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(American Journal of Botany. 2008;95:1606-1620.) doi: 10.3732/ajb.0800148 © 2008 Botanical Society of America, Inc. |
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Systematics and Phytogeography |
2 Departamento de Botânica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre 1181, 4150-191 Porto, Portugal and CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Campus Agrário de Vairão, 4485-661 Vairão, Portugal; 3 Duke University, Department of Biology, Durham, North Carolina 27708 USA 4 Molecular and Environmental Plant Sciences Program, Texas A&M University, College Station, Texas 77840 USA 5 Department of Systematic and Evolutionary Botany, Faculty of Life Sciences, University of Vienna, Rennweg 14, A 1030 Vienna, Austria 6 School of Theoretical & Applied Science, Ramapo College Mahwah, New Jersey 07430 USA
Received for publication 28 June 2008. Accepted for publication 25 August 2008.
ABSTRACT
Allopolyploid speciation is likely the predominant mode of sympatric speciation in plants. The Sphagnum subsecundum complex includes six species in North America. Three have haploid gametophytes, and three are thought to have diploid gametophytes. Microsatellite analyses indicated that some plants of S. inundatum and S. lescurii are heterozygous at most loci, but others have only one allele at each locus. Flow cytometry and Feulgen staining showed that heterozygous plants have twice the genome size as plants with one allele per locus; thus, microsatellite patterns can be used to survey the distribution and abundance of haploid and diploid gametophytes. Microsatellite analyses also revealed that S. carolinianum is consistently diploid, but S. lescurii and S. inundatum include both haploid and diploid populations. The frequency of diploid plants in S. lescurii increases with latitude. In an analysis of one population of S. lescurii, both cytotypes co-occurred but were genetically differentiated with no evidence of interbreeding. The degree of genetic differentiation showed that the diploids were not derived from simple genome duplication of the local haploids. Heterozygosity appears to be fixed or nearly so in diploids, strongly suggesting that although morphologically indistinguishable from the haploids, they are derived by allopolyploidy.
Key Words: allopolyploidy • cytotypes • Feulgen staining • flow cytometry • microsatellites • polyploidy • Sphagnaceae • Sphagnum
Polyploidization is an important evolutionary process (Stebbins, 1950
; Soltis and Soltis, 1999). Speciation via polyploidy is likely the prominent mode of sympatric speciation in plants and can produce immediate shifts in morphology, breeding systems, and ecological tolerances (Otto and Whitton, 2000). Moreover, polyploidy can have profound effects on gene expression, genome structure, and developmental processes (for examples, see Wendel, 2000; Soltis et al., 2004). According to their mode of origin and chromosome behavior during meiosis, polyploids have been classified as autopolyploids and allopolyploids (Stebbins, 1950), but as Grant (1981) and others have noted, these are the extremes of a graded series.
Allopolyploids arise through the processes of interspecific hybridization and chromosome doubling and are generally characterized by fixed heterozygosity because divergent parental chromosomes do not pair (and segregate) at meiosis. Autopolyploids arise from conspecific parents and may form multivalents in meiosis (Stebbins, 1950; Soltis and Soltis, 2000). Tetrasomic inheritance in autopolyploids can lead to complex segregation patterns with a range of homo- and heterozygous genotypes. Nevertheless, cytological behavior is extremely variable (Stebbins, 1950), and it is artificial to distinguish strictly autopolyploid vs. allopolyploid species. Moreover, genomes even among taxonomically conspecific plants may be more or less strongly differentiated, obscuring any sharp distinction between auto- and allopolyploids.
Polyploidy occurs sporadically in animals (for examples and references, see Mable, 2004), but is widespread in plants. Estimates suggest that between 30 and 80% of angiosperms (or 100%, if there was genome doubling deep in their history) have undergone one or more polyploidization events (e.g., Stebbins, 1938; Masterson, 1994); in pteridophytes it could be as high as 95% (Grant, 1981). In bryophytes, the incidence of polyploidy has been estimated as >80% in mosses (Smith, 1979), up to 16% in liverworts (Smith, 1979; Bischler and Boisselier-Dubayle, 1997), and about 2% in hornworts (Smith, 1979). Otto and Whitton (2000) concluded that 2–4% of speciation events in angiosperms and 7% in ferns are associated with polyploidy. Såstad (2005) estimated the incidence of polyploid speciation as from 6.4–18.6% for mosses, 4.9–10.3% for liverworts, and 0% for hornworts (Såstad, 2005).
Bryophytes have a gametophyte-dominant life cycle with a short-lived sporophyte that is nutritionally dependent on the gametophyte. The external environment acts directly on the free-living, haploid gametophyte phase in bryophytes, so polyploidy may be an efficient way to buffer against deleterious alleles in heterozygotes. Diploid gametophyte tissue can develop from cells of (usually immature) sporophytes by apospory, a process known to bryologists since the late 19th century (Pringsheim, 1876). Gametophytes of many species have a high potential for vegetative regeneration, so an unreduced spore or somatic tissue from the sporophyte can develop into a sexually sterile (e.g., triploid) gametophyte that can reproduce vegetatively. Some species of mosses and liverworts have never been observed to reproduce sexually. A single duplication event in the sporophyte, where meiosis occurs, can potentially lead to a bisexual gametophyte able to reproduce by intragametophyte selfing (Såstad, 2005 and references therein).
