Chromosome Botany (2007) 2: 151-158 © Copyright 2007 by the International Society of Chromosome Botany Phylogenetic analysis of Cuban Pinguicula (Lentibulariaceae) based on internal transcribed spacer (ITS) region Hiro Shimai1, Yu Masuda2, Cristina M. Panfet Valdés3 and Katsuhiko Kondo2, 4 1Takamori Orchid Museum, 512-73 Izuhara, Takamori, Nagano 399-3107, Japan; 2Laboratory of Plant Chromosome and Gene Stock, Graduate School of Science, Hiroshima University, l-4-3 Kagamiyama, Higashi-Hiroshima City 739-8526, Japan; 3Cuba National Botanical Garden and University of Habana, Carretera El Rocio, Km 3.5, Calabazar Boyeros, Habana City, C.P. 19230, Cuba 4Author for correspondence: ([email protected]) Received April 23, 2007; accepted September 10, 2007 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 MexicanPinguicula 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. KEYWORDS: Cuba, Distribution, DNA, ITS, Lentibulariaceae, Pinguicula In the West Indies, the Republic of Cuba is the biggest found only in the mountain area of the central regions, island country with more than 4,000 islets, covering and P. lignicola and P. benedicta are seen in eastern Cuba. 110,860km2 all together. It lies just below the Tropic of Due to limited access to their habitat, few works have Cancer. Although Cuba is geographically close to the been carried out, except for several taxonomical works North American continent, e.g. less than 250km to the based on herbarium materials. Florida Peninsular or the Yukatan Peninsular, a remarkably Little information on the ecology of Cuban Pinguicula higher proportion of endemic plant species is seen. In species is available. Most of the species in Pinguicula Cuba, according to Berazaín et al. (2005), approximately grow on wet rocks or sandstones. Several Cuban Pin- 7,000 vascular plant species exist and 50% of them are guicula species also occur in such soils, but exceptionally endemic. P. lignicola grows on tree trunks or branches as an epi- Three genera, Genlisea, Utricularia and Pinguicula, phyte. Casper (1987) observed and reported the mechanism of the Lentibulariaceae are distributed in Cuba (Casper of this unusual life form of P. lignicola. This species is 1966, Taylor 1989, Taylor 1991). Casper (1966) recorded independently placed in section Discoradix, in subgenus five Pinguicula species in Cuba, and more recently Isoloba, together with Pinguicula casabitoana, endemic several other species have been described by Casper to the Dominican Republic in the Hispaniola Island (2003, 2004), and Casper and Urquiola (2003), though (Jiménez 1960). Pinguicula lignicola is morphologically some of them are likely identical with Pinguicula and ecologically different from the other Cuban Pinguicula benedicta. At least seven Pinguicula species are present species. However, it is not yet clear how this species is in Cuba and all of them are endemic. Taxonomically, related to the other Cuban species. Cuban Pinguicula species are divided into two subgenera, Last a few years some molecular data have suggested Isoloba and Pinguicula, with other species distributed in that their phylogenetic evolution largely agree with the North to South America and Eurasia (Casper 1966). geographical distribution patterns rather than the current Further, they are divided into three sections, Agnata, taxonomy based on morphological characteristics (Cieslak Discoradix and Homophyllum (Table 1), based on flower et al. 2005). An earlier molecular work based on the morphology, such as shape of corolla lobe, corolla tube, plastid rps16 intron and trnL-F sequence by Jobson et al. and spur (Casper 1963, 1966). (2003) used no Cuban Pinguicula species. Cieslak et al. Their geographical distribution is often restricted into (2005) used one Cuban Pinguicula species (P. filifolia) in a certain mountain or province. Pinguicula albida, P. their chloroplast gene matK and trnK group II intron se- cubensis, and P. filifolia are seen only in western Cuba. quences, and they suggested a close relationship between Particularly, the distribution of P. cubensis is restricted to Cuban and Mexican Pinguicula. Therefore it is not yet the south-facing slope of the Sierra Cajálbana, Province known the phylogenetic relationships among the Cuban of Pinar del Río (Casper and Urquiola 2003). P. jackii is Pinguicula species, or inadequately so between Cuban 151 152 SHIMAI ET AL. Table 1. Cuban Pinguicula taxa and some other species used in the ITS sequence Taxon Section Origin of material Voucher number Accession number (Cuban Pinguicula) Pinguicula albida Agnata Pinar