Introduction

Speciation by natural selection occurs mainly through two distinct mechanisms; mutation-order and ecological speciation [1,2,3] (Fig 1). Mutation-order speciation occurs when distinct advantageous or neutral mutations are fixed by chance between different populations/entities, while these are under similar ecological conditions or selective pressures [1,3]. The fixation of such mutations by drift, reduces the fitness of hybrids over evolutionary time scales [4]. On the other hand, ecological speciation refers to the evolution of reproductive isolation between species primarily by differential adaptation to distinct environmental or ecological conditions [5,6,7]. In this context, natural selection acts as a divergent mechanism driving to fixation distinct advantageous mutations in each of the different environments [1,2]. Whether ecological divergence may constitute the primary force inducing speciation or simply acts secondarily after an interruption of gene flow due to other causes may be difficult to ascertain in practice by monitoring the ecological traits of clades in a given phylogeny [1]. However, the appraisal of ecological divergence can sometimes serve as a proxy to support species delimitation to understand which ecologically associated factors led to speciation (e.g. [8,9,10,11,12]).

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Fig 1. Illustration of mutation-order and ecological speciation.

In mutation-order speciation, there is the initial fixation of advantageous mutations in the population, which will increase in frequency, leading to the genetic isolation of species in the same ecological niche. Ecological speciation occurs when new environmental conditions appear in the population with the adaptation of some individuals to this novel state and divergence of their niche and consequent genetic diversification from the original population.

https://doi.org/10.1371/journal.pone.0127980.g001

During ecological divergence, natural populations are subjected to different biotic and abiotic factors, such as predation, competition, climate and food resource fluctuations [1,5], which may lead to distinct evolutionary responses and patterns of climatic tolerances (reviewed in [13]) and to the consequent evolution of organisms in new habitats (niche divergence) (e.g. [8,14,15,16,17]). On the contrary, niche conservatism consists of the preservation of ecological similarity among populations over time [18,19], being important in the classic model of allopatric speciation, as it may limit adaptation to the ecological conditions at the geographic barrier and promote genetic isolation and differentiation between vicariant populations (e.g. [15,16,20,21]) (Fig 1). Hence, the assessment of whether the realized niche of natural populations has evolved in a conservative [18,19,20,22] or divergent manner [1,2,5,23] is of paramount importance, in order to evaluate the possibility of an ecological speciation event having occurred.

Not surprisingly, the study of niche evolution has increased in popularity in the last few years, focusing on techniques to estimate overlap in realized niches of different species in an explicit spatial context [24], either based on ordination methods [25,26] or on the output of species distribution models (SDMs) [27,28]. More recently, based on the methodology proposed by Evans et al. [29], SDMs are being combined with calibrated phylogenies in order to study the evolution of realized niches by reconstructing the ancestral environmental tolerances among clades (e.g. [30,31,32]).

The Moorish gecko, Tarentola mauritanica (Linnaeus, 1758), is a species-complex distributed across the Mediterranean Basin, comprising six mitochondrial evolutionary clades, with well-defined geographic ranges (Fig 2); Clade I is present in Central and Southwestern Morocco; Clade II is exclusively Iberian [33]; Clade III is present in Southern Europe and North Africa; Clade IV occupies Central Morocco; T. angustimentalis, endemic to the Eastern Canary Islands (Clade V) lies within the T. mauritanica complex; and, finally, Clade VI is distributed across Southern Iberia, Northeastern Morocco and Northwestern Algeria [33,34,35,36,37]. A recent study by Rato et al. [34] estimated that the oldest cladogenesis event within this group occurred around 5.88 Mya while the most recent one took place 2.47 Mya [34] (Fig 2). These six groups appear to represent several closely related cryptic clades exhibiting high levels of mtDNA diversity, but with nuclear phylogenies demonstrating limited evidence of monophyly due to incomplete lineage sorting [34,37]. However, the paraphyly of T. mauritanica, as currently recognized, together with the obtained high levels of mtDNA divergence, indicate that a taxonomic revision is needed [34]. Surprisingly, low levels of mtDNA variation within the European/North African clade have been observed, when compared to the others. This was initially attributed to a recent colonization [33,36,37], particularly given the frequency with which T. mauritanica is associated with humanized environments, leading to anthropogenic introductions [38]. However, recent molecular investigation of this pattern revealed that nuclear loci do not show this lack of variation and, in fact, they exhibit higher nucleotide diversity values compared to the used mtDNA markers [39]. This is consistent to a process where selection (most likely positive selection) is acting only on the mitochondrial variation of this clade, increasing the frequency of one allele and causing a drop in the overall variability for this marker [39,40]. Similar findings have been reported for pikas [41], monkeys [42] and even teleost fishes [43], where positive selection of the mtDNA has led to an increased capacity to inhabit environments characterized by low temperatures. A similar scenario could be hypothesized for the European/North African clade, which occurs in colder environments. In this study, we aim to: (1) evaluate if fundamental niche evolution is concordant with the currently accepted clades; (2) assess the degree to which phylogenetic proximity predicts niche conservatism (i.e. similarity in environmental tolerances); and (3) test if there is a climatic tolerance divergence of the European/North African clade, with respect to the remaining ones.

