Análise filogenética molecular e biossistemática do complexo Habenaria repens Nutt. (Orchidaceae) no Brasil

Habenaria is one of the largest genera of Orchidaceae, but even though, it is not well studied. Few taxonomic studies and revisions have been done so far, and none of them comprise morphometric or population genetics approaches. One of the many species complexes in the genus is the one formed by H. repens and related morphotypes. Habenaria repens sensu lato occurs from south United States of America to north Argentina and, in Brazil, many morphological variants can be found. In order to clarify the relationships between Brazilian morphotypes and H. repens, as well as the relationships among them, and circumscribe these entities, three different techniques were used: molecular phylogenetic analyses, multivariate morphometric analyses and population genetics. Thirty one populations, encompassing two known species (H. repens e H. aranifera), six Brazilian morphotypes, and one population from the locality where the species type was collected, in the United States of America, were sampled. Two nuclear markers (ITS and ETS) and two plastidial ones (matK and trnK) totalizing 3,250 characters were used in the phylogenetic analyses. In these, the existence of three clades, not directly related and formed exclusively by entities that belong to the complex, was detected. Morphotype H. aff. repens6 belongs to one of these clades, whereas the other morphotypes are separated in two bigger clades. Seventeen floral and three vegetative characters were used in morphometric analyses with multivariate statistic techniques. Little morphological distinction was found between the morphotypes, and not all the groups formed match those obtained with phylogenetic and population genetics approaches. For this last method, five microsatellite loci were amplified for 295 individuals belonging to 20 populations. Five genetic groups were identified and these match partially some of the clades recovered in the phylogenetic analyses. The most consistent patterns found by all three markers were: the reunion of three populations previously classified as the typical form of H. repens (Curitiba-PR, Anguera-BA and Areias-PB), a group with morphotypes H. aff. repens3 and H. aff. repens4, and the discrimination of H. aranifera and of the populations that form H. aff. repens7. Considering all the evidences and the congruence between them, it is suggested that at least five species be recognized in Brazil. Despite the fact that H. aff. repens3 and H. aff. repens4 form a group recovered by all markers employed, they present a large variation in the size of all plant organs (characters, in a great part, not included in the analyses), and are easily distinguishable, which makes it questionable whether they represent a single taxon or not. Therefore, other analyses must be carried out in order to determine if they should be described as one or two new species. Habenaria aff. repens7, which belongs to the same clade as H. aff. repens3 and H. aff. repens4, is distinguishable by morphological and molecular markers and possesses diagnostic features that support its recognition as a different species. Morphotype H. aff. repens6 forms a clade isolated from the others and, although it does not differentiate itself in the morphometric analyses, it was not even included in the population genetic analyses, because it failed to amplify with the primers developed for H. repens. These evidences suggest it represents a distantly related species that resembles morphologically the species that form the rest of the complex due to convergence of floral traits. Thus, this entity must be recognized as a new species supported by characters of habit and size (not included in the morphometric analyses). In regard to the other clade obtained in the phylogenetic analyses, H. aranifera will be maintained as a species, once it was recovered as a consistent group in all the analysis. The remaining terminals, in spite of having a broad morphological variability and some of them have been separated by molecular markers, do not have diagnostic characteristics that sustain more than one species. Hence, they will all be accepted under H. repens, a species with broad geographic distribution, high level of polymorphism as well as all the other attributes of an ochlospecies, probably constituting a progenitor-derivative pair with H. aranifera.