W. Carl Taylor

Habitat, evolution, and speciation in Isoetes

Species of Isoetes are notorious for the difficulties they present in identification. These difficulties are attributable to a series of habitat adaptations that have resulted in morphological simplicity, homoplasy, and reticulate evolution. Internal air chambers in the leaves of all taxa indicate that primitive Isoetes was aquatic. During the breakup of Gondwana, ancestors of modern taxa appear to have passed through a terrestrialization phase, which was accompanied by development of several morphological novelties and reductions including the evolution of sclerotic phyllopodia. This adaptation appears to have evolved after separation of the Indian subcontinent (...)

The Utility of Nuclear ITS, a LEAFY Homolog Intron, and Chloroplast atpB-rbcL Spacer Region Data in Phylogenetic Analyses and Species Delimitation in Isoetes

Abstract Despite its ancient origins, its worldwide distribution, and adaptation to diverse habitats, Isoëtes has a highly conserved morphology. This feature has made it difficult to resolve species and species relationships using morphological characters. In this paper, we report the utility of nucleotide sequences from the nuclear internal transcribed spacer (ITS) regions, chloroplast atpB/rbcL intergenic spacer region, and second intron of a LEAFY (LFY) homolog for identifying species relationships, delimiting basic diploid species, and determining hybrid origins. Variation in the ITS regions and atpB/rbcL spacer is most useful at the family level in Isoëtes and the LFY second intron is appropriate at the species and population level. The tree resulting from an analysis of the combined nuclear ITS and chloroplast atpB/rbcL spacer contains three major well supported clades (bootstrap ≥ 99%): an Old-World/California clade (I. abyssinica, I. longissima, I. velata, I. nuttallii, and I. orcuttii), an Asian/Australian clade (I. taiwanensis, I. japonica, I. kirkii, and I. drummondii), and a poorly resolved clade consisting of nine North American species. To further resolve and delimit the North American species, a combination of the LEAFY second intron and ITS data was used. The resulting consensus tree has limited resolution, supporting the hypothesis that the North American species complex radiated rapidly. The combination of LFY and ITS data provided numerous characters, both substitutions and indels, that are useful in species delimitation and identification of cryptic species. ITS sequence data, through additive banding and sequence misalignment, is also useful in confirming interspecific hybrids and determining their parental origins.

The Species Concept in Pteridophyta with Special Reference to Isoetes

The definition, description, and identification of Isoetes species has always been difficult. This is primarily the result of an overall morphological simplicity and extreme phenotypic plasticity that combine to reduce the number of stable and therefore diagnostic characters. Such aspects of the biology of Isoetes have, historically, resulted in rather diverse specific treatments and circumscriptions. Recent studies in the genus have tended to emphasize previously overlooked characters and to apply both modern experimental techniques and the principles of population genetics in order to resolve many of these inconsistencies. Investigations utilizing cytological data, germination and hybridization studies, and protein electrophoresis have shown that over the range of a species its boundaries vary due to clinal variation, non-patterned geographical variation, interspecific hybridization, and the production of fertile allopolyploids. Such approaches have greatly increased our understanding of the dynamics of this genus. At the outset, it should be stated that it is not the objective of this paper to discuss the theoretical implications, or the limitations and benefits of the various species concepts that are currently in existence. Such discussions are numerous and can be found throughout the pages of many of the major systematic journals such as Systematic Botany, Systematic Zoology and Biology and Philosophy. Rather, this paper is designed to illustrate both the current and the historical application of the various species concepts in pteridophytes with particular reference to Isoetes. It is hoped that the reader will come away with an appreciation of the progressive nature involved in the elucidation of species and for the (practical) necessity of applying multiple species concepts during any given systematic inquiry (see also Zander, 1985). To illustrate such processes the following examples will be presented: the I. storkii aggregate of Costa Rica; the I. melanopoda complex of the southeastern United States; and the Isoetes species of northeastern North America. As a prelude to these examples, a brief review of the species concept and a discussion of the role of herbarium material will be presented. Species concepts.-For the purposes of this paper, the species concepts of consequence are: the typological species concept (TSC), the morphological species concept (MSC) and the biological species concept (BSC). The TSC is associated with all poorly known groups. Typically it is associated with rare or at least rarely collected species and is almost always employed when only a single collection is known for the species; i.e., simply, the species is circumscribed upon the only materials available-the type. The MSC, the traditional concept of

Phylogeny and Biogeography of Isoëtes (Isoëtaceae) Based on Nuclear and Chloroplast DNA Sequence Data

