Epicotyl dormancy in the mesic woodland herb Hexastylis heterophylla (Aristolochiaceae)1
Adams, Christopher A
ADAMS, CHRISTOPHER A., JERRY M. BASKIN (Department of Biology, University of Kentucky, Lexington, KY 40506), AND CAROL C. BASKIN (Department of Biology, University of Kentucky, Lexington, KY 40506 and Department of Agronomy, University of Kentucky, Lexington, KY 40546). Epicotyl dormancy in the mesic woodland herb Hexastylis heterophylla (Aristolochiaceae). J. Torrey Bot. Soc. 130:11-15. 2003.-Seeds of the woodland herb Hexastylis heterophylla (Ashe) Small (Aristolochiaceae) were incubated in two sequences of temperature regimes: (a) warm [arrow right] cool [arrow right] cold [arrow right] cool [arrow right] warm, and (b) cold [arrow right] cool [arrow right] warm [arrow right] cool [arrow right] cold [arrow right] cool [arrow right] warm. In the first sequence, roots emerged during the first cool period (“autumn”) and shoots during the second cool period (“spring”). In the second sequence, roots emerged during the second cool period (“autumn”) and shoots during the third cool period (“spring”). Thus, in seeds of H. heterophylla, a period of warm temperatures is required for subsequent emergence of roots at cool (“autumn”) temperatures and a period of cold (“winter”) temperatures is required for subsequent emergence of epicotyls (shoots) at cool (“spring”) temperatures (in seeds with roots emerged). These dormancy-breaking and germination requirements demonstrate clearly that seeds of this species have deep simple epicotyl morphophysiological dormancy (epicotyl dormancy), like those of Asarum canadense L., another eastern North American woodland herb in this family. This is the first report in the literature on seed dormancy in Hexastylis and only the third one for Aristolochiaceae.
Key words: Aristolochiaceae, epicotyl dormancy, Hexastylis, woodland herb, underdeveloped embryo.
Aristolochiaceae is a predominantly tropical and subtropical family of six (Gonzalez 1999) to twelve (Mabberley 1997) genera and about 475 species (Mabberley 1997). It is represented in the U.S. by three genera: Aristolochia, Asarum, and Hexastylis (Gonzalez 1999). Aristolochia, with approximately 350-400 species, is the most widespread genus; it occurs on every continent except Antarctica (Gonzalez 1999). Asarum consists of approximately 85 species found in North America, Europe, and Asia (Kelly 1998). The genus Hexastylis, which contains approximately ten species, is restricted entirely to the eastern and southeastern United States (Blomquist 1957; Caddy 1987; Whittemore and Gaddy 1993; Kelly 1998).
Very little information has been published on seed dormancy and germination in Aristolochiaceae. Baskin and Baskin (1998) present data for only one member of the family: Asarum canadense L. This species has deep simple epicotyl morphophysiological dormancy (epicotyl dormancy); thus, the radicle emerges in autumn and the epicotyl the following spring (Barton 1944; Baskin and Baskin 1986). Epicotyl dormancy is one of eight types of morphophysiological dormancy (MPD), all of which are characterized by an underdeveloped embryo and a physiological inhibiting mechanism of germination (Baskin and Baskin 1998). Nikolaeva et al. (1985) and Nikolaeva (1988) included only two species of Aristolochiaceae (Asarum canadense and A. europaeum L.) in an extensive compilation of the kinds of seed dormancy that occur among gymnosperms and angiosperms. Following reports in the literature, Nikolaeva recorded A. canadense as having epicotyl dormancy and indicated that A. europaeum may have intermediate simple morphophysiological dormancy. The Ph.D. dissertation of V.C. Gonzalez (1972) contains a short section dealing with germination requirements of Hexastylis arifolia (Michaux) Small seeds. He concluded that seeds of this species require two cold periods (i.e., two winters in its natural habitat) to complete germination (but see our Discussion). Thus, they would have deep simple double morphophysiological dormancy (double dormancy) (Baskin and Baskin 1998).
