BACKGROUND
the eastern arc and coastal forests of east africa have been identified as a biodiversity hotspot: an area featuring exceptional concentrations of endemic species and experiencing severe habitat loss  <cit> .
the extensive botanical endemism and richness is paralleled in several animal groups  <cit>  and has been especially well documented for birds  <cit> .
the eastern arc mountains consists of  <dig> sky islands, which form a chain  of uplifted fault blocks extending from the taita hills in the northeast to the udzungwa mountains in the southwest .
these mountains are under direct climatic influence from the indian ocean and reach an altitude of  <dig> m   <cit> .
typically montane forest occurs above  <dig> m, but there is no sharp change in turnover of arboreal species, and the composition of the botanical communities depend on the inclination of the terrain, rainfall, distance from the coast, height and incidence of cloud cover  <cit> .
at lower elevations , the eastern slopes typically sharply grade into savanna or lowland rainforest characteristic of the coastal forests of the littoral plain of africa that runs from somalia to mozambique.
the drier western and northwestern slopes typically support deciduous woodland at lower elevations.
extensive field research within the eastern arc has taken place over the past  <dig> years which has led to the description of several new species and the development of novel hypotheses concerning patterns of differentiation among species and populations distributed in east africa  <cit> .
despite this accumulating body of knowledge, it still remains to be determined to what extent populations inhabiting montane forest interact with populations distributed in adjacent foothills and along a narrow ribbon of lowland forest abutting the coast of eastern africa .
the tiny greenbul  represents the only described bird species within the eastern arc/coastal forest mosaic, which is polytypic across an altitudinal gradient .
the more common pattern is for polytypic taxa to be distributed as a series of allopatric populations restricted to specific sky islands.
three subspecies are currently recognized  <cit> : albigula in the usambara mountains  as well as in the nguru mts  <dig> km further inland , debilis in the coastal forest zone of southeastern tanzania, mozambique and eastern zimbabwe, and rabai which extends from near dares salaam  north along the coast into kenya, and inland along riverine corridors to the foothill forests od the the uluguru, nguu and nguru mts .
subspecies albigula is the most distinctive form, the top of the head being green and not grey as in debilis and rabai.
unlike the other forms albigula is only found above  <dig> m, mainly in mature forest, and extends up to  <dig> m in the nguru mts  <cit>  and to  <dig> m in the west usambara mts.
this suggests that the green-headed montane birds represent two separate populations  nested inside the geographical range of a more widespread lowland, grey-headed, form.
we therefore have to consider the possibility that two ecologically segregated species are involved.
however, the existence of some greenish feather edges on the nape and crown of some lowland birds could lead to the alternate interpretation that although gene flow occurs, plumage is under directional selection, with greener plumage being favored in the wetter high-altitude habitats.
whether disruptive selection across an altitudinal gradient can lead to parapatric speciation is hotly debated, as it requires natural selection to be sufficiently strong to counteract the effects of gene flow .
although several studies have provided evidence of the importance of natural selection for the formation of new species even when gene flow occurs  <cit> , research that connects presumably adaptive variation in traits  with assessments of the phylogenetic relationships among populations/taxa, extent of genetic variation and gene flow are relatively few  <cit> .
thus, a careful analysis of morphological variation and phylogeographic structure within the tiny greenbul is likely to provide further insight to this debate.
we contend that if populations from the same habitat  are genetically and morphologically more closely related to each other than to population from the other habitat, we would expect historical isolation and secondary contact between the lowland and montane populations to be the prevailing hypothesis for the current contact zone.
in contrast, if disruptive selection has played a major role in driving phenotypic divergence across the altitudinal gradient, then the two populations of the montane form  should be phenotypically more similar despite being genetically closer to their nearest geographically lowland forest population.
in this paper we make use of a multi-locus approach to test the above hypotheses.
RESULTS
morphological variation
birds from the lowland coastal forests of east africa , subspecies rabai, are generally small  with a mouse grey crown, pale grey underparts with bright yellow streaks  contrasting with a very pale grey throat and extensive pale yellow belly.
this description also applies to birds from mt.
kanga , the nguu mts  and the uluguru foothills .
specimens of the montane subspecies albigula from the usambara  and nguru mts  are generally large .
birds of both sexes from the two montane populations of albigula do not differ significantly from each other in any measured morphometric trait , but do differ from lowland birds, with lowland birds being significantly smaller .
the first two principal components  had eigenvalues greater than one and accounted for  <dig> % of the total variation .
principal component loadings on pc <dig> were positive and strongly correlated with weight.
loadings on pc <dig> were positive and strongly correlated with wing- and tail-length; bill- and tarsus-length loaded evenly on both pc <dig> and pc <dig>  in agreement with the bivariate results above, the scatterplot  suggests that montane populations of debilis  are separable from lowland populations  in morphospace, particularly along pc <dig> .
in plumage characters, the dark olive-green color of the back of albigula extends to the crown or even upper forehead.
however, in some individuals the crown feathers are grey with green lateral edges, giving a streaked appearance.
the birds have a distinctive dark grey breast and flanks  and even the throat and chin are rather grey  leaving only a narrow area on the central belly white.
yellow streaks are variably developed but generally weak and there is always a bright yellow axillary patch which, together with the yellow wing-lining, stands out conspicuously.
thus, the most prominent plumage difference between the rabai and albigula specimens is in the color of the underparts rather than the crown as traditionally described.
one bird from mt.
kanga collected at  <dig> m , a locality within the range of lowland rabai, resembles individuals from the adjacent nguru mts  in plumage and size .
the zmuc specimen  from lindi  we sequenced in this study resembled specimens from lowland populations further north on the tanzanian coast  in both plumage and size .
only one specimen from zimbabwe  could be compared directly with tanzanian specimens, and differs from the lindi specimen and most of the other rabai specimens only in having a faint greenish tinge on the crown.
the description in keith et al.
