Evidence for recent gene flow between north-eastern and south-eastern Madagascan poison frogs from a phylogeography of the Mantella cowani group

Background The genus Mantella, endemic poison frogs of Madagascar with 16 described species, are known in the field of international pet trade and entered under the CITES control for the last four years. The phylogeny and phylogeography of this genus have been recently subject of study for conservation purposes. Here we report on the studies of the phylogeography of the Mantella cowani group using a fragment of 453 bp of the mitochondrial cytochrome b gene from 195 individuals from 21 localities. This group is represented by five forms: M. cowani, a critically endangered species, a vulnerable species, M. haraldmeieri, and the non-threatened M. baroni, M. aff. baroni, and M. nigricans. Results The Bayesian phylogenetic and haplotype network analyses revealed the presence of three separated haplotype clades: (1) M. baroni, M. aff. baroni, M. nigricans, and putative hybrids of M. cowani and M. baroni, (2) M. cowani and putative hybrids of M. cowani and M. baroni, and (3) M. haraldmeieri. The putative hybrids were collected from sites where M. cowani and M. baroni live in sympatry. Conclusion These results suggest (a) a probable hybridization between M. cowani and M. baroni, (b) a lack of genetic differentiation between M. baroni/M. aff. baroni and M. nigricans, (c) evidence of recent gene-flow between the northern (M. nigricans), eastern (M. baroni), and south-eastern (M. aff. baroni) forms of distinct coloration, and (d) the existence of at least three units for conservation in the Mantella cowani group.


Background
Madagascar, the fourth largest island of the world, is a hotspot for biodiversity that deserves the highest priority for conservation [1]. Currently, 233 amphibian species are known from this island [2]. All of the native taxa are endemic at the level of species, and all but one also at the level of genera [3][4][5]. Madagascar has so far been spared from one of the causes of the global decline of amphibians [5], the diseases due to dangerous pathogens, especially chytrid fungal infections [6][7][8]. However, the island is not safe from range contractions and extinctions due to habitat destruction and overexploitation of live specimens for the pet trade [5,[9][10][11][12][13][14].
The poison frogs from Madagascar, Mantella, form one of the Malagasy endemic genera of amphibians. This genus contains 16 described species divided into six groups based on morphological and genetic criteria [15]. The genus holds the record in terms of Malagasy amphibians present in the pet trade (>230,000 individuals over 10 years 1994-2003) [16]. Species of Mantella are now included in the appendix II of the Convention on the International Trade in Endangered Species (CITES), and the commerce is thereby better regulated [16].
Genetic analyses to understand phylogeny and phylogeography of the genus Mantella, to solve problems in taxonomy, and to provide a basis for conservation actions to better protect species belonging to this genus are recent and are leading to more and more surprising new discoveries. Initial allozyme studies of a restricted number of taxa unveiled the existence of three lineages among the species from central-eastern rainforests: (1) Mantella baroni and M. cowani, (2) M. madagascariensis and M. pulchra, and (3) M. aurantiaca, M. crocea and M. milotympanum [17], and led to a revised classification of the genus [15]. The use of karyological methods provided some additional hints on the phylogeny of the genus [18], but it were mainly recent studies based on molecular analyses using mitochondrial and nuclear DNA sequences that provided progressively more precise information. In 2002, the use of the 16S rRNA marker in an attempt to elucidate the origin and evolution of the aposematic coloration of frogs in this genus permitted to find clades largely congruent [19] with the species groups defined by Vences et al. [15,17] aurantiaca [20]. Chiari et al. in 2004 [21] then used nuclear as well as mitochondrial DNA sequences (cytochrome b, Rag-1 and Rag-2) to investigate the evolution of coloration and the phylogeographic relationships of the species belonging to the Mantella madagascariensis group. This study strongly confirmed the existence of haplotype sharing between M. milotympanum and M. crocea and gave new insights into the phylogenetic relationships of the genus Mantella. Sequences of cytochrome b were also used to study the phylogeography of M. bernhardi and revealed two distinct clades corresponding to geographically isolated populations [22].
Besides the Mantella madagascariensis and M. bernhardi groups, phylogeographic studies have also been carried out on the M. cowani group [23]. This group is likely to be monophyletic [19,20] and includes five species according to Vences et al. (1999) [15]: M. cowani, which is listed in the IUCN Red List as critically endangered due to small range distribution and anthropogenic pressure [5,14,15], M. baroni and M. nigricans, both non threatened species, M. haraldmeieri, a vulnerable species from the extreme south-east, and Mantella sp. aff. baroni from the southeastern Andringitra region which has been recognized as possibly distinct form by Vences et al. [15] but not formally named. Members of the M. cowani group are characterized by light (mostly yellow or red) flank blotches of variable extension (also found in M. madagascariensis group and M. bernhardi) and single click calls (exclusive to this group).
Cytochrome b-based phylogeographic data by Chiari et al. [23] were limited to two of these five species: Mantella cowani and M. baroni. Sequences of these two species formed separate haplotype networks, with haplotype sharing at one locality of their sympatric occurrence. Most intriguing, and strongly deviating from patterns found for instance in Mantella bernhardi [22], within both mitochondrial networks, specimens from different localities shared identical haplotypes, even those from the most distant sample sites of M. baroni; although most populations were characterized by a rather high haplotype diversity, no haplotype clades exclusive to geographical regions were observed.
In the present study, we provide a phylogeographic analyses that extends to all five species of the Mantella cowani group as recognized by Vences et al. [15]. We provide the first population-level molecular data of Mantella sp. aff. baroni, M. haraldmeieri and M. nigricans, and add numerous additional specimens and localities for M. baroni and M. cowani (figure 1; Table 1). Our results corroborate the status of M. cowani and M. haraldmeieri as distinct evolutionary lineages. However, the new data provide no evidence for differentiation among M. baroni, M. sp. aff.
baroni and M. nigricans and no genetic signature of geographic differentiation within these forms, providing the first evidence for an amphibian with ongoing or recent gene flow between populations occurring across most of Madagascar's eastern rainforests.

