American Fern Journal 93(4):184-190 (2003) an Isoetes tennesseensis Octoploid (Isoetaceae), Quillwort from Tennessee Neil Luebke T. Department of Botany, Milwaukee Public Museum, Milwaukee, WI 53233 M. Budke Jessica OH Department of Botany, Miami University, Oxford, 45056 — = Abstract. Isoetes tennesseensis, an octoploid species with a chromosome count of 2n 88, is described. It occurs in the Hiwassee River in Tennessee. Past collections of this species have been misidentified as Isoetes macrospora (= lacustris). Isoetes tennesseensis differs from lacustris in I. /. chromosome number, megaspore and microspore morphology and distribution. Speculation on the new origin of this species presented. is Eugene Wofford and In 1978, Michael Dennis July collected quillworts from Tennessee the Little River at Jones Ferry, Tomatlo Ford, the southwestern end and of Davis Island, from the Hiwassee River approximately miles 1.1 southeast of the bridge on highway 411 Tennessee. These in collections, as well subsequent ones from Hiwassee as the have been River, identified as macrospora Isoetes Dur. (Dennis Boom, et al, 1979; 1979; Taylor et ah, 1993). The population of lacustris L. (= macrospora) in eastern Tennessee I. /. is roughly 450 from known miles the nearest outlying population Passage at Creek in northern Virginia (Svenson and Griscom, Both 1935). of these populations from more are disjunct the northern lacustris (Taylor et al, /. Dennis 1993). et al. (1979) hypothesized that these outlying populations of/. macrospora could be the result of either long-range dispersal by waterfowl from northern populations or relics of a previously wider distribution. Except for the difference in geography, macrospora and lacustris are /. /. indistinguishable from each Chromosome other. number, as well as leaf and spore morphology the same. Therefore, macrospora is Isoetes has recently been synonymy placed in with the European lacustris (Taylor et 1993). J. al., In North America, lacustris ranges from Greenland and Newfoundland J. west Saskatchewan. to typically occurs It in cool, oligotrophic ponds, lakes, and streams. Isoetes lacustris distinguished by dark is its green, rigid leaves and megaspores large that range from 550 750 to urn in diameter (Taylor et al., Megaspores 1993). have typically a cristate to reticulate ornamentation (Fig. 1 A-C) and densely a papillate below girdle the equatorial ridge Kott (Fig. 1 C). and Britton Taylor and Luebke (1980), and and (1988), Britton Goltz (1991) have chromosome = reported counts of 110 2/7 for lacustris (Fig. 2 A). /. Recent studies of plants from the Tennessee shown populations have that past identifications of these plants as lacustris are These incorrect. populations /. represent an undescribed we species. In this paper present our evidence from ™a „~™~ moroholoeical and rrvtolnpiral stiiHioc anrl Q *u; <-ioor-™v. * LUEBKE & BUDKE: ISOETES TENNESSEENSIS 185 Materials and Methods Mature megaspores and microspores were taken from plants and live herbarium specimens. Photomicrographs of spores were obtained with Measurements megaspore a Hitachi S-570 scanning electron microscope. of diameters and microspore lengths were made using Olympus SZX12 and A Nikon Microphot-FX microscopes with ocular micrometers. outfitted minimum of 20 megaspores and 20 microspores were measured from fertile specimens. Megaspores were measured dry while microspores were placed in a drop of water on a slide and covered with a coverslip before being measured. chromosome Procedures used obtaining counts follow Jong (1997) with for some modifications. Plants of tennesseensis were floated in deionized water 7. in a growth chamber under a cycle of 12 hours of light, 12 hours of darkness New new and a constant 18 °C until roots had formed. roots approximately 6 mm were morning and long harvested in late pretreated in a saturated solution room of paradichlorobenzene (PDB) in the dark at temperature for four hours. 96% Roots were then fixed in Farmer's Solution ethyl alcohol glacial (3:1 : acetic acid), at room temperature for one hour and then stored in the left HCL IN freezer. For staining, roots were hydrolyzed in for ten minutes at 60°C, 96% minutes soaked in three different changes of ethyl alcohol for fifteen each, placed Whitman's hematoxylin approximately one hour, blotted, in stain for and then destained in glacial acetic acid for five minutes. Results and Megaspores of tennesseensis have bold, broad tri-radiate equatorial /. below ridges and an obscure to slightly papillate girdle the equatorial ridge. may Ornamentation on the proximal half be sparse to dense and varies from The muri on bold with even rugate E-G). the distal face are cristate to (Fig. 1 forming broken somewhat regular pattern with areolae of various crests, a to mean shapes. Megaspore size ranges from 616—946 |im in diameter with a of = = SD 753 |im in diameter (N 40; 71.76). These megaspores differ from those of both and ornamentation. Megaspores of lacustris are lacustris in size J. /. slightly smaller ranging in size from 550-750 jam in diameter (Taylor et a/., mean 1993). Kott and Britton (1983) report a diameter of 640 fun. In addition, I. lacustris megaspores have a narrow tri-radiate and equatorial ridge, with smooth The ornamentation pattern a densely papillate to occasionally girdle. on the proximal hemisphere broken, short cristate whereas on the distal face is varies from cristate to nearly reticulate with narrow muri and uneven crests it A-C). (Fig. 1 have and range Microspores tennesseensis a laevigate surface in size of J. = = mean SD from 33-40 jim long with a length of 36 jam (N 40; 2.10) (Fig. 1 H). 