Genetic signature of a north-to-south postglacial expansion

Within the southern third of the historic range of the system (Fig. 1) we investigated genetic diversity within and among populations for each of the three species, in order to test for recent southward range expansion of the symbiosis. The three taxa presented a consistent pattern of reduced within-population genetic diversity from north to south. This pattern was marked for gene diversity, H_E, variance of allele size, V, or both (Fig. 2). For the host plant, L a. africana, both measures of genetic diversity decreased strongly and significantly towards the postulated colonization front. For the mutualist ant P. phylax, clinal loss of diversity was significant for V only. However, the four very southernmost populations showed a strong decrease of H_E, suggesting founder effects. Finally, the ant parasitic on the mutualism, C. mckeyi, exhibited a strong and significant clinal decrease of H_E, while V values only presented a trend towards reduction southward. The global pattern of a north-to-south reduction in genetic diversity, congruent in all three species, is suggestive of a north-to-south colonization route.

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Figure 1. The study area.

A. Distribution of the host-plant Leonardoxa a. africana. B. Location of the 29 populations of trees and associated ants that were sampled.

https://doi.org/10.1371/journal.pone.0005377.g001

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Figure 2. Genetic signatures of southward range expansion in the three species association.

Geographic clines for three measures of within-population genetic variation at microsatellite loci of the host-plant Leonardoxa a. africana, the mutualistic ant Petalomyrmex phylax and the parasitic ant Cataulacus mckeyi.

https://doi.org/10.1371/journal.pone.0005377.g002

Genetic differentiation among populations was marked for each of the three taxa. The global multilocus F_ST values were 0.109 for L a. africana, 0.126 for P. phylax and 0.170 for C. mckeyi. Differentiation between pairs of populations was significant for most comparisons for each of the three taxa (after correction for multiple tests, 161 out of 171 pairwise F_ST values were significant for the host plant L. a. africana; 203 out of 210 for the mutualist ant P. phylax; and 73 out of 91 for the parasitic ant C. mckeyi). Indeed, F_ST values between populations located less than two kilometers apart often revealed significant genetic differentiation, and this for the three species. Finally, a significant genetic pattern of Isolation By Distance (IBD) was detected for each of the three taxa (all P<10⁻⁴; linear regression with respectively intercept/slope/r²: −0.16/0.025/0.264 for L a. africana, −0.20/0.033/0.31 for P. phylax and −0.32/0.052/0.469 for C. mckeyi). Thus, overall differentiation among populations was strong and congruent in all three species, even at short spatial distances, suggesting spatially limited dispersal.

As we had three repeats of the same history, we inferred a rough estimate of the age of the cline using the difference in behavior among microsatellite markers. The effect of stepwise mutations on population divergence at a locus is predicted to be negligible for a divergence time T≪1/μ and to become apparent for T≥1/μ [28]. Three loci for P. phylax (out of 12), and only one for L. a. africana (out of nine) and for C. mckeyi (out of 10) presented statistics of population differentiation based on allele size, R_ST, significantly higher from those based on allele identity, F_ST. Divergence among populations is thus probably recent, as stepwise mutation at microsatellite loci contributed to differentiation between populations for only five out of 31 loci. These loci were used to estimate upper bounds of the confidence intervals for dating colonization of the transect, and the other loci were used to estimate the lower bounds. Estimates of the time of divergence in numbers of generations, T, were of the same order of magnitude for the three taxa: 968≤T≪3,104 for P. phylax, 712≤T≪2,503 for C. mckeyi, and 619≤T≪2,774 for their host-plant. Age estimates of the cline, expressed in numbers of generations, are rather similar, although the plant presents much longer generation time than the ants. We suggest that because more polymorphic loci, presenting higher mutation rates than average, were selected during marker development, the calculated upper bounds of the ranges may be overestimates. Whether the homogeneity in the estimates results from a non-random choice of highly polymorphic microsatellites during marker development requires further studies.

