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Delineation and Distribution of Zephyranthes Species (Amaryllidaceae) Endemic to the Southeastern United States
Raymond O. Flagg1* and Gerald L. Smith2 Carolina Biological Supply Company, Burlington, North Carolina 27215-3398 2 Department of Biology, High Point University, High Point, North Carolina 27262-3598
1

ABSTRACT Three closely related, midwinter to spring-flowering, mesic species of Zephyranthes (Amaryllidaceae) are endemic to the southeastern United States: Z. atamasca, Z. treatiae, and Z. simpsonii. The taxonomic level of the distinctiveness of Z. treatiae from Z. atamasca is discussed. Based on herbarium, cytological, and field studies, and on Principal Component Analysis and pictorialized scatter diagram analysis, we conclude that all three taxa are distinct at the species level. They are distinguished by differences in relative lengths of floral elements. Illustrations, a distribution map, and a key are provided. INTRODUCTION Zephyranthes (Amaryllidaceae) was established by William Herbert (1821), and is an American genus of some 40 species (Mabberley 1997) with native distribution from Maryland to Argentina. Three closely related, midwinter to spring-flowering, mesic species of Zephyranthes are endemic to the southeastern United States: Z. atamasca (L.) Herb. (1821), Z. treatiae S. Watson (1879), and Z. simpsonii Chapm. (1892). All three species have linear stigmatic lobes, green perianth tubes and white perianth segments. The perianth segments may be variously flushed with pink. Zephyranthes atamasca (Figure 1) and Z. treatiae (Figure 2) have been generally treated as though the flowers were not significantly different from one another, and were distinguished by their leaves and their habitats. Although Z. atamasca tends to be associated with hardwoods and to grow in clumps, and Z. treatiae tends to be associated with pine flatwoods and to grow singly or loosely clumped, these general distinctions have been recognized as not always true; therefore, the basic test to tell them apart has been in the leaves, as reflected in the keys of Small (1933), Godfrey and Wooten (1979) and Clewell (1985).
*email address: rflagg@triad.rr.com Received September 4, 2007; Accepted May 24, 2008.

In the seeming absence of recognized significant differences in the flowers of Z. atamasca and Z. treatiae, it has been thought (Flagg 1961, Spencer 1973, and Flory 1978) that Z. treatiae might be only an ecotypic variant of Z. atamasca. Meerow (1985) described differences in the leaves and habits of the two taxa, and agreed that there was no consistent distinguishing floral variation; therefore, he made the combination Zephyranthes atamasco var. treatiae (S. Wats.) Meerow, comb. et stat. nov. [page 94]. Although the perianth of Z. simpsonii (Figure 3) was originally described as pale pink, many flowers observed in nature are often strongly infused with pink or purple distally, grading to white with a yellow-green base. Other flowers are predominantly white and cannot be distinguished from the other two taxa by color. Nevertheless, Z. simpsonii with its narrowly funnelform flowers and little, if any, reflexing of the perianth segments, and with its stigma among (or within 2 mm of) the anthers has always been recognized as florally distinct from Z. atamasca and Z. treatiae with their broadly funnelform flowers, reflexed perianth segments and obviously exserted (3 mm or more beyond the anthers) stigmas. It is interesting to note that Z. simpsonii, which is found in association with pine flatwoods in Florida and Georgia, and maritime forests in North Carolina and South Carolina, tends to grow

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Figure 1. Zephyranthes atamasca (L.) Herb. A. Habit and flowering scape. B. Leaf cross section. C. Flower frontal view. D. Section of flower (l. s.). E. Tepal. (Drawn by Melanie Darst).

singly or loosely clumped and has a leaf type like Z. treatiae, but with larger stomata (ca. 63 mm vs. ca. 43 mm long).

MATERIALS AND METHODS Over a period of decades we examined specimens of

Zephyranthes from a large number of herbaria. In preparing materials for Flora of North America (Flagg, Smith, and Flory 2002), we restudied specimens from The Florida State University (FSU), the Missouri Botanic Garden (MO), the University of Florida (FLAS), The

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Figure 2. Zephyranthes treatiae S. Watson. A. Habit and flowering scape with frontal and side view of flower. B. Leaf cross section. C. Section of flower (l. s.). D. Tepal and stamen. (Drawn by Melanie Darst).

University of Georgia (GA), the University of North Carolina-Chapel Hill (NCU), and the University of North Carolina-Charlotte (UNCC). The preponderance of the morpho-

metric data were taken from specimens at FLAS and MO. As a rule, we quickly determined the species and then recorded data. Zephyranthes simpsonii

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Figure 3. Zephyranthes simpsonii Chapm. A. Habit and flowering scape. B. Leaf cross section. C. Frontal view of flower. D. Section of flower (l. s.). E. Capsule with pedicel and sheath. (Drawn by Jean Putnam Hancock).