<cit>  suggests that plumage and size differences between rabai and debilis are slight.
in conclusion, there exist significant biometric differences between the lowland  and montane  populations in tanzania .
the differences in shape are also coupled with discrete differences in the color of the underparts.
genetic analyses: geographic structure
mitochondrial data
we obtained the complete nd <dig> sequence  for  <dig> individuals.
for some museum samples, we were only able to gather partial sequences.
these partial sequences were very similar to the corresponding fragments obtained from individuals collected at the same locality; however, we did not include individuals with partial sequences in our final analyses.
all sequences had an open reading frame, with no insertions, deletions or unexpected stop codons.
within p. debilis,  <dig> sites were variable, delineating  <dig> haplotypes .
results of the macdonald-kreitman test when comparing the p. debilis  sequences with two of its closest relatives indicate no consistent evidence of selection , although one comparison is marginally significant.
we attribute this to homoplasy as a result of the long divergence time between p. debilis and its closest relatives.
further, comparing the two primary clades  with each other indicated that no selection is acting on the mtdna locus within p. debilis .
both the maximum likelihood  and bayesian inference  analyses, performed using a hky+Γ model, recovered two primary clades ; one clade includes birds collected in the lowland forests of tanzania  and mozambique/zimbabwe  and the other in the montane forests of tanzania  .
birds from the lowland forests are further subdivided into two clades, one restricted to tanzania and one distributed in mozambique/zimbabwe .
we only found three mismatches between subspecies designation and our assignment of individuals to a particular clade.
the three se tanzanian individuals we sampled , which according to the accepted taxonomy, should be associated with the mozambique/zimbabwe lowland populations , were placed together with the more northerly tanzanian lowland populations .
in all subsequent analyses, we considered the individuals sampled in lindi, litipo and pindiro forests as being part of the subspecies rabai and different from the subspecies debilis, which we considered restricted to mozambique and zimbabwe.
in the absence of selection, significant negative values of fu's fs and low values of r <dig> are indicative of population expansion .
for the three nuclear loci, only alleles with phase probabilities greater than  <dig>  were included in the analyses.
the subspecies rabai includes all individuals from lowland tanzania .
greater genetic diversity was observed in the tanzanian lowland clade  than in the montane clade  .
the mozambique/zimbabwe clade is further divided into two subclades  that differ from each other by a minimum of nine substitutions.
one individual, collected in vimba , possesses a mtdna haplotype that is nested within the mozambique subclade .
the three primary clades  could not be connected at the 95% level in the tcs haplotype network .
samples from the two isolated mountains where albigula occurs were segregated genetically; haplotypes from the nguru and usambara mts were reciprocally monophyletic in the ml tree  and tcs network, but not in the 50% majority rule consensus tree resulting from the bayesian analyses, where the nguru haplotypes were paraphyletic with respect to the usambara haplotypes .
we only found evidence for isolation by distance in the tanzanian clade  .
autosomal intron data
the hka test did not detect any evidence of selection between the two autosomal data sets , nor between the autosomal and sex-linked locus .
these results indicate that none of the nuclear introns we sequenced are under selection, or that the selection regime does not differ among them.
selection on introns has recently been highlighted for birds in one locus often used in phylogenetic and phylogeographic studies  <cit> , but even in such rare cases, selective appears to only act on a very few sites .
fgb
we obtained the complete fgb intron- <dig> sequence for  <dig> individuals .
the allelic phase of six individuals  could not be resolved at the  <dig>  pp threshold, even when we incorporated partial sequences from museum specimens.
these six individuals were excluded from further analyses; this resulted in the loss of eight snps, all only present in one out of  <dig> possible copies).
in the final data set, sixteen alleles were found in the lowland group and three in the montane group.
sharing of haplotypes between the lowland and montane population was very limited, involving only one individual: rabai zmuc <dig> from mt.
kanga, with one allele assigned to albigula - morphologically this is a typical rabai specimen.
one further bird from mt.
kanga  resembles albigula in morphology but shared snps with both albigula and rabai .
the alleles within the lowland group  were inter-mixed.
the results of the amova indicated significant structuring of the genetic variability when partitioning by subspecies , with most of the molecular variability being found among the three subspecies .
gapdh
we obtained the complete gapdh intron- <dig> sequence for  <dig> individuals .
one individual  had a three base pair deletion and another  had a single base pair insertion.
we considered the three base pair deletion to be a single mutational event.
eight individuals  could not be phased at the threshold pp of  <dig>  and were excluded from further analyses .
we also excluded the individual which had the autapomorphic deletion of three base pairs due to difficulty in determining the allelic phase.
most of the alleles were shared among the different subspecies .
yet, the amova indicated significant structuring of genetic variability when partitioned by subspecies , with most of the molecular variability being found within the three subspecies .
z-linked locus
we obtained complete brm intron- <dig> sequences from  <dig> individuals .
twelve snps remained in the data set  after excluding the single individual  that could not be satisfactorily phased.
no alleles were shared between albigula and debilis/rabai .
amova indicated significant structuring of genetic variability when the dataset was partitioned by subspecies , with most of the molecular variability being found among the three groups .
structure analysis of the nuclear data set
structure analyses performed on the nuclear data set, revealed two primary genotype clusters  =  <dig> , k = 2), corresponding to the lowland  and montane  mtdna clades .
five intermediate genotypes, with the lowest probability assignment to one or other clade varying from  <dig>  to  <dig> , were sampled from the east usambara mts , nguru mts , and on mt.
kanga .
all the other individuals were assigned to either the montane or lowland clades with a posterior probability greater than  <dig> .
at k =  <dig>  the lowland group is divided in two populations with all individuals being of mixed ancestry .
at k =  <dig>  the pattern is very similar to the one observed at k =  <dig> and k =  <dig>  but the montane group is now considered to be an admixture of two populations, which roughly correspond to the nguru and usambara populations.
multi-locus network and species tree analyses
the multi-locus network derived from the combined analyses of the mitochondrial and nuclear loci recovered: 1) a clear separation of the montane  and lowland  clades, and 2) a greater extent of genetic diversity within the lowland clade relative to the montane clade .
the topology we obtained from the species tree analyses based on the coalescent approach  and information from the mitochondrial and nuclear loci recovered a pattern where the lowland subspecies  are sister to the montane subspecies , although support for the monophyly of the lowland group is limited .
the analyses performed using the species delimitation rjmcmc algorithm implemented in bpp v <dig>  and a ) guide tree indicates that all models visited support at least two lineages: albigula and debilis/rabai ; this result was not dependent on the assumption of a large or small effective population size or a shallow or deep divergence as all prior combinations resulted in the same posterior probability.
a speciation probability greater than  <dig>  was recovered for the node leading to debilis and rabai in three of the four prior combinations; the prior combination of small effective population size and deep divergence resulted in a speciation probability of  <dig>  for debilis and rabai.
genetic analyses: divergence time estimates
divergence date estimates using the mitochondrial neutral mutation rate of the four-fold degenerated sites were quite variable : from a  <dig>  myrs  estimate for the tmrca of debilis to  <dig>  myrs  for the divergence between the lowland  and montane clades .
the divergence between the lowland lineages in tanzania  and mozambique/zimbabwe  is estimated to have occurred about  <dig>  myrs ago .
the estimates obtained using a strict molecular clock hypothesis and the  <dig> %/myr- <dig> rate were very similar to the dates obtained using the neutral four-fold mutation rate and the 95% hpd were largely overlapping.
