lark populations that they encountered as they colonized the area , pointing towards a possible causal mechanism for character displacement.
however, we emphasize that further samples will be required to validate this hypothesis .
color
like in many other bird species  <cit>  , and in agreement with a previous report in morocco  <cit> , plumage variation in galerida larks was in agreement with gloger's rule .
adaptation to desert environments clearly occurred independently in g. theklae  and g. cristata  and probably several times in g. cristata although further samples are needed to confirm this.
in g. theklae on the other hand, a single origin of desert-adapted plumage could not be excluded, as recurrent gene flow was detected among the two desertic populations investigated.
tempo of phenotypic evolution
our data are suggestive of a punctuated-equilibrium mode of evolution : long periods of relative phenotypic stasis, as might be exemplified by the thekla-like morphotype in widely divergent phylogenetic lineages  can sometimes be followed by very rapid episodes of divergence, as a result of e.g.
climate-driven adaptation in expanding lineages .
for instance within senegallensis, morphological differences between populations north and south of the sahara, which exhibit only weak and non-significant genetic differentiation , are much larger than morphological differences found between valid species that belong to widely divergent species complexes .
phenotypic divergence and speciation
phylogeographic analyses of the thekla and especially, crested lark complexes reveal a now classical history  <cit>  of multiple fragmentations followed occasionally by the expansion of some refuge lineages that may lead to secondary contact zones between valid or incipient species .
while incidental divergence during periods of isolation may contribute to the speciation process, it is unlikely to play a dominant role here, given the slow accumulation rate of post-zygotic incompatibilities in birds .
conversely, rapid phenotypic divergence in response to local ecological conditions as demonstrated here could induce at least partial pre-zygotic isolation at secondary contact zones  <cit> , particularly for such passerine birds for which body size and coloration play an important role in mate choice  <cit> .
in the single secondary contact zone studied to date, strong evidence of reproductive isolation was indeed found between cristata and randonii in morocco  <cit> .
role of biogeographic barriers
the sahara
very few studies have addressed the role of the sahara in driving speciation events  <cit>  .
this is particularly true for the last four million years, a period which is characterized by the onset of arid climatic cycles  <cit>  that might have favoured speciation.
our results suggest three different episodes of north-south vicariance during arid phases of late pliocene , mid-pleistocene  and late pleistocene .
rather than simple north-south vicariance between g. cristata and g. randonii, the star-like phylogenetic relationships among the three species of crested larks  suggests that the divergence between g. cristata, g. randonii and g. malabarica occurred fairly simultaneously ~ <dig>  mya.
while g. malabarica and g. randonii are geographically restricted to, respectively, w coastal india and central maghreb, g. cristata is widely distributed in europe, asia and africa .
however, a sub-saharan origin is likely for the latter given that the highest genetic diversity is found there .
taken together, these elements suggests a simple speciation history in the crested lark species complex:  an ancestral species was distributed in africa and asia, occupying part of the current range of the three species g. cristata, g. randonii and g. malabarica;  the expansion of arabo-saharan deserts during an arid episode ~ <dig>  mya might have led to the formation of three refuge species : g. malabarica in india, g. randonii in the maghreb and g. cristata in sub-saharan africa;  while g. malabarica and g. randonii would have remained within the limits of their ancient refuge, g. cristata would have subsequently expanded its range from its sub-saharan refuge.
in agreement with a previous report  <cit> , our data are indeed consistent with two independent events of expansion from sub-saharan africa in g. cristata:  the colonization of the whole eurasia  from the eastern sahel refuge zone ; and  the colonization of the eastern maghreb from the western sahel refuge zone .
a recent range expansion in cristata is indicated by significant fu's fs and the star-like phylogeny of haplotypes .
since this taxon is tightly linked with human-modified habitats in parts of its range , guillaumet et al.
<cit>  proposed that the cristata expansion was facilitated by the development of agriculture that started around ten thousand years ago  <cit> .
when dating this expansion event by mismatch analysis we found a slightly higher but still concordant estimate .
despite the non-significant  negative fs value that we found for senegallensis, a recent expansion event in this group is suggested by the star-like phylogeny of haplotypes  and a mismatch distribution that fitted the sudden expansion model.
in fact, it is likely that the signal of expansion has been partly blurred by subsequent isolation and divergence without gene flow between populations n and s of the sahara, as suggested by mdiv results.
we suggest that the maghreb was colonized from sub-saharan africa because g. cristata is believed to originate from sub-saharan africa .
furthermore, in the case of a recent range expansion, ancestral haplotypes are more likely to be found close to the origin of expansion, whereas derived haplotypes are more likely to be found at the leading edge of range expansion  <cit> .
such a pattern fits the senegallensis haplotype network : two rare haplotypes found in sub-saharan africa tend to occupy an internal position in the network , while one clearly derived haplotype is only found in the maghreb .
however, we emphasize that additional sampling in the maghreb and sub-saharan africa is needed to validate the sub-saharan origin of the senegallensis lineage.
colonisation of the maghreb may have been favoured by steppe-like vegetation in the sahara, possibly during the most recent interglacial around  <dig> kya  <cit>  .
