DONALD M. BRITTON - 1991 RECIPIENT OF THE LAWSON MEDAL

[The following text is reproduced with few changes from a short speech by P.M. Catling, then president of the Canadian Botanical Association, at the annual meeting of the Association in 1991 when Don received the prestigious Lawson Medal.]

Dr. Britton was born in Toronto. He graduated from the University of Toronto with first class honours in science and biology, then took a scholarship in Virginia where he received his Ph.D. from the University of Virginia, Charlottesville in 1950. His thesis was entitled "Cytogenetic studies on the Boraginaceae." Dr. Britton spent one year in the Dept. Plant Sciences at the University of Alberta and then 5 years as Assistant Professor of Horticulture at the University of Maryland where he specialized in the cytogenetics of Rubus. He settled at the University of Guelph in 1958 and became a Professor in the Botany and Genetics Department there in 1971. He continued his association with the University of Guelph after his retirement and up to the present.

Dr. Britton published his first paper on ferns in 1953. This group was to become his specialty and his work on it made hjm an internationally recognized expert. He was one of the pioneers in the use of cytogenetic techniques to reveal relationships, the use of the scanning electron microscope in systematics and the use of cytological data in the systematics of ferns. Whenever evolution of pteridophytes is discussed, his name will be mentioned. Dr. Britton was also a rather early biosystematist in that he integrated micromorphology, morphology, cytology, chemistry and phytogeography in his work.

Dr. Britton soon became recognized as an expert because he studied some of the most intractable groups of ferns including Dryopteris, Woodsia and Isoetes, and also because he was always able and willing to provide an authoritative view on any aspect of fern taxonomy. As a matter of interest, George Lawson, in whose honour the medal is named, was himself very interested in ferns and in 1889, he published a "Fern Flora of Canada". Other Canadian botanists have also made significant contributions to our knowledge of Canadian pteridophytes, but none has contributed in this area to the extent that Dr. Britton has. Among the graduate students he directed were S. J. Rigby (Pellaea), L. Kott (Polypodium, Isoetes}. R E. Newell (Lycopodium) and K. M. Pryer (Gymnocarpium). Dr. Britton has acted as chairman of the Pteridology Section of the Botanical Society of America and has been a leader in the New England Fern Conference since 1970. He is described by his friends, scientists and students as a "pro" and "a friendly person, a little quiet but with a sly sense of humour". His approach to research has been careful and thorough, and it is widely acknowledged that he has contributed substantially to the development of botany in Canada.

In addition to numerous publications on ferns in various journals, Dr. Britton has co-authored The Ferns of Canada with W. J. Cody. Dr. Britton has chosen to publish rather than perish in retirement. He is still publishing at a remarkable rate, mostly in the Canadian Journal of Botany. His recent publications on Isoetes represent a major contribution to our understanding of that group, and are the kind of publications that can only come from a highly skilled taxonomist with a very deep understanding of the organisms he studies. He has served as the regional reviewer for pteridophytes of eastern Canada for the Flora North America Project and a co-author for the treatment of Isoetes. Isoetes x brittonii (Amer. Fern J. 83(3): 85. 1990.) was recently named in his honour. Don, as he prefers to be called, was an active member of the Guelph Field Naturalists Club, serving as Conservation Chairman for many years. He is an outstanding field botanist and has lead a great many field trips. His collection numbers exceed 13,000 and the specimens are deposited mainly at OAC, TRT, DAO, CAN, GH,. H and BM.

DON BRITTON - APPRECIATION

What a huge pleasure it is to be able to say a few words in acknowledging a wonderful milestone - Don Britton's 80th birthday. For Don was my first scientific mentor in systematics and floristics, while I was still an undergraduate at the University of Guelph. I was already deeply interested on native plants, and had taught myself much of the flora (except some of the difficult groups like grasses, and of course, sedges) even starting in high school. Naturally, with those interests and background knowledge already developed, I found introductory courses in biology (and even more so other topics) a little frustrating. And as I had already also developed an interest in ferns (like many people, the first groups of plants I became interested in were orchids and ferns), it was exciting to have Don take me under his wing, and let me accompany him on some of his field trips, trips to other Herbaria, and also do a project with him as an undergraduate.