Overall, the combination of clinal loss of genetic diversity coupled with a genetic pattern of IBD and with recent divergence times between populations for the plant as well as its associated ants, strongly support the hypothesis that the whole system experienced a progressive and relatively recent range expansion towards the south.

Multiple evidence for clinal variation in dispersal ability and the mutualism/parasitism continuum in the ants

We investigated variation of phenotypic traits implicated in species interactions that are likely to be selected during range expansion. For the plant we analyzed size at first reproduction and number of foliar nectaries. For the ants we analyzed investment in reproduction versus investment in colony survival, temporal dynamics of occupation of nest sites and variation in traits correlated with capacity to found new colonies.

(i) The ant-plant.

The host plant Leonardoxa a. africana showed no evidence of a shortened juvenile period towards the south (Table 1). Indeed, the probability of having flowered at least once was correlated strongly with size of trees, but not with spatial distance from the southernmost occurrence of the species, nor with the interaction between tree size and spatial distance. Note that results were similar when the non-significant interaction term was removed from the models. The estimated size at which half the sampled trees had reached sexual maturity varied slightly among populations (from 17 to 26 mm for basal trunk diameter and from 1.7 to 2.5 m for tree height; Table S2) but did not show clinal variation along the transect (basal trunk diameter: P = 0.872, r_S = −0.086, N = 6 populations; tree height: P = 0.913, r_S = 0.058, N = 6 populations).

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Table 1. No geographic cline in host-plant probability of having already flowered at a given size.

https://doi.org/10.1371/journal.pone.0005377.t001

The mean number of observed nectaries per leaf varied little among populations (from 11 to 14) and did not show clinal variation along the transect (P = 0.948; r_S = 0.021, N = 12 populations). Hence there was no evidence for a clinal variation in food resources available to ants (see Table S3 for details). The same result was found when we considered the mean number of nectaries per basal leaflet (P = 0.101; r_S = −0.497, N = 12 populations). Thus, this component of investment of trees in production of food for the mutualists seemed to be independent of geographic location along the north/south transect. Hence, we detected no evidence of change in the host plant's phenotype in response to the colonization process.

(ii) The plant-ants.

In both ant species, multiple regressions explaining the production of individuals of the different castes gave very similar results (Table 2). The main effect was that all parameters of colony productivity increased strongly with colony size. Further, we found for both ant species, P. phylax and C. mckeyi, significant and substantial partial correlations between distance to the southern edge of the distribution and production of both female sexuals and workers. Similar sized colonies produced more sexual females and fewer workers in the south than in the north. In contrast, male production remained constant. Hence our data showed an increasing numerical investment of colonies in female dispersal and a correlated decreasing investment in colony growth and maintenance towards the south for both ant species.

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Table 2. A geographic cline in ant colony production parameters.

https://doi.org/10.1371/journal.pone.0005377.t002

Capture-recapture models applied to change in species occupancy on marked trees over three to seven years indicated a negative correlation between distance to the southern limit of the cline and turnover of inhabitants in C. mckeyi-inhabited trees (P = 0.019; r_S = 0.886, N = 6 populations), but failed to detect clinal variation of turnover of inhabitants in P. phylax inhabited trees (P = 0.623; r_S = 0.257, N = 6 populations, see Table S4). We suggest that, in C. mckeyi, the increased resource allocation to reproduction towards the south, and the associated reduction in worker production, translate directly into reduced colony survival.

In addition to variation at the colony level, P. phylax exhibited traits that varied clinally along the transect at the individual level. These were traits associated with body size of female sexuals. Head size, body weight, and wing size of female sexuals of P. phylax increased from north to south (for all tests P<0.001; −0.964≤r_S≤−0.705; N = 21 populations; Fig. 3, see Table S5 for detailed data). Two geographically extreme populations of P. phylax, one at each end of the transect, EBO (the southernmost) and BOU (among the northernmost) were more thoroughly analyzed. Estimates of relative dry weight of adult females, but not that of adult males or workers, differed markedly between these two extreme populations (Table S6). The difference in female size was thus not a byproduct of a general north-to-south increase in ant size. Finally, the survival of P. phylax females after 60 days in conditions of claustral foundation (see Materials and methods) increased from north to south (P = 0.003; r_S = −0.827, N = 10 populations) (see Table S5), and was positively correlated with mean alate female dry weight per population (P = 0.021; r_S = 0.711, N = 10 populations). Thus, in P. phylax, size and weight of sexual females increased southward and translated into higher capacity to found new colonies.

