_??_1994 The Japan Mendel Society Cytologia 59: 295 -304 , 1994

Cytotypes and Meiotic Behavior in Mexican Populations of Three Species of Echeandia (Liliaceae)

Guadalupe Palomino and Javier Martinez

Laboratorio de Citogenetica, Jardin Botanico , Instituto de Biologia, Apartado Postal 70-614, Universidad Nacional Autonoma de Mexico, D . F. 04510, Mexico

Accepted June 2, 1994

Echeandia Ort. includes herbaceous perennials distributed from the Southwestern United States to South America. More than 60 species have been described from Mexico and Central America, many of which are narrow endemics (Cruden 1986, 1987, 1993, 1994, Cruden and McVaugh 1989). Mexico is considered the center of origin and evolution for this genus (Cruden pers. comm.). They are commonly found in pine and pine/oak forest, grasslands; xerophyte shrublands, and disturbed areas, (Cruden 1981, 1986, 1987, Cruden and McVaugh 1989). Except species based on n=8, as E. longipedicellata n=40, (Cruden 1981); E. altipratensis n=24, and 48; E. luteola n=32, and 64; E. venusta n=84, (Cruden 1986, 1994), chromosomes those exist, little information on interspecific or intraspecific variation in karyo types in Echeandia. In 1988, Palomino and Romo described the of E. flavescens (Benth.) Cruden (as E. leptophylla) and E. nana (Baker) Cruden, both of which are characterized by 2 pairs of chromosomes with satellites. Cytotype variation has been observed commonly in the families Liliaceae, Iridaceae, Commelinaceae, and some Poaceae, Acanthaceae and Leguminosae. It is usually due to the presence of spontaneous aberrations in number or structure, heterozygous inversions, Roberts onian translocations, exchanges, deletions and duplications (Sen 1975, Araki 1975, Araki et al. 1976, Brighton 1976, 1977a, b, Datta and De 1990, Christopher and Jacob 1990, Sakya and Joshi 1990, Vijayavalli and Mathew 1990). Heteromorphic bivalents and/or bridges with or without fragments are evidence of these structural aberrations (Brandham 1970, Brandham and Johnson 1977, Jones 1978, Kenton 1981, Palomino and Vazquez 1991). Cytotype distribution is established by geographical isolation and natural selection (Kenton 1981, Kenton et al. 1988). There is also evidence of polyploid cytotypes reported by Jones et al. (1981), Araki (1985), Kumar and Gohil (1990), Piovano and Bernardello (1991). Sometimes cytotypes can be transmitted via sexual reproduction. They are usually maintained by asexual reproduction forming a cluster of plants with the same cytotype (Haga and Noda 1976, Araki 1985, Araki et al. 1976). We describe variation in the cytotypes of three species, the behavior of meiotic chromo some, and pollen fertility for populations of Echeandia echeandioides, E. tenuis and E. mexicana.

Material and methods

The species studied were: E. echeandioides Cruden (=Anthericum echeandioides Baker), which is endemic species to central part of Mexico, E. tenuis (Weatherby) Cruden and E. mexicana Cruden. Plants were collected from wild populations in pine oak forest (Appendix 1) and voucher specimens were deposited at the National Herbarium (MEXU) of the Universidad Nacional Autonoma de Mexico. 296 Guadalupe Palomino and Javier Martinez Cytologia 59

Three to six individuals from two or three populations of each species (Appendix 1) moved to the Jardin Botanico. The plants were transplanted into pots containing a mixture of vermiculite and organic soil and maintained in a greenhouse. Elongating secondary root tips were placed in a saturated solution of paradichlorobenzene for 6 hr at 4•Ž. They were staining following Feulgen technique (Palomino and Vazquez 1991). Preparations from 3 to 6 plants were made, and ten cells at mitotic were selected from each for examination. Three of the best cells from each plant of each population were photographed using a Zeiss Photomicroscope II. Idiograms were made using a Zeiss Drawing Apparatus. Chromosomes were classified according to Levan et al. (1964) and Naranjo et al. (1986). Index of asymmetry (TF% or TF index) was obtained following Gupta and Gupta (1978). To study meiotic behavior fresh anthers from young buds were squashed in 1.8% aceto-orcein, and 168 pollen mother cells (PMC) in metaphase I (MI) and 913 to 998 PMC in anaphase I (AI) were analyzed. For MI PMCs, from each population the following informa tion was recorded: type of bivalent (IIs) and quadrivalent (IVs), chiasmata frequency and recombination index (RI, White 1973). In AI PMCs recorded data were: single bridge, double bridge, single bridge with fragment and AI with lagging chromosomes. Pollen fertility was estimated by staining samples with cotton blue in lactophenol. Percentage of well-filled stained grains was obtained. This was carried out for 238 to 877 pollen grain cells of three plants in each population studied. The student "t" test, was applied to detect differences among haploid chromatin length in the populations analyzed.