Though known to people worldwide as an expert on Canadian ferns and, especially recently, Isoetes, Don was also deeply interested in phytogeography and floristics of the Great Lakes region generally. To a young undergraduate keenly interested in the flora, it was fascinating to hear about the arctic disjuncts on Lake Superior, and the even more remarkable disjuncts from the far west. But Don was also interested in (with Craig Campbell), regional floristics (the Waterloo region, with a history of study dating back into the late 1800's), and in the distribution of rare plants. He told me about the legendary and long known disjunct station of Woodsia scopulina in Algonquin Park, Ontario which he had visited (and which resulted in my second published scientific paper - for the first, see below). We went out in the field a number of times, and it was always a huge learning experience listening to Don's commentary on the distribution of this or that plant, what similar things to look for, and where to look for them and why fueled my interest greatly in research on floristics and phytogeography (though he can't be blamed for my interest in Cyperaceae).

Don's research often combined systematics and phytogeography, and I the undergraduate project that he had me work on was just such a combination - the distribution of Claytonia chromosome races in Ontario. The two southern Ontario woodland Claytonia are peculiar plants because their chromosome numbers vary hugely, with some geographic pattern but little associated morphological variation. My piece of the puzzle was to fill in Ontario data for the overall documentation of variation. It was exciting to have Don trust me with this, and with his help and advice, I hunted up populations and squashed chromosomes. This was a pretty finger freezing job, actually, as these plants undergo meiosis incredibly early in spring, while everything is still pretty much gripped by winter -- my first experience that research can be uncomfortable! This was also my first published paper and with Don's gracious and patient help, I survived the minefields of reviewers and journal editor's honesty with interest and enthusiasm intact. In Universities now, undergraduate (and even high school) experience in research is a major push. But back then, it was quite unusual, though I realized this only in retrospect.

When I was casting about for graduate schools and people to work under, Don was always helpful with comments, advice, and introductions. He introduced me to the late Arthur Cronquist, when Art was giving a seminar at Guelph. And he took me along to Harvard University on one of his trips, which gave me a chance to look in awe at specimens and books that M.L. Fernald and even Asa Gray - legendary, god-like figures to me at the time - worked with. I had become quite adept at telling Goodyera repens from Goodyera tessellata - not an easy matter in the herbarium, and I wanted to see the specimens at Harvard. I was taken aback, however, since when the people there at the New England Botanical Club Herbarium found out I could name these things, they wanted me to go through their specimens and annotate them! I think I did do some, and hope I didn't make any mistakes. But I did not want to go to the US for graduate work, which many people in my position at that time did. But Don was always accommodating, and I still remember his understated commentary on various people and schools as I continued to try to focus my graduate work I never realized until much later how thoughtful his remarks were, but fortunately, I paid attention to them and (largely) took his advice.

I only wish I could get back to Guelph more often.

Thank you Don, and congratulations.

DON BRITTON (DMB) TURNS 80?

Hard to imagine. I know, we've all gotten older, but my memories of DMB are almost frozen in time when he must have been 57 or so and was my Master's thesis advisor at the University of Guelph where I undertook my first venture doing fern research under his able guidance. I've seen DMB since, of course, but every time I find him to be just the same as ever and not to have aged in any way, and so it's a bit of a shock to learn that he reaches the 80th milestone this week.

I am really proud to have done my M.Sc. with DMB and today, more than ever, I truly appreciate what a wonderful mentor he has been. DMB always listened intently, but didn't always react immediately. This took a bit of getting used to, especially in my early student days when I thought he might just be enjoying his pipe (yes, you could smoke inside in those days!) and perhaps thinking of something more profound than what I was talking about. For example, sometimes I'd mention in these conversations how I'd like to get my hands on "such and such" a rare article and then shuffle back to my office when there was no apparent reaction. The next day whatever I had been looking for would magically appear on my desk. No fanfare, but I knew who had put it there. Often accompanying the article I'd asked about were a couple of others he thought I'd better also have a look at. DMB is a true scholar and has a wonderful gift for sharing his knowledge with others in a very quiet and gentle (and often humorous) manner.

