Chromosome Botany (2013) 8: 23-27 © Copyright 2013 by the International Society of Chromosome Botany

Chromosome numbers of some terrestrial orchids in Chile

Mikio Aoyama1,4, Claudia Alonso2 and Ximena Calderón Baltierra3

1Botanical Garden, Technical Center, Hiroshima University, Japan; 2Laboratory of Higher Biotechnology, Faculty of Natural Renovable Resources, Unversidad Arturo Prat, Chile; and 3Public Relations Manager, Algiz S.A. Enterprise, Chile

4Author for correspondence: ([email protected]) Received March 29, 2013; accepted April 29, 2013

ABSTRACT. Chromosome numbers and ploid levels in 12 species and two hybrids in two orchid genera as Chloraea and Gavilea from Chile were investigated. The chromosome numbers of nine species and one hybrid were commonly 2n=44 or ca.44 (diploid), while those of three species were 2n=88 or ca.88 (tetraploid). One hybrid crossed between plants of diploid and tetraploid was 2n=66 (triploid). Two genera of Chloraea and Gavilea were closely related to each other cytologically with the basic chromosome number of x=22 and the karyomorphology of the simple chromocenter type at resting stage. In addition, the speciation of these members might be occurred and correlated with the diversification of their karyotype and the polyploidization.

KEYWORDS: Chile, Chloraea, Chromosome, Gavilea, , Polyploidy

The orchid flora of Chile consists of seven genera, 49 MATERIALS AND METHODS species and one variety, including 25 endemic species The materials and their sources are listed in Table 1. (Lehnebach 2003). Among them, three major genera of Taxonomic treatment of the materials followed Novoa et Chloraea (30 species), Gavilea (12 species) and Bipinnula al. (2006). They were planted and cultivated in pots in a (four species) are placed in the subtribe Chloraeinae, the greenhouse. tribe (Pridgeon et al. 2003). Since these Growing root tips of those plants were cut into small orchids can be cultivated and bloom pretty flowers, pieces ca 2 mm long and dissected into half longitudinally. horticultural studies on clonal propagation, hybridization Then, they were immersed in 2 mM 8-hydroxyquinoline and so on have been increasing. Although cytological at 15°C for 4 h. They were fixed in Farmer’s solution at information is very important for orchid breeding, only a 5°C for 24 h. The fixed materials were hydrolyzed in a 1:2 few data have still been described in the subtribe mixture of 45% acetic acid and 1 N HCl at 60°C for 30 sec, Chloraeineae with n=8 for Chloraea multiflora (De stained in 2% aceto-orcein by the usual squash method. Nordenflycht 1981) and 2n=42 for C. crispa (Baeza et al. Classification for the chromosome types at resting and 2001). Thus, the present study documents chromosome mitotic prophase stages followed Tanaka (1977). Terminology numbers and karyotypes of 12 species and two hybrids of of chromosome morphology at mitotic metaphase on the basis terrestrial Chilean orchids. of the position of the centromere followed Levan et al. (1964).

Table 1. Chromosome number and ploidy of Chilean Chloraea and Gavilea studied.

Chromosome Karyotype * Material number (2n) Ploidy at metaphase Source Chloraea alpina 44 2x gradual UNAP C. bidentata 88 4x gradual UNAP C. crispa 44 2x gradual E. Matthei C. crispa ‘alba’ 44 2x gradual E. Matthei C. crispa ‘big’ 44 2x gradual E. Matthei C. gavilu 88 4x gradual E. Matthei C. incisa 44 2x gradual E. Matthei C. magellanica 44 2x gradual UNAP C. speciosa 44 2x bimodal UNAP C. volkmanii 44 2x gradual UNAP C. sp (Andes Mts.) 44 2x gradual E. Matthei C. crispa x gavilu 66 3x gradual E. Matthei C. crispa x incisa 44 2x gradual E. Matthei Gavilea grandulifera 44 2x gradual UNAP G. litoralis ca.44 2x bimodal UNAP G. supralabellata ca.88 4x gradual UNAP *UNAP=Unversidad Arturo Prat 24 AOYAMA ET AL.