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Table 1. Widths of leaves and lengths of floral elements in millimeters in herbarium specimens of Zephyranthes species endemic to southeastern United States Z. atamasca Leaf Spathe Pedicel Perianth Perianth tube Filament Style Stigmatic lobe (2) 37 (8) (20) 2232 (36) (0) 416 (34) (55) 6690 (101) (8) 1120 (21) (29) 3244 (47) (52) 5874 (78) (0.8) 1.13.0 (4.0) Z. treatiae 13 (18) 2133 (36) 010 (12) (61) 6695 (108) (17) 1928 (31) (16) 2239 (46) (50) 5582 (87) (1.4) 2.34.3 (5.5) Z. simpsonii 14 (17) 2140 (42) 06 (15) (40) 4385 (101) (14) 1633 (40) (15) 1628 (32) (31) 3357 (62) (1.0) 1.62.9 (4.3)

was readily recognized by its relatively long perianth tube and its stigma among or within 2 mm of the anthers. Zephyranthes atamasca was instantly identified when broad (5 mm or more) leaves were present on the sheet. Zephyranthes treatiae was readily determined when the stigmatic lobes were notably long (over 4 mm). Overall, the flowers of Z. atamasca and Z. treatiae looked different to us; e.g., the free filaments of Z. treatiae seemed notably shorter and the perianth tube seemed longer as compared to Z. atamasca. To obtain some of the data we routinely used strong backlighting of the herbarium specimens. While not always necessary, shining a bright light up through the specimen greatly facilitated many determinations. Many sheets left us with partial data. Parts were missing from some specimens, and in spite of various lighting techniques, we could not always measure each element as desired. We recorded width and length of leaves when they were present, and recorded the lengths of floral elements when we were confident of the measurements. Complete floral data on lengths of spathe, pedicel, total perianth, perianth tube, free filament and style, and position of the stigma relative to the anthers were obtained from 33 specimens of Z. atamasca, 45 specimens of Z. treatiae and 46 specimens of Z. simpsonii, and this data was used in the analysis and is presented in Tables and Figures.

tubes occur in Z. atamasca, and the longest perianth tubes and the shortest styles occur in Z. simpsonii. In comparison with Z. atamasca, longer perianth tubes, shorter free filaments and longer stigmatic lobes occur in Z. treatiae. A Principal Component Analysis (PCA) demonstrated variation with Component 1 containing 44.7% of the variance and Component 2 containing 24.2% of the variance (the remaining percentage is in the other five components). Character weightings are given in Table 2. The PCA analysis (Figure 4) shows the taxa as three clusters with less overlap between Z. atamasca and Z. treatiae than between Z. treatiae and Z. simpsonii. The plot of the first principal component as compared with the second principal component demonstrates that the variation within each taxon is less than the variation between taxa. Individuals of the three taxa can be defined as three clusters supporting a concept of three species. Because relative lengths of floral elements were particularly revealing in analyzing relationships of Zephyranthes species of the Texas Gulf Coast (Flagg 1961, Flagg and Flory 1976), we pursued this with the three southeastern species. Table 3 is a summary of the ratios found when the lengths of specific floral elements were divided by the length of the perianth tube. Restated as simple generalities
Table 2. Principal component matrix (character weightings) for Zephyranthes species endemic to southeastern United States Component 1 Spathe length Perianth length Style length Leaf width Perianth tube length Filament length Pedicel length .179 .887 .906 .559 .008 .915 .576 Component 2 .535 .381 .155 2.428 .904 2.154 2.460

RESULTS Morphometrics
Table 1 is a summary of all relevant measurements obtained in the study. The lengths of each element overlap among the three taxa, but some differences can be noted. Among the three taxa, the widest leaves, the longest pedicels and the shortest perianth

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Figure 4. Principle components analysis of the morphological characters of h: Zephyranthes atamasca, X: Z. treatiae, %: Z. simpsonii.