Abstract Despite its ancient origins, worldwide distribution, and adaptation to diverse habitats, Isoëtes species have a highly conserved morphology, making it difficult to resolve phylogenetic relationships using morphological characters. In this paper, we report the results from various analyses (maximum parsimony, maximum likelihood, and Bayesian inference) for Isoëtes species from around the world based on nucleotide sequences from the nuclear internal transcribed spacer (ITS) and chloroplast atpB/rbcL intergenic spacer regions. The trees resulting from our analyses of the combined data contain six major well-supported clades (bootstrap ≥ 90%, posterior probabilities = 1.00): A clade with possible Gondwanan affinities (I. australis, I. coromandelina, I. panamensis, I. cubana, I. jamaicensis); a South African clade (I. capensis, I. toximontana, I. stellenbossiensis, I. stephansenii); a largely Northern Hemisphere clade (I. nuttallii, I. orcuttii, I. minima, I. dixitei, I. abyssinica, I. olympica, I. longissima, I. velata); an Asian/Australasian clade (I. drummondii, I. gunnii, I. pusilla, I. kirkii, I. muelleri, I. taiwanensis, I. japonica, I. yunguiensis, I. habbemensis); a Mediterranean clade (I. histrix and I. setacea); and a poorly resolved clade consisting of 12 new world species (American species complex). Our results are compared to past classifications and various biogeographical scenarios are explored.

Phylogenetic Relationships of Isoëtes (Isoëtaceae) in China as Revealed by Nucleotide Sequences of the Nuclear Ribosomal ITS Region and the Second Intron of a LEAFY Homolog

Abstract Isoëtes is an ancient lycopod lineage with a highly conserved morphology that provides few morphological characters to resolve the phylogeny of its species. Species appear to have evolved by divergence and allopolyploidy. The basic diploids I. hypsophila, I. taiwanensis, and I. yunguiensis and the tetraploid I. sinensis occur in China. Analysis of ITS sequences indicates that the Chinese Isoëtes species are part of an Australasian clade including I. brevicula from Western Australia and I. kirkii from New Zealand. Two distinct cloned sequences of the second intron of a LEAFY homolog were recovered from I. sinensis supporting the hypothesis that I. sinensis is an allotetraploid. One of the I. sinensis cloned sequences was similar to the I. taiwanensis sequence and the other cloned sequence was similar to the I. yunguiensis sequence identifying I. taiwanensis and I. yunguiensis as the likely parents of I. sinensis. Other cloned sequences recovered from I. sinensis were recombined parts of the two distinct sequences. Morphological evidence supporting an allotetraploid origin of I. sinensis is found in its larger microspore size and intermediate megaspore texture compared to I. taiwanensis, and I. yunguiensis.

A Re-evaluation of Isoetes savatieri Franchet in Argentina and Chile

Abstract Isoetes savatieri has traditionally been interpreted as being a uniform aquatic ranging from the southernmost regions of South America to the central Andes of Chile and Argentina. An examination of herbarium material supports the recognition of two taxa, a southern I. savatieri and a more northern I. chubutiana, from central Chile and Argentina. The latter taxon is hexaploid and described here as a new species. The morphology of these species suggest that they are sister species resulting from divergence following a polyploidy event. These species, and several other species pairs, provide the best and, to date, only examples of allopatric divergence in polyploid Isoetes.

Germinating Spores and Growing Sporelings of Aquatic Isoetes

Several techniques have been described recently for germinating spores of aquatic species of Isoetes which grow in lakes, ponds, and streams in eastern North America. Boom (1980) used glass vials containing sterile sand and pond water in which he successfully raised sporelings of I. engelmannii, I. flaccida, I. macrospora, and I. riparia. Kott and Britton (1982) germinated spores of I. acadiensis, I. echinospora, I. macrospora, I. riparia, and I. tuckermanii in small vials half filled with distilled water. However, many of their vials became contaminated with fungi that destroyed the viability of the megaspores. Sam (1982) germinated megaspores of I. engelmannii in an inorganic nutrient medium supplemented with Nystatin to inhibit fungi. Webster (1979) described a technique for germinating spores of Selaginella that is similar to the following procedure we use for Isoetes. We have been culturing plants from spores of Isoetes acadiensis, I. echinospora, I. engelmannii, I. hieroglyphica, I. macrospora, I. riparia, and I. tuckermanii for use in germination and hybridization experiments for several years. The aquatic species of Isoetes are ideal pteridophytes for germination and hybridization experiments. Their sporangia are large and contain many spores. Megaspores and microspores are borne in different sporangia on separate leaves. If necessary, unopened sporangia can be excised from sporophylls intact and surface cleansed to eliminate extraneous spore contamination. Thus, megaspores and microspores can be completely isolated for controlled breeding experiments. Also, their spores readily germinate in demineralized water and gametophytes and sporophytes develop normally for several months without supplemental nutrients. Spores are obtained from plants collected in September and October, since plants harvested earlier in the year may not have mature, viable spores. Isoetes hieroglyphica and I. macrospora retain their spores through the winter, so mature spores of these species can also be gathered in the spring. Megaspores are cleansed of microspores in a sieving apparatus made of plastic tubing approximately 10 cm long and 2.5 cm in diameter. An 8 cm square of 0.27 mm sifter mesh (available from Carolina Biological Supply Company) is fitted to one end of the tubing with a rubber band. The sieving apparatus, containing megaspores, is attached to a water faucet using plastic strapping tape. Spores are washed with a steady flow of cold water for 30 minutes, followed by a one minute rinse with sterile, demineralized water. Although megaspore surfaces appear clean after the above treatment, unopened sporangia are preferred for crossing experiments because there is less chance of contamination from microspores. Intact sporangia can be excised from