del Río, Cuba 5788-LPCGS (HIRO) AB212095 Pinguicula benedicta Agnata Holguín, Cuba 715-LPCGS (HIRO) AB212097 Pinguicula bissei Agnata Guantánamo, Cuba 5790-LPCGS (HIRO) AB212098 Pinguicula cubensis Agnata Pinar del Río, Cuba 5794-LPCGS (HIRO) AB212102 Pinguicula filifolia Agnata Pinar del Río, Cuba 5795-LPCGS (HIRO) AB212104 Pinguicula jackii var. jackii Homophyllum Cienfuegos, Cuba 5797-LPCGS (HIRO) AB212106 Pinguicula jackii var. parviflora Homophyllum Cienfuegos, Cuba 5798-LPCGS (HIRO) AB212107 Pinguicula lignicola Discoradix Guantánamo, Cuba 5803-LPCGS (HIRO) AB300151 (Southeastern American and Mexican Pinguicula) Pinguicula caerulea Isoloba U.S.A. 5791-LPCGS (HIRO) AB212099 Pinguicula ionantha Isoloba U.S.A. 5796-LPCGS (HIRO) AB212105 Pinguicula lutea Isoloba U.S.A. 5799-LPCGS (HIRO) AB212108 Pinguicula planifolia Isoloba U.S.A. 5800-LPCGS (HIRO) AB212109 Pinguicula primuliflora Isoloba U.S.A. 5801-LPCGS (HIRO) AB212110 Pinguicula pumila Isoloba U.S.A. 5802-LPCGS (HIRO) AB212111 Pinguicula agnata Agnata Mexico 5758-LPCGS (HIRO) AB199752 Pinguicula ibarrae Agnata Mexico 5771-LPCGS (HIRO) AB251603 (Outgroup) Utricularia alpina 708-LPCGS (HIRO) AB212117 Materials used in this study were summarized in this table. Classifications followed Casper (1966). Origin of Cuban materials refers to the province at where the material was collected for the present work. Voucher specimens have been deposited at the Laboratory of Plant Chromosome and Gene Stock, Graduate School of Science, Hiroshima University [LPCGS (HIRO)]. Accession number refers to the sequence data available in the DDBJ/EMBL/GenBank databases. species and morphologically closely related species, primers were 20pmol of ITS 5 or AB 101, and reverse especially those distributed in the southeastern United primers were ITS 4 or AB 102 (White et al. 1990, Douzery States or Mexico. et al. 1999). Usually primers ITS 5 and ITS 4 were used, Therefore, the main objective in this work is to infer a but when amplification was not sufficient, AB 101 and phylogenetic relationship among Cuban Pinguicula and AB 102 were used. The concentration of template DNA some other Pinguicula species distributed in the south- used was 10µg/µl per sample. For PCR, the samples with eastern United States and Mexico, based on the ITS data. ITS primers were incubated for an initial 2 min. at 94°C The results will be discussed with their morphology and followed by 33 cycles of 50 sec. denaturation at 94°C, 1 ecology. min. annealing at 48°C and 30 sec. extension at 72°C. For AB primers, annealing was at 60°C at the same MATERIALS AND METHODS duration as ITS primers. For cycle sequencing, PCR Plant materials The plants used in this study are listed products were purified from collected agarose gels using in Table 1. Fresh leaf materials of the taxa studied were GFX PCR DNA and Gel Band Purification Kit (Amersham obtained from either plants collected at the field or plants Biosciences, NJ, U.S.A.). Cycle sequencing conditions cultivated at the Laboratory of Plant Chromosome and regardless of the type of primer were as follows: 96°C Gene Stock, Graduate School of Science, Hiroshima for 1 min. followed by 35 cycles of 10 sec. denaturation University. The herbarium voucher specimens have been at 96°C, 5 sec. annealing at 50°C, and 80 sec. extension deposited at the laboratory. Total DNA was extracted at 72°C. In some cases, up to 2µl of DMSO per sample from 0.07-0.1g of fresh leaves. Six species (section was added for GC rich samples. PCR products of Pin- Isoloba) from the southeastern United States and two guicula lignicola which showed difficulty in obtaining species (section Agnata) form Mexico were used, and clean sequences from direct PCR sequencing was cloned Utricularia alpina was selected as an outgroup. using pGEM-T easy vector system (Promega, Madison, WI, U.S.A.) following the manufacture’s protocol. The DNA isolation, PCR and sequencing Samples were ITS regions were sequenced using Long-Read Tower DNA ground in liquid nitrogen. Total DNA isolation from fresh Sequencer (Amersham Biosciences, NJ, U.S.A.). leaves was made with ISOPLANT II (Nippon Gene, Tokyo, Japan) following the manufacture’s protocol. The Sequences analysis DNA sequences were aligned by ITS region of the isolated DNA was amplified by poly- Genetyx-Win Version 5.2 (Software Development Co., merase chain reaction (PCR) using TaKaRa LA TaqTM LTD., Tokyo, Japan) and were adjusted manually. The (Takara Bio Inc., Shiga, Japan) with GC buffer II. Forward data included whole 5.8S and partial sequence of 18S ITS OF CUBAN PINGUICULA 153 and 26S.