Chromosome Botany (2007) 2: 151-158 © Copyright 2007 by the International Society of Chromosome Botany

Phylogenetic analysis of Cuban () 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 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 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 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 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 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. Gaps were treated as missing characters. The RESULTS AND DISCUSSION sequence data reported are available in the DDBJ/EMBL/ The NJ and the most parsimonious trees have been GenBank databases. The accession number for each taxon presented in Fig. 1 and Fig. 2, respectively. For the MP is indicated in Table 1. The sequences between Pinguicula method, the number of tree held at each step during benedicta and P. bissei were identical; therefore, these stepwise addition and that retained by heuristic search were treated as one operational taxonomic unit (OTU). was one, respectively. The tree length was 989. Consis- tency index (CI) excluding uninformative characters was Phylogenetic analysis To construct phylogenetic trees, 0.7222. Homoplasy index (HI) excluding uninformative two different methods, neighbor-joining (NJ) and maxi- characters was 0.2778. Retention index (RI) and Rescaled mum parsimony (MP), were used to infer phylogenetic consistency index (RC) was 0.8130 and 0.6305, respec- relationships among the eight Cuban Pinguicula taxa and tively. the other species including an outgroup. For the NJ tree, Both the NJ tree and the most parsimonious tree con- Clustal W was used with the default parameters. The NJ sistently showed that Cuban Pinguicula and southeastern tree was then obtained using Kimura’s two parameter American Pinguicula formed different clades. The method with the bootstrap test at 1,000 replicates. For the geographical distribution of Pinguicula pumila extends MP trees, PAUP* 4.0 beta 10 for Windows (Sinauer from North Carolina down south to Big Pine Key in Associate, MA, U.S.A.; Swofford 2002) was used. The Florida and the Andros Island in the Bahamas (Casper most parsimonious tree was searched by heuristic algo- 1966), but it is likely that there is no direct phylogenetic rithm. The starting tree was obtained via stepwise addition relationship between Cuban Pinguicula and southeastern with random sequence addition. The MulTree option and American Pinguicula. The NJ tree showed a possible tree bisection reconnection (TBR) branch swapping were phylogenetic relationship between Cuban and Mexican in effect. Bootstrap was estimated from 1,000 replicates Pinguicula, but it is only weakly supported by the boot- of heuristic search. The phylogenetic trees were edited by strap value at 75% (Fig. 1). In the most parsimonious TREEVIEW Version 1. 6. 6 (University of Glasgow, tree, Cuban Pinguicula and Mexican Pinguicula formed Scotland: Page 1996). different clades (Fig. 2). Those data imply that Cuban

Fig. 1. Neighbor-joining tree based on the ITS sequence of Cuban Pinguicula and their related species. Numbers below clades show bootstrap value at 75% or higher estimated from 1,000 replicates. Scale bar indicates genetic distance of 0.1. Clade in a box indicates Cuban Pinguicula taxa. 154 SHIMAI ET AL. taxa and some length other = Tree species. 989. CI excluding uninformative characters = 0.7222. Pinguicula . Pinguicula Fig. 2. Most parsimonious tree based on the ITS sequence of Cuban HI excluding uninformative characters = 0.2778. RI = indicates Cuban 0.8130. RC = 0.6305. Bootstrap values above 80% are shown. Classification followed Casper (1966). Clade in a box ITS OF CUBAN PINGUICULA 155

Pinguicula is not only biogeographically, but also phylogenetically closely related to each other, even though phylogenetically isolated from southeastern American they show morphological diversities. A further revision Pinguicula and Mexican Pinguicula (section Agnata). may be necessary within the with several different The ITS results disagree with the current taxonomy approaches, including molecular phylogeny. based on morphology. For example, Cuban Pinguicula All Cuban Pinguicula species are considered to be albida, P. cubensis, P. filifolia and P. benedicta have been endemic. Furthermore, the distribution of each Cuban placed in section Agnata together with Mexican P. agnata Pinguicula species is mostly restricted to the following and P. ibarrae, since they usually have nearly equally three areas; 1) western Cuba (Province of Pinar del Río divided flower lobes forming a radial symmetric corolla and the island of Isla de la Juventud), 2) central Cuba (Casper 1963, 1966). However, these Cuban species (Province of Cienfuegos), and 3) eastern Cuba, or often formed a single clade with other Cuban Pinguicula clas- called Oriente (Provinces of Guantánamo and Holguín). sified in sections Homophyllum or Discoradix rather than The relationships between the ITS phylogeny and the the two Mexican species in the most parsimonious tree. distribution pattern of Cuban Pinguicula have been The result suggests that Cuban Pinguicula species are illustrated in Fig. 3. Altitudinal distribution of Cuban