The purpose of the present study was to determine the kind of dormancy in seeds of Hexastylis heterophylla (Ashe) Small. Hexastylis heterophylla is an herbaceous, perennial, evergreen species found in rich, mesic, deciduous and coniferous-deciduous forests of central and western Virginia, West Virginia, southeastern Kentucky, eastern Tennessee, western North Carolina, and extreme northern Georgia and northwestern South Carolina (Gaddy 1987). According to Gaddy (1987), the flowering period for this species is March to June. In southeastern Kentucky, where seeds were collected for our study, seeds mature in June and are dispersed in June and July. Asarum and Hexastylis are two very closely-related genera (Kelly 1998), and Barringer (1993) suggested that the two should be combined under Asarum. We hypothesized that seeds of Hexastylis heterophylla may have epicotyl dormancy, as do seeds of A. canadense. As such, experiments were designed to identify epicotyl dormancy. This experimental design also would allow us to detect any of the other seven kinds of MPD known to occur in seeds, as well as morphological dormancy (sensu Baskin and Baskin 1998).
Methods and Materials. Mature seeds were collected from Hexastylis heterophylla plants growing in a mesic deciduous forest on the south slope of Pine Mountain (elevation 600 m, latitude 37°04’28”N, longitude 82°46’33”W, USGS Whitesburg Quadrangle KY-VA 7.5′ Series Topographic Map, 1954, photorevised, 1978) in Letcher County, Kentucky. Seeds were collected on 26 June 1999, and germination studies were begun on 8 July 1999. All treatments consisted of three replicates of 30 seeds each, and mean and standard error of percent germination were calculated from the three replicate Petri dishes. Fresh seeds were placed on moist sand in plastic Petri dishes, which were then wrapped in clear plastic film. Seeds were incubated in a 14 h photoperiod of ca. 40 µmol m^sup -2^ s^sup -1^, 400-700 nm, of cool white fluorescent light at 12h/12h daily alternating temperatures of 25/15, 20/10, and 15/ 6°C and at a constant temperature of 5°C. A constant temperature of 5° C was used since this is a near-optimal temperature for breaking dormancy in seeds of many species that require cold stratification to become nondormant (Stokes 1965; Nikolaeva 1969). Seeds were checked for radicle and/or shoot emergence every 5 days throughout the duration of each treatment, at which time water was added to sand in the Petri dishes, if there was a need to do so. Germinated seeds were removed from the Petri dishes only after germination was completed (i.e., both radicle and shoot emergence). There were no problems with fungal infection of the seeds.
Following the “move-along” procedure of Baskin and Baskin (in press), two experiments were done to determine if warm temperatures alone, warm temperatures followed by cold temperatures, cold temperatures alone, or cold temperatures followed by warm temperatures are required for the seeds to complete germination. In the first experiment, seeds were incubated in the following sequence of temperatures: 12 wk at 25/15°C, 4 wk at 20/10°C, 4 wk at 15/6°C, 12 wk at 5°C, 4 wk at 15/6°C, and, finally, 6 wk at 20/10°C (Fig. 1). In the second experiment, seeds were exposed to the following sequence: 12 wk at 5°C, 4 wk at 15/6°C, 4 wk at 20/10°C, 12 wk at 25/15°C, 4 wk at 20/10°C, 4 wk at 15/6° C, 12 wk at 5° C, 4 wk at 15/6° C, and, finally, 6 wk at 20/10°C (Fig. 2).
The first regime was given to mimic the sequence of warm (25/15°C) [arrow right] cool (20/10, 15/6°C) [arrow right] cold (5°C) [arrow right] cool (15/6, 20/10°C) temperatures that seeds experience in the field between dispersal in early summer and germination in early spring. The second regime was given to determine if seeds need only a cold treatment or a warm plus cold treatment for radicle emergence.