Like higher plants, mosses often vary in chromosome numbers, both inter- and intraspecifically (Anderson, 1980; Fritsch, 1982, Fritsch, 1991; Newton, 1984). The frequency of species with polyploid races is high in certain families (e.g., Brachytheciaceae, Amblystegiaceae, Pottiaceae, Bryaceae), whereas polyploid races are practically unknown in others (e.g., Hypnaceae; Anderson, 1980). Anderson (1980) listed 19 moss species with three or more known ploidal levels. Seventeen of these have three ploidal levels, one has four, and one, Physcomitrium pyriforme (Hedw.) Hampe, has five. Most of these infraspecific polyploids are morphologically indistinguishable from the haploids (Anderson, 1980). Because of regular meiosis and high fertility in polyploid bryophytes species and races, it has been assumed that most were formed by autopolyploidy (e.g., Anderson, 1980). Recent molecular studies, however, suggest that allopolyploidy is common in both mosses and liverworts (see Såstad, 2005; Shaw, 2008).
The genus Sphagnum L. is notoriously difficult with regard to species delineation (e.g., Crum, 1984; McQueen and Andrus, 2007). In contrast, chromosome numbers are remarkably uniform in this genus, with gametophytic numbers of either N = 19 or N = 38 (Fritsch, 1991). Recent analyses of genome sizes in Sphagnum using cytometric methods (Temsch et al., 1998; Greilhuber et al., 2003) have shown that variation in genome sizes is closely correlated with chromosome numbers in Sphagnum. Two levels of ploidy, haploid and diploid, could be unambiguously discriminated from the genome sizes obtained (Temsch et al., 1998). These ploidal levels agreed with published data based on chromosome numbers (Newton, 1993; Temsch et al., 1998). Thus, estimates of genome sizes in gametophytic plants of Sphagnum can be used as proxies for chromosome numbers. This is useful because moss chromosomes are small, and it can be difficult to obtain accurate counts without sporophytes undergoing meiosis, which are uncommon in many Sphagnum species.
Allopolyploid origins have been demonstrated with isozyme data for several Sphagnum species with 38 chromosomes: S. russowii Warnst. in the section Acutifolia, S. jensenii H. Lindb., S. majus (Russ.) Jens., and S. troendelagicum Flatb. in the section Cuspidata. In the section Subsecunda, a complex of related dioicous species sometimes called the "S. subsecundum complex" includes both haploid and diploid species. (Bryophytes with unisexual gametophytes are often described as dioicous, in contrast to dioecious seed plants having unisexual sporophytes.) Members of the S. subsecundum complex occur across the northern hemisphere and are common in North America and Europe (Shaw et al., 2008a). They have a distinctive habitat compared to other sphagna—minerotrophic fens instead of ombrotrophic bogs. Among the species of this complex found in eastern North America, S. subsecundum Nees, S. contortum Schultz, and S. platyphyllum (Braithw.) Warnst. have haploid gametophytes, whereas S. inundatum Russow and S. lescurii Sull. are said to have diploid gametophytes (Fritsch, 1982; Newton, 1993; Sliwinska et al., 2000; Melosik et al., 2005). Sphagnum lescurii is widespread in eastern North America but is endemic there, whereas S. inundatum is reported from both North America and Europe. European populations of S. inundatum originated independently of North American plants, based on nucleotide sequence and microsatellite data (Shaw et al., 2008a, c). Sphagnum carolinianum Andrus, also restricted to eastern North America, is a robust species rather similar to S. lescurii. There have been no chromosome counts for S. carolinianum.
Most authors agree that S. contortum, S. platyphyllum, and S. subsecundum rarely present identification problems, but the separation of S. carolinianum, S. inundatum, and S. lescurii is not always easy. They each seem to have their own morphological (mainly quantitative), ecological, and distributional characteristics, but interspecific differences are not sharp and much taxonomic disagreement persists. Crum (1984), for example, considered these three taxa to be conspecific and classified them as varieties of S. subsecundum.
This study was motivated by the observation that some plants of species generally thought to have diploid gametophytes appear to have a single allele at each of more than eight microsatellite loci, whereas most plants are consistently heterozygous at these same loci. This observation raised the possibility that such plants are either highly homozygous or are haploid. We further observed that plants tended to be either heterozygous at many or most microsatellite loci or to have a single allele across virtually all loci (i.e., intermediate levels of heterozygosity are virtually absent), favoring the hypothesis that the latter are in fact haploid. In a survey of over 100 plants of S. lescurii and S. inundatum from Newfoundland, Shaw et al. (2008c) found that all plants had either fixed or nearly fixed heterozygosity, indicating that they all may be diploid. This observation suggested that ploidy in these taxa might be geographically structured. The specific goals of the current study were to (1) determine genome size and ploidy in gametophytes of putative diploid species in the S. subsecundum complex in eastern North America, (2) assay microsatellite allelic patterns in plants of known genome size to confirm that microsatellites can be used as proxies for genome size and ploidy, and (3) use microsatellite markers to assess the distribution of haploid and diploid plants within and between populations. Because S. contortum, S. platyphyllum, and S. subsecundum appear to be consistently haploid on the basis of previous chromosome counts, cytometric estimates of genome sizes, and our unpublished microsatellite analyses, we do not focus on them in this paper although a limited number of plants were included in the genome size estimates for comparison. Similarly, we focus on North American plants, but genome sizes for five European plants were included for comparison.