then, subsequent aridification of the sahara led to a contraction of saharan populations into a few refugia, particularly in mountain areas , that behave as relic populations .
this scenario appears compatible with the estimated time of divergence for populations n and s of the sahara  and with two other phylogeographic studies conducted in the lanner falcon falco biarmicus  <cit>  and in the honeybee apis mellifera  <cit> , which also suggest genetic isolation of populations north and south of the sahara during arid phases  <cit> .
our results demonstrate that the sahara did not constitute a permanent barrier throughout geological times.
the distribution of senegallensis is exemplary, since genetic data still hold the footprints of a recent expansion in the late pleistocene through the current sahara, followed by fragmentation and divergence without gene flow between a northern and a southern refuge, as a probable result of increasingly arid conditions .
interestingly, maghreban populations of senegallensis also showed an accelerated rate of morphological evolution  <cit>  .
galerida larks thus suggest to us a possible general mechanism by which glacial cycles affecting the sahara might promote speciation in birds and other taxa .
e-w vicariance in the sahel zone
the star-like phylogeny for the cristata, senegallensis and somaliensis lineages of g. cristata  suggests once again that they result from a single vicariance event that may have occurred around  <dig> kya .
as we discussed above, g. cristata seems to originate from sub-saharan africa.
accordingly, the divergence between senegallensis and cristata should correspond to e-w vicariance in the sahel zone , even if we cannot exclude the alternative that one or more lineage only secondarily invaded sub-saharan africa.
interestingly, other e-w vicariance events were also evidenced in the sahel zone at similar dates in unrelated species such as the hartebeest , and the roan antelope .
it has been suggested that such eastern and western lineages could have been isolated in refuges north and east  of an expanding central african rainforest belt, during some periods of climate warming  <cit> .
alternatively, we suggest that refuge populations might instead have been isolated by the lake chad during such humid phases that led to lake "mega-chad" episodes  <cit> .
e-w vicariance in the maghreb
g. theklae sensu stricto is structured in two distinct genetic groups  for which the current repartition and divergence date obtained from mdiv analysis suggest a separation in two distinct maghreban refuges during the penultimate pleistocene glacial event around  <dig> kya .
since a recent expansion is strongly supported in theklae , and significantly negative fu's fs) we find likely that genetic exchanges detected by mdiv took place after the widespread western haplogroup theklae underwent an eastwards range expansion ~ <dig> kya , thus creating a zone of contact in e tunisia .
however, we cannot totally exclude the possibility that the presence of one theklae haplotype in tunisia  reflects incomplete lineage sorting instead of gene flow.
the rift valley
the eastern branch of the kenyan rift valley might induce some restrictions to gene flow between sub-saharan populations of g. cristata in the eastern sahel  and kenya .
in addition, our limited data on the former subspecies of g. theklae in ethiopia are indicative of a long-term genetic isolation between populations that live on highland steppes on either side of the rift valley .
hence, these results agree with the observation that the rift valley constitutes a major zoogeographical barrier in mammals  <cit>  and birds  <cit> , but also seems to impact the population genetics of species currently distributed on either side .
systematics and endemism in africa
the present study considerably challenges current taxonomic hypotheses in these two species complexes, and paves the way for future revisions.
first, g. malabarica, an indian endemic taxon, turned out to be closely related to crested lark, and not to thekla lark as previously believed.
hence, apparent disjunction in the distribution of the thekla lark "superspecies", which comprised three sets of populations separated by arabo-saharan deserts , proved here to be an artefact of incorrect taxonomy  <cit> .
interestingly however, the hypothesis of a fragmentation by arabo-saharan deserts of a widely distributed ancestral species was actually supported for the crested lark instead .
secondly, we found a previously unsuspected deep break between thekla lark populations north and south of the sahara that occurred ~ <dig>  mya.
these two sets of populations lack diagnostic morphological  as well as plumage color characteristics, which prevented them from being split by previous authors.
however, as we have shown that phenotypic convergence is overwhelming in these taxa, it is now clear that phenotypes are not appropriate taxonomic clues.
as these two sets of populations are currently separated by a very wide gap of sahara , we believe they should now be recognized as distinct allopatric species.
in addition, the horn of africa seems to harbour several narrow-ranged endemic taxa: praetermissa appears to be endemic to the high plateaus of ethiopia north of the rift valley, hueii replaces it in similar habitats south of the rift valley, while ellioti is restricted to arid landscapes in somalia .
these allopatric taxa might warrant recognition at the species level, but this conclusion is postponed here because of limited sample size for ellioti and hueii, and because this study did not include representatives of three other subspecies described from the horn of africa .
the recognition of new narrow-ranged endemic taxa in the horn of africa might significantly supplement the current list of  <dig> avian species endemic to ethiopia or somalia  <cit> .
interestingly, the only other savanna-dwelling avian species investigated to date, the ostrich, also revealed one previously unexpected endemic species in the horn of africa  <cit> , suggesting that more cryptic species remain to be identified in this part of the world.
this seems to be quite a general phenomenon in sub-saharan africa, where phylogeographical patterns are still poorly understood.
indeed, wherever they are undertaken, molecular phylogenetic studies tend to reveal a more complex biogeographic history than previously recognized, and lead to the recognition of new species .
such findings may have fundamental evolutionary implications for the understanding of large-scale speciation processes, but also for conservation strategies  <cit>  .
