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Revealing unknown or extinct lineages within Isoëtes (Isoëtaceae) using DNA sequencesfrom hybrids¹

³Department of Biological Sciences, University of Wisconsin, Milwaukee, Wisconsin 53201 USA; ⁴Department of Botany, Milwaukee Public Museum, Milwaukee, Wisconsin 53233 USA

Received for publication August 21, 2003. Accepted for publication February 10, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isoëtes, a heterosporous lycopod with a fossil record dating back to the Paleozoic, has numerous putative allopolyploids (resulting from hybridization events coupled with doubling of chromosome number). By using the highly variable nucleotide sequences from the second intron of a LFY homologue in Isoëtes, species could be delimited and hybrid origins determined. The data suggest that reticulate evolution is both common and complex within a more derived species complex of Isoëtes. Sequences of identifiable parentage and sequences that are unlike any diploid species known were recovered, leading to the conclusion that one or both of the putative parents have not yet been discovered or are extinct. A range of observations concerning allopolyploid speciation were categorized as follows: (1) verification of previous hypotheses regarding parentage (e.g., I. riparia, I. appalachiana), (2) determination that two morphologically distinct allotetraploid species can share the same parentage (I. azorica and I. acadiensis), (3) recognition of a cryptic allotetraploid species, indicated by the presence of different parental genomes (I. "appalachiana" from Florida), and (4) identification of allotetraploid species with one or two unknown parents (e.g., I. tuckermanii, I. acadiensis, I. azorica, and I. hyemalis). Some sequences from diploid species are remarkably uniform among populations (e.g., I. echinospora from various locations in North America, Iceland, and Wales), while others are variable at the subspecies level (e.g., northern and southern populations within I. engelmannii).

Key Words: allopolyploidy • hybrid origins • Isoëtes • LEAFY intron

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isoëtes (quillwort) is a cosmopolitan genus of heterosporous lycopod plants containing 200 or more species. Despite their probable ancient Paleozoic origins, worldwide distribution, and diverse ecological adaptations (from seasonal, ephemeral terrestrials to obligate, evergreen aquatics), Isoëtes species are singularly lacking in morphological variation (Taylor and Hickey, 1992 ). The taxonomic difficulties caused by conserved morphology are compounded by the presence of homoplasy and allopolyploid speciation.

In plants, allopolyploidy is a common mechanism of speciation. An interspecific hybridization event coupled with a doubling of chromosome number results in the formation of a new allopolyploid species that contains at least two distinct lineages. Estimates of the frequency of polyploid angiosperm species range beween 30 to 80%; the percentages may be even higher for nonflowering plants (Wagner and Wagner, 1980 ; Masterson, 1994 ; Soltis and Soltis, 2000 ). In Isoëtes, the relatively large number of putative interspecific hybrids and polyploids suggests that allopolyploidy is probably a significant speciation process. In the USA and Canada, 16 of the 31 species of Isoëtes currently recognized are basic diploids (2n = 22). The other 15 are polyploids and include 10 tetraploids (2n = 44), three hexaploids (2n = 66), one octoploid (2n = 88), and one decaploid (2n = 110; Taylor et al., 1993 ; Brunton et al., 1994 ; Brunton and Britton, 1997 , 1998 ; Luebke and Budke, 2003 ).

Many allotetraploid species of Isoëtes have been studied and parental species proposed based on intermediate morphology and collection data. The best documented evidence of allopolyploidy is the allotetraploid I. riparia, found in northeastern North America. Data from distribution patterns, spore morphology and viability, electrophoretic profiles of leaf enzymes, and in vitro hybridization experiments support the hypothesis that the basic diploids I. echinospora (2n = 22) and I. engelmannii (2n = 22) have crossed to form the sterile hybrid I. x eatonii (2n = 22); chromosome doubling gave rise to the fertile allotetraploid, I. riparia (2n = 44; Taylor and Hickey, 1992 ). Isozyme studies by Caplen and Werth (2000a , b) support an Isoetes riparia complex, possibly with mutiple allotetraploid origins.

Traditionally, systematists have used various forms of DNA fragment analysis (e.g., AFLP, RAPD) or isozyme work to determine hybrid origins. Single or low copy nuclear DNA sequence data are increasingly popular for such work (e.g., Sang et al., 1995 ; Campbell et al., 1997 ; Ge et al., 1999 ; Small and Wendel, 2000 ; Ingram and Doyle, 2003 ) but may be problematic for two reasons: (1) it can be difficult to identify and amplify regions of DNA that are sufficiently variable at the species level, and (2) if the hybridization event has not occurred relatively recently, gene conversion and normal substitution events may obliterate or mask the evidence.

