ABSTRACT. The internal transcribed spacer (ITS) region of 18S-26S nuclear ribosomal DNA (nrDNA) in seven species and one variety of Pinguicula (Lentibulariaceae) in Cuba has been sequenced. Although the eight taxa have been taxonomically divided into two subgenera such as Isoloba and Pinguicula with the other species grown in the other regions, and further they are divided into three sections such as Agnata, Discoradix and Homophyllum. The taxa of Pinguicula in Cuba formed a single clade, and it was not consistent with the current taxonomy based on morphology. Six taxa of Pinguicula in Cuba studied have been classified in section Agnata together with Mexican Pinguicula agnata and P. ibarrae, however, they were not ITS-phylogenetically, fully or partially supported by the data. Although the Cuban taxa of Pinguicula were quite different in morphology and life form from each other, they were ITS-phylogenetically close relatives to each other. The species of Pinguicula in Cuba were isolated phytogeographically and ITS-phylogenetically from those in the Southeastern United States and Mexico.

ABSTRACT. The internal transcribed spacer (ITS) region of 18S-26S nuclear ribosomal DNA (nrDNA) in 36 species of Pinguicula (Lentibulariaceae) in Mexico and Central America, most of which are endemic to Mexico, was sequenced. Based on the ITS results, those species of Pinguicula studied were divided into three clades. In general, these cledes do not agree with the current conventional classification proposed by various authors except for Clade I. Clade I includes only two species belonging to section Isoloba, which are widely but sparsely distributed in Mexico and Central America. Clade II included 18 species, which have been conventionally classified in the sections Heterophyllum (six species), Longitubus (three species), Orcheosanthus (eight species), and Temnoceras (one species). Clade III included 16 species, which have been conventionally placed in section Agnata (four species), Crassifolia (four species), Heterophyllum (two species), Microphyllum (three species) and Orcheosanthus (three species). Clade II is widely distributed in the Sierra Madre Occidental, the Sierra Madre del Sur to the southern part of the Sierra Madre Oriental regions and extends down south to Honduras, while the species in clade III were mostly confined to the northern part of the Sierra Madre Oriental regions. In general, the ITS results were largely consistent with biogeographic patterns, but showed disagreements with the current morphological classification.

Earlier phylogenetic studies in the genus Pinguicua (Lentibulariaceae) suggested that the species within a geographical region was rather monophyletic, although the sampling was limited or was restricted to specific regions. Those results conflicted with the floral morphology-based classification, which has been widely accepted to date. In the current study, one nuclear ribosomal DNA (internal transcribed spacer; ITS) and two regions of chloroplast DNA (matK and rpl32-trnL), from up to ca. 80% of the taxa in the genus Pinguicula, covering all three subgenera, were sequenced to demonstrate the inconsistency and explore a possible evolutionary history of the genus. Some incongruence was observed between nuclear and chloroplast topologies and the results from each of the three DNA analyses conflicted with the morphology-based subgeneric divisions. Both the ITS tree and network, however, corresponded with the biogeographical patterns of the genus supported by life-forms (winter rosette or hibernaculum formation) and basic chromosome numbers (haploidy). The dormant strategy evolved in a specific geographical region is a phylogenetic constraint and a synapomorphic characteristic within a lineage. Therefore, the results denied the idea that the Mexican group, morphologically divided into the three subgenera, independently acquired winter rosette formations. Topological incongruence among the trees or reticulations, indicated by parallel edges in phylogenetic networks, implied that some taxa originated by introgressive hybridisation. Although there are exceptions, species within the same geographical region arose from a common ancestor. Therefore, the classification by the floral characteristics is rather unreliable. The results obtained from this study suggest that evolution within the genus Pinguicula has involved; 1) ancient expansions to geographical regions with gene flow and subsequent vicariance with genetic drift, 2) acquirement of a common dormant strategy within a specific lineage to adapt a local climate (i.e., synapomorphic characteristic), 3) recent speciation in a short time span linked to introgressive hybridisation or multiplying the ploidy level (i.e., divergence), and 4) parallel evolution in floral traits among lineages found in different geographical regions (i.e., convergence). As such, the floral morphology masks and obscures the phylogenetic relationships among species in the genus.

We studied the distribution of two Pinguicula (Butterwort) species in Gunma Prefecture, Japan based on our herbarium specimen examinations and field observations. As a result, several localities of Pinguicula macroceras, such as Mt. Akagi-yama or the Tanigawa Mountain Range, were present today. In addition, two new localities of P. macroceras, Mts. Hotaka-yama and Ojikazawa-no-kashira, which had not been previously recorded, were found. However, only a single locality of P. ramosa, a threatened species (Endangered in the Red List of Gunma Prefecture and Vulnerable in the Red List of the Ministry of the Environment of Japan), was confirmed to be present in the prefecture. The two species have extremely narrow environmental preference and are restricted to specific environmental niches. The population size of both species at each microhabitat is small and there is a potential risk of disappearance of those localities in the future by the impacts of environmental stress or human activities. This study documents the current situations of the genus in Gunma Prefecture and suggests that urgent conservation is necessary to protect both the two species and their habitats in the prefecture.

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