in contrast, the divergence times obtained using the traditional  <dig> %/myr- <dig> clock were much older with only slight overlap with the 95% hpd obtained with the other rates .
values in brackets are the 95% hpd.
tmrca were estimated using an uncorrelated lognormal molecular clock model, coalescent prior with constant population size and a mean rate of evolution for the four-fold degenerated sites of  <dig>  s/s/myr ;  <dig> sites were included in the analyses.
genetic analyses: fluctuation in population size
we found evidence of population expansion in the mitochondrial data set for the tanzanian lowland rabai , although the r <dig> test was not significant .
likewise, several significant fu's fs or r <dig> values were found for the nuclear data .
we observed several cases of discrepancies between the two tests, which may be due to differences in power  <cit> .
given our sampling of haplotypes , it is likely that fu's fs will have more power to detect population growth than r <dig>  <cit> .
finally given the difference in the test's significance across data sets and the number of segregating sites per locus , it is very likely that the demographic expansions, when present, were moderate and not sudden  <cit> .
the bayesian skyline plot of the mitochondrial data set for the subspecies rabai also supports a population expansion, a three-fold increase in effective population, but the increase was continuous and not sudden .
the bayesian skyline plot for the subspecies albigula did not show any evidence of population expansion .
genetic analyses: coalescent analyses under the isolation with migration model
the sbp and gard algorithms did not detect any evidence of recombination in the three nuclear loci.
hence we used the complete sequence for the ima analyses.
appropriate mixing and satisfactory effective sampled sizes were achieved using  <dig> chains and a geometric heating scheme  for all parameters except t , where a non-zero probability tail was observed.
hence, for t, we considered the highest point of the posterior distribution to be the most reliable estimate.
values represent the mean of the distribution and the 90% hpd confidence interval.
the posterior distribution of the parameter t possesses a non-zero probability distribution when t increases; the value represents the highest posterior estimate.
for this reason we do not discuss the t value from the isolation with migration analyses in the text.
the estimates for the t parameters are given in million years before present.
for the comparison between the montane populations of albigula from the usambara and nguru mts, some models that assume no gene flow could not be rejected .
for the comparison between the tanzanian lowland and montane clades , all models that assume no gene flow and asymmetrical gene flow, and all models that assume equal effective population sizes in the two extant lineages were strongly rejected .
to determine if gene flow between rabai and albigula was primarily occurring on mt.
kanga as suggested by the structure assignments and morphological data, we conducted a separate ima analysis with the individuals from mt.
kanga excluded.
with birds from mt.
kanga excluded we could no longer reject zero gene flow taking place between rabai and albigula .
this result is consistent with secondary contact and apparent introgression occurring at mid-altitude on mt.
kanga.
for the comparison between the lowland populations from mozambique/zimbabwe  and tanzania , models that assume equal population sizes  were all rejected .
models that assume no gene flow were all rejected, although one was only marginally rejected .
hence, we are unable to decisively exclude the possibility that zero gene flow is occurring between mozambique/zimbabwe and the tanzanian lowland populations.
the coalescent analyses between the tanzanian montane clade  and the mozambique/zimbabwe lowland clade  was not performed because one of the ima assumptions, that is, no gene flow is occurring with an unsampled lineage , was not satisfied.
the highest posterior estimate for the divergence times between populations pairs were very similar or identical to the tmrca we obtained using the mitochondrial data set and the neutral four-fold and  <dig> %/myr- <dig> rate .
bioclimatic data
the principal component analysis performed on the bioclimatic data revealed that the lowland  and montane  localities could primarily be separated by the level of seasonality.
the first two axes explain  <dig> % of the variability .
sites from the usambara mts appear to be very heterogeneous in their distribution .
all sites where gene flow was detected between albigula and rabai are in geographical proximity to each other  and appear to have reduced seasonality, suggesting that this climatic variable may provide a key environmental context under which gene flow among the two lineages may take place.
discussion
secondary contact versus disruptive selection on an altitudinal gradient?
birds from the montane forests of the nguru and usambara mts  represent a distinct clade in the molecular analysis and are also readily distinguishable by their grey underparts.
in addition, albigula is generally larger and individuals tend to have a green rather than grey crown, the later being more characteristic of lowland birds , although some rabai individuals do have some greenish streaks on the nape and crown.
birds of the rabai clade are found in foothill forests at a few hundred meters elevation in the east usambara mts, in the tanzanian coastal lowlands, as well as on mt.
kanga , which rises steeply from the lowlands and is only  <dig> km to the east of the nguru mts, where albigula occurs .
our structure analyses revealed that gene flow or introgression has occurred at three localities: on mt.
kanga, in the foothills forests of the east usambara mts  and in the nguru mts.
however, mt.
kanga is at present the only locality where we have sampled an individual with a genotype/phenotype mismatch and excluding individuals from this locality from the isolation with migration analyses had strong impact on the conclusion concerning gene flow between albigula and rabai.
in the nguu mts to the north of the nguru mts but partly connected by several small hills, rabai is found up to  <dig> m in kilindi forest and to  <dig> m in derema forest.
this results in the montane nguru population of albigula being surrounded to both the north and east by the predominantly lowland rabai.
thus, it appears that rabai is able to ascend into the submontane zone, except where this habitat is already occupied by albigula, which suggests that two mutually exclusive and competing taxa may be involved.
it is noteworthy that birds representing the two clades can be found in close proximity to each other.
we found some evidence of population expansion for rabai , suggesting that the current contact between the two taxa  may be secondary, centered in areas of transitional habitat with low seasonality between montane and lowland forest and not the result of disruptive selection on an altitudinal gradient.
the secondary contact zone hypothesis between once allopatric populations has also been favored by several other studies that have addressed this topic .
our coalescent analyses under the isolation with migration model did allow us to reject the hypothesis of zero gene flow between the lowland  and highland  populations.
yet, when individuals from mt.
kanga were excluded from the analyses, the hypothesis of zero gene flow could no longer be rejected.
this pattern suggests that limited gene flow between albigula and rabai takes place on mt.
kanga.
given that the lowland population likely experienced a demographic expansion, it is likely that mt.
kanga represents a zone of secondary contact and not the initial area where the two species diverged.
consequently, recurrent gene flow appears to be restricted in space to areas of intermediate habitat.
the isolation with migration model implemented in ima has several assumptions that may be violated in empirical data sets, including: no intralocus recombination, no gene flow with an unsampled species/population, no linkage among loci, no selection and no population structure in the ancestral population.
here, we can reasonably reject recombination, selection and linkage among the sampled loci as potential biases.
gene flow with an unsampled species is unlikely, as the tiny greenbul has no close extant relatives  <cit> .
ancestral population structure is thought to have little effect on parameters estimates  <cit> .
it has been shown through simulation study that using an over-simplified substitution model increases the variance in some parameter estimates  <cit> .
the most complex substitution model implemented in ima is hky, which is very close to the best-fit model selected for our data set .
thus, we consider our estimates obtained with the isolation with migration model to not be strongly affected by currently described biases.
given that the two lineages are well differentiated genetically and morphologically, we do not regard limited gene flow at 1- <dig> localities of intermediate habitat as a sufficient reason to reject species rank for the montane clade.
indeed, hybridization and introgression between 'good species' is common, especially for neutral loci and does not preclude them from being genetically distinct.