Results

All the populations of the three species we studied were diploid with 2n=16 and n=8 chromosomes and in which two pairs of chromosomes had satellites. Each species had a different (Table 1, Figs. 1, 2) and their cytotypes were uniform within populations but varied within the species studied. Cytotypes of E. echeandioides varied in the number of metacentric (m) and submetacentric (sm) chromosomes. Population 359 had 3 pairs of heteromorphic chromosomes (Figs. 1A, 2A) whereas population 360 had a single heteromor phic pair of chromosomes (Figs. 1B, 2B). Population 321 the least variable cytotype. All the chromosomes were homomorphic and one pair had subtelocentric chromosomes (st), (Table 1, Figs. 1C, 2C), which were absent from the other populations. The two populations of E. tenuis had cytotypes that varied in quantity of m, sm and st chromosomes (Table 1). Population 356 had a pair heteromorphic chromosomes (Figs. 1D,

Table 1. Cytotypes of three species of Echeandia 1994 Cytotypes and Meiotic Behavior in Mexican Populations of Three Species of Echeandia 297

Fig. 1. Cytotypes for: A. E. echeandioides (359). B. E. echeandioides (360). C. E. echeand ioides (321). D. E. tenuis (356). E. E. tenuis (484). F. E. mexicana (236). G. E. mexicana

(284). H. E. mexicana (357). Numbers 1 and 2 indicate chromosomes with satellites. Scale equals 10ƒÊm.

2D) and population 484 2 pairs (Figs. 1E, 2E). Three populations of E. mexicana were examined. The population 236 and 284 had different cytotypes and exhibited variation in number of m, sm and st chromosomes. Popula tion 357 had only m and sm chromosomes (Table 1). None of the cytotypes of E. mexicana had heteromorphic pairs (Figs. 1F, 2F, 1G, 2G, 1H, 2H). Variation in the genome size was confirmed by the significantly different haploid chromatin lengths (P<0.05, Table 1). Meiotic behavior was shown by the average number of chiasmata per cell and the recombination index. Ring and rod bivalent with variable frequencies were found at MI (Table 2). All the populations analyzed had 1 to 3 heteromorphic bivalents (Figs. 3A, B, C, D). E. echeandioides #359 exhibited a ring and heteromorphic IVs with frequencies of 0.47 and 0.23 (9.7%) respectively. The population 360 had only a heteromorphic chain IVs with a frequency of less than 0.14 (1.7%), (Table 2). All the populations we studied exhibited aberrations at AI with variable frequencies (Table 298 Guadalupe Palomino and Javier Martinez Cytologia 59

3, Fig. 4). E. echeandioides #359 and 360 had cells with bridge and fragment, sometimes one or two bridges, and lagging chromo somes. The population 321, did not display bridges with fragment, or lagging chromo somes (Table 3, Fig 4A). Aberrant AI cells occurred in all populations of all three spe cies that had many shrunken or empty pollen grains, (Table 4). E. tenuis #484 displayed more structural chromosomal aberrations (Table 3, Figs. 4A, C), and many non-viable pollen grains (Table 4) compared to population 356. E. mexicana #284 and 357 had higher percent age of AI aberrations than population 236. No lagging chromosomes were observed (Table 3, Fig. 4B). The highest percentage of non-viable pollen found in E. mexicana occurred in collections 236 and 357 (Table 4).