CONCLUSIONS
in this study, phylogenetic, population genetics and phenotypic data were integrated in a comparative framework to reconstruct the evolutionary history of two widespread sibling species complexes.
in agreement with the climate-driven speciation hypothesis, both past and current climatic factors were found to be influential in driving speciation patterns, as shown respectively by recurrent vicariance mediated by the sahara, and multiple instances of convergence in morphology  and plumage color.

 all models of speciation implicitly recognize two steps, whereby initial divergence is followed by evolution of reproductive isolation. in the standard allopatric model, geographical isolation is viewed as a prerequisite for divergence. while geographical isolation may be due to dispersal or vicariance, the refuge theory argues that climate change is generally responsible for the fragmentation of a species range. for populations that remain isolated a long time, climate-driven vicariance events may be sufficient for reproductive isolation to evolve by drift alone. nevertheless, adaptation to local ecological conditions in different refuges can also accelerate speciation when divergence affects phenotypic traits involved in mate choice. accordingly, climatic fluctuations of the plio-pleistocene may be expected to produce a stasis-punctuation model of evolution, where bursts of speciation are caused by major climatic changes. however, while there is a consensus around the idea of past climatic oscillations having been determinant in shaping the current distribution range and genetic structure of many modern animal and plant species, their importance in driving speciation remains a matter of controversies, a situation that results from several factors. firstly, the timing of speciation events is often inaccurate or too imprecise. secondly, despite an expanding literature on the role of ecology in driving the patterns of phenotypic differentiation and the levels of gene flow between sympatric or parapatric populations, the relationship between ecology and allopatric speciation remains poorly understood. thirdly, recent analyses suggested that the importance of the pleistocene for speciation events might actually vary among regions and habitats. however, the majority of studies focused on european and north american species, even if climatic cycles were as pronounced elsewhere. in africa for instance, frequent climatic shifts of moderate amplitude before million years ago were followed by three major steplike shifts towards more arid conditions near, and mya. then, the climate changed quite abruptly around mya with an accentuation of amplitude and duration of climatic cycles. periods of increased aridity were characterized by a contraction of forests and an expansion of savannas and deserts, and this have affected the biotas that occupy these biomes. fossil african bovid and rodent assemblages shifted towards arid-adapted species between and mya, and a further increase in the relative abundance of arid-adapted bovid species was noticed in africa near both and mya. also, these periods correspond to key junctures in early hominid evolution, including the emergence of the genus homo. in addition, some recent phylogeographical studies in africa revealed a good congruence between divergence times among species or phylogroups and the peak of aridification that occurred mya. furthermore, several intraspecific radiations appear to have occurred within the past 200– ky, corresponding to the last two or three major pleistocene glaciations. however, generality of these results might be affected by biases in the current sampling of groups and biomes, with savanna-dwelling mammals and forest avian species being over represented in comparison with arid regions which are somewhat neglected. as a consequence the role of the sahara in driving speciation events is still poorly understood, while we may expect that periods of increased aridity, that actually started more than million years ago and corresponded to an expansion of the sahara, promoted north-south vicariance events. since climatic fluctuations appeared to affect arid regions in the arabian peninsula and south-east asia in a similar fashion, it is further possible that plio-pleistocene climatic fluctuations were responsible for additional east-west vicariance events. in this paper, we investigated the applicability of the climate-driven speciation hypothesis to avian populations distributed around the desert belt in north africa and eurasia. in particular, we tested whether vicariance events were concomitant with plio-pleistocene climatic changes and current ecological factors contribute to phenotypic divergence between allopatric populations. we choose the crested and thekla lark species complexes as model because: they have a very large old world distribution north and south of the sahara in africa, but also to the east within and beyond the arid belt of asia; they show a high level of phenotypic diversification, resulting in a large number of described subspecies; the crested and the thekla lark species group have diverged ~ mya, and thus represent two independent evolutionary trajectories through plio-pleistocene climatic fluctuations. in addition, they are species of open grounds, for which relevant phylogeographic data in africa are scarce, particularly in comparison with mammals and forest birds. prior analyses in the crested lark complex revealed the existence of two reproductively isolated and essentially allopatric species. galerida randonii is an endemic of central maghreb while g. cristata has a much wider distribution. limited geographic sampling further revealed the existence of two distinct genetic groups in g. cristata: cristata has the largest distribution from western morocco to the middle east and through eurasia, and possibly also in eastern sahel. the senegallensis group was found on both sides of the saharan desert, in the western sahel and in the eastern maghreb. the distribution of the thekla lark is disjoint, with one set of populations in the western mediterranean region, and the other one in the horn of africa. in addition, g. malabarica of coastal india might form a superspecies with the thekla lark. since these three sets of populations are currently separated by arabo-saharan deserts, we may speculate that they became isolated following one or several episodes of increased aridity in these areas. however, a single study has examined genetic variation in g. theklae, but it was limited to morocco and did not detect any phylogeographical structure among five described subspecies. in this study, important new sampling and new analyses were used to investigate the evolutionary history of modern taxa belonging to the