.
hence, based on the molecular, morphological and altitudinal distribution patterns, we suggest that the taxa albigula and debilis  be considered distinct species.
genetic structure among the highlands
the nguru and usambara mts are separated by a substantial 100- <dig> km lowland gap of dry savannah.
as these two sky islands hold the only two populations of albigula, it is important to know if gene flow is occurring between the two populations.
our coalescent-based analyses could not exclude the hypothesis of zero gene flow between the two populations.
our result is consistent with phylogeographic studies of other eastern arc mountain birds where the usambara-nguru gap has consistently been recovered as the major phylogeographic break among clades .
in contrast to the pattern of strong genetic structuring in birds, and several other vertebrates  <cit> , weak genetic differentiation  was found between two populations of a frog, arthroleptis xenodactylus, from the usambara and nguru mts  <cit> .
these results are not easy to compare as these organisms have different dispersal capacities, yet it is surprising that several montane bird lineages appear to be more geographically structured than the single frog study published to date.
however, one striking pattern among all of the above studies remains that, vertebrate populations from the northern  and central mountains  are genetically differentiated from each other across the usambara-nguru lowland gap.
yet, only a few of the estimated divergence times  are compatible with each other, suggesting several periods where gene flow was interrupted among species from the same community.
hence, whereas the pleistocene climatic oscillations likely constrained the distribution of mountain birds in the eastern arc due to aridification, the response of individual species to these climatic fluctuations appears to have been varied, suggesting that a model assuming a single 'common' vicariant event is unlikely  <cit> .
genetic structure in the tanzanian lowland
birds from lindi district, located on the coast in se tanzania are more closely related to the northern tanzanian-kenyan lowland populations, than to the central mozambique and zimbabwe populations, as traditionally thought.
the coastal zone of northern mozambique is fairly dry, and there may be a significant gap in the distribution of this species.
recent surveys  in ne mozambique did not detect the species
our analyses revealed substantial genetic structure and genetic diversity in the lowland clade, as inferred from the mean genetic distance among haplotypes and nucleotide diversity.
part of this genetic diversity may be explained by isolation-by-distance.
yet, we also observed substantial mitochondrial differentiation within some localities .
this would either imply that the distribution of the lowland forest population has remained stable and large for a considerable period of time, or that these localities represent areas of secondary contact after population expansion from different lowland refugia.
there is evidence in our data set for a signature of population expansion in the rabai clade, with individuals sampled from inland localities  primarily having haplotypes shared or derived from coastal populations.
this pattern would be consistent with constant and large population size through time  in coastal forests and a signature of population expansion towards the interior.
the validation of this hypothesis would require further sampling from within the distributional range of rabai, which is challenging due to many coastal forests having been transformed by humans.
patterns of genetic differentiation in mozambique/zimbabwe
our analyses indicate that populations from mozambique/zimbabwe are genetically differentiated from the lowland populations in tanzania .
dating analyses using the mitochondrial data set indicate that the two lineages  diverged about  <dig> mya, using the neutral four-fold rate and 6%/myr clocks, or  <dig>  mya using the more conventional 2%/myr mitochondrial clock.
the divergence time obtained using the four-fold degenerated rate and 6%/myr rate corresponds to a peak of aridification in africa, a consequence of glaciation at higher latitudes  <cit> .
this aridification peak, together with the general drying of africa since the miocene, may have altered the distribution of coastal lowland forest in northern mozambique, thus promoting the divergence between the mozambique/zimbabwe and tanzanian clades of tiny greenbul.
the pattern of disjunct populations distributed in lowland tanzania and zimbabwe/mozambique has been observed for some other vertebrate species, although zimbabwe is often not sampled for species associated with densely wooded habitats.
moodley and bruford  <cit>  found some differentiation of populations of bushbuck  in this region, with diverged about  <dig>  <dig> years ago.
our analyses revealed the existence of two well-differentiated mitochondrial lineages within the mozambique/zimbabwe clade.
the first clade consists of individuals collected in mozambique  and zimbabwe .
the second clade only includes individuals collected in zimbabwe .
thus, that one individual from vimba  has a mitochondrial haplotype characteristic of the mozambique clade is intriguing, especially since the two mitochondrial lineages are rather divergent.
through several checks in the laboratory and because the collector visited vimba five years later than central mozambique , we can rule out sample mix-up or contamination.
therefore, the most plausible explanation is recent gene flow from central mozambique to vimba.
this result also supports the fact that seasonality of the sites may play an important role in facilitating gene flow, as vimba is the site with the lowest seasonality.
we emphasise that further sampling in this area is needed to confirm this result.
CONCLUSIONS
our study suggests that the single case of altitudinal morphological segregation within a species in the eastern arc mountains is likely the result of secondary contact of two highly differentiated lineages instead of disruptive selection across an altitudinal gradient.
our analyses support three main lineages within phyllastrephus debilis sensu lato and stress the recognition of two species of 'tiny greenbul', p. albigula restricted to the nguru and usambara mts, and p. debilis distributed in the tanzanian lowland forest  and in mozambique/zimbabwe .
introgression of some alleles  appears to have occurred at three different localities , in mid-altitude areas with a transition between foothill and montane forest, especially in areas which experience low seasonality.
the extent of this introgression is limited and appears to be the result of the population expansion of the coastal rabai clade further inland.
the pattern of genetic divergence we found for the montane clade  is similar to what has been described in other vertebrates for mtdna , although our results from the nuclear genome emphasize the need for more studies from east africa to include a diverse set of markers in phylogeographic studies.
greater genetic variability was recovered in the tanzanian lowland clade, suggesting a greater long-term effective population size or the existence of distinct refugia in the past, which have now merged as lowland populations expanded.