Results. In the first experiment (Fig. 1), radicles began to emerge during the 12 wk of incubation at 25/15°C. Radicle emergence began on day 70, and by the end of 12 wk radicles had emerged in 27% of the seeds. At the end of the 20/10 and 15/6°C periods of the temperature sequence, radicles had emerged from 77 and 83% of the seeds, respectively. There was no radicle emergence while seeds were at 5°C (i.e., the cold stratification period). Shoot emergence began only after seeds were moved to 15/6°C. Of those seeds in which radicles had emerged, shoots emerged in 28% of them at 15/6° C and in the other 72% at 20/10°C (i.e., total shoot emergence of 100%).
In the second experiment (Fig. 2), radicle emergence did not occur during the first exposure of the seeds to 5, 15/6, or 20/10°C. Radicles emerged only after seeds were moved to 25/ 15°C. The cumulative percentage of seeds in which radicles emerged during their first exposure to 25/15 C, followed by their second exposures to 20/10 and to 15/6 °C was 21, 74, and 81%, respectively. No additional radicle emergence occurred at 5°C. Shoot emergence began during the third exposure of the seeds (with radicles emerged) to 15/6°C. Twenty-three percent of the seeds in which radicles emerged produced shoots at 15/6°C, and 75% of them produced shoots at 20/10°C (i.e., total shoot emergence was 98%).
Discussion. Seeds of species that have epicotyl dormancy are characterized by radicles and epicotyls that have different dormancy-breaking requirements. Warm stratification is required to break dormancy of the radicles, while cold stratification is necessary to break dormancy of the epicotyl. Further, dormancy in the epicotyl can be broken only after radicles have emerged (Baskin and Baskin 1998). Thus, it follows that for seeds in this dormancy class that are dispersed in spring or summer, radicles will emerge in autumn and shoots in spring. In general, MPD is a very important dormancy class among herbaceous species of mesic woodlands. Baskin and Baskin (1998) list 58 species in 12 families that have one of the eight types of MPD. From this group, 23 species in eight families (Judd et. al 1999), including the Araceae, Araliaceae, Aristolochiaceae, Berberidaceae, Gentianaceae, Liliaceae, Papaveraceae, and Ranunculaceae, are known to have epicotyl dormancy (Baskin and Baskin 1998). In addition, seven Viburnum species (all shrubs) found in temperate deciduous forests, and several Quercus species have epicotyl dormancy (Allen and Farmer 1977; Farmer 1977). In contrast to herbaceous and Viburnum species, in which a warm period is required to break radicle dormancy, seeds of Quercus may or may not require a warm pretreatment to germinate, depending on the species (Olson 1974; Alien and Farmer 1977; Farmer 1977). Furthermore, whereas embryos of herbs and Viburnum species have underdeveloped embryos at seed maturity, those of the oaks are fully developed.
Seeds of Hexastylis heterophylla exhibit dormancy and germination patterns very similar to those of Asarum canadense (Barton 1944; Baskin and Baskin 1986). In the first “move-along” experiment (Fig. 1), radicles of H. heterophylla began to emerge only after a long period of warm stratification. After these seeds were moved to lower alternating temperatures (20/10°C), radicle emergence increased dramatically, from 27 to 77%. Although the 25/15°C regime breaks radicle dormancy, it is too high for radicle emergence in most seeds. Radicles came out of dormancy at the higher temperatures and were able to emerge as temperatures decreased. Of seeds that produced radicles, epicotyls emerged in 100% of them following 12 wk of cold stratification and subsequent movement to warmer temperatures. No emergence of roots or shoots occurred at 5°C. However, 28% of the seeds with radicles emerged produced shoots by the end of the 4 wk period at 15/6°C. Epicotyl emergence was rapid after seeds were moved to 20/10°C; all seeds with emerged radicles produced shoots within 6 weeks. Although low temperatures clearly break dormancy in the epicotyl, epicotyls emerge only at higher temperatures. In the field, this shoot dormancy would be broken during winter in seeds with emerged radicles. As temperatures increase in spring, epicotyls can emerge, and thus seeds can complete germination. Note that seeds cannot be “tricked” into producing epicotyls before radicles emerge. In the second “move-along” experiment, seeds initially cold stratified produced no radicles or epicotyls during the 12 wk period (Fig. 2). Then, seeds were moved sequentially through a series of increasingly warmer alternating temperatures (i.e., 5 to 15/6 to 20/10°C), and still no radicle emergence occurred. Radicles began to emerge near the end of the 25/15°C period, as they did in the previous experiment (Fig. 1). From this point on, seeds were moved sequentially through the same temperature regimes as in the first experiment, and the results essentially were the same. Thus, it can be concluded that in seeds of H. heterophylla, as in most other species with epicotyl dormancy, radicle dormancy must be broken first in order for shoots to come out of dormancy.