Fig. 3. Relationships between the ITS phylogeny and distribution pattern in Cuban Pinguicula taxa. Approximate distribution pattern of Cuban Pinguicula studied was mapped and combined with a clade of Cuban Pinguicula obtained from the most parsimonious tree in Fig. 2. Pinguicula albida, P. cubensis and P. filifolia group is distributed in western Cuba. P. jackii var. jackii and P. jackii var. parviflora group is distributed in Central Cuba. P. benedicta and P. lignicola are distributed in Eastern Cuba, but they did not form a sister group within the clade. 156 SHIMAI ET AL.

Pinguicula is comparatively lower (sea level to 1,100m) leaves (Fig. 4-A). Pinguicula filifolia have distinctively than that of some Mexican or South American Pinguicula filiform leaves (Fig. 4-B). These two species can be species occurring at altitudes of 4,000m or even higher. found in wet fine sand on the coastal pine savanna of All Pinguicula species in southeastern United States Pinar del Río, and P. filifolia is also found in Isla de la occur at low altitudes (sea level to 300m) in the coastal Juventud. Pinguicula filifolia tolerates direct sun, but P. regions without exception (Wood and Godfrey 1957, albida usually restricted to under juvenile trees of Col- Casper 1966, Godfrey and Stripling 1966). pothrinax wrightii (Palmaceae), providing lower light In western Cuba, Pinguicula albida, P. cubensis and P. intensity. Pinguicula cubensis, possessing oblong- filifolia are seen and the three species phylogenetically lanceolate leaves (Fig. 4-C), is found only in wet limestone form a sister group (see Figs. 1 and 2) within the clade. cliffs (220m altitude) of the Sierra Cajálbana in Pinar del Pinguicula albida has obovate to rotundate pale green Río (Casper and Urquiola 2003). The corolla lobes of the

Fig. 4. Morphological diversities of Cuban Pinguicula studied in the ITS phylogeny. A: P. albida. B: P. filifolia. C. P. cubensis. D: P. jackii var. jackii. E: P. jackii var. parviflora (flower). F: P. benedicta. G: P. bissei (flower). H: P. lignicola. Drawings were made from the live materials used in the ITS sequence. ITS OF CUBAN PINGUICULA 157