Discussion

The populations of the three species we examined were diploid with 2n=16 and n=8 (x=8) chromosomes. Each species had a distinctive karyotype that varied among pop Fig. 2. Idiograms for: A. E. echeandioides (359). B. ulations. Intraspecific cytotype variation was E. echeandioides (360). C. E. echeandioides (321). D. manifest as heteromorphic chromosome E. tenuis (356). E. E. tenuis (484). F. E. mexicana

(236). G. E. mexicana (284). H. E. mexicana (357). pairs in E. echeandioides and E. tenuis, num Asterisks indicate pairs of heteromorphic chromo bers of metacentric, submetacentric, and sub somes. Scale equals 10ƒÊm. telocentric chromosomes, and chromosomes with satellites. In addition, Cruden (1981) reported tetraploid populations of E. mexicana from Michoacan and Jalisco. Similar intraspecific cytotype variation has been reported in other Liliaceae. Heteromor phic chromosome pairs resulting from asymmetrical exchanges were reported in Scilla (Sato 1942, Gimenez-Martin 1959, Haga and Noda 1958, Noda 1961), Gloriosa superba (Vijayavalli and Mathew 1990), and in tribe Aloineae (Brandham 1974, 1976). In Scilla, the heteromorphic chromosome pairs were attributed to translocations and deletions. In Scilla scilloides, Araki (1975, 1977, 1985), Araki et al. (1976) studied 46 natural populations, and reporting diploid, polyploid and aneuploid cytogenetic types. Clusters of plants with the same karyotype were sexually unstable. The only effective mode of propagation was vegetative reproduction, via bulb multiplication as those reported by Haga and Noda (1976). In Smilacina and Dianella cytotype variation is supported by vegetative reproduction, which is very common (Sen 1975). In Haworthia browniana several asymmetrical exchanges were reported in a single large clone (Brandham and Johnson 1977). Polyploid and aneuploid cytotypes are known in many Liliaceae, including Scilla (Araki 1975, 1977, 1985, Araki et al. 1986) and Polygonatum (Tamura 1990). Heteromorphic bivalents and/or bridges with or without fragment reflect structural 1994 Cytotypes and Meiotic Behavior in Mexican Populations of Three Species of Echeandia 299

Table 2. Type and frequency of bivalents, (IIs), quadrivalents, (IVs), chiasmata frequency and recombination index (RI) for eight populations of three species of Echeandia

Fig. 3. PMCs in: A. E. echeandioides (359) with 5 IIs+heteromorphic ring II+heteromorphic ring IV. B. E. echeandioides (359) with 5 IIs+heteromorphic ring II+heteromorphic chain IV. C. E. tenuis (484) with 5 IIs+3 heteromorphic rod IIs. D. E. mexicana (357) with 7 IIs+ heteromorphic rod II. Numbers 1- indicate heteromorphic ring IIs; 2- heteromorphic ring IV; 3- heteromorphic chain IV; 4- heteromorphic rod IIs. Scale equals 10ƒÊm.

changes such as heterozygous inversions, Robertsonian translocations, exchanges, deletions and duplications (Brandham 1970, Brandham and Johnson 1977, Jones 1978, Jones et al. 1975, Kenton 1981, Palomino and Vazquez 1991). The heteromorphic chromosomes we observed in all populations we studied, also the quadrivalents, both ring and chains, we observed in the two 300 Guadalupe Palomino and Javier Martinez Cytologia 59

Table 3. Normal and irregular Al for eight populations of three species of Echeandia

Fig. 4. PMCs showing irregular AI: A. E. tenuis (484) AI with bridge and fragment . B. E. mexicana (357) AI with bridge. C. E. tenuis (356) AI with 2 bridges. D. E. echeandioides

(360) AI with lagging chromosome. b, indicate bridges; f, indicate fragments and l, indicate lagging chromosome. Scale equals 10ƒÊm. 1994 Cytotypes and Meiotic Behavior in Mexican Populations of Three Species of Echeandia 301

Table 4. Shrunken or empty pollen grains for eight populations of three species of Echeandia