Doing fieldwork with DMB is a treat. He has radar for finding plants in the field that is awesome - he goes right to what he's looking for. If he can't find it - it's not there - and he won't waste anyone's time beating the bushes for it. We'd just get back in the car and race off to the next hopeful spot. Field trips with DMB were so meticulously planned - everything happened on schedule, ALWAYS with good humor, and without a hitch. Except for once. We were somewhere in Wellington Co. in search of Gymnocarpium hybrids. It was May - lilacs were blooming - just the right time of year to "pickle" these ferns for getting good chromosome squashes. We parked the car next to a farmhouse and began to cross a huge pasture to get to the patch of woods in question. Suddenly DMB pointed out that there was a bull at the far end of the pasture. I peered in the distance and saw it pawing at the ground and looking intently in our direction. I quietly asked DMB what we should do. When he didn't answer, I turned to look at him - - just in time to see his lanky 6 ft.+ frame hightailing it over the fence some 50 feet away. Nice guy, but so damn quiet, even in emergencies. I never ran so fast in all my life. We still chuckle over that "incident" to this day.

DMB always has time for everyone, many with whom he has communicated regularly on his famous typewriter (did he ever own one that didn't have maladjusted keys?). If you ever received a letter from DMB - you know what I'm talking about. His peers such as Rolla Tryon at Harvard and Herb Wagner at Michigan communicated with him regularly; graduate students everywhere, including his own (Laima Kott, Ruth Hersey, and me at that particular time) were always looking for his insight into their projects; and every amateur botanist within a day's drive of Guelph, and many more far beyond, all eventually found their way to his office door. DMB's patience and good humor with everyone, no matter what their official affiliation, was a wonderful example. I know that his example and the joy he gets from working with ferns is what inspired me to study ferns for the long haul.

In the summer of 2000, I had the good fortune to race around the southern Ontario countryside with DMB again - this time in search of 9 species of Equisetum that are within a 20- mile radius of Guelph. (Nine of the 15 species of Equisetum that are known in the world are found right around Guelph - isn't that amazing?!) This time I was the one driving, while DMB instructed me to turn right, then left, then right and so on, all the while telling me (Qu‚bec driver) to slow down - "now we don't want them throwing us in the slammer!" he'd exclaim every once in a while. In 2 short days, we zeroed in on variegatum, laevigatum, hyemale, arvense, scirpoides, fluviatile, pratense, sylvaticum, and palustre and I had all the material I needed to set about doing a phylogenetic study of the genus (the other 6 taxa, bogotense, ramosissimum, telmateia, giganteum, diffusum, and myriochaetum, were sent to me from various botanists across the globe). I handed the material to a young Berkeley undergraduate student (Dave Des Marais) who was doing an internship with me that summer and he ran with the project by sequencing two chloroplast genes for each of the 15 species, doing the phylogenetic analyses, and even managing to date the time of divergence of the living members of Equisetum. The manuscript is currently is press in the International Journal of Plant Sciences and I append an abstract below.

Happy 80th Birthday DMB!!! Thank you for being such a great mentor, and when is our next field trip, by the way?

Des Marais, D.L., A.R. Smith, D.M. Britton, & K.M. Pryer. 2003.

Phylogenetic relationships and evolution of extant horsetails, Equisetum, based on chloroplast DNA sequence data (rbcL and trnL-F). International Journal of Plant Sciences 164: in press.