We have found that the second intron region of the nuclear floral meristem identity gene, LFY, is variable enough within a more recently derived species complex of Isoëtes to delimit species and determine hybrid origins. The relatively large number of LFY molecular markers (both substitutions and gaps) and the greater likelihood of homology that are characteristic of sequence data allow us to delimit species with a precision and confidence that is often absent from studies at this taxonomic level. While the LFY intron is highly variable in Isoëtes, the surrounding exon regions are relatively conserved across all vascular plants (Frohlich and Meyerowitz, 1997 ), facilitating primer design. By cloning and sequencing, we can separate homoeologous LFY loci contributed by the diploid parents for inclusion in a data set consisting of all possible diploid parents for subsequent phylogenetic analyses.

In this study, we compare LFY nucleotide sequences for nine allotetraploids with all known diploid species within a more derived, well-supported clade of Isoëtes, the American species complex (Fig. 1; Hoot and Taylor, 2001 ), to unravel the results of reticulate evolution. We also demonstate the usefulness of LFY data in delimiting species and exploring regional variation at the intraspecific level.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Phylogeny of selected Isoëtes showing the placement and nearest outgroup of the American species complex (Hoot and Taylor, 2001 ). Tree is based on combined nuclear ITS and plastid atpB-rbcL spacer data and is rooted with I. coromandelina

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sampling, DNA extraction, PCR, and cloning
The species sampled for this study are listed in the Appendix (see Supplemental Data accompanying online version of this article), along with their provenance, voucher information, and GenBank numbers. Our sampling included all 15 commonly recognized diploid species found in the United States, excluding the two western diploids (I. orcuttii and I. nuttalii) that are not within the American species complex (Fig. 1).

Total cellular DNA was isolated from fresh leaf material using the miniprep method of Doyle and Doyle (1987) . The LFY regions were amplified under conditions reported previously (Hoot and Taylor, 2001 ). PCR products were purified using QIAquick PCR purification columns (Qiagen, Valencia, California, USA). The purified PCR product was sequenced with an ABI automated sequencer (Applied Biosystems, Model 373A-Stretch, Boston, Massachusetts, USA). Both 5' and 3' strands of DNA were sequenced.

Before species of hybrid origin were cloned, the PCR product was purified on a 2% agarose gel with subsequent removal of agarose using the QIAquick® gel extraction kit (Qiagen). The purified PCR product was ligated and subsequently transformed using a T-overhang vector kit (pGem-T Easy Vector System II, Promega, Madison, Wisconsin, USA). The QIAPrep spin miniprep kit (Qiagen) was used to isolate, purify, and concentrate the vector DNA for use in automated sequencing. In the case of hybrid taxa, cloning was used to separate the putative homoeologous LFY intron loci. To reduce the amount of sequencing, length variation and/or a regimen of diagnostic restriction digests (identified from the diploid sequences) were used to help identify unique clones. We sequenced five to 15 clones for each hybrid to recover all potential homoeologous loci.

Data analysis
Alignment of DNA sequences was approximated using Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, Michigan, USA) with subsequent manual corrections. Alignment procedures were as described in Hoot and Douglas (1998) , with careful attention to repeated motifs (Type Ib indels) and runs of the same nucleotide (Type Ia indels). Using MacClade (Maddison and Maddison, 1992 ), we scored gaps using the conservative simple gap coding method (Simmons and Ochoterena, 2000 ). Regions of ambiguous alignment were removed from the data set without gap scoring. The sequences resulting from putative hybrids were also scanned carefully for recombination or concerted evolution.

Fitch parsimony of the data was performed with PAUP* version 4.0b2a (Swofford, 1998 ) using the heuristic search option, maximum number of trees = 8000. To assess branch support, PAUP* was used to perform bootstrap analyses (Felsenstein, 1985 ) using "fast" stepwise-addition and number of replications = 5000. Based on previous broader analyses of Isoëtes from around the world using ITS data (Fig. 1), I. setacea from Spain was chosen as the outgroup.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The region amplified and sequenced/cloned included 80 bases within LFY exon 2, approximately 1000 bases within intron 2 and 19 bases within exon 3; exon/intron boundaries were estimated using several flowering plant alignments. The final data set consisted of 1164 characters; 205 characters were variable and 187 were parsimony informative. Estimated against one of the shortest trees, Ts/Tv was 1.57. Of the 46 gaps scored, 21 were informative. Based on the outgroup condition, 43 of the 46 gaps were deletions. Two regions of 8 and 21 bp were removed from the data set because of ambiguous alignments associated with nucleotide runs of differing lengths (Type Ia indels; Hoot and Douglas, 1998 ).