crested and thekla lark species complexes and its links with past climatic events. in addition to examining historical patterns of diversification events in these two partially co-distributed species complexes, phylogenetic and population genetics data were used to understand the processes which underlie complex patterns of phenotypic differentiation in these taxa. in particular, we tested the hypothesis that recurrent adaptation to similar living environments provides a tenet for long-standing systematic controversies. by studying traits that are prominent in mate choice in passerine birds, this study also brought insights into speciation patterns in these taxa and biogeographic zones. firstly, previous analyses in the crested lark complex revealed a striking syndrome of body size increase in the maghreb for two different lineages. however, previous studies did not make further attempts to explain this pattern, which could be explained either by climatic effect, and/or by interspecific competition. the design of the present study allowed us to address the climatic component of the variation while simultaneously controlling for the possible influence of interspecific competition and phylogenetic effects. secondly, subspecies that live in desertic areas tend to have a very pale sandy plumage, seemingly mainly as a result of drastic reduction in the amount of melanin deposition in feathers centre of the head, back, wings and breast. however, such phenotypic similarity might reflect close phylogenetic relationships rather than independent adaptation to desert conditions. for instance, no mtdna divergence was found between two desert subspecies of the thekla lark in a previous study in morocco. in this study, phylogenetic differentiation of desert subspecies was investigated for geographically remote populations, both in the thekla lark complex and in the crested complex. in addition, a coalescence-based method was used to investigate the possible influence of gene flow in establishing similarity between desert subspecies. in summary, this paper addresses the influence of the climate on speciation and phenotypic variation in two widespread species complexes of larks. incidentally, this study should also bring new insights into the biogeography of poorly-known regions such as the sahara, and to the phylogenetic status of currently isolated populations in india or the horn of africa.  phylogeny as expected, two major groups broadly corresponding to the thekla lark complex on one hand, and to the crested lark complex on the other hand were identified: for nuclear data, 6– mutations were fixed between the two species complexes, as compared to 0– mutation among sequences within groups, and for mtdna, both groups were found to be monophyletic with strong bootstrap support. however, despite its thekla-like morphology, g. malabarica turned out to be a member of the crested lark complex. this result is supported by identical mtdna sequences of three different specimens of g. malabarica, and by the bp of the β-fibrinogen gene obtained for four different g. malabarica: the four specimens shared three mutations that are diagnostic of crested larks, but they were not fixed for any mutation diagnostic of the thekla lark complex. bootstrap support and estimates of divergence times in million years ago derived for various nodes of the mitochondrial phylogeny using the standard molecular clock of 2% per million year. possible coincidence with some climatic events in mya is indicated, as well as modified estimates derived from slightly different molecular clocks. haplotypes for the short cytochrome b fragment. alleles for intron of the β-fibrinogen gene; s = c and g; y = c and t. the geographic origin of samples is indicated as follows: mo = morocco; tu = tunisia; ch = chad; se = senegal; ke = kenya; ir = iran. further resolution is revealed by mtdna data. within the crested lark complex, the phylogenetic tree revealed three monophyletic groups corresponding to three previously recognized species: g. randonii, g. malabarica and g. cristata. lack of resolution for the basal nodes is suggestive of a fairly simultaneous vicariant event for these three species, coincident with the second aridification event at ~ mya. g. randonii and g. malabarica show little intraspecific genetic variation, which might be due to their narrow geographic distribution and/or small sample size. within the more widely distributed g. cristata, three haplotypes cluster with allopatric distribution could be identified the cristata group is by far the most widespread, being present throughout eurasia, but also in ne africa and nw morocco; the senegallensis group is located in the western sahel and eastern maghreb; the type specimen of the subspecies helenae in the central sahara clearly belongs to that group; the somaliensis group is only found in kenya. geographic distribution of haplotypes for the short - bp- cytochrome b fragment. within the thekla lark complex, populations currently separated by the sahara form two highly divergent monophyletic assemblages that diverged concomitantly with the first aridification event at ~ mya and table 5). within g. theklae sensu stricto, we only found a shallow genetic division among the whole sample. by contrast, in the horn of africa, we found two old mitochondrial lineages, namely praetermissa and ellioti whose split coincided with the second aridification event at ~ mya. they correspond to two previously unrecognized evolutionary units. the sequence obtained for a single specimen of the race hueii turned out to be most closely related to g. praetermissa, even if it is quite divergent. geographic distribution of haplotypes for the short - bp- cytochrome b fragment. population genetics population subdivision in both g. cristata and g. theklae, none pairwise Φst value between populations within lineages was significant, while as expected, all pairwise Φst among lineages were significant. population pairwise Φst using kimura two parameter distance. p-values, without bonferonni correction: ns p >; * p <; ** p <; *** p <. population pairwise Φst using kimura two parameter distance. p-values, without bonferonni correction: ns p >; * p <; ** p <; *** p <. test of demographic stability taken separately, each geographic population of g. cristata or g. theklae showed non-significant fu's fs value, with the exception of moroccan populations of g. theklae. evidence of expansion was found in the theklae lineage of g. theklae and in the cristata lineage of g. cristata. for the senegallensis lineage of g. cristata, evidence of expansion was weaker, with nearly significant result. mismatch-distribution analyses were performed for the theklae, cristata and senegallensis lineages. all three distributions fitted the