The Phylogeny of Japanese Enkianthus Species (Ericaceae)
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Bull. Natl. Mus. Nat. Sci., Ser. B, 38(1), pp. 11–17, February 22, 2012 The Phylogeny of Japanese Enkianthus species (Ericaceae) Chie Tsutsumi* and Yumiko Hirayama Department of Botany, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan * E-mail: [email protected] (Received 16 November 2011; accepted 28 December 2011) Abstract The genus Enkianthus consists of 12–17 species, most of which are distributed in China and Japan. We performed molecular phylogenetic analyses for Enkianthus species of Japan, using nuclear ITS, chloroplast matK with trnK intron, and trnS-trnG spacer regions. The result suggested that Enkianthus was separated into two groups; i.e., sect. Enkianthus with umbellate inflorescences and a clade of sect. Andromedina, sect. Enkiantella and sect. Meisteria with race- mose inflorescences. It also inferred that the ancestral Enkianthus seems likely to be shrubs with tubular or urceolate corollas and 4–5-apertured pollen, whereas the more derived species may pos- sess campanulate corollas and 3-apertured pollen. Kew words : Enkianthus, ITS, matK, molecular phylogeny, trnSG. Introduction ture, inflorescences structure, corolla shape, and morphologies of anther, pollen and seed (Ander- The genus Enkianthus Lour. (Ericaceae) is a berg, 1994; Kron et al., 2002). Infrageneric rela- genus of shrubs or small trees and often used as tionships of Enkianthus were studied by Ander- ornamental plants in the gardens. The genus con- berg (1994), who proposed a classification sists of 12–17 species (Anderberg, 1994; Kron et comprising four sections. Sect. Enkianthus has al., 2002; Fang and Stevens, 2005). All of the more or less coriaceous leaves, tubular or urceo- species are distributed from the Himalayas to late flowers, umbellate inflorescences, 4–5-aper- Japan (Fang and Stevens, 2005) and most of tured pollen, glabrous pedicels and smooth them are in China and Japan. Recent molecular anthers (except for E. serotinus Chun et Fang phylogenetic studies showed that Enkianthus is with papillose anthers). Sect. Andromedina sister to all other representatives of Ericaceae s.l. shares several features with sect. Enkianthus but including Empetraceae, Epacridaceae, Monotro- is distinct in racemose inflorescences and non- paceae and Pyrolaceae (Kron and Chase, 1993; winged seeds. Sect. Meisteria is characterized by Kron, 1996; Morton et al., 1996; Anderberg et racemose inflorescences, 3-apertured pollen, al., 2002; Kron et al., 2002). A cladistic analysis campanulate corollas, and a papillose corolla sur- using morphological, anatomical, embryological face, and sect. Enkiantella by racemose inflores- and cytological characters suggested monophyly cences (except for E. pauciflorus Wils. with soli- of the genus (Anderberg, 1994). The species of tary flowers), 3-apertured pollen, campanulate Enkianthus are distinct from the other Ericaceae corollas, conspicuously winged and lamellate in several characters, such as embryological fea- seeds, and anthers without a prolonged connec- tures and morphologies of anther, pollen, seed tive. Anderberg (1994) also suggested that and hair (reviewed by Kron et al., 2002). sect. Enkianthus is sister to sect. Andromedina, The species of Enkianthus differ in leaf tex- then they are sister to sect. Meisteria, and sect. 12 Chie Tsutsumi and Yumiko Hirayama Enkiantella is firstly diverged group in the genus The internal transcribed spacer regions of 18S- based on morphology. Therefore, the ancestral 26S nuclear ribosomal DNA (ITS), and two chlo- Enkianthus seems to have deciduous leaves, rac- roplast regions, matK (the maturase-encoding emose inflorescences, campanulate corollas, and gene) with trnK intron, and trnS-trnG spacer, winged seeds, whereas the more derived species were analyzed. Primers for amplification were likely possess coriaceous leaves, umbellate inflo- 17SE (Sun et al., 1994) and ‘26SE’ (Topik et al., rescences, flowers with tubular or urceolate 2005, modified 26SE in Sun et al., 1994) in ITS corollas, and non-winged seeds (Anderberg, region, and trnK-3914F and trnK-2R (Johnson 1994). The classification was supported by a pol- and Soltis, 1994) in matK with internal primers len morphological study (Golam Sarwar and MK-F1 (Koi et al., 2008), matK-E728F (5′-ATA- Takahashi, 2007). The molecular phylogeny of GATCTCAGCAACATGAC-3′), matK-E1166F the genus, however, remains to be examined. (5′-CATCCATCTGGAAATCTTGG-3′), matK- In Japan, six species and two varieties of Enki- E780R (5′-GTGGATATATGAAGTCATGTT- anthus are recognized (Yamazaki, 1993). GCTG-3′), and matK-E1391R (5′-TTGGCAC- According to the section concept sensu Ander- TATGGTATCGAACT-3′), and trnS (GCU) and berg (1994), Enkianthus perulatus (Miq.) trnG (UCC) in