The phylogenetic analysis produced >8000 shortest trees, each with a consistency index excluding uninformative characters (CI) of 0.77 and a retention index (RI) of 0.91. The strict consensus and bootstrap consensus trees (Fig. 2) were not well resolved. Most of the resolution is due to populations and/or clones from various hybrids grouping together, indicating species identity.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Bootstrap consensus tree of the American species complex based on LFY intron 2 data. Taxa with hybrid and allopolyploid origins are in bold. Unknown or extinct species are identified by letters W–Z. Numbers above the lines are bootstrap values. Acronyms after species names are abbreviations for collections sites (NS = Nova Scotia, NB = New Brunswick). * indicates a new allopolyploid species that was misidentified in the field

The clades containing cloned sequences from putative hybrid or allotetraploid species (Fig. 2, indicated in bold) are well supported. To help understand the results for species of hybrid origin, a network diagram was manually constructed, showing diploid progenitors and the resulting hybrid species (Fig. 3). For the time being, letters W–Z indicate unknown species with sequences that differ substantially from any known diploid sequences in our data set. Table 1 shows the number of unique characters (substitutions or gaps not found in any other species) that are potential molecular markers for each species, including unknown species W–Z.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Diagram showing reticulate evolution in various Isoëtes species. Lines indicate a hybridization event between two diploid species; resulting hybrid taxa are indicated in bold midway along the connecting lines. The two listings for I. engelmannii indicate northern and southern populations

The allotetraploid I. maritima from western United States results from a hybridization event between either I. bolanderi or I. howellii, also of the western United States and I. echinospora, which has a circumboreal distribution (Figs. 2, 3).

Within the I. engelmannii clade, there is evidence for two well-supported groups, one consisting of populations with a more northern distribution in North America (Figs. 2, 3; I. engelmannii N) and the other with a more southern distribution (I. engelmannii S). Surprisingly, I. acadiensis from Nova Scotia and I. azorica from the Azores share the same parental species: I. engelmannii N and an unknown species labeled "Y" (not similar to any known diploid in our data set). One parent of I. tuckermanii is also from this northern engelmannii group, the other parental sequence is unknown species "Z." Thus, the only evidence we have so far for a northern engelmannii subspecies or variety comes from the allotetraploid sequence data; we have no sequences from known diploids that are similar to these (Fig. 2, Table 1).

Two allotetraploid species in our sampling have no known diploid parents: I. louisianensis (unknown parents represented by "W" and "X") and I. hyemalis (sharing parent "X" with I. louisianensis and parent "Y" with I. acadiensis/I. azorica; Fig. 3). Isoëtes hyemalis was the only species in which recombination was evident. The last approximately 400 bases (of approximately 1190 for this sequence) of the hyemalis "Y" parent converted to the same sequence as the hyemalis "X" parent. Because this recombinant confounds the evidence for parentage, these last 400 bases were removed from the appropriate sequence for the phylogenetic analysis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Because of the conserved morphology among Isoëtes species, species identification and delimitation have been problematic. The number of molecular markers and the greater likelihood of homology characteristic of the LFY intron sequence data allows us to delimit species in the North American species complex with a degree of certainty often absent from studies at this level (Table 1). This greatly enhances our ability to determine hybrid origins, whether of infertile diploid hybrids or allopolyploids.

Our work to date confirms that reticulate evolution is common in Isoëtes and that the situation is complicated by many "missing" parents (Fig. 2), that is, parents not in our data set. There are three possible explanations for these missing lineages: (1) the missing parental species may be extant but not yet discovered and/or collected; (2) the missing parental species is now extinct; or (3) the parental and hybrid taxa are present in our data set, but subsequent evolution in the intron sequences of both diploids and polyploids have obscured the parentage.

The first explanation, that the "missing" parents have not yet been discovered, is certainly possible. Due to their morphology (grass- or rush-like appearance) and habitat preferences (often underwater), Isoëtes are easily overlooked in the field. Botanists frequently identify new species of Isoëtes (e.g., Brunton and Britton, 1998 ; Musselman et al., 2001 ; Luebke and Budke, 2003 ).

The second explanation, that some of the missing parental species are now extinct, is also possible. Extinction may have occurred naturally or resulted from the frequent destruction of suitable wetland habitats throughout North America. Three species (out of approximately 30 species in North America) are currently listed as federally endangered.