The only other information we are aware of on seed germination in Hexastylis is found in V.C. Gonzalez’s Ph.D. dissertation (1972). Gonzalez investigated the autecology of H. arifolia var. arifolia (hereafter H. arifolia) in North Carolina, a species of upland and mixed coniferousdeciduous forests (Gaddy 1987). Gonzalez reported that seeds of this species require two cold periods (i.e., two winters) to complete germination; that is, they have deep simple double MPD (double dormancy) (Baskin and Baskin 1998). However, Gonzalez’s methodology for identifying dormancy classes was inadequate for detecting epicotyl dormancy. Seeds collected in June were cold stratified at 4°C (without first being incubated at warm temperatures) for 1 mo and then moved to 20/10, 15/10, 20/5, and 20/ 15°C for 2 mo, at which point the experiments were discontinued. No seeds germinated in any of these four temperature treatments. Seeds of H. arifolia also were buried in pots of soil in a (not specified, but apparently nonheated) greenhouse and exposed to 3 mo of winter temperatures. After the seeds were moved to an unspecified warmer temperature, radicles of some seeds began to emerge after 95 days, but no shoot emergence occurred. Gonzalez concluded that 12 wk of winter temperatures are necessary to the latter stage of seed dormancy break in the spring.
Since H. arifolia and H. heterophylla are congeners that grow in essentially the same type of habitat and have similar flowering/fruiting periods (Gaddy 1987), it is reasonable to expect that their dormancy and germination characteristics are very similar. The two “move-along” experiments conducted on seeds of H. heterophylla demonstrated that the warm summer temperatures following dispersal are necessary for radicles to come out of dormancy and emerge their first autumn. Only then is cold stratification effective in breaking shoot dormancy. Thus, the cold treatment given to seeds of H. arifolia did not break either radicle or shoot dormancy in this species. In Gonzalez’s four laboratory treatmerits, seeds were not incubated long enough or at high enough temperatures to allow radicles to come out of dormancy and emerge. If fresh seeds of H. arifolia had been incubated at 25/15°C, for example, instead of at 5°C, for 12 wk, it is reasonable to expect radicle emergence would have occurred in a high percentage of them. If seeds of H. arifolia had then been moved through the same or a similar sequence of temperatures as those of H. heterophylla in the first “move-along” experiment (which started at 25/15°C, Fig. 1), it is very likely that these requirements would have been fulfilled and epicotyl dormancy identified.
Our study appears to be the first known published paper on the seed dormancy and germination of Hexastylis. Furthermore, it is only the third report (see Introduction) that has been published on seed dormancy and germination for this large, predominantly tropical and subtropical family (Mabberley 1997; Gonzalez 1999). Evidence clearly indicates that seeds of H. heterophylla (present study) and Asarum canadense (Baskin and Baskin 1986) have epicotyl dormancy; thus double dormancy in H. arifolia (Gonzalez 1972) would appear to be unlikely. The present study contributes to the sparse amount of information available on the seed germination ecology of both the genus and its family. Furthermore, identification of MPD in H. heterophylla adds another genus and species to the long list of herbs found in mesic deciduous woodlands whose seeds are known to be in this dormancy class (Baskin and Baskin 1998).
1 We sincerely thank James E. Padgett and Zack E. Murrell, Appalachian State University, for help with species identification.
ALLEN, R. AND R.E. FARMER, JR. 1977. Germination characteristics of bear oak. South. J. Appl. For. 1: 19-20.