RESULTS 2n=88 was counted for this species at prometaphase and The results of chromosome numbers, ploid levels and reported here for the first time. The chromosomes in the karyotypes at metaphase were shown in Table 1. complement at prometaphase varied gradually in length (Fig. 2B). This species showed a homogeneous and gradual Chloraea crispa Lindl. (2n=44, Table 1, Figs. 1 and 3A) variation of karyotype in length. At the resting stage, the chromosomes contained numerous chromomeric granules, fibrous threads and approximately Chloraea gavilu Lindl. (2n=88, Table 1, Fig. 2C) The 20 small chromatin blocks scattered throughout the chromosome number of 2n=88 was counted for this nucleus. The chromatin blocks were round shaped, darkly species for the first time. The chromosomes in the stained and varied in size from 1.8-0.8 µm diameter. The complement varied gradually in length from 2.4 to 1.0 µm chromosome features at resting stage were identified as (Fig. 2C). This species showed a homogeneous and gradual the simple chromocenter type (Fig. 1A) and were similarly variation of karyotype in length at mitotic metaphase. observed in those of the other taxa during the course of study. Early condensed segments were located in the Chloraea incisa Poepp. (2n=44, Table 1, Fig. 2D) This proximal regions of the chromosomes at mitotic prophase species was treated as a synonym of C. piquichen (Lam.) (Fig. 1B). Morphological similarity at mitotic prophase Lindl. by Govaerts (2003). The chromosome number of was also observed in the other taxa. 2n=44 was counted and was reported here for this for the The chromosome number of C. crispa was not 2n=42 first time. The chromosomes in the complement varied previously documented by Baeza et al. (2001) but 2n=44 gradually in length from 2.6 to 1.4 µm (Fig. 2D). This uniformly counted at mitotic metaphase in a wild type species showed a homogeneous and gradual variation of clone and two selected clones (Table 1, Fig. 1C). karyotype in length at mitotic metaphase. The chromosomes in the complement were gradually decreased in length from 2.7 to 1.1 µm (Fig. 3A). Most of Chloraea magellanica Hook.f. (2n=44, Table 1, Fig. 2E) the chromosomes in the complement were median or The chromosome number of 2n=44 was counted in this submedian-centromeric. Two smallest chromosomes were species for the first time. The chromosomes in the subterminal-centromeric, and formed small satellites on complement varied gradually in length (Fig. 2E). This the distal region of the short arm. Thus, this species species showed a homogeneous and gradual variation of showed a homogeneous and a gradual decrease in karyotype in length at mitotic metaphase. chromosome size of karyotype in length and a symmetric karyotype in centromeric position. Chloraea speciosa Poepp. (2n=44, Table 1, Fig. 2F and 3B) The chromosome number of 2n=44 was counted in Chloraea alpina Poepp., (2n=44, Table 1, Fig. 2A) The this species for the first time (Fig. 2F). The chromosome chromosome number of 2n=44 was counted in this species complement exhibited a bimodality in length with a group for the first time. The chromosomes in the complement of four large chromosomes between 3.8 and 3.1 µm and varied gradually in length from 2.8 to 1.5 µm (Fig. 2A). the other group between 2.3 and 1.0 µm (Fig. 3B). Four This species showed a homogeneous and gradual variation large chromosomes were submedian or subterminal of karyotype in length at mitotic metaphase. centromeric and the others were median or submedian centromeric. This species showed a heterogeneous and Chloraea bidentata (Poepp. & Endl.) M. N. Correa bimodal variation of karyotype in length at mitotic (2n=88, Table 1, Fig. 2B) The chromosome number of metaphase.

Fig. 1. Photomicrographs of somatic chromosomes in Chloraea crispa, 2n=44.A. resting stage. B. prophase. C. metaphase. CHROMOSOME NUMBERS OF SOME CHILEAN TERRESTRIAL ORCHIDS 25

Fig. 2. Photomicrographs of somatic chromosomes in Chilean orchids studied. A. Chloraea alpina, 2n=44. B. C. bidentata, 2n=88. C. C. gavilu, 2n=88. D. C. incisa, 2n=44. E. C. magellanica, 2n=44. F. C. speciosa, 2n=44. G. C. volkmanii, 2n=44. H. C. crispa x gavilu, 2n=66. I. C. crispa x incisa, 2n=44. J. Gavilea grandulifera, 2n=44. K. G. litoralis, 2n=ca.44. L. G. supralabellata, 2n=ca.88.

Chloraea volkmanii Phil. ex Kraenzl. (2n=44, Table 1, Chloraea crispa x C. gavilu (2n=66, Table 1, Fig. 2H) Fig. 2G) The chromosome number of 2n=44 was counted The chromosome number of 2n=66 was counted in the in this species for the first time. The chromosomes in the artificial hybrid between C. crispa and C. gavilu. The complement varied in gradation of length from 2.7 to 1.6 µm chromosomes in the complement varied gradually in (Fig. 2G). This species showed a homogeneous and gradual length (Fig. 2H). This hybrid showed a homogeneous and variation of karyotype in length at mitotic metaphase. gradual variation of karyotype in length at mitotic metaphase.