Table 2. Ecology and morphology of Cuban Pinguicula studied in the ITS sequence Taxon Soil type Environment Life form Leaf shape Flower color P. albida fine sand savanna terrestrial obovate to rotundate white P. benedicta clay rain forest terrestrial ovate to obovate crimson, pink, white P. bissei limestone rain forest lithophyte obovate crimson, pink, white P. cubensis limestone mountain slope lithophyte oblong-lanceolate white P. filifolia fine sand savanna terrestrial filiform white, pale purple, pale yellow P. jackii var. jackii lime sandstone rain forest lithophyte obovate to rotundate dark blue P. jackii var. parviflora lime sandstone rain forest lithophyte obovate to rotundate white with pale blue margin P. lignicola bark rain forest epiphyte oblong-lanceolate white Data from Casper (1966), Casper (1987), Casper (2004), Casper and Urquiola (2003), and the field observations by C. Panfet Valdés and H. Shimai in June, 2004. three species are almost equally divided in both shape morphological similarities, geographical distributions, and size. The flower color in this group is basically and the identical ITS sequences between the two taxa, white, but Pinguicula filifolia also has pale purple or pale these might be conspecific. A taxonomical review of the yellow flower (Table 2). The ITS data supported the theory so-called Pinguicula benedicta complex, including P. by Casper and Urquiola (2003), stating that Pinguicula bissei and P. caryophyllaceae, is needed. cubensis was closely related to P. albida and P. filifolia. Pinguicula lignicola is also distributed in eastern Cuba, In Central Cuba, Pinguicula jackii var. jackii and P. but the altitudinal distribution range is often higher than jackii var. parviflora are seen and they are phylogenetically that of P. benedicta. The leaf of Pinguicula lignicola is very closely related to each other (Figs. 1 and 2). These oblong-lanceolate (Fig. 4-H), and the flower color is white. two taxa show no morphological differences on the The roots directly anchor to the surface of tree trunks or vegetative organs, having obovate to rotundate leaves, branches. It is unclear why this species possesses some but the conspicuous difference appears in the flowers unusual characteristics, such as an epiphytic life form, (Ernst 1961). The corolla of Pinguicula jackii var. jackii root systems, which cannot be seen in other Cuban species. is dark blue but fades day by day after opening the corolla, Although Casper (1987) reported the detailed morphology approximately 3.0-3.5cm across in diameter (Fig. 4-D), of Pinguicula lignicola, it is still poorly understood their and that of P. jackii var. parviflora is white with faintly ecological system with a relation to the environment. pale blue margin, approximately 2.0cm across in diameter This species is often found on tree trunks or branches of (Fig. 4-E). These two taxa grow on vertical lime sandstone Podocarpus spp. (observation by C. Panfet Valdés) in a cliffs (600-800m altitude) particularly around the Pico de mountain range (at altitudes of mostly 800-1,100m) of San Juan in the Alturas de Trinidad, a part of the Sierra the Provinces of Guantánamo and Holguín. Mexican de Guamuhaya (or also known as Sierra de Escambray), Pinguicula hemiepiphytica (section Longitubus) also Province of Cienfuegos. A limited number of populations grows on mossy tree trunks (Zamudio and Rzedowski and plants in each variety have been confirmed in this area. 1991), but not directly rooted on the exposed bark. One Pinguicula benedicta and P. bissei are distributed in remark should be addressed is that P. casabitoana, eastern Cuba. Pinguicula benedicta is recorded from the morphologically similar to P. lignicola, is found in the Sierra Cristal in the Province of Holguín to Camp San Alto de Casabito, the Dominican Republic, geographically Benito (La Vega de Taco) in the Province of Guantánamo, very close to Eastern Cuba (Jiménez 1960). The distribu- at altitudes of mostly below 900m. Pinguicula benedicta tion of Pinguicula lignicola is partly overlapped with that is a morphologically variable species even within the of P. benedicta, but they did not form a sister group same population. The leaf is ovate to obovate. The shapes within a clade (Fig. 3). of corolla lobes vary from oblong to obovate in the upper As discussed above, the Cuban Pinguicula group is lip and from narrowly oblong to obovate or nearly rotun- isolated both biogeographically and phylogenetically from date in the lower lip (Fig. 4-F) (Britton 1920, Casper southeastern American or Mexican Pinguicula. Within 1966). Flower color is crimson to pale pink, or rarely the Cuban Pinguicula examined in this study, P. lignicola white. Pinguicula benedicta is often found in wet reddish divided at the base of phylogenetic trees (Figs. 1 and 2), clay along the rivers or streems. Pinguicula bissei was and it suggests that all Cuban Pinguicula have evolved described from the river of Río Báez, Province of Guan- from the common ancestor. It is probable that Cuban tánamo by Casper (2004). It has thinner corolla lobes and Pinguicula has been isolated from the other species, except the margin of the middle