37-50 The microspores in I lacustris (Fig. 1 D) are larger in size ranging from = = SD mean and have |im long with a of 43 |im long (N 20; 3.25) papillose ornamentation (Kott and Britton, 1983; Taylor et aL, 1993). showed Chromosome counts from the squashed root of eight plants that tips VOLUME NUMBER AMERICAN FERN JOURNAL: 93 4 (2003) 186 LUEBKE & BUDKE: ISOETES TENNESSEENSIS 187 = = I. tennesseensis is an octoploid, 2n 88 (Fig. 2 B), not the 2n 110 characteristic of lacustris. This is the first octoploid species of Isoetes 7. reported North America. for Discussion Based on our examination and of recent past collections from the Little Tennessee and Hiwassee we new Rivers describe the following species: TYPE- Isoetes tennesseensis N. Luebke & M. Budke, T. sp. nov. U.S.A. J. Tennessee: Polk Hiwassee downstream Co., River, ca. 1 mile of the crossing of Tellico-Reliance Road, 15 July 2001, Budke, K. Heafner, Lickey and E. /. K. Gustafson 17 (holotype: MIL; isotype: MU). Figs. 1 E-H, 2 B, C. Caudex Planta aquatica. bilobatus. Folia 15-35, usque ad atro-olivacea, 11 cm alta, rigida; subula recta usque recurvata apicem versus; alae basim versus, pallida brunneae. Ligula anguste elongata usque triangulata. Labium spathu- Velum latum. tegens sporangium <20%. Sporangium cum basale, ovale, maculis brunneis. Megasporae 616-946 albidae, jam diametro, cristato- Microsp 33-40 88. Plant aquatic. Rootstock 2-lobed. Leaves 15-35, dark olive-green, up 11 to cm Subula recurved toward tall, rigid. straight to tip, terete in cross-section, ca. mm wide mid 1.5 at length (Fig. 2 C). Alae on either side of the base of the cm up microphyll to 4.5 pale brown. Ligule narrowly elongate tall, to < Labium Velum 20% triangular. spathulate. covering of sporangium. mm mm Sporangium 4-6 and basal, oval, long 1-1.5 wide, brown- lightly = Megaspore 616-946 streaked. white, |im in diameter, x 753 |im; cristate to rugate proximally, the ornamentation sparse dense; to cristate to reticulate distally, with thick- walled muri of even height; proximal surface with tall, bold tri-radiate ridges; equatorial ridge with an obscure to slightly papillate = girdle below. Microspores light gray in mass, 33-40 jim long, x 36 |im; = Chromosome number 2n laevigate. 88. — Monroe Paratypes. U.S.A. Tennessee: Tennessee Co., Little River: Jones Ferry, B.E. Wofford et 78-133 (TENN); Tomatlo Ford, B.E. Wofford and W. al. nnis, 78-134 (TENN); southwest end of Davis Island, B.E. Wofford and W. M, WM, Mile 15, A-C Fig. 1. Taylor 4902 (MIL): A: proximal view of megaspore; B. distal view of megaspore; view C. lateral of megaspore. D. Taylor 5010 (MIL): Microspore. E-H. tennesseensis; Budke 17 (MIL— /. et al. holotype): E. proximal view of megaspore; F. distal view of megaspore; G. lateral view of megaspore; H. microspore. AMERICAN FERN JOURNAL: VOLUME NUMBER 188 93 4 (2003) and Fig. 2. Isoetes lacustris I. tennesseensis. A. Somatic chromosomes in mitotic root tip squash of Isoetes lacustris, Jermy 22931 (MIL). B. Somatic chromosomes in mitotic root tip squash of /. tennesseensis, Taylor 6153 (MIL). C. Plants of tennesseensis. D. Habitat shot of Hiwassee River, I. mi downstream from 1.6 Reliance, Tennessee. Dennis (TENN). Polk Hiwassee et al. Co., River: shallow shoals intersection at and d 2518, B.E. Wofford A.M. Evans 78-168 (TEN: NW of bridge Reliance, W.C. Taylor 5189 at (MIL): ice and scattered mi downstream, 1.6 K.D. Heafi MU) (MIL, Budke MU) jad, et 8 (MIL, al. /. — At Distribution. Dresent. Tsoeh kn Monroe River Polk County. Specimens in of/, tennesseensis have not been found in the Tennessee dam Little River since the construction of a and the permanent raising of the water However, level. likely that plants could some it is persist in areas Isoetes tenneseensis grows in the cool waters of the Hiwassee River 2 (Fig. An dam upstream D). results in water levels rising and on falling a regular On basis. average the water is two meters deep but can vary across the river. Plants of tennesseensis are constantly submerged and appear be /. to obligate LUEBKE & BUDKE: ISOETES TENNESSEENSIS 189 aquatic evidenced by as their lack of stomata. River substrate varies, including and cobble, sand, crevice-ridden shale. Plants were found growing wedged