Chloraea sp. (2n=44, Table 1) The chromosome number Chloraea crispa x C. incisa (2n=44, Table 1, Fig. 2I) of 2n=44 was counted in an unidentified species collected The chromosome number of 2n=44 was counted in the from the Andes mountains. The chromosomes in the artificial hybrid between C. crispa and C. incisa. The complement varied gradually in length from 2.9 to 1.5 µm, chromosomes in the complement varied gradually in and their positions of centromere were median or length from 2.7 to 1.1 µm, and their positions of centromere submedian. This species showed a homogeneous and were median or submedian (Fig. 2I). This hybrid showed a gradual variation of karyotype in length at mitotic homogeneous and gradual variation of karyotype in length metaphase. at mitotic metaphase. 26 AOYAMA ET AL.

Fig. 3. Karyotype alignments of metaphase chromosomes in gradual decrease order in length. A. Chloraea crispa with a monomodal karyotype. B. C. speciosa with a bimodal karyotype including four large chromosomes. Bar=5µm.

Gavilea grandulifera (Poepp. & Endl.) M. N. Correa compared with the gradual karyotype was commonly (2n=44, Table 1, Fig. 2J) The chromosome number of observed in the other taxa studied. Thus, Chloraea and 2n=44 was counted in this species for the first time. The Gavilea studied could be closely related in cytology chromosomes in the complement varied gradually in regarding basic chromosome number and karyotype at length from 2.5 to 1.3 µm (Fig. 2J). This species showed resting stage and mitotic prophase and chromosome a homogeneous and gradual variation of karyotype in length. The various karyotype differentiations of length at mitotic metaphase. polyploidization and structure changes might be correlated with the speciation in both genera. Gavilea litoralis (Phil.) M. N. Correa (2n=ca.44, Table 1, Recent phylogenetic analyses based on DNA sequences Fig. 2K) The chromosome number of 2n=ca.44 was have shown that the monophyly of the subtribe Chloraeinae speculated in this species at mitotic prometaphase. The is considered in an expanded concept of the tribe chromosome complement exhibited a bimodality in length Cranicideae (Chemisquy and Morrone 2012; Cisternas et with a group of ten large chromosomes and the other al. 2012). Gavilea nested with Chloraea chica and group (Fig. 3K). This species showed a heterogeneous Bipinnula nested with the monotypic taxon of Geoblasta and bimodal variation of karyotype in length. penicillata are paraphyletic. Chloraea is polyphyletic nested with the clades of Gavilea and Bipinnula. Thus, the Gavilea supralabellata M. N. Correa (2n=ca.88, Table 1, generic revision in whole subtribe Chloraeinea is Fig. 2L) The chromosome number of 2n=ca.88 was necessary. speculated in this species at mitotic prometaphase. The Further information on chromosome numbers and chromosomes in the complement varied gradually in detailed karyotypes are necessary to understand length (Fig. 2L). This species showed a homogeneous and cytological relationships and phylogeny of the members gradual variation of karyotype in length. of the subtribe Chloraeinea.

CKNOWLEDGMENT. The authors sincerely thank to Dr. Katsuhiko ISCUSSION A D Kondo, Tokyo University of Agriculture, for critically reading the Although two species among the 12 species and two manuscript, to Dr. Enrique Matthei in Concepción, for his kind help for hybrids in the Chilean terrestrial orchid genera studied did sampling the materials. not show their chromosome numbers during the course of investigation, they have a polyploidy series of 2n=44, 66 LITERATURE CITED and 88 with the basic chromosome number of x=22 in the Baeza, C. M., Kottirsch, G., Espejo, J. and Reinoso, R. 2001. genera of Chloraea and Gavilea. The triploid hybrid Recuento cromosomicos en plantas que crecen en Chile. I. Gayana, Bot. 58(2): 133-137. occurred by crossing between diploid species and Chemisquy, M. A. and Morrone, O. 2012. Molecular phylogeny tetraploid one. It suggests that the tetraploid species may of Gavilea (Chloraeinae: Orchidaceae) using plastid and reproduce by normal germination in native habitat. nuclear markers. Molec. Phylog. Evol. 62: 889-897. Karyotypes at resting stage and mitotic prophase and Cisternas, M. A., Salazar, G. A., Verdugo, G., Novoa, P., Calderon, X. and Negritto, M. A. 2012. Phylogenetic analysis of chromosome length are almost similar in the taxa studied. Chloraeinae (Orchidaceae) based on plastid and nuclear Thus, the bimodal karyotype at mitotic metaphase were DNA sequences. Bot. Jour. Linnean Soc. 168: 258-277. significantly observed in C. speciosa and G. litoralis in De Nordenflycht, G. 1981. Chromosome number reports CHROMOSOME NUMBERS OF SOME CHILEAN TERRESTRIAL ORCHIDS 27

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