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Fig 2. Map illustrating the occurrence records of all six clades of Tarentola mauritanica used for the SDMs and a calibrated phylogenetic tree with node ages (in Ma), modified from Rato et al. [34].

The tree was obtained using concatenated nuclear (ACM4, MC1R, PDC, and RAG2) and mtDNA (12S rRNA and 16S rRNA) loci. Calibration was performed with the software BEAST [87], considering the mean substitution rates and the standard error of the mean values for 12S, extracted from a fully calibrated phylogeny of Tarentola from the Canary Islands [70,88].

https://doi.org/10.1371/journal.pone.0127980.g002

To achieve these goals, we used ordination methods and SDMs to estimate niche differences and similarities between the clades of T. mauritanica. Subsequently, the evolutionary history of niche occupancy of each clade of T. mauritanica was reconstructed by merging the SDMs with a published calibrated multilocus phylogeny of the genus Tarentola [34].

Materials and Methods

Results

Species distribution models (SDMs)

The dimensionality of the 30 variables listed in S1 Table was reduced into four principal components, with the first two explaining 83.15% of the total variance (PC1 explaining 71% and PC2 12% of the total variance). As for PC1, most of the variation along the axis was explained by 21 variables (|r| ≥ 0.8) pertaining to different temporal transformations of humidity (Middle Infra-Red), Land Surface Temperature, Normalised Difference Vegetation Index (NDVI) and Enhanced Vegetation Index (EVI), while PC2 was mainly associated with seasonality (X4) and annual range (X7) of the Middle Infra-Red and by the seasonality of the Land Surface Temperature (X13), (for details see S1 Table).

Visual inspection of the potential occurrence of each clade in space (Fig 3) showed that for some of them, the areas of high probability of occurrence are quite similar to their currently known distribution (Clades III and V; see details in Fig 2). In contrast, for the South/Central Moroccan clade (Clade I), the Iberian (Clade II), the Central Moroccan clade (Clade IV) and the North African Southern Iberian clade (Clade VI), the SDMs predicted a much larger area of potential occurrence. Specifically, for Clade I suitable conditions occur in Northern Maghreb and the Southeastern portion of the Iberian Peninsula; Clade II potentially ranged from the Iberian Peninsula all the way to the Balkans and Northern Maghreb; Clade IV also potentially included the Northern border between Morocco and Algeria and Northeastern Tunisia; and Clade VI potentially covered the entire Western Mediterranean region. It is noteworthy that the European/North African clade (Clade III) was mostly restricted to Mediterranean environments, being predicted to occur primarily near the coast. The same holds true for Clade VI.

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Fig 3. Species distribution models (SDMs) for the six clades of Tarentola mauritanica.

Warmer colours indicate higher environmental suitability.