in the sand-filled crevices of the shale or partially buried in sandy cobble. To date, Isoetes tennesseensis has only been found in a few locations along Hiwassee known the River. Searches for the plant farther upstream from the and locations in other river systems have not revealed other populations. is It unknown whether tennesseensis occurs in the Tennessee. Further still Little /. more known field investigation is necessary. Until is about the species' known distribution, is suggested that the populations be afforded protection. it does not appear that the population Passage Creek, Virginia new It at this is species. Rebecca Bray has counted the chromosomes from these plants and = reports that they are 2n 110 (Personal Communication). Examination of specimens from this population also reveals that they differ from /. tennesseensis in leaf and spore morphology, but are similar to lacustris. /. Megaspores range in size from 580-705 fim and fall within the range for lacustris. /. Kott and Britton (1983) found that spore size can be correlated with ploidy seem level in Isoetes. This does not to hold with this species since megaspore = size of the octoploid, tennesseensis (x 753 (im) is larger than that for the de- J. = 640 However, between caploid, lacustris (x jim). this correlation ploidy level J. and where spore microspore size is reflected in the size those of/, tennesseensis = = comparison are smaller (x 36 jim) in to those of/, lacustris (x 43 |im). Isoetes tennesseensis the only octoploid quillwort reported North is for America and only the third worldwide. The others are pseudojaponica M. /. Takamiya, Watan. & Ono which Mitsu. K. occurs in Japan (Takamiya, 1999; Troia, 2001) and andina Hook, from South America (Taylor 2002). et /. a/., Preliminary studies of comparisons of nuclear ribosomal ITS nucleotide sequences suggest a possible origin of tennesseensis. The comparison /. indicates that tennesseensis similar engelmannii A. Braun and to is /. /. /. valida (Engelm.) Clute and shares several ITS nucleotide and indels with sites = each. Isoetes engelmannii and valida, both diploids and their (2/7 22), /. = appalachiana Brunton & M. allotetraploid [2n 44), D. F. D. Britton (Napier /. et ah, 2002) are sympatric within the area of tennesseensis. Further /. comparison cloned ITS genomic sequences showed were of six six similar all From to engelmannii. these preliminary studies pedigree proposed a for is /. /. tennesseensis that suggests the result of the backcrossing of engelmannii it is /. = with appalachiana form which doubled to a sterile triploid (2n 33) its /. = chromosomes form hexaploid The engelmannii to a fertile (2n 66). result of/, would backcrossing with hexaploid produce with this a sterile tetraploid that chromosomes would the doubling of its produce a fertile octoploid. Further may molecular more investigations of/, tennesseensis reveal information about new the origin of this species. Acknowledgements acknowl specimens Owen, loan of for this study. Special thanks to Heather University of Wisconsin- VOLUME 190 Milwaukee for the scanning electron micrographs and to Pat Cox, Jim Hickey, Dan Brunton, Carl Taylor, Angel Lekschas, Kerry Heafner, Joanne Peterson and Mary Ann Polasek for their We assistance. also thank the reviewers for their comments and suggestions which strengthened the paper. Literature Cited Boom, M. B. 1979. Systematic studies of the genus Isoetes in the southeastern United States. Thesis. University of Tennessee, Knoxville. Britton, D. M. and P. Goltz. 1991. Isoetes prototypus, a new diploid species from eastern Canada. J. Can. 69:277-281. Bot. J. W. Dennis, M., A. M. Evans and B. E. Wofford. 1979. Disjunct populations of Isoetes macrospora in southeastern Tennessee. Amer. Fern 69:97-99. J. Manual Jong, K. 1997. Laboratory of Plant Cytological Techniques. Royal Botanic Garden Edinburgh. Kott, L. S. and D. M. Britton. 1980. Chromosome numbers for Isoetes in northeastern North America. Can. Bot. 58:980-984. J. Kott, L. S. and D. M. Britton. 1983. Spore morphology and taxonomy of Isoetes in northeastern North America. Can. 61:3140-3163. Bot. J. Napier, N. Hoot and W. S., S. B. C. Taylor. 2002. Unraveling tangled web: hybrid and a allopolyploid origins of Isoetes. Botany 2002 Abstracts, 142 Abstr. Takamiya, M. 1999. Natural history and taxonomy of Isoetes (Isoetaceae). Acta Phytotax. Geobot. 50:101-138. Svenson, H. K. and L. Griscom. 1935. Isoetes macrospora in the Shenandoah Valley. Amer. Fern J. 25:70-71. Taylor, W. and N. C. T. Luebke. 1988. Isoetes xhickeyi: a naturally occurring hybrid between /. echinospora and macrospora. Amer. Fern 78:6-13. /. J. Taylor, W. C, N. T. Luebke, D. M. Britton, R. Hickey and D. F. Brunton. 1993. Isoetaceae, 64- pp. J. FNA 75. In: Editorial Committee, eds., Flora of North America, North Mexico, Volume of 2. New Oxford University Press, York. Taylor, W. C, N. T. 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