MATERIALS AND METHODS
Plant sampling
A total of 453 samples of S. carolinianum, S. inundatum, and S. lescurii were included in the molecular analyses, representing 182 North American populations (Appendix 1). Samples are vouchered in the Duke University herbarium (DUKE) and in the State University of New York Herbarium (BING). The sampling goal was to include plants from throughout eastern North America, and in addition, multiple plants from some populations were included to assess variation in genome size/ploidy at more local scales. For collections made by other researchers and preserved in DUKE, plants from the same area (based on locality names and/or geographical coordinates) were considered to belong to the same population. Most populations were represented by one or two plants, but for 30 populations we sampled between four and 28 plants.
At one population, from Wake County, North Carolina, plants were sampled in the field more intensively to document local-scale variation in genome size/ploidy and to assess any spatially structured genetic patterning among plants. Individual gametophytes were sampled from six more or less discrete patches of S. lescurii at the site. The patches were separated by two to 25 m. During the first sampling, in early July 2006, 10 plants were haphazardly collected (i.e., sampling was not formally randomized) from each patch. Individual samples were taken from across each patch to reduce the probability of repeatedly sampling individual clones. After initial analyses showed that the patches, and plants within patches, were genetically variable, sampling of additional plants was conducted about two weeks later, in late July. The total sample size was 72 plants. Patch 6 turned out to be highly divergent from the remaining patches, and preliminary assessments suggested that these plants were either highly homozygous or haploid, so sampling was increased there. Because of the elimination of some plants from the data and more intensive sampling from patch 6, the overall sampling across patches was unbalanced.
Herbarium specimens sampled for the molecular analysis were chosen based in part on collection date. Previous molecular work on Sphagnum suggested that plants sampled within the last 25 years are most likely to yield useable DNA. Our oldest sample from which microsatellites were amplified was collected in 1976. Sphagnum species generally grow in interspecific mixtures, and herbarium specimens often contain multiple species, so to accurately voucher plants used in the analyses, the individual stem sampled for our analyses was placed in a small envelope and returned to the herbarium packet from which it was derived. Only a small piece of the capitulum was needed for the analyses, leaving most of the plant for microscopic examination if necessary. Complete information about these voucher plants and all other Sphagnum collections in the Duke University herbarium can be obtained online at http://www.biology.duke.edu/herbarium/bryodata.html
Genome size determinations
To estimate ploidal levels from nuclear DNA contents (genome sizes), we took 60 measurements on 45 of the samples included in the microsatellite study (genome sizes were also measured for 13 samples of S. contortum, S. platyphyllum, and S. subsecundum). All genome sizes were estimated from herbarium specimens in the laboratory of J. Greilhuber in the Department of Systematic and Evolutionary Botany, Faculty of Life Sciences, University of Vienna, Vienna, Austria.
Flow cytometry
About 8 mg of the youngest Sphagnum branches of a capitulum were chopped together in 1.1 mL of Otto I isolation buffer solution (Otto, 1990; Dole
el and Göhde, 1995; Greilhuber et al., 2007; for herbarium material: Suda and Trávni
ek, 2006) with about 25 mg fresh young leaves of Solanum pseudocapsicum (2.609 pg DNA/2C, unpublished) as an internal reference. The homogenate was filtered through a 30-µm nylon mesh and incubated at 37°C for 30 min with 0.15 mg/mL RNase. Propidium iodide (PI) dissolved in Otto II buffer solution was added to a final concentration of 50 mg PI/mL. Staining occurred in a refrigerator for 60 min. Measurements were done with a flow cytometer (CyFlow ML, Partec, Münster, Germany) equipped with a green laser (532 ± 0.2 nm, 100 mW, Cobolt Samba, Cobolt AB, Stockholm, Sweden) as the excitation light source.
Histograms and fluorescence/side scatter cytograms were first evaluated visually for presence of clear nuclear populations of Sphagnum and the standard. Then manual gating was done, and the function "Gaussian peak analysis" provided by the program FloMax version 2.4 was applied to calculate peak means. Because the Sphagnum material consisted of dried specimens of various ages, the coefficients of variation (CVs) of the Sphagnum peaks were higher than usually obtained with fresh material and typically ranged between 5 and 8%, with those of the standard between 2 and 4%. Runs with dubious or negative results, as occurred with some herbarium specimens older than two years, were rejected. Such samples upon repetition mostly yielded results but with higher CVs between 10 and 20% and were then remeasured with Feulgen densitometry. The 1C-values of Sphagnum samples in picograms (pg) were calculated from histograms using to following formula: 2C pg of standard x Sphagnum peak position ÷ standard peak position. The relatively narrow ranges of genome sizes in haploid and diploid Sphagnum species, respectively, as known from previous studies in numerous taxa (Temsch et al., 1998; Greilhuber et al., 2003) served as a guideline to estimate ploidal levels.