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Realized niche overlap analyses

Results from PCA-env analyses demonstrated that for all pairwise comparisons among clades, the sum of the first two axes of the PCA explained between 65.97% and 77.05% of the total variance. This information, as well as the contribution of the 30 variables to each PC for all pairs of clades compared, is illustrated in more detail in S1 Fig. Regarding the pairwise assessment of niche overlap using the metric D [57] (Table 1), this measure varied between 0.03 (Clade III—Clade IV) and 0.43 (Clade III—Clade VI), corresponding to indices of no or very limited overlap and moderate overlap, respectively, according to the metrics suggested by Rödder and Engler [58]. For the pairs I-II, I-III, I-VI, II-VI, II-VI and IV-V, niche overlap is higher than expected based on the environmental conditions available to each but significant results were obtained in only one direction.

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Table 1. Niche overlap analyses.

https://doi.org/10.1371/journal.pone.0127980.t001

In agreement with these relatively low values of the Schoener´s D metric for most pairs of clades, the niche equivalence hypothesis was rejected for all pairwise comparisons (in all cases P<0.05, Table 1), suggesting that all clade pairs possess more significantly distinct realized niches than expected by chance.

History of niche occupancy

Results of the ancestral niche occupancy profiles (Fig 4) showed evidence of cladogenesis associated with both niche divergence and conservatism.

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Fig 4. Inferred history of the evolution of climatic tolerances within the Tarentola mauritanica species-complex.

The calibrated phylogenetic tree used, was modified from Rato et al. [34]. Interior nodes represent the mean of climatic tolerances inferred for the most recent common ancestor of the extant clades defined by that node. Solid lines connect ancestors with their descendants. A vertical dashed line indicates the 80% central density of climatic tolerance for each extant taxon, and the taxon label, to the right of each graph, indicates the mean. Lines and labels are coloured according to clades defined in Fig 2.

https://doi.org/10.1371/journal.pone.0127980.g004

Niche divergence was most noticeable for Clade V, and particularly for climatic variables associated with PC1, PC2 and PC3, while niche conservatism was mainly observed between clades I and IV and clades II and III along PC1. Surprisingly, this clear evidence of ancestral niche divergence for T. angustimentalis (Clade V) is not in agreement with the results obtained for the niche overlap in Table 1. For PC1 (which explains 71% of environmental variance), the closely related clades II and III exhibited highly similar environmental affinities, as well as clades I and IV, while clades V and VI showed evidences of niche divergence from the remaining clades, although their current environmental tolerances exhibit a high level of overlap with Clade I, and clades II and III, respectively. Most of these results are in agreement with the ones obtained for the niche overlap analyses in Table 1, as most clades displayed some levels of niche overlap, except the pairs III-IV and IV-VI. PC2 predicts a slight ancestral niche divergence of the mean tolerances of Clade III from the remaining groups, although its current tolerance range still largely overlaps. Since humidity and temperature seasonality were the variables most strongly correlated with PC2, these had the highest influence on the niche tolerance pattern exhibited by all clades.

Discussion

The study of niche evolution is of paramount importance in order to understand the speciation mechanisms behind the divergence of species. Specifically, the evaluation of whether the realized niche of natural populations has evolved in a conservative or divergent way, allows us to infer if an ecological speciation event may have taken place.

The moderate overlap value observed for the Clade III—Clade VI comparison is related to the fact that individuals from both the European/North Africa (Clade III) and the North Africa/Southern Iberian (Clade VI) clades are mostly distributed along Mediterranean climate areas (Fig 2) and, therefore, it is reasonable that their potential niches tend to overlap. The moderate niche overlap of these two clades is also supported by the results obtained with the niche similarity tests, as this comparison, along with the pair composed by clades II and III, were the only ones where a two-way significantly higher similarity than expected based on their available environmental conditions was found. Nevertheless, some authors have argued that comparison of SDMs is probably not the best surrogate for a comparison of environmental requirements (reviewed in [65]). Not surprisingly, the results obtained from this test differ from some of the niche similarity tests. For example, for clades III-IV and IV-VI niche similarity was significantly smaller than expected based on the environmental conditions available to each clade.