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Figure 3. Body size of female sexuals of P. phylax increases southward.

Geographic cline of within-population variation (mean±standard deviation) of head length, a surrogate of queen size, measured over 1,036 individuals from 256 colonies in 21 populations of the ant P. phylax.

https://doi.org/10.1371/journal.pone.0005377.g003

Recent range expansion

In all three species we observed genetic isolation by distance and decreasing genetic diversity towards the south. This provides strong data supporting recent southward expansion of the association. Indeed, the three species are very tightly associated and their population-genetic patterns tell the same story. We aimed at estimating a rough age of population divergence using microsatellite data. Despite large differences in generation time between tree and ants, we obtained similar rough estimates of elapsed numbers of generations, which suggests a bias. Nevertheless, as stated above the bias is likely directional, so that numbers of generations are likely to be overestimated. This strongly suggests a postglacial expansion, as upper bound estimates of divergence times are hardly compatible with the hypothesis of an older, Pleistocene, expansion. Understanding the individual origin, age and phylogeographic history of currently interacting species is central to understanding the forces that have shaped community structure and species evolution [31]. The idea of using parasites (and mutualists) to help analyze host history [32] is validated in this case study.

During the late Pleistocene and the Holocene, the rainforest of Atlantic equatorial Africa experienced recurrent cycles of contraction and expansion, driven by changes in climate [see e.g. 33], [34], [35], [36], [37]. Two episodes of more humid and warmer conditions may have driven the expansion of the Leonardoxa system. The first one corresponds to the end of the last glacial maximum (12,000 years BP), and the second one followed an arid episode centered 3,000–2,500 years BP. The data on C. mckeyi with an estimate of divergence time along the transect ≪2,500 generations, and a colony mortality of at least 25% per year (Table S4), suggest that the cline originated after the more recent arid climate episode.

Although data and models of past vegetation suggest recent events of contraction-expansion of lowland rain forest in central Africa, the consequences of these climatic episodes on current within-species genetic structure have hardly been explored. Most studies in tropical Africa connecting genetic structuring with ORD have investigated afromontane species [38], [39], [40]. The very few data available for species of lowland rainforests such as shrews [41], mandrill [42] and the tree Aucoumea klaineana [43] suggest that climate-driven range expansion may be common in Atlantic equatorial Africa –including Cameroon. The present study provides congruent data for three species involved in symbiotic interactions that have shared a common history, and thus adds strong support to the controversial issue of the impact of past climatic oscillations on the current structuring of biodiversity in African lowland equatorial rain forests, supposedly one of the most stable ecosystems on earth [8].

Evolution of dispersal strategies during range expansion

A prediction about evolutionary consequences of colonization processes is that selection favors more dispersive strategies on the colonization front [12]. This evolutionary trend has been documented for a few species subjected to range shifts [e.g. 15], [16], [17], [18], but to our knowledge never at equatorial latitudes and never at such a small scale as in our three-species system. Petalomyrmex phylax exhibits a gradient of gyny (number of nestmate egg-laying queens per colony) along the transect, from populations exhibiting secondarily polygynous mature colonies in the northern part of the study area to populations in the south, only 86 km distant, presenting almost exclusively monogynous colonies [44]. Previous genetic analyses at the colony and population scales have shown that secondary polygyny in P. phylax mostly results from lack of queen dispersal associated with intra-nidal mating, whereas monogyny occurs through outbred queens dispersing and founding new colonies independently from the assistance of workers [44]. Dalecky et al. [44] have shown that the variation in social structure of P. phylax –along with its associated mating system– correlates with a historical process corresponding to a progressive colonization of coastal southern Cameroon. Gyny is one of several life history traits classically associated with dispersal in ants [e.g. 45]. In ants in general, a lower queen number is associated with larger queens as a result of their contrasted reproductive and dispersal strategies [e.g. 45]. We have documented here variation at the individual level in a series of other associated traits that are predicted to be selected for on a colonization front, including body size of P. phylax queens and a component of their capacity to found new colonies. It is noteworthy that clinal variation in body size was expressed in females but not in males and workers, which do not participate in foundation of new colonies. We note that the effect of wing load ( = body mass per unit wing area) variation on dispersal capability of P. phylax queens needs further study (see [45] p. 12). Furthermore, we observed very similar geographic clines for sexual investment at the colony level and an investment tradeoff between sexual reproduction and growth/maintenance in the two ant species, as well as a geographic cline in colony turnover in the parasitic ant C. mckeyi.