Feulgen DNA image densitometry
When samples did not yield clear results for genome size by the flow cytometric method, we used Feulgen staining to obtain estimates. A few samples were run with both methods to confirm the accuracy of our determinations. Young branch tips of herbarium specimens were used for the Feulgen staining. Embryo tissue, i.e., roots, hypocotyls, and shoot tips from dry mature seeds of Pisum sativum L., cv. Kleine Rheinländerin (2C = 8.84 pg, Greilhuber and Ebert, 1994) served as internal (exceptionally external) references to monitor nuclear staining intensity. Internal standards are prepared (isolated and stained) and analyzed simultaneously with the sample, whereas for external references the root tips of P. sativum and Sphagnum meristems were prepared separately under identical conditions and analyzed successively (Doleel et al., 2007). The tissues were processed either including a fixation with formaldehyde (method 1) before hydrolysis or without such a fixation (method 2).
Method 1
A few (1–3) Sphagnum branch tips from one capitulum and embryo tissue from one pea seed were added to about 1 mL of 4% phosphate buffered formaldehyde (pH 7). The Sphagnum leaves were removed until the very youngest leaves were exposed. The pea tissue was broken to smaller pieces. After 1.5 h at room temperature (22–25°C), the material was thoroughly rinsed several times in acetic methanol (1:3) for about 1 h to remove the formaldehyde and then with distilled water for 30 min to remove the acetic methanol. After hydrolysis in 5 N HCl at 20°C for 1.5 h, then a wash in distilled water for 10–15 min, the material was stained in Schiff’s reagent (Merck 1.09033.0500). Unbound Schiff’s reagent was then thoroughly washed out with SO2-water (0.5 g Na2S2O5 dissolved in 95 ml distilled water plus 5 mL 1 N HCl added) for 30 min. The tissue was placed in distilled water, then softened for about 10 min in 45% acetic acid. Sphagnum and pea tissues were squashed under separate cover slips on the same slide, which was frozen over a cold plate. The cover slips were removed and the slides air-dried.
Method 2
The tissues were placed in distilled water for dissection and were then hydrolyzed, or they were directly dissected in 5 N HCl at the beginning of hydrolysis, with further processing as described in method 1.
The advantage of method 1 was a cleaner background than usually obtained with method 2. Slides were measured with the Cell Image Retrieval and Evaluation System (CIRES, release 3.1, Kontron, Munich), using 63x oil immersion lens, a green interference filter, the green channel of the CCD camera, and "local background determination" around the periphery of the segmented nucleus. No cover slips were used. According to availability, a variable number of Sphagnum nuclei in 1C, on average about 160, were measured. Embryonal leaves before or shortly after the last mitotic divisions were the best source of nuclei. Most Sphagnum nuclei were unreplicated (1C), as found previously (Temsch et al., 1998; Greilhuber et al., 2003); S-phase nuclei were infrequent. In pea, unreplicated nuclei in 2C with 8.84 pg DNA (Greilhuber and Ebert, 1994) were measured until a consolidated peak was established, on average with about 87 nuclei. Mostly only one slide per sample was measured. The 1C peaks in Sphagnum had CVs between 2.2 and 5.3% (in one case 8.8%), and 2C peaks in pea were between 1.3 and 3.1%.
Molecular methods
DNA extraction was accomplished according to the protocol of Doyle and Doyle (1987), modified as described in Shaw (2000).
Primer sequences for all microsatellites included in this study were provided by Shaw et al. (2008b). Microsatellites were amplified in 8-µL multiplexed reactions, each targeting a set of three loci, for a total of 15 loci for our study of populations from throughout eastern North America (loci 1, 4, 9, 10, 12, 14–20, 22, 26, and 28 in Shaw et al., 2008b). Nineteen loci were resolved for the study of ploidy and genetic structure in the Wake County, North Carolina population (loci 1, 4, 5, 7, 9, 10, 12, 14–20, 22, 26, 28–30). Primer sets were arrayed for multiplexing according to expected fragment sizes (for nonoverlapping amplification products) and alternating fluorophores. Each primer pair included a forward primer fluorescently labeled with hexachlorofluorescein (HEX) or 6-carboxyfluorescein (6-FAM) (Integrated DNA Technologies, Coralville, Iowa, USA). Multiplexing was accomplished using a Qiagen Multiplex PCR kit (Valencia, California, USA), scaled for smaller reactions, but otherwise used according to the manufacturer’s recommendations. Five to 20 ng of genomic DNA in 3 µL dH2O served as template in each reaction. A standard thermocycling regime was implemented for all primer sets, with no additional optimization. It consisted of an initial denaturation and hot-start activation at 95°C for 15 min, then 30 cycles of 94°C for 30 s, 54°C for 90 s, and 72°C for 60 s. A final extension at 60°C for 30 min was performed. PCR products were diluted in sterile water, and 1.2 µL of the dilution was mixed with the GS500 size standard and Hi-Di Formamide (Applied Biosystems, Foster City, California, USA) for electrophoresis on an ABI 3730 sequencer. Size determinations and genotype assignments were made using GeneMarker 1.50 software (Softgenetics, State College, Pennsylvania, USA).