BARRINGER, K. 1993. New combinations in North American Asarum (Aristolochiaceae). Novon 23: 225-227. BARTON
, L.V. 1944. Some seeds showing special dormancy. Contrib. Boyce Thompson Inst. 5: 451460.
BASKIN, C.C. AND J.M. BASKIN. 1998. Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. Academic Press, San Diego. California.
BASKIN, C.C. AND J.M. BASKIN. “How to get the most information on dormancy-breaking and germination requirements from the fewest seeds.” In: E. Guerrant, K. Havens, and M. Maunder (eds.). Strategies for Survival. Island Press, Covelo, California. In press.
BASKIN, J.M. AND C.C. BASKIN. 1986. Seed germination ecophysiology of the woodland herb Asarum canadense. Am. MidL. Nat. 116: 132-139.
BLOMQUIST, H.L. 1957. A revision of Hexastylis in North America. Brittonia 8: 225-281.
FARMER, R.E. JR. 1977. Epicotyl dormancy in white and chestnut oaks. For. Sci. 23: 329-332.
GADDY, L.L. 1987. A review of the taxonomy and biogeography of Hexastylis (Aristolochiaceae). Castanea 52: 186-196.
GONZALEZ, F. 1999. A phylogenetic analysis of the subfamily Aristolochioideae (Aristolochiaceae). Ph.D. thesis. City University of New York, New York, New York.
GONZALEZ, V.C. 1972. The ecology of Hexastylis arifolla, an evergreen herb in the North Carolina deciduous forest. Ph.D. thesis. Duke University, Durham, North Carolina.
JUDD, W.S., C.S. CAMPBELL, E.A. KELLOGG, AND P.P. STEVENS. 1999. Plant systematics: A phylogenetic approach. Sinauer Associates, Inc., Sunderland, Massachusetts.
KELLY, L.M. 1998. Phylogenetic relationships in Asarum (Aristolochiaceae) based on morphology and ITS sequences. Amer. J. Bot. 85: 1454-1467.
MABBERLEY, D.J. 1997. The Plant Book: A Portable Dictionary of the Vascular Plants. Second edition. Cambridge University Press, Cambridge, United Kingdom.
NIKOLAEVA, M.G. 1969. Physiology of Deep Dormancy in seeds. Izdatel’stvo “Nanka”, Leningrad (Translated from Russian by Z. Shapiro, NSF, Washington, DC).
______. 1988. The characters of seed germination of plants in the subclasses Magnoliidae, Ranunculidae, Caryophyllidae, and Hamamelididae. Botanicheskii Zhurnal 73: 508-521. (Russian with English summary)
______. M.V. RASUMOV, AND V.N. GLADKOVA. 1985. Reference Book on Dormant Seed Germination. Nauka Publishers, Leningrad. (Russian)
OLSON, D.F., JR. 1974. Quercus L. Oak, pages 692-703. In Seeds of Woody Plants in the United States (C.S. Schopmeyer, Tech. Coord.). Agriculture Handbook, No. 450. USDA Forest Service, Washington, District of Columbia.
STOKES, P. 1965. Temperature and seed dormancy, pages 746-803. In W. Ruhland (ed.). Encyclopedia of Plant Physiology, vol. 15/2. Springer-Verlag, New York, New York.
WHITTEMORE, A.T. AND L.L. GADDY. 1997. Hexastylis, pages 54-58. In Flora of North America Editorial Committee (eds.). Flora of North America north of Mexico, vol. 3, Oxford University Press, New York, New York.
Christopher A. Adams2,4, Jerry M. Baskin2, and Carol C. Baskin2,3
2Department of Biology, University of Kentucky, Lexington, KY 40506
3Department of Agronomy, University of Kentucky, Lexington, KY 40546
4 Corresponding author: Christopher A. Adams, Telephone (859) 257-3996; FAX (859) 257-1717; E-mail: email@example.com
Received for publication February 5, 2002, and in revised form August 12, 2002.
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