in relationship to perianth-tube length: total perianth length in Z. atamasca is always more than 4 times the perianth-tube length, in Z. treatiae it is 3 to 4 times, and in Z. simpsonii it is usually less than 3 1/5 times perianth-tube length; filament length in Z. atamasca is at least 1 2/3 times and usually 2 to 3 times (or more) perianth-tube length, in Z. treatiae it is usually 1 to 1 1/2 times and occasionally as much as 1 2/3 times, and in Z. simpsonii it is 2/3 to 1 1/3 times; the style in Z. atamasca is always more than 3 times the perianth-tube length, in Z. treatiae it is about 2 1/2 to 3 1/3 times, and in Z. simpsonii is always less than 2 1/2 times; in Z. atamasca spathe length is

usually at least 1 1/2 times the perianth-tube length, in Z. treatiae it is usually less than 1 1/2 times, and in Z. simpsonii it ranges from 2/3 to 2 times. Using records of specimens under study for which there were complete data on lengths of perianth, perianth tube, filament, style and spathe, we created a pictorialized scatter diagram (Figure 5) based on the relative lengths of the floral elements in individual specimens. Within each taxon every specimen could not be depicted because on occasion one glyph would partially obscure another. If the overlapping glyphs of a taxon were not identical, we used the one most

Table 3. Ratios of lengths of floral elements to length of perianth tube in Zephyranthes species endemic to southeastern United States Z. atamasca Perianth/Perianth tube Filament/Perianth tube Style/Perianth tube Spathe/Perianth tube (4.1)4.36.4 (8.5) 1.73.4 (4.6) 3.25.3 (7.5) 1.42.4 (3.0) Z. treatiae (3.0)3.14.0(4.1) (0.8) 1.01.5(1.7) 2.53.3(3.4) (0.7)0.91.4(1.5) Z. simpsonii (2.2)2.43.1(3.2) 0.71.1(1.3) (1.4)1.62.4 (0.7)0.81.7(2.0)

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Figure 5. Pictorialized scatter diagram using floral characteristics of Zephyranthes endemic to southeastern United States.

different from the majority of glyphs for that taxon, which illustrates the morphological diversity but also somewhat exaggerates the amount of variation within a taxon. The pictorialized scatter diagram produced a pattern similar to the PCA cluster analysis, but with even less indication of morphological overlap between species. We concluded that there are three closely related species

distinguished by suites of floral characteristics.

Cytology
The somatic chromosome number of both Z. atamasca and Z. treatiae is 2n 5 24 (this paper, Flory 1940 and 1943, Flagg 1961) (Figure 6A and 6B). We have prepared karyotypes of both species and conclude that the chromo-

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Figure 6. Somatic chromosomes of (A) Zephyranthes atamasca (Smith 1398, Bladen County, North Carolina) (B) Z. treatiae (Smith s.n., Brooks County, Georgia) and (C) Z. simpsonii (Flory s.n., De Soto County, Florida, Greenhouse accession 849).

some types are remarkably similar in size and centromere position. Even though some slight chromosomal differences are observable between Z. atamasca and Z. treatiae, we think that the morphological distinctions discussed in this paper are largely the result of gene mutations that have become established in populations through selection in each species. The divergence of Z. atamasca and Z. treatiae into distinctive habitats suggests to us that they have speciated as a response to their environmental conditions. We believe that gene sequencing of variable genes and DNA fingerprinting techniques (ISSR) are likely to reveal those suspected genetic differences as demonstrated by Meerow et al. (2000) for other species of Zephyranthes. The cytological distinction of Z. simpsonii is a more clear-cut case. The somatic number is 2n 5 48 (this paper, Flory 1940 and 1943, Flagg 1961) (Figure 6C). We hypothesize that Z. simpsonii is of autopolyploid origin from Z. treatiae. They have nearly indistinguishable leaf forms, although Z. simpsonii has larger stomata and may possess a thicker leaf. Further supporting our idea of Z. simpsonii origin is that floral distinctions between Z. simpsonii and Z. treatiae are less than those of Z. simpsonii and Z. atamasca (Figures 4 and 5). It is conceivable to us that, as Z. simpsonii evolved, the perianth tube continued to elongate and effectively shortened the free segments of the perianth resulting in the trumpet form. There is widespread evidence in Zephyranthes that as chromosome numbers increase style exsertion decreases placing the stigma at or below the anthers and supporting self pollination (Flagg and Smith, pers. obs.). We believe that gene studies will also

reveal genetic differences between Z. simpsonii and Z. treatiae.