sudden expansion model, as indicated by non-significant tests of goodness of fit, supporting the hypothesis of a recent expansion in senegallensis also. the time to expansion event t estimated from mismatch-distribution analyses was t ~ kya for senegallensis, t ~ kya for theklae and t ~ kya for cristata. because expansions are detected in three distinct lineages that are also the most widespread, and because three independent and fairly simultaneous selective sweeps appear unlikely, we feel confident that these patterns reflect demography and not selection. test of recurrent gene flow across the sahara – eight independent mdiv runs were conducted and yielded highly consistent results. the data are most consistent with a non-null period of divergence, but with complete isolation. the latter result is indicated by a strictly decreasing curve for proba as long as the prior for tmax is set < which seems reasonable given the estimated mode of t. phenotypic evolution morphometry the topology of the tree obtained from the six morphological traits is very different from the genetic one. such a discrepancy can be explained by two factors: taxa do not always cluster according to the species complex they belong to. for instance, g. malabarica cluster with members of the thekla lark complex and not with the more closely related g. cristata or g. randonii; and within species complexes, populations tend to cluster on the basis of geographic -and not phylogenetic-affinities, suggesting morphological convergence. a multiple regression analysis revealed the role of ecological competition and current climate in driving the patterns of body size variation. within both species complexes, populations tended to evolve a larger body size in the presence of a competitor and in more arid areas. however, this latter effect was eclipsed by a significant interaction between aridity and competition. generalized estimating equations were used to control for phylogenetic relationships between populations. they yielded significant results consistent with ordinary regression. in addition, the significant interaction between species complex and competition factors suggested that body size shift in the presence of competition affects mainly the crested larks, thus supporting the character displacement hypothesis. however, this latter effect was not found again when the analysis was limited to males. in contrast with these results for body size, neither competition nor climate explained a significant part of the variation in body shape, as no factor was retained in the multiple regression procedure. anova table presenting factors included in the best regression model for body size: n = observations; r =; f =, p <. for the sc and sc × comp effects, the sign of student's t ) is given here with the thekla lark complex as a standpoint. for body shape, no factor was included in the best model. color variation in g. cristata, the three chadian light-plumaged isabellina specimens shared the most common haplotype of the cristata lineage, while the algerian light-plumaged type specimen of helenae had the most common senegallensis haplotype. the hypothesis of reciprocal monophyly of cristata and senegallensis is weakly supported. nevertheless, mdiv results suggested that they diverged around kya, seemingly without any subsequent gene flow, while the origin of helenae and isabellina within each lineage is probably much more recent. the cristata lineage mostly includes dark-plumaged populations, e.g. in s europe or turkey and the same is true for the senegallensis lineage. hence, each subspecies holds widespread haplotypes of two genetic groups that are mainly made of typical/dark populations. our molecular results are thus consistent with the hypothesis that several convergence events explain the evolution of plumage color patterns in g. cristata. in g. theklae, all three light-plumaged carolinae from se morocco had haplotypes from the theklae haplogroup. by contrast, each light-plumaged deichleri of s tunisia had a distinct haplotype that belonged either to theklae or to superflua haplogroups. rather than incomplete lineage sorting, mdiv favours a model of recent divergence between populations in morocco and tunisia, associated with weak but non-null gene flow. hence, it is not possible to exclude a single origin of the light colored plumage that characterizes desertic phenotypes in the thekla lark. discussion support for the climate-driven speciation hypothesis vicariance diversification events coincide with past climatic events as could be predicted on the basis of traditional systematic hypotheses and results of a previous study, both mitochondrial and nuclear dna data revealed an old division between members of two well-supported species groups corresponding to two traditionally recognised species: the thekla lark and the crested lark. all subsequent diversification events were remarkably congruent with major late pliocene and pleistocene climatic events in africa, particularly steplike shifts towards more arid conditions near, and mya, and with radiations in co-distributed unrelated species groups, bringing support to the climate-driven speciation hypothesis. periodic expansion of the sahara appears to have played a major role in cladogenesis, by repeatedly isolating vicariant populations respectively north and south of the main desert landmass, but periods of aridification also impacted populations of the thekla lark complex living in the horn of africa, with a split between populations in highland steppes and xeric grasslands that occurred ~ mya; the separation of highland steppes populations by the rift valley dated at ~ mya. this scenario should of course be refined in the future and more accurate tests performed. first, the general history of climate change in africa as described by demenocal shows local or even regional exceptions, which could be taken into account with wider geographical sampling. perhaps more importantly, the coincidence between divergence and climatic events rely on an assumed cytochrome b mutation rate of 2% per my, which has not been calibrated for these larks. although the widespread use of this rate has been criticized, there is no alternative to date when no fossil record is available, and this rate seems to be largely consistent among avian groups, being also relevant in the single songbird clade calibrated to date. in any case, if we except unlikely low values, all diversification events we report would still be consistent with a late pliocene or pleistocene influence, with the possible exception of the palearctic/ethiopian split in the thekla lark. finally, confidence intervals around estimated values of divergence times are quite large. in the future, a multilocus approach could be used to