All these traits are predicted to be associated with selection for dispersal in ants, and we thus propose that we effectively observed a selected north-south gradient of vagility in both ant species. Such a gradient may result from range expansion, but it could alternatively result from variation in the ecological conditions encountered by the ants. Given that the habitat of these symbiotic ants is typically restricted to their host plant, we searched for variation in plant traits. However, the phenotypic homogeneity of Leonardoxa along the transect, in terms of life history strategy and number of extrafloral nectaries per leaf –a principal resource on which the ants feed– suggests that ant colonies benefit from equivalent resources all along the transect. Further, a careful search for indicators of variation in environmental characteristics that could be associated with disturbance and colony turnover, such as the amount of light reaching the forest floor, percentage of trees with trunks that had been broken by disturbances, or the availability of empty nest sites, did not identify any habitat characteristic that varied clinally from north to south [44]. Hence we propose that the best explanation available for the north-south variation in ant traits is selection for dispersive phenotypes on the colonization front. These dispersive traits seem to be lost very slowly. We therefore suggest that these traits may involve relatively limited selective costs. Furthermore, low gene flow among populations, as evidenced by the strong genetic isolation by distance, must have limited subsequent colonization by less dispersive genotypes.

The clines in adaptive traits we observe here concern species moving out from a postulated refugium zone in a biodiversity hotspot. Within hotspots, in areas of marked elevational relief, species or assemblages of species may move very slowly during climatic oscillations [46] so that selection for dispersal will be very limited. Our suggestion is that as soon as species move out of such buffered zones selection for dispersal during range expansion or range shift becomes an active force, a feature which can be expressed as within-species selection and as species sorting [9]. Indeed, a global analysis of Upper Guinean endemic forest species gives a pattern in which relatively widespread endemics are ruderal while species with very restricted ranges are linked to particularly humid places, i.e., probably refugia [47].

General conclusion

The progression of these species has most likely been slow. The distance between the northernmost historically documented populations and today's southern limit hardly exceeds 200 km. This should be compared with some temperate-zone species which have expanded over thousands of kilometers after the last glacial maximum. Although the three studied species may have moved extremely slowly, we have shown here that ORD, as a historical process, is highly relevant to understanding contemporary local biology of the symbiosis. The role of past, contemporary and future climate change in shaping the biology of species in general and of symbiotic interactions in particular should not be neglected.

Distribution of study system and population sampling

The distribution of the plant is restricted to a band, 250 km long and 80 km wide, of coastal rain forests of Cameroon, from southeast of the Cameroonian volcanic arc to near the border with Equatorial Guinea [48]. It is an understory plant of mature rainforests, where it grows in patchily distributed, well-defined stands of up to some hundreds of individuals, near rivers and wet sites [48], [49]. Given this natural history and the low water-holding capacity of the sandy to clayey-sandy soils of coastal rain forests [35], the distribution of the plant is predicted to be highly sensitive to fluctuations in water availability, rainfall, and seasonality, and must have been strongly affected by climate changes. On the basis of biogeography and a cladistic analysis of morphological characters in the L. africana complex, McKey [48] proposed that L. a. africana ‘colonized coastal forests, spreading southward’.