From an overall inspection of the environmental tolerance profiles by PC1 and PC2, the general speciation pattern within the Tarentola mauritanica complex, seems to have led to both niche divergence and conservatism. Niche divergence seems to be clear for Clade V, while conservatism is noticeable in clades I and IV (in both PC1 and PC2) and between clades II and III (in PC1 but not PC2). Furthermore, there is no evidence of similar environmental tolerances with phylogenetic proximity (except for clades II and III in PC1). However, when analysing the results obtained for the niche overlap analyses, it is apparent that currently some of the clades occupy quite similar ecological conditions.

Around 5.88 Mya, the North African/Southern Iberian Clade (Clade VI) became separated from the remaining clades, matching very closely the closing of the Strait of Gibraltar, and the Messinian Salinity Crisis (5.959 to 5.33 Mya) [66,67,68]. Since there are no fossil records of Tarentola in the Iberian Peninsula from this period [69], we can only assume that individuals did not cross from North Africa to Europe during the Miocene. Results from the niche overlap analyses confirm that Clade VI still occupies a relatively distinct realized niche from Clade IV and the ancestral climatic tolerance profile suggests cladogenesis by ecological divergence, most probably in North Africa.

Later, the separation of the ancestor of Tarentola angustimentalis (Clade V) took place, when these reached the Eastern Canary Islands (Lanzarote and Fuerteventura) around 4.82 Mya, by transmarine dispersal [34,70]. In this new environment, the ancestors of T. angustimentalis were subject to novel environmental conditions, leading to their ecological divergence in allopatry with respect to their continental relatives, as suggested by their ancestral climatic tolerances. Studies on the paleoclimatology of the Canaries indicate that during the Mio-Pliocene these islands had a tropical climate similar to the present day conditions along the coast of the Gulf of Guinea and Caribbean Sea [71]. Currently, due to their old geological ages determining the erosion of the relief, Lanzarote and Fuerteventura are no longer able to retain precipitation brought by trade winds [72]. In consequence, the climate of both islands has become heavily influenced by the North African Saharan winds, presenting annual temperatures ranging from 17–25°C and 22–35°C, respectively and very low levels of precipitation per year (147 mm in Fuerteventura) [73]. Hence, it was expected that individuals of T. angustimentalis would inhabit completely distinct ecological conditions, yet the acceptance of the null hypothesis of niche background similarity test suggests that the low levels of niche overlap between this clade and the others is most likely a result of the available background of Clade V; when defining a buffer of 100 km around the species records, mostly ocean will be selected and the background will consist mainly of unsuitable environment, making it difficult to determine whether there is active niche search or not.

Around 4.32 Mya, the South-Central Moroccan clade (Clade I) split from the remaining elements, as did later the Central Moroccan clade (Clade IV), approximately 4.14 Mya. However, these clades conserved the same niche and currently show no signs of niche divergence between them. The most probable hypothesis to explain this pattern is that both clades evolved in allopatry, likely caused by vicariant barriers, resulting in a high-level of niche conservatism. This would imply that in the past their distribution was distinct from today, since they are presently in contact. Unfortunately, past SDMs can only be produced for the available palaeoscenarios, namely up to the Last inter-glacial (~120.000–140.000 years BP) [74], making it difficult to validate this hypothesis.

By contrast, the ancestor of clades II (Iberian Clade) and III (European/North African Clade) split from Clade IV, approximately 4.14 Mya, with a strong niche divergence between them. Such divergence mainly concerned the mean temperature and humidity of the areas occupied (PC1). Subsequently, around 2.47 Mya, Clade III diverged from Clade II (and from the remaining clades) occurring in areas undergoing distinct humidity-related and temperature seasonality conditions (PC2). We interpret this as an ecological shift of Tarentola geckos from arid to Mediterranean habitats, which indeed started to spread during the Miocene-Pliocene transition [75], and subsequent adaptation to Mediterranean continental climate of Central Iberian Peninsula. Whether the first transition took place in North Africa or in the Iberian Peninsula is debatable but certainly the second transition should have been exclusively Iberian [34], with Clade II diverging from Clade III in allopatry due to the Mediterranean acting as a barrier. Depending on the contribution of the geographic barriers (i.e. the Gibraltar Strait), such niche shifts may have played a substantial role in the speciation processes of this group, as well as allowing it to occupy more northerly regions in Europe that would have been previously unavailable. Indeed, a recent ecophysiological study comparing multiple Iberian populations belonging to these two clades [76] reports clear differences in water loss rates. Overall, the European lineage displayed a trend for higher water loss when compared to the Iberian lineage. The lack of correspondence between ecophysiological traits and local climatic conditions, support the existence of a phylogenetic signal rather than local adaptation. These results suggest that divergent evolutionary responses to the environment in both lineages, mainly acting on water ecology, may account for the differences in their range expansion [76]. These findings reinforce the evidence that humidity-related conditions led to the divergence between these two clades, at least in the Iberian Peninsula. Nonetheless, such a shift may have permitted both clades to cope with the successive expansions and retractions during the Pleistocenic climatic oscillations [77,78] and, more recently, allowed Clade III to colonize (through human-mediation) other continental and insular regions of the Mediterranean Basin [34].