Abstract: Equisetum is a small and morphologically distinct genus with a rich fossil record. Two subgenera have been recognized based principally on stomatal position and stem branching: subg. Equisetum (8 species; superficial stomates; stems branched) and subg. Hippochaete (7 species; sunken stomates; stems generally unbranched). Prior attempts at understanding Equisetum systematics, phylogeny, and character evolution have been hampered by the high degree of morphological plasticity in the genus, as well as frequent hybridization among members within each subgenus. We present the first explicit phylogenetic study of Equisetum, including all 15 species and two samples of one widespread hybrid, E. x ferrissii, based on a combined analysis of two chloroplast markers, rbcL and trnL-F. Our robustly supported phylogeny identifies two monophyletic clades corresponding to the two subgenera recognized by earlier workers. The phylogenetic placement of E. bogotense, however, is ambiguous. In maximum likelihood analyses, it allies with subg. Hippochaete as the most basal member, while maximum parsimony places it as sister to the rest of the genus. A consensus phylogeny from the two analyses is presented as a basal trichotomy (E. bogotense, subg. Hippochaete, subg. Equisetum) and morphological character evolution is discussed. We detected rate heterogeneity in the rbcL locus between the two subgenera that can be attributed to an increased rate of nucleotide substitution (transversions) in subg. Hippochaete. We calculated molecular-based age estimates for the Equisetaceae using the penalized likelihood approach, which accounts for rate heterogeneity and does not assume a molecular clock. Extant species of Equisetum appear to have diversified in the early Cenozoic, which is in remarkable agreement with current interpretations of the fossil record.

FERNS & FERN ALLIES OF SOUTHEAST ALASKA: A PRELIMINARY CHECKLIST

The following is a preliminary list of 65 ferns and fern allies known from southeastern Alaska. This portion of the state is located between the latitudes of 54°40' and 60°22' degrees north and is bounded on the west by the North Pacific Ocean, on the north by the Wrangell-St. Elias Mountains, on the east by the Coast Range, and on the south by Dixon Entrance.

Plant names are listed alphabetically by family and follow the family arrangement of the Flora of North America (Flora of North America Editorial Committee, 1993). Scientific names were generally taken from treatments in Flora of North America. Synonymous names from older floras covering southeastern Alaska are also listed: however, this is not a complete synonymy. These floras include: Anderson's Flora of Alaska and adjacent parts of Canada (Welsh, 1974), Flora of Alaska and Neighboring Territories (Hulten, 1968), Vascular Plants of the Pacific Northwest (Hitchcock, et al. 1955- 61), Flora of the Queen Charlotte Islands (Calder & Taylor, 1968).

LYCOPODIACEAE
Diphasiastrum alpinum (L.) Holub
   Lycopodium alpinum L.
Diphasiastrum complanatum (L.) Holub
   Lycopodium complanatum L.
Diaphiastrum sitchense (Ruprecht) Holub
   Lycopodium sitchense Ruprecht
Huperzia haleakalae (Brackenridge) Holub
   Lycopodium selago L. var. haleakalae Brackenridge
   Lycopodium selago L. var. selago
   Lycopodium selago L. var. appressum Desv.
Huperzia chinensis (Christens.) Ching
   Huperzia miyoshiana (Makino) Ching
   Lycopodium selago L. var. miyoshianum Makino
   Lycopodium selago L. ssp. chinense (Christens.) Hult.
Huperzia occidentalis (Clute) Kartesz & Gandhi
   Lycopodium lucidulum Michx. var. occidentale (Clute) L.R. Wilson
   Lycopodium selago L. ssp. patens (Beauv.) Calder & Taylor
Lycopodium annotinum L.
Lycopodium clavatum L.
Lycopodium dendroideum Michaux
   Lycopodium obscurum L. var. dendroideum (Michx.) D.C. Eat.
Lycopodium lagopus (Laestad. ex Hartm.) G. Zinserl. ex Kuzen
   Lycopodium clavatum L. var. monostachyon Grev. & Hook.
Lycopodiella inundata (L.) Holub
   Lycopodium inundatum L.