lobe in lower lip is often irregu- for Dominican P. casabitoana, at an early evolutional larly waved (Fig. 4-G). Pinguicula bissei occurs in wet stage, and evolved morphologically different species limestone rocks along the Ráo Báez. Concerning the presently seen. It is also presumable that the direction of 158 SHIMAI ET AL. expansion in Cuban Pinguicula might have been roughly Casper, S. J. 1987. On Pinguicula lignicola, an epiphytic east to west. However, there is little evidence that they heterophyllic member of the Lentibulariaceae in Cuba. Pl. System. Evol. 155: 349-354. evolved from epiphyte to terrestrial. No natural hybrid- Casper, S. J. 2003. Two new Pinguicula species (Lentibulari- ization has been reported even though the distribution of aceae) from eastern Cuba (Cuba occidental). some species overlapped each other. The distribution of Haussknechtia 9: 141-155. Cuban Pinguicula is restricted to particular regions, so Casper, S. J. 2004. Two new Pinguicula species (Lentibulari- aceae; P. benedicta-group) from the eastern mountain there is a higher risk of extinction by climate changes. In range of Cuba (Grater Antilles) with reddish flowers. the meanwhile, six species have been listed in the Red Wulfenia 11: 1-13. List of Cuban Vascular Plants (Berazaín et al. 2005), but Casper, S. J. and Urquiola Cruz, A. J. 2003. Pinguicula cubensis (Lentibulariaceae)-a new insectivorous species from no practical conservation programs are in action. Several western Cuba (Cuba occidental). Willdenowia 33: 167- different methods should be necessary to save Cuban 172. Pinguicula, such as habitat protection, in vitro propaga- Douzery, E. J. P., Pridgeon A. M., Kores P., Linder H. P., tion, and introduction to cultivation. Also, phylogenetic Kurzweil H. and Chase, M. W. 1999. Molecular phylo- genetics of Diseae (): a contribution from relationship between Cuban Pinguicula and all the other nuclear ribosomal ITS sequences. Am. Journ. Bot. 86: species are unknown due to limited samplings in this 887-899. work. A further investigation is necessary to see the ITS Ernst, A. 1961. Revision der Gattung Pinguicula. Bot. Jb. 80: 145-194. phylogeny of overall the genus. Godfrey, R. K. and Stripling, H. L. 1961. A synopsis of Pin- guicula (Lentibulariaceae) in the southeastern United ACKNOWLEDGEMENTS. We would like to gratefully thank States. Am. Midland Nat. 22,2: 395-409. the Japan Society for the Promotion of Science (JSPS) for support- Jiménez, J. 1960. Novelties in the Dominican flora. Rhodora ing Cristina M. Panfet Valdés with a JSPS Invitation Fellowship 62: 235-238. (short term) for Research in Japan in 2006 (S-06232) (December 6, Jobson, R. W., Playford, J., Cameron, K. M. and Albert, V. A. 2006 February 5, 2007). This study was supported by the Grant- 2003. of Lentibulariaceae in-Aid for Scientific Research Program (A) No.19255004 of the inferred from plastid rps16 intron and trnL-F DNA JSPS (Representative: Katsuhiko Kondo). The authors wish to sequences: implications for character evolution and express sincere appreciation to Dr. Armando J. Urquiola Cruz, biogeography. Syst. Bot. 28: 157-171. Jardín Botánico de Pinar del Río, Pinar del Río, and Mr. Omar Page, R. D. M. 1996. TREEVIEW: an application to display Alomá Moreno, Cienfuegos, for their assistance for collecting phylogenetic trees on personal computers. Computer materials. Thanks are also due to Prof. Dr. Angela T. Leiva Applications in the Biosciences 12: 357-358. Sánchez, Jardín Botánico Nacional, Havana, for arranging the Swofford, D. L. 2002. PAUP*. Phylogenetic Analysis Using permissions for collecting and exporting materials. Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, MA. Taylor, P. 1989. The Genus Utricularia-a taxonomic mono- LITERATURE CITED graph. HMSO. London. Berazaín Iturralde, R., Areces Berazaín, F., Lazcano Lara, J. Taylor, P. 1991. The Genus Genlisea. Carn. Pl. News. 20, 1 & C., and González Torres, L. R. 2005. Lista roja de la flora 2. 20-26. vascular cubana. Jardín Botánico Atlántico de Gijón. White, T. J., Bruns T., Lee S., and Taylor, J. 1990. Amplification Britton, N. L. 1920. Descriptions of Cuban plants new to and direct sequencing of fungal ribosomal RNA genus science. Memoirs Torrey Bot. Clb. 16(2): 57-118. for phylogenetics. In M. Inns, D. Gelfand, J. Sninsky, Cieslak, T., Polepalli J. S., White A., Müller, K., Borsch, T., and T. White [eds.], PCR protocols: a guide to methods Barthlott, W., Steiger, J., Marchant, A. and Legendre, L. and amplifications, p. 315-322. Academic Press, San 2005. Phylogenetic Analysis of Pinguicula (Lentibulari- Diago, CA. aceae): Chloroplast DNA sequences and morphology Wood, C. E. and Godfrey R. K. 1957. Pinguicula (Lentibula- support several geographically distinct radiations. Am. riaceae) in the southeastern United States. Rhodora 59: Journ. Bot. 92, 10: 1723-1736. 217-230. Casper, S. J. 1963. Gedanken zur gliederung der Gattung Zamudio, S. and Rzedowski, J. 1991. Dos especies de Pin- Pinguicula L. Bot. Jb. 82(3): 321-335. guicula (Lentibulariaceae) del estado de Oaxaca, Mexico. Casper, S. J. 1966. Monographie der Gattung Pinguicula L. Acta Bot. Mex. 14: 23-32. Biblioth. Bot. 127/128.