reduce these confidence limits. prior to this work, the role of past climatic fluctuations was essentially noticed for savanna-dwelling mammals and forest avian species. the present study suggests that the same general pattern also applies for ground-dwelling birds adapted to open habitats. interestingly, the diversification pattern we describe here for species of steppe-like and montane habitats in and near the tropics is in accordance with the standard model for temperate species, but differs from what has been reported for lowland tropical forest species, where most speciation events seem to predate the late pliocene and pleistocene epochs. overall, these results highlight the importance of comparative phylogeographic approaches to unravel the factors underlying diversification processes. current ecological factors shape phenotypic variation in agreement with previous reports in these taxa, we found overall a strong discordance between phylogenetic relationships inferred from neutral molecular markers and phenetic similitude among groups based on phenotypic data sets. this suggests a major role for natural selection in driving the patterns of phenotypic evolution in galerida larks, especially given that in birds a strong genetic component has been reported in both morphological traits and melanin-based plumage traits. however, the exact contribution of phenotypic plasticity cannot be addressed with present data. morphology the most striking example concerns morphological convergence in the maghreb. for the crested lark, convergence is found between g. randonii and both senegallensis and cristata lineages of g. cristata. for the thekla lark, convergence is found between theklae and superflua. as the trend in both complexes is for increasing size, this suggested a common, possibly climatic, cause. however, a multiple regression analysis indicated that variations in body size – such as the trends towards larger body size in the maghreb- are mainly driven by interspecific competitive interactions between sympatric members of each species complex. nevertheless, this response to competition is stronger in more arid areas, which is consistent with the view that phenotypic response to natural selection should be strongest during periods or in areas where resources are most limiting. our analysis also provided some evidence that competition might affect primarily representatives of the crested lark complex, thereby supporting the character displacement hypothesis. mitochondrial data suggest that in g. cristata, the maghreb was invaded twice: 1) by the cristata lineage, from southern europe to the western maghreb, and 2) by the senegallensis lineage, from sub-saharan africa to eastern maghreb. in presumed source areas, g. cristata is entirely or predominantly allopatric with g. theklae. interestingly, the body size of source g. cristata populations are strikingly similar to the body size of maghreban thekla lark populations that they encountered as they colonized the area, pointing towards a possible causal mechanism for character displacement. however, we emphasize that further samples will be required to validate this hypothesis. color like in many other bird species, and in agreement with a previous report in morocco, plumage variation in galerida larks was in agreement with gloger's rule. adaptation to desert environments clearly occurred independently in g. theklae and g. cristata and probably several times in g. cristata although further samples are needed to confirm this. in g. theklae on the other hand, a single origin of desert-adapted plumage could not be excluded, as recurrent gene flow was detected among the two desertic populations investigated. tempo of phenotypic evolution our data are suggestive of a punctuated-equilibrium mode of evolution: long periods of relative phenotypic stasis, as might be exemplified by the thekla-like morphotype in widely divergent phylogenetic lineages can sometimes be followed by very rapid episodes of divergence, as a result of e.g. climate-driven adaptation in expanding lineages. for instance within senegallensis, morphological differences between populations north and south of the sahara, which exhibit only weak and non-significant genetic differentiation, are much larger than morphological differences found between valid species that belong to widely divergent species complexes. phenotypic divergence and speciation phylogeographic analyses of the thekla and especially, crested lark complexes reveal a now classical history of multiple fragmentations followed occasionally by the expansion of some refuge lineages that may lead to secondary contact zones between valid or incipient species. while incidental divergence during periods of isolation may contribute to the speciation process, it is unlikely to play a dominant role here, given the slow accumulation rate of post-zygotic incompatibilities in birds. conversely, rapid phenotypic divergence in response to local ecological conditions as demonstrated here could induce at least partial pre-zygotic isolation at secondary contact zones, particularly for such passerine birds for which body size and coloration play an important role in mate choice. in the single secondary contact zone studied to date, strong evidence of reproductive isolation was indeed found between cristata and randonii in morocco. role of biogeographic barriers the sahara very few studies have addressed the role of the sahara in driving speciation events. this is particularly true for the last four million years, a period which is characterized by the onset of arid climatic cycles that might have favoured speciation. our results suggest three different episodes of north-south vicariance during arid phases of late pliocene, mid-pleistocene and late pleistocene. rather than simple north-south vicariance between g. cristata and g. randonii, the star-like phylogenetic relationships among the three species of crested larks suggests that the divergence between g. cristata, g. randonii and g. malabarica occurred fairly simultaneously ~ mya. while g. malabarica and g. randonii are geographically restricted to, respectively, w coastal india and central maghreb, g. cristata is widely distributed in europe, asia and africa. however, a sub-saharan origin is likely for the latter given that the highest genetic diversity is found there. taken together, these elements suggests a simple speciation history in the crested lark species complex: an ancestral species was distributed in africa and asia, occupying part of the current range of the three species g. cristata, g. randonii and g. malabarica; the expansion of arabo-saharan deserts during an arid episode ~ mya might have led to the formation of three refuge species: g. malabarica in india, g. randonii