The northern part of the current distribution of L. a. africana has been strongly affected by agriculture and other human activities and there are few places where the species still survives. In contrast, in the south of its range L. a. africana is locally abundant, representing a characteristic element of the caesalpinioid-rich flora of coastal rainforest. This is compatible with the hypothesis that L. a. africana is such a slow colonizer that it has yet to reach the southern limit of its potential range. This would also be compatible with the very slow growth of the host plant, for which qualitative observations from marked trees followed for several years (1989–1997) suggest that they will not flower before reaching tens of years in age.

For this study 29 populations, constituting a roughly north-south transect, were sampled for genetic studies, phenotypic studies or both (Fig. 1, see also Table S1 for detailed coordinates). This transect covers the southern half of the range of L. a. africana, and the population EBO (near the village Ebodjé) is the southernmost known population of L. a. africana. Despite the abundance of the plant and its ant occupants in the vicinity of Ebodjé, neither field nor herbarium research led to the discovery of more southerly populations of L. a. africana. EBO was thus considered to represent the probable front of colonization of the system, and in the following all statistical treatments involving geographic distance along the transect are based on geographic distance (in km) from the southernmost documented occurrence of the three species (population EBO). Unless stated otherwise, to assess the existence of clinal variation of each measure, we tested for a correlation between the spatial distance to EBO and the mean values per population using Spearman rank correlation coefficient (r_S). Correlations were tested using the software Analyse-it version 2.12 (Analyse-it Software, Ltd. http://www.analyse-it.com/) or the VassarStats web site for statistical computation (http://faculty.vassar.edu/lowry/corr_rank.html).

Testing for recent range expansion

Several genetic features are expected to be signs of recent range expansion. During the period of colonization, each successive foundation of a new population induces a bottleneck, thus reducing genetic diversity progressively from the place of origin of the expansion towards the front of colonization [5], [50], [51]. Furthermore, repeated foundation events along a colonization front result in adjacent populations being genetically more similar than distant populations, a pattern termed isolation by distance (IBD) [51], [52]. The patchy distribution of populations of this symbiotic system should not only facilitate bottleneck effects, but also the appearance of a genetic pattern of IBD. Hence, to test the hypothesis that the whole system has undergone a recent range expansion, we investigated for each species: (1) the spatial pattern of genetic diversity; (2) the pattern of genetic differentiation between pairs of populations (IBD); and (3) the time elapsed since divergence of populations.

We investigated genetic variation in host plant, parasitic ant and mutualistic ant using nuclear microsatellite markers: (i) Genetic variation in L. a. africana was analyzed for 594 diploid individuals from 19 populations (mean = 31 individuals per population, range = 13–80) sampled in 2000–2007, and individual genotypes were inferred using 9 markers amplified following Léotard et al. [53]. (ii) Genetic variation in C. mckeyi was analyzed for 206 diploid individuals from 14 populations (mean = 15 individuals per population, range = 5–62, each from a different colony) sampled in 2000–2002, and genotypic data from 10 markers were already available for this sample [54]. (iii) Genetic variation in P. phylax was analyzed for 475 diploid individuals from 21 populations (mean = 23 individuals per population, range = 13–66, each from a different colony) sampled in 2000–2002, and genotypic data from 12 markers were already available for this sample [55]. Within species, all genetic markers were shown to be statistically independent with the program GENEPOP 3.3 [56].

Using the software SPAGeDi 1.1 [57], we computed for each taxon and population two estimators of diversity across loci, one based on allele identity, the expected heterozygosity H_E [58], and the other based on allele size, the variance of allele size V [59]. If the dynamics of return to equilibrium are driven by local mutations involving a stepwise mutation model, then V returns to equilibrium much more slowly than H_E. Thus V is expected to enable detection of older (in terms of numbers of generations) colonization processes than is H_E [59], [60]. Three other allele identity-based estimators of diversity (n_p, the number of polymorphic loci; n_a, the mean number of observed alleles; and A, the mean allelic richness using the rarefaction method to provide unbiased estimates corrected for unequal sample size [61]) were computed using the program FSTAT 2.9.3.2. [62] and their mean values across loci per population are presented as supporting information (Table S1).