 the eastern arc and coastal forests of east africa have been identified as a biodiversity hotspot: an area featuring exceptional concentrations of endemic species and experiencing severe habitat loss. the extensive botanical endemism and richness is paralleled in several animal groups and has been especially well documented for birds. the eastern arc mountains consists of sky islands, which form a chain of uplifted fault blocks extending from the taita hills in the northeast to the udzungwa mountains in the southwest. these mountains are under direct climatic influence from the indian ocean and reach an altitude of m. typically montane forest occurs above m, but there is no sharp change in turnover of arboreal species, and the composition of the botanical communities depend on the inclination of the terrain, rainfall, distance from the coast, height and incidence of cloud cover. at lower elevations, the eastern slopes typically sharply grade into savanna or lowland rainforest characteristic of the coastal forests of the littoral plain of africa that runs from somalia to mozambique. the drier western and northwestern slopes typically support deciduous woodland at lower elevations. extensive field research within the eastern arc has taken place over the past years which has led to the description of several new species and the development of novel hypotheses concerning patterns of differentiation among species and populations distributed in east africa. despite this accumulating body of knowledge, it still remains to be determined to what extent populations inhabiting montane forest interact with populations distributed in adjacent foothills and along a narrow ribbon of lowland forest abutting the coast of eastern africa. the tiny greenbul represents the only described bird species within the eastern arc/coastal forest mosaic, which is polytypic across an altitudinal gradient. the more common pattern is for polytypic taxa to be distributed as a series of allopatric populations restricted to specific sky islands. three subspecies are currently recognized: albigula in the usambara mountains as well as in the nguru mts km further inland, debilis in the coastal forest zone of southeastern tanzania, mozambique and eastern zimbabwe, and rabai which extends from near dares salaam north along the coast into kenya, and inland along riverine corridors to the foothill forests od the the uluguru, nguu and nguru mts. subspecies albigula is the most distinctive form, the top of the head being green and not grey as in debilis and rabai. unlike the other forms albigula is only found above m, mainly in mature forest, and extends up to m in the nguru mts and to m in the west usambara mts. this suggests that the green-headed montane birds represent two separate populations nested inside the geographical range of a more widespread lowland, grey-headed, form. we therefore have to consider the possibility that two ecologically segregated species are involved. however, the existence of some greenish feather edges on the nape and crown of some lowland birds could lead to the alternate interpretation that although gene flow occurs, plumage is under directional selection, with greener plumage being favored in the wetter high-altitude habitats. whether disruptive selection across an altitudinal gradient can lead to parapatric speciation is hotly debated, as it requires natural selection to be sufficiently strong to counteract the effects of gene flow. although several studies have provided evidence of the importance of natural selection for the formation of new species even when gene flow occurs, research that connects presumably adaptive variation in traits with assessments of the phylogenetic relationships among populations/taxa, extent of genetic variation and gene flow are relatively few. thus, a careful analysis of morphological variation and phylogeographic structure within the tiny greenbul is likely to provide further insight to this debate. we contend that if populations from the same habitat are genetically and morphologically more closely related to each other than to population from the other habitat, we would expect historical isolation and secondary contact between the lowland and montane populations to be the prevailing hypothesis for the current contact zone. in contrast, if disruptive selection has played a major role in driving phenotypic divergence across the altitudinal gradient, then the two populations of the montane form should be phenotypically more similar despite being genetically closer to their nearest geographically lowland forest population. in this paper we make use of a multi-locus approach to test the above hypotheses.  morphological variation birds from the lowland coastal forests of east africa, subspecies rabai, are generally small with a mouse grey crown, pale grey underparts with bright yellow streaks contrasting with a very pale grey throat and extensive pale yellow belly. this description also applies to birds from mt. kanga, the nguu mts and the uluguru foothills. specimens of the montane subspecies albigula from the usambara and nguru mts are generally large. birds of both sexes from the two montane populations of albigula do not differ significantly from each other in any measured morphometric trait, but do differ from lowland birds, with lowland birds being significantly smaller. the first two principal components had eigenvalues greater than one and accounted for % of the total variation. principal component loadings on pc were positive and strongly correlated with weight. loadings on pc were positive and strongly correlated with wing- and tail-length; bill- and tarsus-length loaded evenly on both pc and pc in agreement with the bivariate results above, the scatterplot suggests that montane populations of debilis are separable from lowland populations in morphospace, particularly along pc. in plumage characters, the dark olive-green color of the back of albigula extends to the crown or even upper forehead. however, in some individuals the crown feathers are grey with green lateral edges, giving a streaked appearance. the birds have a distinctive dark grey breast and flanks and even the throat and chin are rather grey leaving only a narrow area on the central belly white. yellow streaks are variably developed but generally weak and there is always a bright yellow axillary patch which, together with the yellow wing-lining, stands out conspicuously. thus, the most prominent plumage difference between the rabai and albigula specimens is in the color of the underparts rather than the crown as traditionally described. one bird from mt. kanga collected at m, a locality within the range of lowland rabai, resembles individuals from the adjacent nguru mts in plumage and size. the zmuc specimen from lindi we sequenced in this study resembled specimens from lowland populations further north on the tanzanian coast in both plumage and size. only one specimen from zimbabwe could be compared directly with tanzanian specimens, and differs from the lindi specimen and most of the other rabai specimens only in having a faint greenish tinge on the crown. the description in keith et al. suggests that plumage and size differences between rabai and debilis are slight. in conclusion, there exist significant biometric differences between the lowland and montane populations in tanzania. the differences in shape are also coupled with discrete differences in the color of the underparts. genetic analyses: geographic structure mitochondrial data we obtained the complete nd sequence for individuals. for some museum samples, we were only able to gather partial sequences. these partial sequences were very similar to the corresponding fragments obtained from individuals collected at the same locality; however, we did not include individuals with partial sequences in our final analyses. all sequences had an open reading frame, with no insertions, deletions or unexpected stop codons. within p. debilis, sites were variable, delineating haplotypes. results of the macdonald-kreitman test when comparing the p. debilis sequences with two of its closest relatives indicate no consistent evidence of selection, although one comparison is marginally significant. we attribute this to homoplasy as a result of the long divergence time between p. debilis and its closest relatives. further, comparing the two primary clades with each other indicated that no selection is acting on the mtdna locus within p. debilis. both the maximum likelihood and bayesian inference analyses, performed using a hky+Γ model, recovered two primary clades; one clade includes birds collected in the lowland forests of tanzania and mozambique/zimbabwe and the other in the montane forests of tanzania. birds from the lowland forests are further subdivided into two clades, one restricted to tanzania and one distributed in mozambique/zimbabwe. we only found three mismatches between subspecies designation and our assignment of individuals to a particular clade. the three se tanzanian