Results
The total dataset consisted of 195 individual sequences of 453 bp that were distributed among 82 distinct haplotypes (of which 70 unique to single individuals) divided into three separated haplotype networks (figure 2). One  (Table 2). Two individuals of M. cowani (two "putative hybrids") from Farimazava were also included in this haplotype network differing from the most common haplotype (Mbn1) by one and two mutations. Three out of seven individuals of M. nigricans from Marojejy and one out of five of M. nigricans from Andranomenabe share the common haplotype Mbn1. The maximum distance of M. nigricans noticed from this common haplotype is four mutations. The second network includes individuals of M. cowani (and putative hybrids between this species and M. baroni) with one common haplotype (Mc1; frequency = 65%). The individuals of M. cowani included in this network were representative of all the sampled localities, while it also contains one specimen initially identified as M. baroni from South-Ampasimpotsy and three M. baroni from Farimazava. Hence, these four individuals of M. baroni are to be considered as a posteriori hybrids (cf. definitions in [23]). The M. haraldmeieri (mh) network is separated from all other species and no indication for hybridization of this with any other species was detected.
The partitioned and non-partitioned Bayesian analyses gave a consensus tree in which three clades were recovered (not shown). One internally paraphyletic clade, supported by 36% posterior probability for the non partitioned analyses and 72% for the partitioned analyses, included individuals of Mantella baroni, M. aff. baroni, M. cowani/baroni hybrids, and Mantella nigricans, with inter-nal nodes supported by low posterior probability. Monophyletic groups containing all specimens of Mantella cowani and M. haraldmeieri, respectively, were recovered (with some hybrid specimens of M. baroni in the M. cowani clade) as monophyletic groups supported by 100% posterior probabilities, but these clades were nested within a clade containing sequences of M. baroni and M. nigricans.