Data analysis
The following analyses were all computed using the program Excel (Microsoft, Redmond, Washington, USA). The percentage of heterozygous loci per individual was calculated for S. carolinianum, S. inundatum, and S. lescurii combined and for each species separately. After we discovered that S. inundatum, and S. lescurii includes both haploid and diploid plants, we sought to determine if there is a relationship between polyploidy (i.e., diploid gametophytes) and latitude. Many of the herbarium specimens were not georeferenced with latitude–longitude coordinates, so instead of determining this information for each collection, the relative frequencies of diploid and haploid plants in relation to latitude was estimated first by grouping samples by state. We then used the geographical center of each state as a rough proxy for latitude of the sample. The geographical center of each state was obtained from the website http://www.netstate.com/states/index.html. Haploids and diploids were distinguished by their microsatellite profiles after we determined that there is a nearly perfect correlation between the percentage heterozygous loci and genome size (ploidy) (see Results).
For each microsatellite locus screened in the interpopulational analysis, the percentage of heterozygous individuals was calculated using Excel. Deviations from the Hardy–Weinberg equilibrium were tested using the program GENALEX version 6.1 (Peakall and Smouse, 2006). Fragment sizes were coded as codominant data.
PHiPT between haploids and diploids (analogous to FST, see Peakall and Smouse, 2006) and between diploid patches within the intensively sampled Wake County population, AMOVAs, principal coordinates analysis (PCO), Nei’s genetic identity, and private allele frequencies were computed using GENALEX 6.1 (Peakall and Smouse, 2006). Fragment sizes were coded as codominant data, and microsatellite repeat numbers were not calculated, because it was clear from sequencing selected fragments that some of the allelic variation could be attributed to indels in flanking regions rather than to variation in repeat number alone (data not shown).
RESULTS
Estimation of genome size and ploidy
Two groups of samples could be distinguished with regard to genome size (Table 1, Fig. 1). One group had 1C-values (Greilhuber et al., 2005) ranging between 0.36 and 0.48 pg, with a mean of 0.42 pg and coefficient of variation (CV) of 7%. The second group had 1C-values between 0.82 and 0.94 pg, a mean of 0.85 pg, and a CV of 6%. Thus one holoploid genome size (Greilhuber et al., 2005) is twice the size of the other.
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Percentage of heterozygous loci per individual
For Sphagnum carolinianum, S. inundatum, and S. lescurii combined, the percentage of heterozygous loci per sample followed a bimodal distribution (Fig. 2A). In one cluster, there were 120 samples with no heterozygous loci, 10 samples with 10% of the loci heterozygous, and five samples with 15% of the loci heterozygous. In the second cluster, 252 samples had between 47% and 100% heterozygous loci (mean = 72%).
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Relation between C-value and percentage heterozygous loci
Figure 3 shows the 1C-value of each sample in relation to the percentage of heterozygous loci. The average 1C-value of the samples with 0–15% of heterozygous loci was 0.42 pg, whereas samples with more than 50% loci heterozygous had an average 1C-value of 0.85 pg. There was no exception to this relation between the microsatellite data and genome size. Therefore, for each individual plant, we could infer genome size, and thus ploidy, based on the microsatellite results. For the remaining analyses, we defined samples with more than 50% heterozygous loci as diploids and samples with less than 15% heterozygous loci as haploids. This ploidal level refers to the gametophyte because all the analyses were done using gametophytes. Haploid plants would presumably have diploid sporophytes, and diploid gametophytes would have tetraploid sporophytes.
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0.01). This pattern is also valid for S. lescurii (Fig. 4D) but not for S. carolinianum (Fig. 4B) and S. inundatum (Fig. 4C), probably due to limited sampling.
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Genetic relationships among haploids and diploids
Principal coordinates analysis (PCO) of the microsatellite data (Fig. 6) grouped the samples in three distinct clusters. All samples of S. carolinianum formed an isolated cluster. Diploid S. lescurii and S. inundatum formed a cluster with little or no evidence of differentiation from each other. Some samples of the diploids, however, were present in the cluster formed mainly by haploid S. lescurii. Haploid S. inundatum appeared in both clusters.
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Nei’s genetic identities and PhiPT values (Table 2) corroborated the results from the PCO. The average genetic identity between diploid S. lescurii and diploid S. inundatum was the highest value (0.926) among all comparisons, followed by 0.789 between haploid S. inundatum and haploid S. lescurii. The genetic identities between the diploids S. inundatum and S. lescurii and the haploids varied between 0.403 and 0.591. Sphagnum carolinianum had lower genetic identities and higher PhiPT values relative to other species.
