The JapaneseSocietyJapanese Society forforPlant Plant Systematics ISSN OOOI-6799 Acta Phytotax. Geobot 50 (2): 173-178 (1999) A Cytological Study of Three Genera of Neotropical Bombacaceae (Clades Bombacoideae and Malvoideae) i, 2 3 KAZUO OGINUMA WILLIAM S. AL:VERSON and DirvID A, BAUM i Department of Ehviommental Science, Fkec"tty ofH"man Lde and Environmental 2Ehvironmentaland Science, Kbchi -bments' [lhiversit.ll Kbchi 780-8515, jopan; 3Department Conservation Programs, 77ze Field Museum, Chicago llL 6060524% USA; ofBotanM Uhiversiry of wrseonsin. Mddison, wr,yconsin 53706, Usu AbstracL Somatic chromoserne numbers for representatives of three genera of neotropical Bombacaceae were investigated using meristematic cells of root tips, [[Xve of the three genera were investigated for the first time cytologically, and for the third genus, the chromosome count reported here is the first for the species studied. The chromosome numbers were 2n=96 in (lyrantheru cdribensis, 2n=88 in euaruribea aurantiocalyx, and 2n=88 in opirotheca rosea, In view of these results, the range of chromosome numbers for the traditional Bombacaceae, from 2n=86 to 2n=92, proposed by Baum and Oginuma (1994), should be extended to 2n=86 to 2n=96. For the entire Bombacoideae clade, which appears to include the genera Chiranthodendron and Fremontodendron, the reported numbers range from 2n=.40 to 2n=98, pending new counts from these two genera, Key words: Bombacaceae, Bombacoideae, chromosome number, cytology, Malvoideae ReceivedAugust 10, 1999; acceptedAugust 3a 1999 As traditionally circumscribed, the family Bombacaceae (order Malvales) comprises approximately 30 genera and 250 species of mainly tropical trees. Historically, the family was classified into four to five tribes, e.g., Bombaceae, Catostemateae, Ceibeae, Durioneae, and Matisieae (Thkhtajan, 1997). However, recent rnolecular work strongly suggests that traditional Bombacaceae are not monophyletic (Baum et al., 1998; Alverson et al., l999; Bayer et al., 1999). For example, Durio, and all other Durioneae (except Cbvaniltesia and Scleivnema) fall in the clade Helicteroideae, a separate lineage from other traditional Bombacaceae. Most members of the traditional family fal1 into the clade Bombacoideae, which is nested within the 1arger clade Malvatheca. The Bombacoideae clade comprises the majority of traditional Bombacaceae and includes mostly neotropical taxa, often with palmately compound leaves. The few remaining traditional Bombacaceae (Camptostemon, Matisia, Pqpuodendron, Phragmotheca, and euararibea) fall within the clade Malvoideae, which also includes all raditional Malvaceae and also is nested within the Malvatheca clade. In this paper we provide new counts for members of the Bombacoideae and Malvoideae clades and discuss the pattem of evolution of chromosome numbers in these clades. Previous cytological studies in Bombacoideae have noted the presence of numerous and small chromsomes. Chromosome numbers of 13 genera NII-Electronic Library Service The JapaneseSocietyJapanese Society forforPlant Plant Systematics l74 Acta Phytotax. Geobot. Vbl. 50 in this group have been reported including diploid complements of 2n=28, 48, 56, 60-64, 72, 74, 75, 76, 80, 80-84, 86, 88, 92, 96, 128, 144, 150, 160, 210, 270 and 276. This hypervariability of chromsome numbers (Baum and Oginuma, 1994) may be due in part to the occurrence of accessory chromosomes (Baker and Baker, 1968; Bawa, 1973), aneusomatic mitosis in root tip cells (Gibbs et al., 1988), and the practical ditTiculties of counting small chromosomes, For example, early work in the genus Adansonia suggested 2n=48, 60-64, 72, 80-84, 88, 96, 128 and 144. However, and exhaustive study by Baum and Oginuma (1994) found that seven species had 2n=88 whereas A. digitata L. had 2n=160. Similarly, Gibbs et al. (1988) reported a conservative chromosome number of n=43 in fbur species of Ceiba and in three species of Chorisia. Baum and Oginuma (1994) reviewed "Bombacaceae" chromosome numbers published in and found that all the gametophytic chromosome numbers previously reported in 14 species of eight genera fa11 between n=43 and n=46 (except for two genera of Durioneae, which we now know do not belong to the Bombacoid clade). Consequently, they suggested that many of the published chromosome numbers appear to be erroneous, and the chromosome numbers from 2n=86 to 2n=92 are general in ''Bombacaceae" (except for some polyp]oid species). Subsequent molecular phylogenetic studies indicate that Chiranthodendron and Fnemontodendron, two genera usually placed in the Sterculiaceae, fall within the Bombacoid clade (Alverson et al., 1999; Bayer et al., 1999). TVvo coullts for one of these genera have been reported, n=20 in Fnemontodendron calijbrnicum (Tbrrey) Cov. (Lenz, 1950) and n= ca. 49 in E decumbens (Lloyd, 1965). In contrast to BQmbacoideae, Malvoideae generally have lower chromosome counts (Fryxell, 1968), mostly in the range of n=5 to n=22 (though a few counts range as high as n=42). Therefbre, we were interested in determining whether those traditional Bombacaceae that appear, based on molecular･data, to be closely related to Malvoideae have simi1arly low chromosome counts. In this paper we obtained the first chromosome count for a member of the Matisieae clade, which falls within Malvoideae. Additionally, we obtained counts for two new taxa in Bombacoideae. Materials and Methods The plants representing these three genera are cultivated at the Olbrich Botanical Garden in Madison, Wisconsin, U.S.A. These are (including voucher information): euararibea aurantiocalyx W. S. Alverson (Alverson s. n. [at the "spirotheca'' WIS Herbarium]) and rosea (Alverson 2185 [WIS]) (= Ceiha rosea [Seem.] K. Schum.), both originally wild-collected in Costa Rica, and G);ranthera caribensis Pittier (Ittis and Ruiz 3058i [WISI), originally collected in Venezuela. The somatic chromosomes were examined in the meristematic cells of root tips. These root tips were pretreated in a saturated aqueous solution of PDB -20 (paradichlorobenzen) at ℃ for about 6 hrs and then fixed in Jackson's Solution (4 parts of concentrated ethanol, 2 parts of concentrated methanol, 2 parts of -20 chlorofbrm, 1part of propionic acid and 1 part of acetone) at ℃ fbr about 24 hrs, and stored in 70% ethanol at -20 ℃ . The methods fbr squashing and observing chromosomes have been described elsewhere (Oginuma and Nakata, l988). From NII-Electronic Library Service The JapaneseSocietyJapanese Society for Plant Systematics Decembcr 1999 0GINUMA et at: Cytology of Bombacaceae 175･ each species, a minimum of three metaphase plates were examined. Some species of Spirotheca have synonyms under the generic name Ceiba, but recent molecular studies suggest that Elpirotheca is less closely related to Ceiha than are some other genera of traditional Bombacaceae (Alverson et al., 1999; Alverson et al., in prep.). However, since the new combination has not yet been published fbr the "Spirotheca" species used in this study (Ceiba rosea), we refer to it here as rosea. Results and Discllssion (lyranthera caribensis Pittier (Bombacoideae clade) Chromosomes at metaphase are 2n=96. The variation in chromosome length is gradual; the longest chromosomes are al)out 1.3 pm long, and the shortest about O.4 ptm long. Some of the chromosomes appear to have centromeres in median or submedian positions, but others the exact position of centromeres is uncertain '(Figs. 1-2). (Ilyranthera comprises two or three species distributed from Panama to Peru apd Venezuela. This is the first report of chromosome number for the genus. The same chromosome number of 2n=96 in Clyranthera is found in two other genera of Bombacoideae, Adansonia and Bomh(vt. e"araribea aurantiocalyx W. S. Alverson (Matisieae clade, within the Malvoideae clade) Chromosomes at metaphase are 2n=88. The variation in chromosome length is gradual; the longest chromosomes are about 1.3 pm long, and the shortest about O.4 t.tm long. Most of larger and medium-sized chromosomes have centromeres at median position, and some chromosomes at submedian position (Figs. 3-4). As regards some shorter chromosomes, the positions of their centromeres are uncertain. euararihea comprises approximately 30 species widely distributed in Central and South America. This is the first report of chromosome nurnber for the genus."Spirotheca" nosea (= Ceiba rosea [Seem.] K. Schum.), Chromosomes at metaphase are 2n=88. The variation in chromosome length is gradual; the longest chromosomes are about 1.5 ym long, and the shortest about O.4 rmi long. Most of 1arger and medium-sized chromosomes have centromeres at median position, but others the exact position of centromeres is uncertain (Figs. S- 6). Spirotheca comprises six or more species distributed in tropical South America north to Costa Rica and is notable for its hemiepiphytic habit. Our previous knowledge of the chromosomes of Spirotheca was restricted to only one species; n=46 of Eij?irotheca passijlonoides (Gibbs et al., 1 988). "Spirotheca'" The present observations of rosea, with 2n=88, and Gyranthera caribensis with 2n=96, are consistent with the range of chromosome numbers in the core Bombacoideae clade reported by Baum and Oginuma (1994), but indicates that this range needs to be broadened slightly, to 2n=86 to 2n=96. Additional research and re-counts of previously reported species may clarify the chromosomal evolution of all Bombacoideae, including Chiranthodendron and Fnemontodendron, and may suggest relationships between genera. NII-Electronic