individuals we sampled, which according to the accepted taxonomy, should be associated with the mozambique/zimbabwe lowland populations, were placed together with the more northerly tanzanian lowland populations. in all subsequent analyses, we considered the individuals sampled in lindi, litipo and pindiro forests as being part of the subspecies rabai and different from the subspecies debilis, which we considered restricted to mozambique and zimbabwe. in the absence of selection, significant negative values of fu's fs and low values of r are indicative of population expansion. for the three nuclear loci, only alleles with phase probabilities greater than were included in the analyses. the subspecies rabai includes all individuals from lowland tanzania. greater genetic diversity was observed in the tanzanian lowland clade than in the montane clade. the mozambique/zimbabwe clade is further divided into two subclades that differ from each other by a minimum of nine substitutions. one individual, collected in vimba, possesses a mtdna haplotype that is nested within the mozambique subclade. the three primary clades could not be connected at the 95% level in the tcs haplotype network. samples from the two isolated mountains where albigula occurs were segregated genetically; haplotypes from the nguru and usambara mts were reciprocally monophyletic in the ml tree and tcs network, but not in the 50% majority rule consensus tree resulting from the bayesian analyses, where the nguru haplotypes were paraphyletic with respect to the usambara haplotypes. we only found evidence for isolation by distance in the tanzanian clade. autosomal intron data the hka test did not detect any evidence of selection between the two autosomal data sets, nor between the autosomal and sex-linked locus. these results indicate that none of the nuclear introns we sequenced are under selection, or that the selection regime does not differ among them. selection on introns has recently been highlighted for birds in one locus often used in phylogenetic and phylogeographic studies, but even in such rare cases, selective appears to only act on a very few sites. fgb we obtained the complete fgb intron- sequence for individuals. the allelic phase of six individuals could not be resolved at the pp threshold, even when we incorporated partial sequences from museum specimens. these six individuals were excluded from further analyses; this resulted in the loss of eight snps, all only present in one out of possible copies). in the final data set, sixteen alleles were found in the lowland group and three in the montane group. sharing of haplotypes between the lowland and montane population was very limited, involving only one individual: rabai zmuc from mt. kanga, with one allele assigned to albigula - morphologically this is a typical rabai specimen. one further bird from mt. kanga resembles albigula in morphology but shared snps with both albigula and rabai. the alleles within the lowland group were inter-mixed. the results of the amova indicated significant structuring of the genetic variability when partitioning by subspecies, with most of the molecular variability being found among the three subspecies. gapdh we obtained the complete gapdh intron- sequence for individuals. one individual had a three base pair deletion and another had a single base pair insertion. we considered the three base pair deletion to be a single mutational event. eight individuals could not be phased at the threshold pp of and were excluded from further analyses. we also excluded the individual which had the autapomorphic deletion of three base pairs due to difficulty in determining the allelic phase. most of the alleles were shared among the different subspecies. yet, the amova indicated significant structuring of genetic variability when partitioned by subspecies, with most of the molecular variability being found within the three subspecies. z-linked locus we obtained complete brm intron- sequences from individuals. twelve snps remained in the data set after excluding the single individual that could not be satisfactorily phased. no alleles were shared between albigula and debilis/rabai. amova indicated significant structuring of genetic variability when the dataset was partitioned by subspecies, with most of the molecular variability being found among the three groups. structure analysis of the nuclear data set structure analyses performed on the nuclear data set, revealed two primary genotype clusters =, k = 2), corresponding to the lowland and montane mtdna clades. five intermediate genotypes, with the lowest probability assignment to one or other clade varying from to, were sampled from the east usambara mts, nguru mts, and on mt. kanga. all the other individuals were assigned to either the montane or lowland clades with a posterior probability greater than. at k = the lowland group is divided in two populations with all individuals being of mixed ancestry. at k = the pattern is very similar to the one observed at k = and k = but the montane group is now considered to be an admixture of two populations, which roughly correspond to the nguru and usambara populations. multi-locus network and species tree analyses the multi-locus network derived from the combined analyses of the mitochondrial and nuclear loci recovered: 1) a clear separation of the montane and lowland clades, and 2) a greater extent of genetic diversity within the lowland clade relative to the montane clade. the topology we obtained from the species tree analyses based on the coalescent approach and information from the mitochondrial and nuclear loci recovered a pattern where the lowland subspecies are sister to the montane subspecies, although support for the monophyly of the lowland group is limited. the analyses performed using the species delimitation rjmcmc algorithm implemented in bpp v and a ) guide tree indicates that all models visited support at least two lineages: albigula and debilis/rabai; this result was not dependent on the assumption of a large or small effective population size or a shallow or deep divergence as all prior combinations resulted in the same posterior probability. a speciation probability greater than was recovered for the node leading to debilis and rabai in three of the four prior combinations; the prior combination of small effective population size and deep divergence resulted in a speciation probability of for debilis and rabai. genetic analyses: divergence time estimates divergence date estimates using the mitochondrial neutral mutation rate of the four-fold degenerated sites were quite variable: from a myrs estimate for the tmrca of debilis to myrs for the divergence between the lowland and montane clades. the divergence between the lowland lineages in tanzania and mozambique/zimbabwe is estimated to have occurred about myrs ago. the estimates obtained using a strict molecular clock hypothesis and the %/myr- rate were very similar to the dates obtained using the neutral four-fold mutation rate and the 95% hpd were largely overlapping. in contrast, the divergence times obtained using the traditional %/myr- clock were much older with only slight overlap with the 95% hpd obtained with the other rates. values in brackets are the 95% hpd. tmrca were estimated using an uncorrelated lognormal molecular clock model, coalescent prior with constant population size and a mean rate of evolution for the four-fold degenerated sites of s/s/myr; sites were included in the analyses. genetic analyses: fluctuation in population size we found evidence of population expansion in the mitochondrial data set for the tanzanian lowland rabai, although the r test was not significant. likewise, several significant fu's fs or r values were found for the nuclear data. we observed several cases of discrepancies between the two tests, which may be due to differences in power. given our sampling of haplotypes, it is likely that fu's fs will have more power to detect population growth than r. finally given the difference in the test's significance across data sets and the number of segregating sites per locus, it is very likely that the demographic expansions, when present, were moderate and not sudden. the bayesian skyline plot of the mitochondrial data set for the subspecies rabai also supports a population expansion, a three-fold increase in effective population, but the increase was continuous and not sudden. the bayesian skyline plot for the subspecies albigula did not show any evidence of population expansion. genetic analyses: coalescent analyses under the isolation with migration model the sbp and gard algorithms did not detect any evidence of recombination in the three nuclear loci. hence we used the complete sequence for the ima analyses. appropriate mixing and satisfactory effective sampled sizes were achieved using chains and a geometric heating scheme for all parameters except t, where a non-zero probability tail was observed. hence, for t, we considered the highest point of the posterior distribution to be the