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0.001); thus, 75% of the total genetic variation at the site was attributable to differences between the cytotypes (Table 4).
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Ploidy and microsatellite heterozygosity
We found that plants identified as Sphagnum inundatum and S. lescurii consist of both haploid and diploid cytotypes and that microsatellites are a useful tool to infer the ploidy of these plants. Populations can be entirely haploid or diploid or a mix of both. In the North Carolina population of S. lescurii where we sampled intensively, a complex population structure was revealed with a high degree of genetic differentiation between haploid and diploid plants in terms of microsatellite allelic profiles. Overall, diploids had fixed or nearly fixed heterozygosity for eight of the 15 loci analyzed, indicating that the diploids are likely allopolyploids derived from hybridization between taxa with sufficiently differentiated genomes such that homeologous chromosomes do not pair and segregate at meiosis.
The C-values obtained were in concordance with those published by Temsch et al. (1998). Across the genus Sphagnum, they found two unambiguous gametophytic genome sizes that agreed with data based on chromosome numbers. Temsch et al. (1998) and Greilhuber et al. (2003) reported a range of 0.39–0.49 pg among haploid species and a range of 0.81–0.95 among diploids. Because our results on genome sizes are consistent with theirs, we also designated as haploid those samples with C-values between 0.36 and 0.48 pg and as diploid those samples with C-values ranging from 0.72 to 0.94 pg.
Some of the plants we interpreted as haploid had low frequencies of heterozygous loci, from either aneuploidy or local duplications. At this time, we cannot distinguish between these explanations, but it is worth noting that aneuploidy has not been reported in Sphagnum. In addition, while heterozygosity in the putative diploid species is much higher than expected under Hardy–Weinberg equilibrium, some apparent homozygotes were observed. There are several possible explanations, including multiple origins sometimes involving haploids that did not differ at these loci. The occurrence of null alleles—failure of one allele to amplify—could also account for occasional "homozygotes." Such null alleles are well known in microsatellite studies (Dakin and Avise, 2004). Finally, we cannot eliminate the possibility that some pairing of homeologous chromosomes occurs, segregating homozygotes at low frequencies. Autopolyploids would be expected to yield high frequencies of heterozygotes by polysomic segregation, and our analyses of heterozygosity cannot definitively exclude the possibility of autopolyploid origin(s) for the diploids. Some level of limited segregation, an intermediate situation between auto- and allopolyploidy, further complicates any attempt to reject specific hypotheses. The possibility, even probability, of multiple polyploid origins provides yet another complication. Experimental studies involving single spore isolates from tetraploid sporophytes are needed to critically determine if segregation occurs.
Based on nucleotide sequence data, Shaw et al. (2008c) showed that two divergent alleles at three anonymous nuclear loci could be cloned from heterozygous plants, supporting the view that the diploids are of allopolyploid origin. They further showed that European diploids originated independently of North American diploids but that diploids from both continents shared high levels of nucleotide and microsatellite similarities with another haploid species, S. subsecundum.
We did not find any evidence of higher polyploids from either microsatellite analyses or genome size estimates. If genome doubling of any heterozygous diploid plants occurred, yielding autoallotetraploids, our microsatellite-based analyses would likely fail to detect them. Given that our genome size estimates revealed no plants with genomes larger than diploids, we assume that any such plants must be rare. In general, microsatellite data appear to provide reliable proxies for establishing ploidy in allopolyploids where heterozygotes are fixed or nearly so. This approach would not be appropriate for detecting autopolyploids, but in the case of allopolyploids, using microsatellite markers provides simultaneous information about genetic variation, population structure, and ploidal level. Whether microsatellites, flow cytometry, or Feulgen staining are used to infer ploidy, comparison to direct chromosome counts is of course prerequisite.
Taxonomic implications
Our results show that S. carolinianum is genetically divergent from S. lescurii and S. inundatum. The latter two taxonomic species, however, present a complicated evolutionary picture and a difficult taxonomic problem. Diploid S. lescurii is scarcely differentiated from diploid S. inundatum. Likewise, haploid S. lescurii is genetically very close to haploid S. inundatum. We cannot at present identify the parents of the diploids, but it is likely that the haploids generally considered conspecific based on morphology were involved in the origins of the allodiploids. Whether S. inundatum and S. lescurii represent mutiple derivatives of crosses between related (but genetically distinct) haploids cannot presently be determined. Allelic diversity within the diploids and the occurrence of some apparent homozygotes at otherwise heterozygous loci is consistent with multiple origins. Moreover, the degree of allelic differentiation between haploids and diploids of S. lescurii at the Wake Co. site demonstrate unequivocally that the diploids there are not derived from simple genome doubling in sympatric haploids.