Interestingly, Tarentola responded to the environmental changes during the Miocene resulting in a combination of niche conservatism under unchanged conditions and niche shift to newly arisen habitats (e.g. [79]). By contrast, responses to the faster climate oscillations during the Pleistocene and the Holocene are dominated by niche conservatism suggesting a degree of phylogenetic inertia (e.g. clades II and III and clades I and IV for PC1). During the glaciations, Tarentola populations probably survived in Iberia in various isolated “microclimate pockets” [80], which is in agreement with the “refugia-within-refugia” model, hypothesized for the Iberian Peninsula (but see [81], e.g. [82]). It is noteworthy that Clade III, the most recently divergent in terms of realized niche, shows evidence of selection, which led to the existence of a single mtDNA variant [39]. Support that individuals from this clade have chosen unique environmental conditions is corroborated not only by its ancestral niche occupancy profile in PC2, but also by the fact that none of the other clades were ever found in the range occupied by the European/North Africa clade. Taking into account that individuals of Tarentola are frequently associated with humanized habitats, and their accidental introductions are common [38], especially around the Mediterranean, we would expect other clades to be introduced in Europe as well. Competitive interactions between clades (e.g. [83]) can also be hypothesized, and this should be validated with extensive fieldwork and modelling in the contact areas [84] as well as by experimental tests [85].

Conclusions

The integration of spatial ecological models with phylogenies provides a very powerful tool, helping in the delineation of cryptic species [8,9,10], or to at least giving additional support to their specific status [86]. According to the results from this study, the diversification of the clades within the Tarentola mauritanica species-complex fits both the niche divergence and conservatism profiles, with individuals of some clades still maintaining a realized niche distinct from their relatives. These results, allied with the high levels of mitochondrial divergence observed between clades [34], provide additional evidence for the need of a taxonomic revision of the whole complex, using a battery of nuclear markers to test species boundaries. Likewise, the niche evolution study and overlap patterns of the European/North African clade corroborate that climatic variables related to humidity and temperature seasonality (and hence, natural selection) resulted in the divergence of this clade and to the maintenance of the individuals in a realized niche distinct from the remaining members of Tarentola mauritanica.

Supporting Information

Acknowledgments

Both CR and SBC are funded through a Post-doctoral grant (SFRH/BPD/92343/2013 and SFRH/BPD/74423/2010, respectively) and AP by an IF contract (IF/01257/2012) supported by the Fundação para a Ciência e a Tecnologia (FCT) under the Programa Operacional Potencial Humano—Quadro de Referência Estratégico Nacional funds from the European Social Fund and Portuguese Ministério da Educação e Ciência. DJH was supported by the Project “Genomics and Evolutionary Biology”, cofinanced by North Portugal Regional Operational Programme 2007/2013 (ON.2 —O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). Research was funded by FEDER funds through the Operational Programme for Competitiveness Factors—COMPETE and by National Funds through FCT—under the projects PTDC/BIA-BEC/105327/2008 and FCOMP-01-0124-FEDER-008970 (to AP) and PTDC/BIA-BIC/118624/2010 and FCOMP-01-0124-FEDER-019676 (to SBC).