in the maghreb and g. cristata in sub-saharan africa; while g. malabarica and g. randonii would have remained within the limits of their ancient refuge, g. cristata would have subsequently expanded its range from its sub-saharan refuge. in agreement with a previous report, our data are indeed consistent with two independent events of expansion from sub-saharan africa in g. cristata: the colonization of the whole eurasia from the eastern sahel refuge zone; and the colonization of the eastern maghreb from the western sahel refuge zone. a recent range expansion in cristata is indicated by significant fu's fs and the star-like phylogeny of haplotypes. since this taxon is tightly linked with human-modified habitats in parts of its range, guillaumet et al. proposed that the cristata expansion was facilitated by the development of agriculture that started around ten thousand years ago. when dating this expansion event by mismatch analysis we found a slightly higher but still concordant estimate. despite the non-significant negative fs value that we found for senegallensis, a recent expansion event in this group is suggested by the star-like phylogeny of haplotypes and a mismatch distribution that fitted the sudden expansion model. in fact, it is likely that the signal of expansion has been partly blurred by subsequent isolation and divergence without gene flow between populations n and s of the sahara, as suggested by mdiv results. we suggest that the maghreb was colonized from sub-saharan africa because g. cristata is believed to originate from sub-saharan africa. furthermore, in the case of a recent range expansion, ancestral haplotypes are more likely to be found close to the origin of expansion, whereas derived haplotypes are more likely to be found at the leading edge of range expansion. such a pattern fits the senegallensis haplotype network: two rare haplotypes found in sub-saharan africa tend to occupy an internal position in the network, while one clearly derived haplotype is only found in the maghreb. however, we emphasize that additional sampling in the maghreb and sub-saharan africa is needed to validate the sub-saharan origin of the senegallensis lineage. colonisation of the maghreb may have been favoured by steppe-like vegetation in the sahara, possibly during the most recent interglacial around kya. then, subsequent aridification of the sahara led to a contraction of saharan populations into a few refugia, particularly in mountain areas, that behave as relic populations. this scenario appears compatible with the estimated time of divergence for populations n and s of the sahara and with two other phylogeographic studies conducted in the lanner falcon falco biarmicus and in the honeybee apis mellifera, which also suggest genetic isolation of populations north and south of the sahara during arid phases. our results demonstrate that the sahara did not constitute a permanent barrier throughout geological times. the distribution of senegallensis is exemplary, since genetic data still hold the footprints of a recent expansion in the late pleistocene through the current sahara, followed by fragmentation and divergence without gene flow between a northern and a southern refuge, as a probable result of increasingly arid conditions. interestingly, maghreban populations of senegallensis also showed an accelerated rate of morphological evolution. galerida larks thus suggest to us a possible general mechanism by which glacial cycles affecting the sahara might promote speciation in birds and other taxa. e-w vicariance in the sahel zone the star-like phylogeny for the cristata, senegallensis and somaliensis lineages of g. cristata suggests once again that they result from a single vicariance event that may have occurred around kya. as we discussed above, g. cristata seems to originate from sub-saharan africa. accordingly, the divergence between senegallensis and cristata should correspond to e-w vicariance in the sahel zone, even if we cannot exclude the alternative that one or more lineage only secondarily invaded sub-saharan africa. interestingly, other e-w vicariance events were also evidenced in the sahel zone at similar dates in unrelated species such as the hartebeest, and the roan antelope. it has been suggested that such eastern and western lineages could have been isolated in refuges north and east of an expanding central african rainforest belt, during some periods of climate warming. alternatively, we suggest that refuge populations might instead have been isolated by the lake chad during such humid phases that led to lake "mega-chad" episodes. e-w vicariance in the maghreb g. theklae sensu stricto is structured in two distinct genetic groups for which the current repartition and divergence date obtained from mdiv analysis suggest a separation in two distinct maghreban refuges during the penultimate pleistocene glacial event around kya. since a recent expansion is strongly supported in theklae, and significantly negative fu's fs) we find likely that genetic exchanges detected by mdiv took place after the widespread western haplogroup theklae underwent an eastwards range expansion ~ kya, thus creating a zone of contact in e tunisia. however, we cannot totally exclude the possibility that the presence of one theklae haplotype in tunisia reflects incomplete lineage sorting instead of gene flow. the rift valley the eastern branch of the kenyan rift valley might induce some restrictions to gene flow between sub-saharan populations of g. cristata in the eastern sahel and kenya. in addition, our limited data on the former subspecies of g. theklae in ethiopia are indicative of a long-term genetic isolation between populations that live on highland steppes on either side of the rift valley. hence, these results agree with the observation that the rift valley constitutes a major zoogeographical barrier in mammals and birds, but also seems to impact the population genetics of species currently distributed on either side. systematics and endemism in africa the present study considerably challenges current taxonomic hypotheses in these two species complexes, and paves the way for future revisions. first, g. malabarica, an indian endemic taxon, turned out to be closely related to crested lark, and not to thekla lark as previously believed. hence, apparent disjunction in the distribution of the thekla lark "superspecies", which comprised three sets of populations separated by arabo-saharan deserts, proved here to be an artefact of incorrect taxonomy. interestingly however, the hypothesis of a fragmentation by arabo-saharan deserts of a widely distributed ancestral species was actually supported for the crested lark instead. secondly, we found a previously unsuspected deep break between