Cytologia 43: 671-678, 1978

Somatic Karyotype Analysis of Uvularia floridana Chapman (Liliaceae)

Frederick H. Utech

Carnegie Museum of Natural History, Section of Plants, Pittsburgh, PA. 15213, U. S. A.

Received April 30, 1977

The small genus Uvularia L. with 5 species is confined to eastern North America and has been investigated by both classical taxonomic methods (Wilbur 1963) and cytotaxonomic comparison (Kawano and Iltis 1964). In the latter study, material for karyological analysis of U. floridana Chapman was not available. It is a member of the sessile-leaved section Oakesiella (Small) Wilbur. This rare endemic is confined exclusively to the southeastern Coastal Plain (Johnson 1969, Wilbur 1963). Since its range is wholly within areas only available since the Createous and since its mor phology is derived within the section, an investigation of its somatic karyology was undertaken to complete the cytotaxonomy of the genus.

Materials and methods

Materials for this study consisted of two clones of U. floridana collected ca.

10 km. apart along the terraced floodplain of the Chipola River, north and north west of Marianna, Jackson Co., Florida. This area is the southern most known site for this species. Herbarium specimens prepared from both clones (24 March 1977, Utech 77-101a and 77-101b) have been distributed as cyto-vouchers (CM, WIS, GA, MO, KYO, TI). Excised root tips from the two isolated clones were pretreated with 0.02 colchicine for 4hrs. at room temperatures (19-21•Ž) and fixed 3:1 (acetic ethanol) for 1 hr. All resulting squash preparations were made using a modification of the aceto-orcein procedure of Tjio and Levan (1950) (cf. Kawano 1965). Chromo somal measurements and photographs were made at 1250•~ using an Olympus microscope equipped with a standard 10•~ Leitz occular micrometer. Ten cells with well spread and condensed chromosomes from each clone were selected for measurement and inter-clonal comparison (Table 1; Fig. 1). Re presentative squashes from both clones are presented (Fig. 2) as well as somatic idiograms of these cells (Fig. 3). Standard deviations are indicated for the follow ing pooled clonal measurements: long arm, short arm, total chromosome length, relative length and centromeric index (Table 2). These measurements were used to construct a composite haploid idiogram for the species (Fig. 4).

Observations and results

The interphase nuclei of Uvularia floridana stain uniformly with no indication 672 Frederick H. Utech Cytologia 43 of heteropycnotic bodies or chromocenters. The colchicine pretreated root tips had numerous metaphase figures. For each clone a total of 100 cells were counted. All 200 counts were 2n=12. This reduced aneuploid base number for U. floridana was totally unexpected since the four other members of the genus are 2n=14 (Kawano and Iltis 1964).

Table 1. Clonal comparison showing the average total genome length (u) and the ratio of the longest (L) to the shortest (S) complement chromosome

Fig. 1. Karyotype comparison of ten selected cells from each of two clones of Uvularia floridana. The comparison is between total genome length and the length ratio of the longest/shortest chromosome which estimates the degree of condensational uniformity and complement homogeneity.

Two different checks on the degree of chromosomal condensation and com plement homogeneity were used for each cell. The ratio of the longest to shortest (L/S) chromosome of the complement and the total genome length (a) were cal 1978 Somatic Karyotype Analysis of Uvularia floridana Chapman (Liliaceae) 673

culated (Table 1; Fig. 1) for each of the 20 selected cells , i.e. 10 cells per clone. These two standardized measures are extremely useful for complement comparison

(Utech and Kawano 1974, 1976).

The average genome length for clone 1 was 83 .90ƒÊ (range 78.19-86.14ƒÊ),

Fig. 2. Representative cells from the two clones. A. Clone 1, 2n=12. B. Clone 2, 2n=12. (Fla.: Jackson Co., Chipola River floodplain, Marianna; Scale indicated).

Table 2. Measurements for a composite haploid karyotype of Uvularia floridana based on 20 colchicine pretreated, somatic complements (cf. Fig. 4)

while that for clone 2 was a similar 83.94ƒÊ (range 80,25-85.72ƒÊ). The average L/S ratio was 2.361 for clone 1 and 2.367 for clone 2. A representative cell from each clone is presented in Fig. 2 (A-B), while the chromosomes of each cell have 674 Frederick H. Utech Cytologia 43 been rearranged and presented in an associated diploid idiogram (Fig. 3). Since there were no reportable significant differences between the measured complements of the two clones (Table 1), the data from both clones were pooled (Table 2) and used to construct a composite haploid idiogram (Fig. 4). Based on the 20 diploid cells measured, i.e. 40 measures per homolog, the fol lowing average chromosomal measurements (long arm, short arm and total length)

Fig. 3. Somatic chromosomal complements of Clones I and 2. Reproduced from Fig. 2 A and B. (Scale indicated).