populations of E. echeandioides, may reflect nonreciprocal translocations. The heteromorphic chromosome pairs observed in tribe Aloineae resulted from asymmetrical exchanges and involved Robertsonian fusions (Brandham 1976). In Gibasis pulchella, chromosomes of heterozygous plants with a single exchange formed a ring or chain of four and half the pollen was sterile (Kenton et al. 1987). However, in Astroloba foliolosa the formation of quadribalents has been attributed to a hybrid origin (Brandham 1973). Additional evidence for translocations and chromatid exchange is the low level of meiotic irregularities observed during , including U-type bridges associated with acentric fragments in AI, side-arm bridges (SAB) without acentric fragment, and lagging chromosomes at AI. Except one population of E. echeandioides (#321), all populations we studied exhibited U-type bridges. Frequencies ranged from 3.91% (E. echeandioides, #359) to 0.30% (E. mexicana, #236). SABs occurred at higher frequency than U-type bridges with a range of 1.91% (E. mexicana, #236) to 12.78% (E. mexicana, #284). Likewise is Aloineae, although the frequency of U-type bridges was higher, 1% to 20%, they were less common that SABs. Only in 41/186 plants chromatid errors U-type was observed and subchromatid errors (SAB) occurred together in 104/186 plants. No plants produced SAB errors alone (Brandham 1970). Lagging chromosomes occurred at low frequencies in two populations of E. echeandioides and in two populations of E. tenuis (Table 3), but were not observed in E. mexicana. The presence of lagging chromosomes is less frequent in wild populations. They have been observed in a mitotic anaphase in pollen grains of Tradescantia commelinoides (Kenton et al. 1988). This clone displayed a spontaneous breakage, reunion and disturbance of mechanisms of control, similar to those that produce lagging chromosomes. A period of instability, leading the establishment of new equilibrium, may represent an important source of variability in species with vegetative reproduction (Kenton et al. 1988). The various types of meiotic irregularities observed during meiosis undoubtedly account for the high levels of shrunken, emptied, and/or small pollen grains observed. Likewise, in Gibasis pulchella, plant heterozygous for translocations exhibited a 50% reduction in pollen viability (Kenton et al. 1987) The presence of same cytotype in populations of 3 Echeandia species suggests, they were originated from a single plant via vegetative reproduction, common in species of the genus. It can be assumed that various aberrant cytotypes confer advantages on the individuals carrying them in the colonization of new habitats. In summary, we suggest that chromosomal rearrangements may have played an important role in the evolution of Echeandia. Observation of low quadrivalent levels and bridge with anc without fragment, also the presence of heteromorphic chromosome pairs suggests that trans locations and chromatid exchanges have played a mayor role in shaping the karyotypes of species and populations. 302 Guadalupe Palomino and Javier Martinez Cytologia 59

Summary

Analysis of 8 populations of 3 species of Echeandia echeandioides, E. tenuis and E. mexicana showed that all were diploid, chromosome counts were 2n=16, n=8 (x=8). Each species had a different karyotype. The three species also presented different cytotypes in all populations. However, the presence of the same cytotype in each population suggested that all populations originated from a single clone by vegetative reproduction. Cytotype variation was observed in heteromorphic pairs of metacentric, submetacentric, subtelocentric and chromo somes with satellite. The meiotic analysis had heterozygotic exchanges. MI analysis showed heteromorphic IIs and IVs. AI analysis displayed U-type chromatid exchange (3.91%-0.30%) and sub-chromatid aberrations (12.78%-1.91%). Lagging chromosomes were recorded in AI in 4 populations of E. echeandioides and E. tenuis. E.mexicana did not present this type of aberration. Based on these results, we can suggest that exchanges had played an important role in the evolution of the genus.

Appendix 1

E. echeandioides. 1- Mexico state, 43km Toluca to Temascaltepec, Palomino and Marti- nez 359 (MEXU). 2- Mexico state, 2.6km Temascaltepec to Valle de Bravo, Palomino and Martinez 360 (MEXU). 3- Guerrero State, 33km Chipalcingo to Chichihualco, Palomino and Martinez 321 (MEXU). E. tenuis. 1- Mexico state, 21.3km Temascaltepec to Tejupilco, Palomino and Martinez 356 (MEXU). 2- Mexico State, 4km Temascaltepec to Valle de Bravo, Palomino and Martinez 484 (MEXU). Echeandia mexicana. 1- Mexico State, Cerro Tetzcutzingo, Palomino and Martinez 236 (MEXU). 2- Hidalgo State, 67km Atotonilco to Molango, Palomino and Martinez 284 (MEXU). 3- Mexico State, 4km Temascaltepec to Valle de Bravo, Palomino and Martinez 357 (MEXU).

Acknowledgements

This study was supported by OEA:"Estudios Biosistematicos en algunos generos de Leguminosas, Liliaceae y Palmas de Mexico": Citogenetica 88-89, PRDCyT; CONACyT and Jardin Botanico of the Instituto de Biologia of the Universidad Nacional Autonoma de Mexico. We are grateful to Dr. Peter Brandham and Dr. Robert Cruden for their comments and suggestions to the manuscript. We also thank Dr. Cruden for the identification names of the plants used in this research and Rocio Cid for help in table's edition.

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