Distribution
Zephyranthes atamasca is found in the Coastal Plain and Piedmont from southeastern Maryland to northern Florida and west to the southeastern corner of Mississippi (Figure 7). Zephyranthes treatiae occurs between central Florida and southern Georgia (Figure 7). Zephyranthes simpsonii occurs disjunctly in the Coastal Plain in southeastern North Carolina and northeastern South Carolina, from southeastern Georgia to south-central Florida, and with two very small populations on the Alabama-Mississippi border (Figure 7). In reviewing the morphological data across the distribution, we did not find a general tendency for Z. atamasca to be more like Z. treatiae in north-to-south or west-to-east directions. Our studies indicate that there is not widespread hybridization between Z. atamasca and Z. treatiae. Nevertheless, in Darien (McIntosh Co.), Georgia, where it was apparent that both Z. atamasca and Z. treatiae had been brought in and planted together, a single specimen of Z. atamasca found in a lawn had perianth element ratios that nearly proximated the most extreme ratios found in Z. treatiae. The leaf width and short stigmatic lobes of that specimen placed it in Z. atamasca, but we suspect some hybridization has been involved in Darien, Georgia, and we cannot rule out natural hybridization in the vicinity of Jasper, Florida in ecotonal zones between the two species. This is likely to occur in other counties where Z. atamasca and Z. treatiae are both present (Figure 7) and in which habitats are adjacent to one another.

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Figure 7.

Distributions of Zephyranthes species endemic to southeastern United States.

However, it is also important to point out that there are counties where populations of each occur, but that they are spatially isolated in different habitats, and the opportunity for hybridization is not as likely. Measurements of specimens, which have been taken from vouchers documenting populations as reported above, support the distinctiveness of each species.

SUMMARY/DISCUSSION The three midwinter to spring-flowering, mesic species of Zephyranthes endemic to the southeastern United States, were described in the 19th century: Z. atamasca in 1821 establishing the genus, Z. treatiae in 1879, and Z. simpsonii in 1892. Since Chapman (1892) described Z. simpsonii, as stamens and style equal, included, it has been recognized as florally

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Figure 8.

Floral diagrams of Zephyranthes species endemic to southeastern United States.

distinct from the other two species. Zephyranthes atamasca and Z. treatiae have been distinguished from one another primarily by their leaves: broad, shiny green, slightly channeled, margins sharp-edged in Z. atamasca; and, narrow, dull green, semi-terete, margins rounded in Z. treatiae. For some plant taxonomists, the seeming lack of floral differences has called into question the specific status of Z. treatiae. While we suspect that some hybridization has occurred between Z. atamasca and Z. treatiae, we dont accept that as justification for lumping together the two taxa. Broyles and Wyatt (1991) reported that Z. atamasca is fully self-compatible [self-compatibility is most common in the genus (Flagg and Smith, personal observation)], but also sets a similar number of viable seed in cross-pollinations. This breeding system would offer the potential for hybridization, which would not be unexpected considering the natural hybridization (Flagg 1961, Flagg and Flory 1976) that has occurred between such morphologically different rain-lilies as Z. pulchella J. G. Sm. (1895) and Z. chlorosolen (Herb.) D. Dietr. (1840). Because of the great floral morphological differences between these parents, and in spite of their recognized natural hybridization, some authors continue to recognize Cooperia including Z. chlorosolen [5 Cooperia drummondii Herb. (1836)] as a separate genus (Diggs et al. 2006, Correll and Johnston 1970); although we considered Cooperia to be congeneric with Zephyranthes (Flagg, Smith, and Flory 2002). Clearly, putative natural hybridization alone

would not be sufficient reason to deny specific status to a rain-lily. Broad leaves (5 mm or more) generally distinguish Z. atamasca from Z. treatiae and Z. simpsonii. A stigma among or near (within 2 mm) the anthers distinguishes Z. simpsonii from Z. atamasca and Z. treatiae. The three species are further distinguished by floral characteristics related, by and large, to the degree of fusion of the perianth into a tube (Figure 8). PCA of the taxa demonstrates that the variation between Z. atamasca and Z. treatiae is greater than the variation between Z. treatiae and Z. simpsonii. A pictorialized scatter diagram using lengths of floral elements relative to perianth-tube length supports the concept of three species. Our conclusion supports taxonomists, such as Chapman (1892), Small (1933), Hume (1935), Godfrey and Wooten (1979), Clewell (1985) and Wunderlin (1998), who treated Z. atamasca, Z. treatiae and Z. simpsonii as three distinct species.

KEY TO ZEPHYRANTHES SPECIES ENDEMIC TO THE SOUTHEASTERN UNITED STATES

1. Stigma exserted more than 4 mm beyond anthers; perianth-tube length usually less than 1/3 of the total perianth length . . . . . . . . . . . . . . . . . . 2 2. Leaf greater than 4.5 mm in width . . . . . . Zephyranthes atamasca 2. Leaf less than 4.5 mm in width . . . . 3

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3. Perianth-tube length less than 1/4 of the total perianth length, about 1/2 (1/32/3) of the filament length . . . . Zephyranthes atamasca 3. Perianth-tube length at least 1/4 of the total perianth length, about equal (2/31J) to the filament length . . . . . . Zephyranthes treatiae 1. Stigma very near, among or below anthers; perianth-tube length usually 1/3 or more of the total perianth length . . . . . . . . Zephyranthes simpsonii