Regardless of the precise parentage of diploid plants in S. carolinianum, S. inundatum, and S. lescurii, our inference that these taxa are allopolyploids rather than autopolyploid races of the haploid plants currently classified under the same binomials presents a difficult taxonomic situation. There has been much discussion of how to delineate species (for a review of species concepts, see Coyne and Orr, 2004; Shaw, 2008), but it is fair to say that many or most systematists believe that species should be monophyletic, even if the evidence for species monophyly is indirect and the reasoning is implicit (i.e., morphological similarity within taxa and discontinuity between them). A monophyletic origin is clearly not the case for most allopolyploids (Soltis and Soltis, 1999), even if they currently function as biologically meaningful units of biodiversity (e.g., as gene pools in which genetic recombination occurs, or they have their own distinctive ecological characteristics). Sphagnum carolinianum is genetically and morphologically distinguishable from the other diploids and is consistently diploid, but the case of S. inundatum and S. lescurii is incompatible with their current taxonomic status. As currently defined, each species includes both haploid and diploid populations, and the diploids are, in a genealogical sense, related to two different haploid taxa and are not simply polyploid races of the haploids. Haploid and diploid plants of S. inundatum and S. lescurii, respectively, cannot be considered conspecific in any evolutionarily meaningful way. This taxonomic situation is paralleled in other groups where a single taxonomic species has been shown to include one or more allopolyploids as well as one of their ancestors (e.g., Doyle et al., 2004). In Glycine L., like in Sphagnum, the taxonomy has had to change as our understanding of evolutionary history progresses. We cannot at present identify any consistent morphological differences between haploid and diploid plants in either of the two Sphagnum "species." For the current time, we make no nomenclatural changes pending additional genetic and morphological work currently in progress.
Evolution of polyploidy in mosses
The mechanisms through which polyploidization can occur in mosses are thought to be apospory (regeneration of a diploid gametophyte from sporophyte tissue), diplospory (production of unreduced diploid spores), syndiploidy (fusion of sporocytes prior to meiosis), and/or somatic doubling in gametophyte tissue (Såstad, 2005.) Unfortunately, we know virtually nothing about the actual mechanisms underlying the origins of polyploidy in mosses.
Cytological studies over the last forty years have shown numerous cases in bryophytes of infraspecific and infrageneric polyploidy. In many cases, the different cytotypes are morphologically indistinguishable and have been treated as conspecific chromosome races without taxonomic recognition (examples in Anderson, 1980). Historically, it was thought that bryophyte polyploids were usually formed by apospory (Anderson, 1980; Wyatt and Anderson, 1984), although Smith (1979) concluded that in liverworts diplospory is likely to be the most important mechanism because the sporophyte is short lived with a very delicate seta that elongates rapidly and perishes quickly after spores are shed from the capsule (sporangium). The ephemeral setae of liverworts, unlike those of mosses, are apparently unable to regenerate diploid gametophytes (Smith, 1979). The assumption that polyploid mosses are usually (or always) formed by apospory and of autopolyploid origin was based on the observation that there is often no morphological difference between different cytotypes and on the assumption that hybridization is rare in bryophytes. However, examples of interspecific hybridization have been documented (Shaw, 1994; Cronberg, 1996; Cronberg and Natcheva, 2002; Flatberg et al., 2006; Natcheva and Cronberg, 2007), and some now argue that hybridization may be grossly underrecognized in bryophytes.
One possible reason for the apparent rarity of hybridization in bryophytes is likely because closely related moss and liverwort species often have nearly identical sporophytes, so F1 hybrids are difficult to detect on the basis of morphology. Recent evidence from molecular markers has revealed gametophytes in natural populations with mixtures of isozyme and/or DNA markers that are characteristic of related sympatric moss species, and the gametophytes have been interpreted as recombinants derived from hybrid sporophytes (Shaw, 1994; Natcheva and Cronberg, 2007). Moreover, hybrids and recombinants are not necessarily intermediate in morphology between the two parental species (Shaw, 1994; Natcheva and Cronberg, 2007). The current study provides another such example, given that there is little or no morphological differentiation between haploid and diploid plants of S. inundatum and S. lescurii despite the clear indication (i.e., nearly fixed heterozygosity) that the diploids are derived from interspecific hybridization. Thus, it could be that some "morphologically indistinguishable chromosome races" known in other moss species may well be derived via allo- rather than autopolyploidy. Allopolyploids have now been documented in at least five moss genera and seven liverwort genera (Såstad, 2005; Shaw, 2008). Moreover, Soltis et al. (2007) recently reviewed the importance of autopolyploid speciation in angiosperms and concluded that the occurrence and significance of autopolyploidy has likely been grossly underestimated. Biosystematic analyses of bryophytes known to include multiple "chromosome races" would be highly worthwhile.