most reliable estimate. values represent the mean of the distribution and the 90% hpd confidence interval. the posterior distribution of the parameter t possesses a non-zero probability distribution when t increases; the value represents the highest posterior estimate. for this reason we do not discuss the t value from the isolation with migration analyses in the text. the estimates for the t parameters are given in million years before present. for the comparison between the montane populations of albigula from the usambara and nguru mts, some models that assume no gene flow could not be rejected. for the comparison between the tanzanian lowland and montane clades, all models that assume no gene flow and asymmetrical gene flow, and all models that assume equal effective population sizes in the two extant lineages were strongly rejected. to determine if gene flow between rabai and albigula was primarily occurring on mt. kanga as suggested by the structure assignments and morphological data, we conducted a separate ima analysis with the individuals from mt. kanga excluded. with birds from mt. kanga excluded we could no longer reject zero gene flow taking place between rabai and albigula. this result is consistent with secondary contact and apparent introgression occurring at mid-altitude on mt. kanga. for the comparison between the lowland populations from mozambique/zimbabwe and tanzania, models that assume equal population sizes were all rejected. models that assume no gene flow were all rejected, although one was only marginally rejected. hence, we are unable to decisively exclude the possibility that zero gene flow is occurring between mozambique/zimbabwe and the tanzanian lowland populations. the coalescent analyses between the tanzanian montane clade and the mozambique/zimbabwe lowland clade was not performed because one of the ima assumptions, that is, no gene flow is occurring with an unsampled lineage, was not satisfied. the highest posterior estimate for the divergence times between populations pairs were very similar or identical to the tmrca we obtained using the mitochondrial data set and the neutral four-fold and %/myr- rate. bioclimatic data the principal component analysis performed on the bioclimatic data revealed that the lowland and montane localities could primarily be separated by the level of seasonality. the first two axes explain % of the variability. sites from the usambara mts appear to be very heterogeneous in their distribution. all sites where gene flow was detected between albigula and rabai are in geographical proximity to each other and appear to have reduced seasonality, suggesting that this climatic variable may provide a key environmental context under which gene flow among the two lineages may take place. discussion secondary contact versus disruptive selection on an altitudinal gradient? birds from the montane forests of the nguru and usambara mts represent a distinct clade in the molecular analysis and are also readily distinguishable by their grey underparts. in addition, albigula is generally larger and individuals tend to have a green rather than grey crown, the later being more characteristic of lowland birds, although some rabai individuals do have some greenish streaks on the nape and crown. birds of the rabai clade are found in foothill forests at a few hundred meters elevation in the east usambara mts, in the tanzanian coastal lowlands, as well as on mt. kanga, which rises steeply from the lowlands and is only km to the east of the nguru mts, where albigula occurs. our structure analyses revealed that gene flow or introgression has occurred at three localities: on mt. kanga, in the foothills forests of the east usambara mts and in the nguru mts. however, mt. kanga is at present the only locality where we have sampled an individual with a genotype/phenotype mismatch and excluding individuals from this locality from the isolation with migration analyses had strong impact on the conclusion concerning gene flow between albigula and rabai. in the nguu mts to the north of the nguru mts but partly connected by several small hills, rabai is found up to m in kilindi forest and to m in derema forest. this results in the montane nguru population of albigula being surrounded to both the north and east by the predominantly lowland rabai. thus, it appears that rabai is able to ascend into the submontane zone, except where this habitat is already occupied by albigula, which suggests that two mutually exclusive and competing taxa may be involved. it is noteworthy that birds representing the two clades can be found in close proximity to each other. we found some evidence of population expansion for rabai, suggesting that the current contact between the two taxa may be secondary, centered in areas of transitional habitat with low seasonality between montane and lowland forest and not the result of disruptive selection on an altitudinal gradient. the secondary contact zone hypothesis between once allopatric populations has also been favored by several other studies that have addressed this topic. our coalescent analyses under the isolation with migration model did allow us to reject the hypothesis of zero gene flow between the lowland and highland populations. yet, when individuals from mt. kanga were excluded from the analyses, the hypothesis of zero gene flow could no longer be rejected. this pattern suggests that limited gene flow between albigula and rabai takes place on mt. kanga. given that the lowland population likely experienced a demographic expansion, it is likely that mt. kanga represents a zone of secondary contact and not the initial area where the two species diverged. consequently, recurrent gene flow appears to be restricted in space to areas of intermediate habitat. the isolation with migration model implemented in ima has several assumptions that may be violated in empirical data sets, including: no intralocus recombination, no gene flow with an unsampled species/population, no linkage among loci, no selection and no population structure in the ancestral population. here, we can reasonably reject recombination, selection and linkage among the sampled loci as potential biases. gene flow with an unsampled species is unlikely, as the tiny greenbul has no close extant relatives. ancestral population structure is thought to have little effect on parameters estimates. it has been shown through simulation study that using an over-simplified substitution model increases the variance in some parameter estimates. the most complex substitution model implemented in ima is hky, which is very close to the best-fit model selected for our data set. thus, we consider our estimates obtained with the isolation with migration model to not be strongly affected by currently described biases. given that the two lineages are well differentiated genetically and morphologically, we do not regard limited gene flow at 1- localities of intermediate habitat as a sufficient reason to reject species rank for the montane clade. indeed, hybridization and introgression between'good species' is common, especially for neutral loci and does not preclude them from being genetically distinct.. hence, based on the molecular, morphological and altitudinal distribution patterns, we suggest that the taxa albigula and debilis be considered distinct species. genetic structure among the highlands the nguru and usambara mts are separated by a substantial 100- km lowland gap of dry savannah. as these two sky islands hold the only two populations of albigula, it is important to know if gene flow is occurring between the two populations. our coalescent-based analyses could not exclude the hypothesis of zero gene flow between the two populations. our result is consistent with phylogeographic studies of other eastern arc mountain birds where the usambara-nguru gap has consistently been recovered as the major phylogeographic break among clades. in contrast to the pattern of strong genetic structuring in birds, and several other vertebrates, weak genetic differentiation was found between two populations of a frog, arthroleptis xenodactylus, from the usambara and nguru mts. these results are not easy to compare as these organisms have different dispersal capacities, yet it is surprising that several montane bird lineages appear to be more geographically structured than the single frog study published to date. however, one striking pattern among all of the above studies remains that, vertebrate populations from the northern and central mountains are genetically differentiated from each other across the usambara-nguru lowland gap. yet, only a few of the estimated divergence times are compatible with each other, suggesting several periods where gene flow was interrupted among species from the same community. hence, whereas the pleistocene climatic oscillations likely constrained the distribution of mountain birds in the eastern arc due to aridification, the response of individual species to these climatic fluctuations appears to have been