SELAGINELLACEAE
Selaginella selaginoides (L.) Palisot de Beauvois ex Martius & Schrank

ISOETACEAE
Isoetes echinospora Drieu
Isoetes maritima L.
Isoetes occidentalis L. F. Henderson
Isoetes x truncata (A. A. Eaton) Clute

EQUISETACEAE
Equisetum arvense L.
Equisetum fluviatile L.
Equisetum hyemale L. subsp. affine (Engelmann) Calder & Taylor
Equisetum x litorale Kuehlewein ex Ruprecht
Equisetum palustre L.
Equisetum pratense Ehrhart
Equisetum scirpoides Michaux
Equisetum sylvaticum L.
Equisetum telmateia subsp. braunii (J. Milde) Hauke
Equisetum variegatum Schleicher ex F. Weber & D. Mohr subsp. alaskanum (A. A. Eaton) Hulten
Equisetum variegatum Schleicher ex F. Weber & D. Mohr subsp. variegatum

OPHIOGLOSSACEAE
Botrychium alaskense W. H. Wagner & J. R. Grant
Botrychium ascendens W. H. Wagner
Botrychium lanceolatum (S. G. Gmelin) Angstroem ssp. lanceolatum
Botrychium lunaria (L.) Swartz
Botrychium minganense Vict.
   Botrychium lunaria (L.) Swartz var. minganense (Vict.) D”ll
Botrychium multifidum (S. G. Gmelin) Rupr.
Botrychium pinnatum H. St. John
   Botrychium boreale (Fries) Milde
Botrychium tunux Stensvold & Farrar
Botrychium virginianum (L.) Swartz
Botrychium yaaxudakeit Stensvold & Farrar

PTERIDACEAE
Adiantum aleuticum (Rupr.) Paris
   Adiantum pedatum L. var. aleuticum Ruprecht
Cryptogramma acrostichoides R. Br.
   Cryptogramma crispa (L.) R. Br. ex Hook. ssp. acrostichoides (R. Br.) Hulten
Cryptogramma sitchensis (Rupr.) T. Moore
   Cryptogramma crispa (L.) R. Br. ex Hook. var. sitchensis (Rupr.) C. Christens
Cryptogramma stelleri (S. G. Gmelin) Prantl in Engler

HYMENOPHYLLACEAE
Hymenophyllum wrightii Bosch
   Mecodium wrightii (Bosch) Copeland

DENNSTAEDTIACEAE
Pteridium aquilinum (L.) Kuhn var. pubescens Underwood

THELYPTERIDACEAE
Phegopteris connectilis (Michx.) Watt
   Thelypteris phegopteris (L.) Slosson
Thelypteris quelpaertensis (Christ) Ching
   Thelypteris limbosperma auct. non (All.) Fuchs

BLECHNACEAE
Blechnum spicant (L.) Smith

ASPLENIACEAE
Asplenium viride Huds.
   Asplenium trichomanes-ramosum L.
Asplenium trichomanes L. ssp. trichomanes

DRYOPTERIDACEAE
Athyrium americanum (Butters) Maxon
   Athyrium alpestre (Hoppe) Milde var. americanum Butters
   Athyrium distentifolium Tausch ex Opiz var. americanum (Butters) Cronq.
Athyrium filix-femina (L.) Roth ssp. cyclosorum (Rupr.) C. Christens.
Cystopteris fragilis (L.) Bernh.
Cystopteris montana (Lam.) Bernh. ex Desv.
Dryopteris expansa (C. Presl) Fraser-Jenkins & Jermy
   Dryopteris dilatata (Hoffmann) Gray ssp. americana (Fisch.) Hult‚n
   Dryopteris austriaca (Jacq.) Woynor ex Schinz & Thell.
Gymnocarpium disjunctum (Ruprecht) Ching
   Gymnocarpium dryopteris (L.) Newman var. disjunctum (Rupr.) Ching
Gymnocarpium dryopteris (L.) Newman
Polystichum andersonii Hopkins
   Polystichum braunii (Spenner) F‚e ssp. andersonii (Hopkins) Calder & Taylor
Polystichum braunii (Spenner) F‚e
   Polystichum braunii (Spenner) F‚e ssp. purshii (Fern.) Calder & Taylor
Polystichum kruckebergii W.H. Wagner
Polystichum lonchitis (L.) Roth
Polystichum munitum (Kaulf.) C. Presl
Polystichum setigerum (C. Presl) C. Presl
   Polystichum braunii (Spenner) F‚e var. alaskense (Maxon) Hult‚n
Woodsia alpina (Bolton) S.F. Gray
Woodsia ilvensis (L.) R. Br.
Woodsia scopulina D.C. Eat. ssp. scopulina