It has long been recognized that the frequency of polyploid plant taxa (i.e., species, subspecies) tends to increase with latitude in the northern hemisphere (Stebbins, 1950). The pattern has been related to general climatic harshness, historical fluctuations in climate, shorter-term temperature fluctuations, and to Pleistocene glaciation per se, among other factors (e.g., Ehrendorfer, 1980; Stebbins, 1984, 1985; Stebbins and Dawe, 1987). In a thorough analysis of geographic patterns as they relate to the arctic flora, Brochmann et al. (2004) found that while the frequency of higher level polyploid taxa (above the tetraploid level) increases dramatically along a south–north arctic transect, the frequency of tetraploids does not. They also found that, contrary to earlier arguments by Stebbins (1984, 1985), there does not appear to be a general relationship between polyploid frequency and areas that were glaciated vs. areas that were not. The frequency of diploid S. lescurii gametophytes (and therefore presumably tetraploid sporophytes) increases with latitude, but this pattern was not recovered in the other species, probably due to small sample sizes. It is difficult to draw a general inference from this observation, but it seems that latitude has some importance in the distribution of the diploids. In a sample of over 200 plants from Newfoundland, near the northern limit of S. carolinianum, S. inundatum, and S. lescurii, not a single haploid was detected (Shaw et al., 2008c). It should be kept in mind that the taxonomy of species in the S. subsecundum complex is currently misleading. We are not dealing with gametophytically haploid vs. diploid cytotypes of a single species, but rather haploid species vs. allodiploids in which the haploids are one of at least two parents. Nevertheless, our data do show that haploid S. lescurii sensu stricto is rare or absent in Newfoundland and is replaced ecologically by the allodiploid for which it likely served as a parent.
The latitudinal gradient we detected can be explained by a greater colonizing ability of polyploids (Stebbins, 1985), by an origin of the allodiploid(s) at high latitudes (and subsequent spread southward), or by better competitive abilities of the diploids in harsh environments relative to their parental species. We do not yet know some critical pieces of information that are necessary to fully understand the geographic pattern, including especially whether the allopolyploids originated at lower vs. high latitudes, and their time(s) of origin. We did not investigate the distribution of haploids and diploids across different elevations, which might help sort out historical explanations such as a high latitude origin for polyploids vs. ecological explanations for their increasing frequency with latitude.
It has been observed repeatedly in auto- and allopolyploids of angiosperms that diploids and tetraploids are geographically separated for the majority of their distribution (see references in Husband and Schemske, 1998). These differences in their distribution can be the result of divergent ecology between the diploids and tetraploids, differences in their colonizing ability, or the geographic origin(s) of the polyploids. Analyses of the local distribution of diploid and tetraploid cytotypes sometimes reveal complex patterns. In several studies (e.g., Husband and Schemske, 1998; Burton and Husband, 1999; Stuessy et al., 2004), both cytotypes co-occurred, but their relative abundance varied from site to site, which can be explained either by microhabitat differences or by competition between the two cytotypes. In our study there were 11 populations that had more than one cytotype, but the relative frequencies of diploids and haploids could not be calculated due to small sample sizes. In Wake Co., North Carolina, the population structure was rather complex. The two cytotypes appear mixed in one of the six patches, but their allelic profiles were highly differentiated, indicating that the diploids were not derived from those local haploids and that they are reproductively isolated. Abundant sporophytes were detected in that population in 2008, and genetic analyses of the sporophytes and their maternal gametophytes are underway.
Newly formed polyploids would be subject to "minority cytotype" negative frequency-dependent selection, with decreased fitness because they have fewer potential mates (Levin, 1975). However, other factors may be involved in the establishment of polyploids, such as the so-called triploid bridge, higher relative fitnesses of polyploids, niche separation between cytotypes, and prezygotic isolation (see Rausch and Morgan, 2005, for references). Additionally, newly formed bryophyte polyploids may overcome the lack of compatible mates by clonal reproduction and thus establish a stable population. Further studies are needed to understand the factors that determine the distribution of polyploids in Sphagnum, and the local scale dynamics of sympatric populations having both cytotypes.
Both auto and allopolyploid origins have been hypothesized for the diploid taxa in the S. subsecundum complex (e.g., Maass and Harvey, 1973; Eddy, 1977). Some evidence of heterozygosity at isozyme loci has previously been described (Melosik et al., 2005), and our results corroborate an interpretation that diploid plants of S. carolinianum, S. inundatum, and S. lescurii have fixed or nearly fixed heterozygosity, a pattern consistent with allopolyploid origins. Nevertheless, significant questions remain regarding the evolutionary origin(s) of these diploid plants. In particular, our ongoing research on the S. subsecundum complex is designed to determine how many times the allodiploids have originated, whether the morphologically defined species (S. carolinianum, S. inundatum, and S. lescurii) are individually monophyletic, and precisely which haploids are involved as parents. One of the most often repeated quotes in bryophyte evolutionary biology is Crum’s (1972, p. 279) statement that mosses are evolutionary dead ends that are "unchanging, unmoving sphinxes of the past." Our observations that reveal a complicated reticulate evolutionary history for the S. subsecundum complex provide yet more evidence that Crum’s assumption was dead wrong.
Appendix 1. List of collections included in this study.
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FOOTNOTES
1 This research was supported by FCT grant no. SFRH/BD/21643/2005 to MR and NSF grant no. DEB-0515749 to A.J.S. The authors thank R. Andrus (and BING) for the loan of herbarium specimens. 
7 Author for correspondence (e-mail: mdf7{at}duke.edu)
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