Of the three explanations, we feel the third, post-hybridization mutations that mask species identity, is least likely for the following reasons. First, the American species complex appears to have evolved relatively recently. This is supported by the placement of the complex as one of the most derived clades on our tree, based on a combination of nuclear ITS and plastid atpB-rbcL spacer region data (Fig. 1; Hoot and Taylor, 2001 ). It is also supported by the relatively limited amount of homoplasy present in our trees (CI = 0.77, RI = 0.91); one would expect the number of homoplastic events to increase with time. Second, when the known diploid parental species are present in our sampling, the cloned sequences from the putative parental species are strongly supported as members of the same clade, both by bootstrap values and numbers of unique mutations, indicating limited molecular evolution in the LFY intron region after the hybridization event (Fig. 2, Table 1). Third, our results agree with past work on hybrid origins. For example, the parentage for I. x eatonii (a known sterile diploid hybrid of I. echinospora and I. engelmannii) and I. riparia (a fertile allotetraploid) confirm the parental species (Fig. 2). Finally, in our study of nine hybrid taxa, we found evidence of recombination in only one case, I. hyemalis.

One of the most interesting findings has been the discovery of two allotetraploid species of different range and morphology with identical parentage. Isoëtes acadiensis, ranging in northeastern North America, and I. azorica, endemic to the Azores, contain the same two parental genomes, but they differ in leaf color, sporangium wall pigmentation, megaspore ornamentation, and microspore color. In addition, their LFY intron sequences differ by nine mutations (including one 3-bp gap). These differences may indicate that the two species resulted from crosses between two different populations of the same species (e.g., different populations of I. engelmannii that differed at a few sites before the hybridization event occurred) and/or that there has been enough time since the hybridization and polyploidization events for considerable divergent molecular (and morphological) evolution.

The well-supported sister relationship of I. acadiensis and I. azorica for both parental species, parent "Y" and the northern I. engelmannii, raises interesting biogeographical questions. It is more parsimonious to assume that the allotetraploid species I. acadienensis made its way to the Azores, possibly by a chance bird migratory event. An alternative, less parsimonious explanation is that the two parents were previously in Europe, hybridized to eventually produce I. azorica with subsequent extinction of both parents from Europe. Britton and Brunton (1996) speculated that, in spite of the affinities of the Azore flora with that of Europe, I. azorica was most like the allotetraploid I. tuckermanii of northeastern North America. Our molecular data indicates they were on the right track: I. tuckermanii and I. acadiensis/azorica share one parent, the northern I. engelmannii (Figs. 2, 3).

Sequences recovered from hybrid species offer not only a unique opportunity to detect the presence of unknown or extinct diploid species, but also to hypothesize about the relationships and characteristics of these unknown species. While intermediate morphology is not always the rule when dealing with hybrids (e.g., Rieseberg and Ellstrand, 1993 ), our morphological work on hybrids indicates it is very common in Isoëtes. Therefore, the allotetraploid I. tuckermanii from Nova Scotia is probably intermediate in morphology between the northern I. engelmannii and unknown parent "Z" (Fig. 3). We can speculate that the unknown parent "Z" was probably found in the northern United States, had olive-green to reddish leaves less than 20 cm long, a velum covering more than half the sporangium, and a reticulate, irregularly crested spore morphology. In addition, because I. engelmannii is an emergent aquatic and I. tuckermanii is a shallowly submerged aquatic, we speculate that parent "Z" may be a deeply submerged aquatic.

Our work confirms that not only is reticulate evolution frequent in Isoëtes, but that we are only beginning to uncover the complexities of allopolyploid speciation. As we continue adding new populations to our data set, we are finding more new species (both diploid and polyploid), more undiscovered parents, and more cases of misidentification (preliminary data). It is clear that we need to increase our sampling of Isoëtes (preferably with multiple populations/species) from the Americas, looking for undescribed, missing species and additional intraspecific variation.

	FOOTNOTES

 
¹ The authors thank D. Britton, R. Brooks, D. Brunton, D. Bilderback, C. Caplen, P. Cox, J. Hickey, C. Jermy, J. Lark, S. Leonard, N. Luebke, L. Musselman, C. Prada, R. Small, and M. Voge for providing plant material used in this study. This work was supported in part by NSF grants DEB-9981460 to SBH and DEB-9981501 to WCT.

² E-mail: hoot{at}uwm.edu

	LITERATURE CITED

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This Article Abstract Full Text (PDF) Supplementary Data Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Hoot, S. B. Articles by Taylor, W. C. Search for Related Content PubMed Articles by Hoot, S. B. Articles by Taylor, W. C. Agricola Articles by Hoot, S. B. Articles by Taylor, W. C.