POLYPODIACEAE
Polypodium glycyrrhiza D.C. Eat.

References

Calder, James A. & R.L. Taylor. 1968.

Flora of the Queen Charlotte Islands, Part 1. Canada Department of Agriculture, Research Branch, Monograph No. 4 Part 1. Ottawa.

Flora of North America Editorial Committee, eds. 1993.

Flora of North America, north of Mexico, Part 2: Pteridophytes and gymnosperms. Oxford University Press, New York.

Hitchcock, C.L., A. Cronquist, M. Ownbey, and J.W. Thompson. 1955, 1959, 1961, 1964, 1969.

Vascular Plants of the Pacific Northwest. Parts 1-5. University of Washington Press, Seattle, Washington.

Hulten, Eric. 1968.

Flora of Alaska and Neighboring Territories. Stanford University Press, Stanford, California.

Welsh, S.L. 1974.

Anderson's Flora of Alaska and adjacent parts of Canada. Brigham Young University Press, Provo, Utah.

TAXONOMIC STATUS AND EVOLUTIONARY RELATIONSHIP OF ISOETES MINIMA A. A. EATON (ISOETACEAE) BASED ON NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Introduction

On 16 May 1889, W. N. Suksdorf collected a small Isoetes from a moist depression on a prairie near the town of Waverly, Spokane County, Washington. Specimens (Suksdorf 2365) were sent to A. A. Eaton, who named these plants Isoetes minima and described them as species new to science (Eaton 1898).

Eaton (1898) characterized his Isoetes minima as unique in several ways. First, at the time, it was the smallest quillwort known in America with leaves up to only 4 cm long. Second, it was the only American Isoetes species that had the combined characters of a 3-lobed, corm-like rhizomorph and a partial velum. Isoetes nuttallii and I. orcuttii also have 3-lobed rhizomorphs, but both of them have a complete velum. Third, he noted that the spinulose textured megaspores of I. minima also had a spinulose equatorial ridge that in outline "resembles a ship's wheel with the spinules for handspikes." In addition to its short leaves, 3-lobed corm, and partial indusium, the spinulose megaspores of Isoetes minima are unique among all other seasonal terrestrial quillworts in North America.

On 30 June 1985, Oldriska Ceska discovered a small Isoetes growing in wet swales in sagebrush steppe at Colockum Pass, Kititas County, Washington (Ceska 19754). On 5 July 1996, Adolf and Oldriska Ceska found similar plants in wet depressions among rocky ledges, 12 km west of Salmo, British Columbia (Ceska 30000) and on 12 July 1996 Hans Roemer at Lloyd's Meadows made collections of comparable plants, 8.2 km west of Castlegar, British Columbia (Roemer 96-164).

Scanning electron photomicrographs of spores from specimens collected at each of these three sites were made by Adolf Ceska and Lesley Manning on 19 March 1997 at the Pacific Forestry Centre in Victoria, British Columbia. In these photomicrographs, the megaspores from each of these collections are clearly seen to be "covered with short, slender, blunt distinct spinules" just as described by Eaton a hundred years earlier for his I. minima. Furthermore, the microspores textures are "papillose or sparingly spinulose" also as described by Eaton.