Isoetes Xbrittonii hyb. nov. (Isoetaceae): A Naturally Occurring Hybrid (I. engelmannii X I. riparia) in the Eastern United States

Although Jeffrey (1937) provided a convincing cytological and morphological argument for the existence of hybrids between natural populations of Isoetes in North America over 50 years ago, a systematic and broadly-based investigation of this phenomena has been undertaken only recently. Boom (1980; 1982) showed that various eastern North American species could be hybridized in culture. On the basis of herbarium studies he also reported several naturally occurring hybrid combinations. Taylor et al. (1985) established geographical, cytological and electrophoretic evidence for the existence of naturally occurring Iso6tes hybrids. They showed that the uncommon northeastern North American diploid taxon I. x eatonii Dodge is a sterile hybrid between I. echinospora Dur. and I. engelmannii A. Br., and they made in vitro hybridizations between these parental species. Two other naturally occurring Iso6tes hybrids from northeastern North America have been recently discovered. Taylor and Luebke (1988) reported I. x hickeyi (I. echinospora x macrospora [lacustris]) from Wisconsin and Britton & Brunton (1989) identified I. x dodgei A. A. Eaton (pro sp.) as I. echinospora x riparia Dur. from Ontario, Quebec, Vermont and New Hampshire. All of these interspecific hybrids were initially detected by their production of irregular megaspores which are more varied in size, shape and surface ornamentation than megaspores of parental species. This paper describes another Isoetes hybrid from northeastern North America. it is believed to be the result of hybridization between I. engelmannii and I. riparia. Nine plants of this hybrid have been found, each demonstrating combinations of the characters expressed by Isoetes hybrids, viz, plants with hybrid vigour, aborted spores of variable size and shape, and spores with surface ornamentation patterns more or less intermediate between the putative parents (Jeffrey, 1937; Taylor & Luebke, 1988; Britton & Brunton, 1989). Further evidence supporting the hybrid nature of this taxon comes from its chromosome number, its sporadic occurrence within the partially sympatric ranges of its putative parents and its constant association with one of its parents at each station.

Morphological and Anatomical Variation in Sporophylls of Isoetes sinensis Palmer (Isoetaceae), an Endangered Quillwort in China

ABSTRACT Isoetes sinensis is a rare and endangered plant in wetlands of southeastern China. Previous studies have reported the chromosome number, geographic distribution, and ecology of this Asian endemic, but there has not been an analysis of diagnostic characters associated with the sporophylls of I. sinensis in China. Therefore, morphological and anatomical variation of sporophylls in the three known Chinese populations of I. sinensis were evaluated and compared. The variation found is discussed in relation to the present taxonomy of I. sinensis.

Relationship between Water Chemistry and the Distribution of the Endangered Aquatic Quillwort Isoetes sinensis Palmer in China

ABSTRACT The progressive decline in populations of Isoetes sinensis Palmer in China has been attributed to deteriorating water quality; however, no data have been adduced to support this assumption. In this study we identified locations in China that presently have populations of I. sinensis and locations where the species has been extirpated. We measured various water chemistry parameters at these sites, as well as at similar sites within the distribution range of I. sinensis but from where the species has never been recorded. The results indicate that distribution and occurrence of I. sinensis are closely correlated with water chemistry. Water samples drawn from habitats from where I. sinensis has been extirpated had higher concentrations of heavy metal ions, particularly chronium VI (Cr6+. In addition, these samples consistently yielded higher readings for dissolved carbon dioxide, water hardness, and concentration of nitrates.