For each species, genotypic population differentiation was analyzed for each pair of populations and over all populations by estimating F_ST values following Weir and Cockerham [63], and tested using exact tests with the program GENEPOP 3.3 [56]. Tests of IBD were performed over all populations by regressing multilocus pairwise estimates of F_ST/(1−F_ST) on pairwise ln-geographical distances [64], and tested with the Spearman rank correlation coefficient using Mantel-like tests based on 10,000 permutations of locations with SPAGeDi.

In an attempt to produce a rough estimate of the age of the cline, we tried to estimate the mutation rate for each locus and then used the hypothesis of Hardy et al. [28] that the effect of stepwise mutations on population divergence will be negligible (R_ST = F_ST) for a divergence time T≪1/μ generations and will become apparent (R_ST>F_ST) for T≥1/μ generations. First, the mutation rate at locus i was estimated as [65], where the average mutation rate of microsatellite loci, , was set at 5×10⁻⁴ [e.g. 66], V_i was the observed average variance of allele size at locus i over all populations and the average population mean (across loci) of the observed variance of allele size. Then to assess whether stepwise mutation at microsatellite loci contributed to differentiation between populations, statistics of population differentiation based on allele identity (F_ST) and based on allele size (R_ST) were compared following Hardy et al. [28], using SPAGeDi. Finally, for each species, we averaged the rate of mutation of the loci presenting a significant difference between R_ST and F_ST to compute a rough lower bound of the time elapsed since the divergence of populations, and we used the average rate of mutation of the other loci to compute a rough upper bound of this time.

Testing for evolution of life history traits in the host-plant

Some north-temperate plants are known to reproduce earlier in life in sites in the northern part of their range, far from glacial refugia [19]. This is interpreted as the result of selection for dispersal on the colonization front. To test whether Leonardoxa reached sexual maturity at progressively smaller size when getting closer to the postulated colonization front we measured, for a total of 402 trees in six populations (41 to 126 trees per population), the diameter at the trunk base (in mm) and estimated the total height (in m). We then scored the stage of maturity (immature, i.e., that had never flowered yet, or mature, i.e., that had already flowered) of each individual. Indeed Leonardoxa a. africana is cauliflorous and the inflorescences leave detectable scars on the trunk, so that each tree could be categorized as immature versus mature. The relationship between the stage of maturity and geographical distance to the colonization front, controlling for the effect of tree size (measured either as diameter at the trunk base or as total height), was assessed using multiple logistic regression analysis with the program SYSTAT 9. Size of sampled trees and geographical distance were slightly correlated (all P<10⁻³; diameter: r_S = −0.346, height: r_S = −0.271, N = 402 trees), so we included an interaction term in the statistical model. For each population sample separately we then used multiple logistic regressions to estimate the size at which half the trees had reached sexual maturity and tested whether these estimates varied clinally.

To assess spatial variation in a main component of food resources provided by the plants to ant colonies, we counted the number of nectaries per leaf for 1,293 leaves (4–11 leaves per tree) from a total of 161 trees occupied by P. phylax in 12 populations (10–30 trees per population) in 2000–2002. L. a. africana possesses paripinnate leaves with four to eight leaflets. For this analysis missing leaflets were thus considered as bearing no nectaries. This analysis provides an estimate of this component of actual resources per leaf provided by the trees to the ants. In a separate analysis of the dataset, we quantified the mean number of nectaries produced per basal leaflet and we considered missing leaflets as missing data. This analysis provides an estimate of the potential, ontogenetic, investment of the plants in production of food per leaf for ant colonies. We only used the basal leaflets because the number of nectaries varies according to leaflet position, basal leaflets bear the greatest number of nectaries, and basal leaflets are less often missing than those in other positions.

Testing for evolution of life history traits in the ants