Pfeiffer (1922) reduced I. minima to a variety of I. howellii stating, "Eaton founded his species I. minima on a 3-lobed specimen of this form. Since most of the material cited proves to be 2-lobed, it is supposed that the occurrence of 3 lobes is like the chance development reported occasionally in other regularly 2-lobed species." Pfeiffer also modified Eaton's original description to include plants with leaves up to 10 cm long and "with vague short crests, tubercles, like species" and megaspores 320-420 mm in diameter. A recent treatment of North American Isoetes has followed Pfeiffer's taxonomy (Taylor et al. 1993).

In June 2002, the Ceska's kindly sent the author fresh specimens of "Isoetes minima" collected from the Colochum Pass, Salmo, and Lloyd's Meadows sites. Having living specimens of this taxon afforded an opportunity to make chromosome counts and isolate DNA to obtain nucleotide sequences that might provide information about the relationship of I. howellii var. minima to other western North American taxa.

Methods and Materials

Chromosome counts of I. howellii var. minima were made from root tip squashes using procedures recorded by Jong (1997) with some modifications. Roots of I. howellii var. minima were harvested from newly arrived plants. New roots, approximately 6 mm long, were pretreated in a saturated solution of paradichlorobenzene in the dark at room temperature for 4 hrs. Roots were then fixed in Farmer's Solution (3:1 96% EtOH:glacial HAc), left at room temperature for 1 hr and stored at -20°. For staining, roots were hydrolyzed in 1N HCl for 10 min at 60°C, soaked in three changes of 96% EtOH for fifteen minutes each, blotted, placed in Whitman's hematoxylin stain for approximately 1 hr, and then destained in glacial HAc for five minutes.

For DNA analysis, total cellular DNA was extracted from fresh, silica gel dried, or herbarium samples using a Dneasy Plant Mini Kit (Qiagen, Valencia, California, USA) and quantitated using a Biophotometer (Eppendorf, Westbury, New York, USA).

The internal transcribed spacer (ITS) region (ITS-1, 5.8S, ITS-2 nrDNA) was amplified by PCR (polymerase chain reaction) using primers ITS-I (5'- GTCCACTGAACCTTATCATTTAG-3'; Urbatsch, Baldwin, and Donoghue, 2000) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'; White et al. 1990) in 25-mL reactions using puReTaqT Ready-To -GoT PCR Beads (Amersham, Piscataway, New Jersey, USA) with approximately 5mol of each primer. PCR reaction conditions in a Hybaid PCR Sprint thermal cycler (Hybaid, Middlesex, UK) were 40 cycles of denaturation at 97°C for 10 sec, primer annealing at 48°C for 30 sec, and primer extension at 72°C, initially for 20 sec (with an increase of 4 sec to each successive extension). Thermal-cycling was followed by a final extension at 72°C for 7 min. (Baldwin and Wessa 2000).

PCR products were purified using a QIAquickT PCR Purification Kit (Qiagen). Purified PCR products and primers were sent to the DNA Sequencing and Synthesis Facility, Iowa State University, Ames, Iowa, USA for sequencing. SequencherT 4.1 (Gene Codes Corporation, Ann Arbor, Michigan, USA) was used to roughly align sequences with subsequent manual editing and alignment.

After its chromosome number was determined, Isoetes howellii var. minima sequences were aligned and compared with other basic diploid species (2n = 2x = 22) that occur in western North America, i.e., I. bolanderi, I. echinospora, I. howellii, I. nuttallii, and I. orcuttii.

Fitch parsimony analysis of the data was performed with PAUP* version 4.0b10 (Swofford 2002) using the exhaustive search option, maximum number of trees = 1000. To assess branch support, PAUP* was also used to perform bootstrap analyses (Felsenstein 1985) using full heuristic search and number of replications = 1000. Based on previous analyses of Isoetes worldwide using ITS data, I. australis from Western Australia was chosen as the outgroup.

Results

Chromosome counts from the root tip squashes yielded several countable figures. Twenty-two chromosomes were counted in each of the well- spread figures, indicating that I. howellii var. minima is a basic diploid species with 2n = 2x = 22.