thekla lark populations north and south of the sahara that occurred ~ mya. these two sets of populations lack diagnostic morphological as well as plumage color characteristics, which prevented them from being split by previous authors. however, as we have shown that phenotypic convergence is overwhelming in these taxa, it is now clear that phenotypes are not appropriate taxonomic clues. as these two sets of populations are currently separated by a very wide gap of sahara, we believe they should now be recognized as distinct allopatric species. in addition, the horn of africa seems to harbour several narrow-ranged endemic taxa: praetermissa appears to be endemic to the high plateaus of ethiopia north of the rift valley, hueii replaces it in similar habitats south of the rift valley, while ellioti is restricted to arid landscapes in somalia. these allopatric taxa might warrant recognition at the species level, but this conclusion is postponed here because of limited sample size for ellioti and hueii, and because this study did not include representatives of three other subspecies described from the horn of africa. the recognition of new narrow-ranged endemic taxa in the horn of africa might significantly supplement the current list of avian species endemic to ethiopia or somalia. interestingly, the only other savanna-dwelling avian species investigated to date, the ostrich, also revealed one previously unexpected endemic species in the horn of africa, suggesting that more cryptic species remain to be identified in this part of the world. this seems to be quite a general phenomenon in sub-saharan africa, where phylogeographical patterns are still poorly understood. indeed, wherever they are undertaken, molecular phylogenetic studies tend to reveal a more complex biogeographic history than previously recognized, and lead to the recognition of new species. such findings may have fundamental evolutionary implications for the understanding of large-scale speciation processes, but also for conservation strategies.  in this study, phylogenetic, population genetics and phenotypic data were integrated in a comparative framework to reconstruct the evolutionary history of two widespread sibling species complexes. in agreement with the climate-driven speciation hypothesis, both past and current climatic factors were found to be influential in driving speciation patterns, as shown respectively by recurrent vicariance mediated by the sahara, and multiple instances of convergence in morphology and plumage color. 
in particular, we tested whether: 1) vicariance diversification events are concomitant with past climatic events; and 2) current ecological factors contribute to phenotypic divergence between allopatric populations. in particular, our results suggested that desert-adapted plumage evolved at least three times and variation in body size was mainly driven by interspecific competition, but the response to competition was stronger in more arid areas. phenotypic variation did not correlate with the history of populations, but was strongly influenced by current ecological conditions. mitochondrial and nuclear sequence data indicated that the crested and thekla lark species groups diverged in the early pliocene and that subsequent speciation events were congruent with major late pliocene and pleistocene climatic events. climatic fluctuations of the plio-pleistocene strongly impacted diversification patterns in the galerida larks. in addition, since most studies focused on europe and north america, the influence of many other biogeographic barriers such as the sahara remains poorly understood. in particular, steep increase in aridity in africa near and million years ago were coincident with two north-south vicariance speciation events mediated by the sahara. firstly, we found that cladogenesis coincides with major climatic changes, and the sahara appears to have played a key role in driving speciation events. subsequent glacial cycles of the last million years seem to have shaped patterns of genetic variation within the two widespread species. the sahara appears to have allowed dispersal from the tropical areas during climatic optima but to have isolated populations north and south of it during more arid phases. in this paper, climate-driven diversification was investigated by using a comparative phylogeographic approach in combination with phenotypic data in two avian species groups distributed on both sides of the deserts belt of africa and asia.
 the major impact of plio-pleistocene climatic oscillations on the current genetic structure of many species is widely recognised but their importance in driving speciation remains a matter of controversies. in addition, since most studies focused on europe and north america, the influence of many other biogeographic barriers such as the sahara remains poorly understood. in this paper, climate-driven diversification was investigated by using a comparative phylogeographic approach in combination with phenotypic data in two avian species groups distributed on both sides of the deserts belt of africa and asia. in particular, we tested whether: 1) vicariance diversification events are concomitant with past climatic events; and 2) current ecological factors contribute to phenotypic divergence between allopatric populations.  mitochondrial and nuclear sequence data indicated that the crested and thekla lark species groups diverged in the early pliocene and that subsequent speciation events were congruent with major late pliocene and pleistocene climatic events. in particular, steep increase in aridity in africa near and million years ago were coincident with two north-south vicariance speciation events mediated by the sahara. subsequent glacial cycles of the last million years seem to have shaped patterns of genetic variation within the two widespread species. the sahara appears to have allowed dispersal from the tropical areas during climatic optima but to have isolated populations north and south of it during more arid phases. phenotypic variation did not correlate with the history of populations, but was strongly influenced by current ecological conditions. in particular, our results suggested that desert-adapted plumage evolved at least three times and variation in body size was mainly driven by interspecific competition, but the response to competition was stronger in more arid areas.  climatic fluctuations of the plio-pleistocene strongly impacted diversification patterns in the galerida larks. firstly, we found that cladogenesis coincides with major climatic changes, and the sahara appears to have played a key role in driving speciation events. secondly, we found that morphology and plumage were strongly determined by ecological factors following vicariance. 