Fig. 4. Haploid idiogram for Uvularia floridana based on Table 2. (N=40 measures per homolog). with their respective standard deviations, relative chromosomal lengths and centro meric indices are presented (Table 2). A centromeric index which defines a median chromosome (m) by a 1.000-0.850 ratio, a submedian (sm) by 0.850-0.450 and a subtelocentric (st) by 0.450 to zero was used (Utech and Kawano 1974, 1976). Using the above centromeric index, chromosome pairs 1-4 and 6 are clearly classifiable as subtelocentric (st), while the nuclear organizing region (NOR) chromo 1978 Somatic Karyotype Analysis of Uvularia floridana Chapman (Liliaceae) 675 some, i.e. pair 5, is of the submetacentric (sm) type. In all the cells examined the NOR pair consistently had the secondary constriction. Furthermore within a given cell, each pair is readily distinguishable (on size and/or centromere position) from

Fig. 5. Cytotaxonomic position of Uvularia floridana (2n=12) in relationship to the other mem bers of the genus (2n=14). The taxonomic framework is based on Wilbur (1963) and indicates the two sections: the perfoliated species-section Uvularia and the sessile-leaved species-section Oakesiella (Small) Wilbur. The idiograms for the other four species which are drawn to the same scale as U. floridana are based on Kawano and Iltis (1964). 676 Frederick H. Utech Cytologia 43

the other pairs (Figs. 2, 3). Within the complement, pairs 1-3 are the most obvious, basic on size and show a decreasing size gradient in their long arms. Though their short arms are similar (Table 2; Figs. 2-4), the short arms of pair 2 are consistently shorter than those of pairs 1 and 3. Pair 4 is readily distinguishable as the most subtelocentric of the complement (Table 2), while pair 6 is the smallest of the com plement. Neither polyploidy, aneuploidy, aneusomaty (i.e. 2n=14) nor B-chromo somes, including fragments, were encountered in the somatic material from the two studied clones of U. floridana.