varied, suggesting that a model assuming a single'common' vicariant event is unlikely. genetic structure in the tanzanian lowland birds from lindi district, located on the coast in se tanzania are more closely related to the northern tanzanian-kenyan lowland populations, than to the central mozambique and zimbabwe populations, as traditionally thought. the coastal zone of northern mozambique is fairly dry, and there may be a significant gap in the distribution of this species. recent surveys in ne mozambique did not detect the species our analyses revealed substantial genetic structure and genetic diversity in the lowland clade, as inferred from the mean genetic distance among haplotypes and nucleotide diversity. part of this genetic diversity may be explained by isolation-by-distance. yet, we also observed substantial mitochondrial differentiation within some localities. this would either imply that the distribution of the lowland forest population has remained stable and large for a considerable period of time, or that these localities represent areas of secondary contact after population expansion from different lowland refugia. there is evidence in our data set for a signature of population expansion in the rabai clade, with individuals sampled from inland localities primarily having haplotypes shared or derived from coastal populations. this pattern would be consistent with constant and large population size through time in coastal forests and a signature of population expansion towards the interior. the validation of this hypothesis would require further sampling from within the distributional range of rabai, which is challenging due to many coastal forests having been transformed by humans. patterns of genetic differentiation in mozambique/zimbabwe our analyses indicate that populations from mozambique/zimbabwe are genetically differentiated from the lowland populations in tanzania. dating analyses using the mitochondrial data set indicate that the two lineages diverged about mya, using the neutral four-fold rate and 6%/myr clocks, or mya using the more conventional 2%/myr mitochondrial clock. the divergence time obtained using the four-fold degenerated rate and 6%/myr rate corresponds to a peak of aridification in africa, a consequence of glaciation at higher latitudes. this aridification peak, together with the general drying of africa since the miocene, may have altered the distribution of coastal lowland forest in northern mozambique, thus promoting the divergence between the mozambique/zimbabwe and tanzanian clades of tiny greenbul. the pattern of disjunct populations distributed in lowland tanzania and zimbabwe/mozambique has been observed for some other vertebrate species, although zimbabwe is often not sampled for species associated with densely wooded habitats. moodley and bruford found some differentiation of populations of bushbuck in this region, with diverged about years ago. our analyses revealed the existence of two well-differentiated mitochondrial lineages within the mozambique/zimbabwe clade. the first clade consists of individuals collected in mozambique and zimbabwe. the second clade only includes individuals collected in zimbabwe. thus, that one individual from vimba has a mitochondrial haplotype characteristic of the mozambique clade is intriguing, especially since the two mitochondrial lineages are rather divergent. through several checks in the laboratory and because the collector visited vimba five years later than central mozambique, we can rule out sample mix-up or contamination. therefore, the most plausible explanation is recent gene flow from central mozambique to vimba. this result also supports the fact that seasonality of the sites may play an important role in facilitating gene flow, as vimba is the site with the lowest seasonality. we emphasise that further sampling in this area is needed to confirm this result.  our study suggests that the single case of altitudinal morphological segregation within a species in the eastern arc mountains is likely the result of secondary contact of two highly differentiated lineages instead of disruptive selection across an altitudinal gradient. our analyses support three main lineages within phyllastrephus debilis sensu lato and stress the recognition of two species of'tiny greenbul', p. albigula restricted to the nguru and usambara mts, and p. debilis distributed in the tanzanian lowland forest and in mozambique/zimbabwe. introgression of some alleles appears to have occurred at three different localities, in mid-altitude areas with a transition between foothill and montane forest, especially in areas which experience low seasonality. the extent of this introgression is limited and appears to be the result of the population expansion of the coastal rabai clade further inland. the pattern of genetic divergence we found for the montane clade is similar to what has been described in other vertebrates for mtdna, although our results from the nuclear genome emphasize the need for more studies from east africa to include a diverse set of markers in phylogeographic studies. greater genetic variability was recovered in the tanzanian lowland clade, suggesting a greater long-term effective population size or the existence of distinct refugia in the past, which have now merged as lowland populations expanded. 
the tiny greenbul represents the only described bird species within the eastern arc/coastal forest mosaic, which is polytypic across an altitudinal gradient: the subspecies albigula is distributed in the montane usambara and nguru mountains whereas the subspecies rabai is found in tanzanian lowland and foothill forest. the differences in shape are coupled with discrete differences in the coloration of the underparts. our coalescent analyses suggest that limited gene flow, mostly from the subspecies rabai to albigula, is taking place at three mid-altitude localities, where lowland and montane rainforest directly abut. we found significant biometric differences between the lowland and montane populations in tanzania. using a combination of morphological and genetic data, we aim to establish if the pattern of morphological differentiation in the tiny greenbul is the result of disruptive selection along an altitudinal gradient or a consequence of secondary contact following population expansion of two differentiated lineages. the clear altitudinal segregation in morphology found within the tiny greenbul is the result of secondary contact of two highly differentiated lineages rather than disruptive selection in plumage pattern across an altitudinal gradient. the eastern arc mountains of africa have become one of the focal systems with which to explore the patterns and mechanisms of diversification among montane species and populations. one unresolved question is the extent to which populations inhabiting montane forest interact with those of adjacent lowland forest abutting the coast of eastern africa. based on our results, we recommend albigula be elevated to species rank.
 the eastern arc mountains of africa have become one of the focal systems with which to explore the patterns and mechanisms of diversification among montane species and populations. one unresolved question is the extent to which populations inhabiting montane forest interact with those of adjacent lowland forest abutting the coast of eastern africa. the tiny greenbul represents the only described bird species within the eastern arc/coastal forest mosaic, which is polytypic across an altitudinal gradient: the subspecies albigula is distributed in the montane usambara and nguru mountains whereas the subspecies rabai is found in tanzanian lowland and foothill forest. using a combination of morphological and genetic data, we aim to establish if the pattern of morphological differentiation in the tiny greenbul is the result of disruptive selection along an altitudinal gradient or a consequence of secondary contact following population expansion of two differentiated lineages.  we found significant biometric differences between the lowland and montane populations in tanzania. the differences in shape are coupled with discrete differences in the coloration of the underparts. using multi-locus data gathered from individuals, we show that lowland and montane birds form two distinct genetic lineages. the divergence between the two forms occurred between and myrs ago. our coalescent analyses suggest that limited gene flow, mostly from the subspecies rabai to albigula, is taking place at three mid-altitude localities, where lowland and montane rainforest directly abut. the extent of this introgression appears to be limited and is likely a consequence of the recent expansion of rabai further inland.  the clear altitudinal segregation in morphology found within the tiny greenbul is the result of secondary contact of two highly differentiated lineages rather than disruptive selection in plumage pattern across an altitudinal gradient. based on our results, we recommend albigula be elevated to species rank. 