One most parsimonious ITS tree resulted from the analysis of I. bolanderi, I. echinospora, I. howellii, I. nuttallii, and I. orcuttii rooted with I. australis. There were 205 variable characters and 93 were parsimony informative with a consistency index excluding uninformative characters of 0.94 and a retention index of 0.96.

Two well-supported clades (bootstraps = 100%) were resolved. One clade, supported by 55 nucleotide changes, contained I. bolanderi, I. echinospora, and I. howellii. The other clade, supported by 47 nucleotide changes, contained I. howellii var. minima, I. nuttallii and I. orcuttii. The branch leading to I. orcuttii was supported by 16 nucleotide changes whereas the branch connecting I. howellii var. minima and I. nuttallii was supported by four nucleotide changes. Four and three nucleotide changes distinguished I. howellii var. minima and I. nuttallii from each other, respectively.

Discussion

ITS sequences indicate that Pfeiffer's I. howellii var. minima is more closely related to I. nuttallii and I. orcuttii than it is to I. howellii. Isoetes howellii var. minima appears to be most closely related to I. nuttallii (bootstrap = 64%), but distinct enough from I. nuttallii to be recognized as a species, I. minima.

Several field characters distinguish plants of I. minima from plant of I. nuttallii. First, I. minima has an incomplete velum covering up to 75% of the sporangium whereas, I. nuttallii has a complete velum covering 100% of the sporangium. Second, I. minima has spinulose textured megaspores ranging 290-350 mm in diameter. In contrast, I. nuttallii has smooth to tuberculate textured megaspores ranging 360-600 mm in diameter. Third, I. minima is generally a smaller plant with leaves up to only 4 cm long whereas, I. nuttallii is usually larger plant with leaves up to 20 cm long.

Thus, Eaton's original description of I. minima as a species of Isoetes appears correct. Isoetes minima is a basic diploid species distinguishable from the species it is most nearly related to by DNA sequence, I. nutallii, by several characters. ITS sequence data indicates I. minima is only distantly related to I. howellii and therefore it should not be treated as a variety of I. howellii.

Acknowledgements

Thanks to Adolf Ceska for providing living and dried specimens of I. minima and SEM photomicrographs of I. minima spores. We are especially grateful to Oldriska Ceska, without whose rediscovery of I. minima, this study would never have been possible. Last, but certainly not least, thanks to Professor Donald Britton, who has graciously shared his insight and inspiration about Isoetes for many years.

Funding for this research was provided by NSF grant DEB9981501 to W. Carl Taylor.

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ISOETES MINIMA - GUESS WHO IDENTIFIED IT!

When we first collected this strange terrestrial quillwort in sage brush steppe at several spots on Colockum Pass in central Washington, we were convinced that this was a new, yet undescribed species. If you see this plant in the field, it lacks any similarity to Isoetes howellii (although they grow together in the area of the type locality near Spangle, WA), and it did not resemble any species we knew. We discussed this with Don Britton when we were hunting for Polystichum root tips in British Columbia in the spring of 1987. A few weeks later we received a letter from him - typed on his famous typewriter - that said: "Adolf, I wonder, why didn't you consider Eaton's Isoetes minima?" How do you translate Italian "sotto voce" into English?

This reminds me of another of Don's insights that helped us to make another discovery. On one Wednesday we received a letter from Don: "I read Robbin Moran's paper on Asplenium trichomanes and I think you should look for Asplenium adulterinum on Vancouver Island. That species was described in Bohemia and you should know it, since you mentioned Bohemia and bohemians to me several times before." The following Monday we went with Bob Ogilvie for a field trip to northern Vancouver Island. On Tuesday we landed in a carst region and the first plant we saw was Asplenium adulterinum, a species new to North America. The next day I had the pleasure to buy the ugliest post card I could find in a small fishing town and I wrote to Don: "Yes, Don, we have tons of Asplenium adulterinum on Vancouver Island."