trnS-trnG spacer (Hamilton, C.K.Schneid. is assigned to sect. Enkianthus, E. 1999). PCR for ITS region was performed using nudipes (Honda) Ueno and E. subsessilis (Miq.) a DNA thermal cycler (Perkin-Elmer 9700, Makino are assigned to sect. Andromedina, Applied Biosystems, Foster, CA) with LA Taq E. campanulatus (Miq.) G.Nicholson, and E. DNA polymerase (TaKaRa Bio, Tokyo, Japan) in sikokianus (Palib.) Ohwi and E. cernuus (Siebold 35 denaturation, annealing, and elongation cycles et Zucc.) Makino belong to sect. Meisteria. (30 sec at 95°C, 30 sec at 55°C and 90 sec at Enkianthus perulatus is distributed in Japan and 72°C) with a final elongation step (5 min at Taiwan, although plants in Taiwan are sometimes 72°C). PCR for matK with trnK intron and trnS- treated as a distinct species, E. taiwanianus Ying. trnG spacer regions was conducted with Ex Taq The other five species are endemic to Japan. In DNA polymerase (TaKaRa Bio, Tokyo, Japan) in this study, the molecular phylogeny of the six 35 denaturation, annealing, and elongation cycles Japanese Enkianthus species was analyzed using (30 sec at 95°C, 30 sec at 55°C and 90 sec at nuclear ITS regions and two chloroplast DNA 72°C) with a final elongation step (7 min at regions, matK with trnK intron and trnS-trnG 72°C). The PCR products were purified with spacer. Enkianthus quinqueflorus Lour. distrib- ExoSAP-IT (USB corporation, Cleveland, OH) uted in China to Vietnam, and Clethra species, following the manufacturer’s instruction. one of sister candidates to Enkianthus, were also Sequencing was conducted using an ABI PRISM analyzed in this study. 3130xl Genetic Analyzer (Applied Biosystems). The sequences obtained were assembled using Materials and Methods Seqman II (Dnastar, Madison, WI). The assem- bled sequences were aligned by Clustal X pro- Leaf samples of seven Enkianthus species gram (Thompson et al., 1997) and then aligned were collected in the field or from cultivated manually. In addition to the sequences analyzed, plants in the Tsukuba Botanical Garden, National several registered sequences of Enkianthus and Museum of Nature and Science (Table 1). Cle- Cyrilla racemiflora L. (Cyrillaceae) in GenBank thra barbinervis Siebold et Zucc. was also ana- were added for phylogenetic analyses. lyzed (Table 1). DNA was extracted from fresh Phylogenetic analyses were performed by or silica-gel-dried material using a QUIAGEN Bayesian analyses and maximum parsimony DNeasy Mini Kit (QUIAGEN, Valencia, CA) (MP). Bayesian analyses were performed by following the manufacturer’s instruction. MrBayes 3.1.2 (Huelsenbeck and Ronquist, Phylogeny of Enkianthus 13 Table 1. Species used in molecular phylogenetic analysis, sources and GenBank accession no. of ITS, matK with trnK intron and trnS-trnG spacer regions. GenBank accession no. Species Source ITS matK trnSG Sect. Meisteria E. campanulatus (Miq.) Cultivated in Tsukuba Botanical Garden; AB697666 AB697674 AB697682 G.Nicholson Tsutsumi s.n. E. campanulatus from Genbank AF091940 U61344 GU176714 (Miq.)*1 E. campanulatus from Genbank AF133752 (Miq.)*2 E. sikokianus (Palib.) Ohwi Cultivated in Tsukuba Botanical Garden AB697667 AB697675 AB697683 (Origin; Kochi, Japan); Tsutsumi s.n. E. cernuus (Siebold et Cultivated in Tsukuba Botanical Garden; AB697668 AB697676 AB697684 Zucc.) Makino f. cernuus Tsutsumi s.n. Sect. Andromedina E. nudipes (Honda) Ueno Shinshiro, Aichi, Japan; Tsutsumi AB697669 AB697677 AB697685 Ho3-20100514 E. subsessilis (Miq.) Makino Cultivated in Tsukuba Botanical Garden AB697670 AB697678 AB697686 (Origin; Tochigi, Japan); Tsutsumi s.n. Sect. Enkianthus E. perulatus (Miq.) Cultivated in Tsukuba Botanical Garden; AB697671 AB697679 AB697687 C.K.Schneid. Tsutsumi s.n. E. quinqueflorus Lour. Cultivated in Tsukuba Botanical Garden AB697672 AB697680 AB697688 (Origin; Hong Kong); Tsutsumi s.n. E. quinqueflorus Lour.* from Genbank FJ378572 FJ357264 Sect. Enkiantella E. chinensis Franch.* from Genbank FJ378571 FJ357263 Clethraceae Clethra barbinervis Siebold et Kagoshima Pref. Japan; Tagane & Fuse AB697673 AB697681 AB697689 Zucc. (TNS763118) Cyrillaceae Cyrilla racemiflora L.* from Genbank AF396236 AJ429282 * Asterisks show the data derived from registered sequence in Genbank. 2001; Ronquist and Huelsenbeck, 2003). involving TBR branch swapping. Clethra MrModeltest 2.0 (Nylander, 2004) was used to barbinervis and/or Cyrilla racemiflora were used determine the nucleotide substitution model for as outgroups (Anderberg et al., 2002; Soltis et Bayesian analysis. Bayesian searches were con- al., 2011). ducted by mcmc with four chains over one mil- Homogeneity among the different datasets was lion generations for each of ITS, matK with trnK calculated with the ILD test (Farris et al., 1995; intron, trnS-trnG spacer regions, and over two Johnson and Soltis, 1998) as implemented in