Discussion
The cytochrome b marker has been shown to be a good marker to identify species and hybridization phenomena [24] and to highlight genetically isolated populations [22]. Our results using the cytochrome b marker confirm the results of Chiari et al. [23] on the probable hybridization between M. cowani and M. baroni which, however, in general appear to be well separated species by morphology and ecology [15,23], with an uncorrected pairwise distance of about 3.5% among most of their cytochrome b haplotypes, and a clustering of these haplotypes in two unconnected networks. This distance appears to be in a similar order of magnitude as that between other closely related Mantella species, e.g., M. aurantiaca, M. crocea and M. madagascariensis (4.5-5.3% [20]).
However, the situation may be different in the case of M. baroni, M. sp. aff. baroni from Andringitra, and M. nigricans. The cytochrome b sequences used here were unable to provide any indication of genetic differentiation between these three forms, which indeed all share their most common haplotype. The structure of network 1 in figure 2 suggests that there is a high level of gene flow between these three forms. An example is offered by specimens of M. nigricans from Andranomenabe and M. baroni from Vohiparara, which shared one haplotype (Mbn3; see Table 2 and figure 2), in addition to the common haplotype Mbn1 that is present in all localities in these taxa.
The haplotypes sequenced from individuals of M. aff. baroni from Andringitra (Korokoto) do not present any significant differentiation from the most common haplotype Mbn1 in the M. baroni/nigricans network (maximum 1 mutation). This indicates that despite the larger extent of yellow colour in these specimens [15], they are not likely to represent a distinct taxonomical unit.   within the sampling gap of our study, was mentioned by Vences et al. [15], but the available specimens from voucher collections had largely faded colour patterns and could not be attributed to either species with certainty. The contact zone of these two forms could be around these sites but more sampling in the vast area between Masoala and Moramanga is suggested to better resolve this question.
However, in general terms, the absence of a geographical structure in the haplotype differentiation of the M. baroni/ M. nigricans complex over its distribution area indicates that these frogs have colonized their entire range very recently, and/or that gene flow between their populations is an ongoing or at least very recent phenomenon. In turn, the data would favour the hypothesis that chromatic differences between M. nigricans and M. baroni may have evolved by disruptive selective pressures and not just by genetic drift in the context of historical barriers to gene flow between a northern and southern population group, although such barriers may have evaded the resolution of our mitochondrial analyses if they had been too recent (post-Pleistocene) in age.
Taxonomically, our results suggest that M. nigricans may be best seen as the northern colour morph of M. baroni, similar to Mantella crocea and M. milotympanum [21]. Indeed, in Mantella nigricans and M. sp. aff. baroni, a certain chromatic differentiation between individuals of the same population (mainly regarding the extent of brown, green or yellowish colour on the dorsum) is also observed [15], confirming that in some cases conspecific Mantella specimens may bear different colour patterns. However, we propose not to formalize these taxonomic changes before they are confirmed by analyses of nuclear markers, and before a more stable and complete framework of Mantella systematics can emerge from a comprehensive analyses.
In terms of conservation, M. baroni, M. sp. aff. baroni and M. nigricans could be seen as a single unit of conservation based on the mitochondrial marker we used. However, it is necessary to consider the chromatic differences (of possible adaptive value) before issuing precise recommendations. In fact, different populations (or in this case different colour forms) within a species could justify specific conservation measures to preserve their genetic, ecological, and/or morphological diversity. Different definitions of conservation units have been proposed according to the parameters used to define them. From an initially broader concept of evolutionary significant unit (ESU) including ecological and genetic data, a more molecular based concept is currently mostly used (see Box 1 in [25]). However, since no ESU concepts can universally be applied, a more comprehensive adaptive evolutionary conservation (AEC) concept has been proposed [26]. The aim of the AEC concept is to preserve the evolutionary potential, thus the adaptive variance within a species (indicated under the AEC concept as ecological and or morphological as well as genetic differences) [26], which Haplotype networks for the Mantella cowani group The hybridization mentioned in Chiari et al. [23] between M. baroni and M. cowani is confirmed by our study, with a slightly shorter cytochrome b fragment but using more samples. Hybridization is a recognized phenomenon in amphibians as mentioned by numerous authors [27][28][29]. However, the use of nuclear marker is necessary to clearly assess the presence of hybridization between the two above mentioned species and exclude the alternative scenario of ancient haplotype sharing by incomplete lineage sorting. The present study extends records of hybridization between these two species to the locality of South Ampasimpotsy, very close to Farimazava where this phenomenon has been recorded before [23]. At these sites, the hybridization detectable at the mitochondrial level appears to affect up to 10% of the population of M. cowani. As observed by Andreone et al. [5], M. cowani deserves a special attention in terms of conservation strategy. Before 2004 this species provided a non negligible income for the pet trade [5,16]. For this purpose, we suggest: (1) in situ breeding programs, where parts of the original habitat are protected and promoted to stabilize the populations and in the long term possibly allow a sustainable harvesting, as described by Vines et al. [28] and (2)   The new sequences were combined with sequences from a previous study [23] corresponding to individuals from twelve populations: four populations of M. cowani and eight populations of M. baroni with the one locality (Farimazava) of sympatry. As in Chiari et al. [23], two putative hybrids between M. cowani and M. baroni were included in our study, defined as individuals with an orange-yellowish coloration, more extended lateral spots (versus small and rounded spots in M. cowani), residual of cephalic lines (clearly delimited in M. baroni and lacking in M. cowani) and presence of yellowish shading on tibiae (versus red bands in M. cowani and black-orange patterned tibiae in M. baroni. Table 1 summarizes the localities, the species and the sample size of each species used in this study. The map of the localities of each species is presented in figure  1.

Laboratory techniques
Total genomic DNA was extracted from the tissue samples or toe clips using proteinase K digestion (1 mg/ml concentration) followed by a standard salt extraction protocol

Analysis techniques
The sequences were checked and aligned using the software Sequence Navigator (Applied Biosystems). Sequences were deposited in GenBank (accession numbers DQ889341-DQ889429). The program Collapse v3.1 [33] was used to merge sequences into haplotypes. Modeltest version 3.7 [34,35] was used in conjunction with PAUP*, version 4.0b10 [37] to estimate the best fitting models for our complete dataset and for each partition corresponding to the different codon positions of the cytochrome b gene. Based on the Akaike Information Criterion (AIC), the following models of sequence evolution were determined: (1) for the first codon positions, a TrNef model with a proportion of invariable sites of 0 and equal rates for all sites; (2) for the second codon positions, a F81 model with a proportion of invariable sites of 0 and equal rates for all sites; (3) for the third codon position, a GTR model with a proportion of invariable sites of 0 and equal rates for all sites; (4) for the complete dataset, a GTR model with a proportion of invariable sites of 0 and variable sites distributed according to a gamma distribution shape parameter of 0.3438. Bayesian phylogenetic inference using the program MrBayes 3.1.1.
[36] was carried out with substitution settings adjusted according to these substitution models, and with four chains with the default heating values, 1,000,000 generations, saving the tree at every tenth generation and discarding the initial 10,000 trees as burn-in. We ran both partitioned and non partitioned analyses, both of which yielded almost identical results. Mantella aurantiaca was used as the outgroup.
Haplotype networks were constructed using the TCS software package [38], which employs the method of Templeton et al. [39]. It calculates the number of mutational steps by which pairwise haplotypes differ and computes the probability of parsimony [39] for pairwise differences until the probability exceeds 0.95.