Discussion and conclusion The occurrence of 2n=12 for U. floridana, reported here for the first time, re presents a new base number for the genus. From the sampled clones and the 200 counts from them, this number does not appear to be a chance aneuploid reduction, but rather a stablized species specific number different from the other members of the genus. Fig. 5 provides an overall view of the known karyotypes in the genus, and these karyotypes have been superimposed on a classical taxonomic phylogeny (Wilbur 1963) which exhibits their hypothetical origin and relationship. The haploid karyotypes of Fig. 5 for the 4 species of Uvularia, other than U. floridana, are from Kawano and Iltis (1964). There are some reasonable limitations to their data. One to several (no number given) cells were probably measured for each species and on this their karyotypes are based. Their original paper presented several anaphase figures and presumably if measurements were made from them, they would not necessarily be directly comparable to the C-metaphase cells of U. floridana. No satellited chromosomes (NOR) were reported for the 4 species (Kawano and Iltis 1969) which does not necessarily negate their occurrence. While secondary constrictions are difficult to see during anaphase, telocentric chromosome, on the other hand, are readily discernible. Furthermore, the two perfoliate-leaved species, U. grandiflora and U. perfoliata, and the two sessile leaved species, U. puberula and U. sessilifolia, are extremely wide spread. The possibility that chromosomal races (within these species) with different somatic karyologies exist must also be considered. Within each pair the distribution is more or less allopatric (Wilbur 1963, Kawano nad I1tis 1964, Johnson 1969). Withstanding these considerations, the current knowledge of the karyology of Uvularia is summarized in Fig. 5. The karyotypic similarity of the two members of section Uvularia, i.e. U. grandiflora and U. perfoliata, are shown (Fig. 5), and have been previously discussed (Anderson and Whitaker 1934, Kawano and Iltis 1964). On the other hand, the dissimilarity between U. sessilifolia and U. puberula of section Oakesiella, and between them and section Uvularia is most significant. The specialized karyotype of the primitive U. puberula involving a telocentric pair is most noteworthy. The here reported karyotype of Uvularia floridana adds further to the disparity within section Oakesiella and between the sections. No direct clue as to the chromosomal evolution within section Oakesiella is apparent. Two estimates of karyotype asymmetry are frequently used (Stebbins 1958, 1966, Utech 1975). One is the longest/shortest (L/S) chromosome ratio and the other is 1978 Somatic Karyotype Analysis of Uvularia floridana Chapman (Liliaceae) 677 the proportion of the karyotype with greater than 0.450 arm ratios. A primitive karyotype is assumed to have equal sized chromosomes and a L/S ratio near zero which would indicate the asymmetry magnitude within the complement. A primi tive karyotype is also assumed to have metacentric chromosomes, i.e. all the chromosomes of the complement would have short/long arm ratios between 1.000 0.850, and the overall karyotype proportion would be near 1. The asymmetry magnitude due to rearrangements and translocations within the complement would be indicated by a progressively lower proportion using the second measure. The following asymmetry values can be calculated for the species studied by Kawano and Ilitis (1964): Section Uvularia with U. grandiflora-L/S=5.60, pro portion=4/7 or 0.571, and U. perfoliata-L/S=5.35, proportion=4/7 or 0.571; Section Oakesiella with U. puberula-L/S=4.33, proportion=4/7 or 0.571 and U. sessilifolia-L/S=3.92, proportion=5.7 or 0.714. For U. floridana, these com parable values are available: L/S=2.37 (significant) and proportion=l/6 or 0.166 (significant). That chromosomal evolution within the genus has occurred not via polyploidy has been discussed by Anderson and Whitaker (1934) for section Uvularia and for 4 of the 5 species of the genus by Kawano and Iltis (1964). To the statement that chromosomal speciation in the genus has occurred by recombination of the different basic genomes and/or by the accumulation of point mutations (Kawano and Iltis 1964) can be added the aneuploid reduction and associated asymmetry reported here for U. floridana. This type of chromosomal evolution still repre sents a functional rearrangement at the lower ploidy level. Largely due to the apparent rearrangements within the genus and the dif ferent base number for U. floridana, the assignment of a karyotypic formulae to U. floridana which parallels those given by Kawano and Iltis (1964) to the other species is here avoided. Breeding results within the genus would be needed to establish a parallel letter-per-chromosome code that directly implied genetic homologies. Since both the presented cytology of U.floridana and the gross floral morphology (bract present, rostrate upper ovary with a sessile to sub-sessile base, not stipitate) clearly represent a derived condition within section Oakesiella and the genus (Wilbur 1963), an examination of its geographical distribution is whorthwhile. The known Coastal Plain range of this species (Johnson 1969, Wilbur 1963) can be further pin-pointed by the use of area geological surface maps (Stose 1932, Bennison 1975, Renfro and Feray 1970, Toulmin 1955). The present known range occurs entirely within areas available (emergent) after the early Oligocene (38,000,000 BP), since the substrate is composed of marine rocks of the Eocene Jackson, Clai borne and Wilcox Groups. That the derived origin of U. floridana can be dated to this time and area is speculative for it could also have had a prior origin and distribution on the older uplands and migrated to its present range since. Neverthe less, there is an interesting, time-dependant boundary to its present distribution.

Summary

This, the first investigation of the somatic karyotype of Uvularia floridana, was based on 200 cells from 2 clones and has shown a stablized 2n=12 number 678 Frederick H. Utech Cytologia 43

which differs from the 2n=14 counts that characterize the other species of Uvularia. This 2n=12 number represents a derived aneuploid reduction in the genus. For U. floridana, each of the 6 pairs is readily distinguishable. Pairs 1-4 form a sub telocentric series with decreasing size. Pair 5 is submetacentric and also the nucleolar organizing (NOR) pair. (NOR chromosomes have not been reported for the other species of the genus.) Pair 6 is the smallest and subtelocentric. The karyotype is highly asymmetrical. To the derived karyology of U. floridana can be added its derived morphology and restricted geographical distribution.

Acknowledgment This work was supported by a grant from the M. Graham Netting Research Fund (Carnegie Museum of Natural History) whose funding is deeply appreciated and acknowledged. Special thanks are also due to my old friends and teachers, Dr. Hugh H. Iltis (University of Wisconsin-Madison) and Dr. Shoichi Kawano (Toyama University, Japan) for their continued encouragement and discussion on the problems in the lily family, and to Dr. Mary R. Dawson (Carnegie Museum - Vertebrate Paleontology) for interpretive assistance on the surface geology of the southeast.

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