Diggs, G.M., B.L. Lipcomb, M.D. Reed, and R.J. OKennon. 2006. Illustrated flora of east Texas. Volume 1. Botanical Research Institute of Texas, Fort Worth, Texas. Flagg, R.O. 1961. Investigations in the tribe Zephyrantheae of the Amaryllidaceae. Ph.D. dissertation, University of Virginia, Charlottesville, Virginia. Flagg, R.O. and W.S. Flory. 1976. Origins of three Texas species of Zephyranthes. Pl. Life 32:6780. Flagg, R.O., G.L. Smith, and W.S. Flory. 2002. Zephyranthes. p. 296303. In: Flora of North America Editorial Committee (eds.). Flora of North America North of Mexico. Magnoliophyta: Liliidae: Liliales and Orchidales. New York and Oxford. Volume 26. Flory, W.S. 1940. Cytology of Zephyranthes. Texas Agric. Exp. Sta. Ann. Rpt. 53:29. Flory, W.S. 1943. Chromosome numbers reported in recent years for Hemerocallideae, Alstroemeriales and Amaryllicales. Herbertia 10:114123. Flory, W.S. 1978. Walter S. Flory, Jr., an Autobiography. Pl. Life 34:1127. Godfrey, R.K. and J.W. Wooten. 1979. Aquatic and wetland plants of southeastern United States, Monocotyledons. University of Georgia Press, Athens, Georgia. Herbert, W.m. 1821. Appendix. 36. Herbert, W.m. 1838. Cooperia Drummondi in Lindley. Bot. Reg. 22:pl. 1835. Hume, H.H. 1935. The bulbs called Zephyranthes. Garden, March:1520. Mabberley, D.J. 1997. The Plant-Book, ed. 2. Cambridge University Press, Cambridge, United Kingdom. Meerow, A.W. 1985. Notes of Florida Zephyranthes. Herbertia 41:8694. Meerow, A.W., C.L. Guy, Q. Li, and S. Yang. 2000. Phylogeny of the American Amaryllidaceae based on nrDNA ITS sequences. Syst. Bot. 25:708726. Small, J.K. 1933. Manual of the southeastern flora. University of North Carolina Press, Chapel Hill, North Carolina. Smith, J.G. 1895. Zephyranthes pulchella sp. nov. Annual Rep. Missouri Bot. Gard. 6:114115.

ACKNOWLEDGEMENTS We are particularly grateful to Walter S. Flory, Jr. (deceased June 8, 1998) for his steady encouragement and for his contributions to the formative stages of this paper. We thank the Curators of FLAS, FSU, GA, MO, NCU, USF, UNCC for making loans available; and, we particularly thank Richard Wunderlin and Bruce Hansen (USF), and Kent Perkins (FLAS) for their patience in granting to us several specific loan requests. We thank Charles K. Smith (Biology Department, High Point University) for assistance in performing the Principal Component Analysis of our data. Jacquelyn Reaves and several taxonomy classes at HPU collected data in the early stages of this project from herbarium and field collected specimens that proved helpful to the artists in developing the illustrations. Robert Godfrey (deceased), Angus Gholson, Melanie Darst, Mark Garland, and Gary Knight participated in field studies in March of 1992 to collect Zephyranthes specimens. Melanie Darst and Jean Putnam-Hancock provided the fine illustrations. Funding for the field work and artwork was provided by NC Board of Science and Technology grant, no. 90SE010.

LITERATURE CITED
Broyles, S.B. and R. Wyatt. 1991. The breeding system of Zephyranthes atamasco (Amaryllidaceae). Bull. Torrey Bot. Club 118:137140. Chapman, A.W. 1892. Fl. South. U. S. ed., 2, Suppl. 2:696. Clewell, A.F. 1985. Guide to the vascular plants of the Florida Panhandle. Florida State University Press, Tallahassee, Florida. Correll, S. and M.C. Johnston. 1970. The manual of the vascular plants of Texas. Texas Research Foundation, Renner, Texas. Dietrich, D. 1840. Sy. Pl. 2; 1176.

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Spencer, L.B. 1973. A monograph of the Genus Zephyranthes (Amaryllidaceae) in North and Central America. Ph.D. dissertation, Wake Forest University, WinstonSalem, North Carolina.

Watson, S. 1879. Proc. Amer. Acad. Arts 14:300. Wunderlin, R.P. 1998. Guide to the vascular plants of Florida. University Press of Florida, Gainesville, Florida.