Diferenciação de duas espécies crípticas de planárias terrestres do gênero Geoplana (Platyhelminthes: Tricladida: Continenticola) ocorrentes em diferentes formações florestais do sul do Brasil

Abstract

According to its original description, the type-specimens of Geoplana ladislavii Graff, 1899 are characterized by having a green-colored dorsum. When redescribed, specimens with greenish-brown color were also considered members of this species. In several forest formations in Rio Grande do Sul, specimens with brown or greenish- brown dorsum have been registered occurring sympatrically with specimens of Geoplana ladislavii Graff, 1899, both in forest and gardened areas with several degrees of human impact. In this study, morphological and molecular analyses of both morphospecies were conducted in order to verify if they belong or not to the same species. The specimens analyzed were collected in areas of mixed ombrophilous forest, seasonal semideciduous forest and seasonal deciduous forest, as well as from gardened areas from several municipalities in southern Brazil. The identification was conducted initially by analysis of external morphology, observing shape and size of the body, color pattern, eyes distribution and position of the mouth and the gonopore. After histological processing, the internal morphology was analyzed in body fragments corresponding to the anterior, pre-pharyngeal, pharyngeal and copulatory apparatus regions. Two specimens of each morphospecies were submitted to reactions of Alcian Blue-PAS, bromophenol blue, DMAB and Ninhydrin-Schiff for histochemical analyses of the secretory cells. The DNA extraction was conducted by the non-phenolic method and proteinase K. The hypothesis of phylogenetic relationship was recovered using the mitochondrial gene cytochrome c oxidase subunit I (COI) and the region of the nuclear gene of the internal transcribed spacer 1 (ITS-1), amplified by PCR with specific primers. Glands with protein and glycoprotein secretions open through the epidermis of anterior and pre-pharyngeal regions of both morphos species, as well as glands secreting neutral mucopolysaccharides in Geoplana sp. and glycosaminoglycans in G. ladislavii. Geoplana sp. has a conspicuous glandular border consisting of three types of secretory cells: erythrophil with protein, cyanophil with glycoprotein and xanthophil with proteic secretions. G. ladislavii has a less conspicuous glandular border constituting of erythrophil glands with protein and cyanophil glands with glycosaminoglycan secretions. Both morphospecies have a cylindrical pharynx with cyanophil, erythrophil and anthophil/erythrophil glands, as well as a fourth type (cyanophil cells with protein secretion) in G. ladislavii. The secretion type from cyanophil and xanthophil and/or erythrophil cells in pharyngeal glands is different between the two morphospecies, showing basic protein or glycoprotein. The prostatic vesicle is tubular with a forked ental portion, being intrabulbar in Geoplana sp. and extrabulbar in G. ladislavii. Both morphospecies show glands with proteic secretion opening into the prostatic vesicle. The penis papilla is oblique, projecting ventrally from the roof and anterior wall of the male atrium and having cells with protein, glycoprotein and glycosaminoglycan secretions. G. ladislavii is differentiated from Geoplana sp. by having a large amount of cyanophil cells ending close to the ventral insertion of the penis papilla. In the female atrium, G. ladislavii has proteic secretions while Geoplana sp. has glycoproteic secretions. Both morphospecies have shell glands with erythrophil cells, constituting glycoproteic secretions in G. ladislavii and proteic secretions in Geoplana sp. Molecular analyses indicate that Geoplana sp. constitutes a taxonomic group close to Geoplana ladislavii, but independent from it. Thus, morphological and molecular analyses indicate that Geoplana sp. and G. ladislavii are distinct species.