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Nuclear genome size in Selaginella Damon P. Little, Robbin C. Moran, Eric D. Brenner, and Dennis Wm. Stevenson

Abstract: Estimates of nuclear genome size for 9 Selaginella species were obtained using flow cytometry, and measurements for 7 of these species are reported for the first time. Estimates range from 0.086 to 0.112 pg per holoploid genome (84–110 Mb). The data presented here agree with the previously published flow cytometric results for S. moellendorffii. Within the 9 species sampled here, chromosome number varies from 2n = 16 to 2n = 27. Nuclear genome size appears to be strongly correlated with chromosome number (Spearman’s rank correlation; p = 0.00003725). Cultivated S. moellendorffii lacks sexual reproduction—manifest by the production of abortive megasporangia. Flow cytometric data generated from a herbarium specimen of a fertile wild-collected S. moellendorffii are virtually indistinguishable from the data generated from fresh material (0.088 vs. 0.089 pg/1C). Therefore, the limited fertility observed in cultivated plants is probably not the result of abnormal chromosome number (e.g., induced by interspecific hybridization). Key words: flow cytometry, genome size. Re´sume´ : Une estimation de la taille du ge´nome nucle´aire a e´te´ obtenue par cytome´trie en flux pour neuf espe`ces du genre Selaginella; dans 7 cas, il s’agit d’une premie`re. Les estimations varient entre 0,086 et 0,112 pg par ge´nome holoploı¨de (84 a` 110 Mb). Les donne´es pre´sente´es sont en accord avec les re´sultats obtenus pre´ce´demment pour le S. moellendorffii. Au sein des neuf espe`ces e´tudie´es, le nombre de chromosomes varie entre 2n = 16 a` 2n = 27. La taille du ge´nome nucle´aire semble fortement corre´le´e avec le nombre de chromosomes (corre´lation de rang de Spearman; p = 0,00003725). En culture, le S. moellendorffii ne se reproduit pas sexuellement tel que manifeste´ par la production de me´gasporangies avorte´es. Les donne´es de cytome´trie en flux ge´ne´re´es a` partir de spe´cimens d’herbier d’un S. moellendorffii fertile collectionne´ en nature sont virtuellement identiques aux donne´es obtenues a` partir de mate´riel frais (0,088 vs. 0,089 pg/1C). Ainsi, la fe´condite´ limite´e observe´e chez les plantes cultive´es ne re´sulte vraisemblablement pas d’un nombre anormal de chromosomes (e.g., induit par une hybridation interspe´cifique). Mots-cle´s : cytome´trie en flux, taille du ge´nome. [Traduit par la Re´daction]

Introduction Selaginella (‘‘spike moss’’) is a cosmopolitan genus of small, spreading, herbaceous, spore-bearing vascular plants. Approximately 700 species are known to science (Mabberley 1997). Although Selaginella species occupy a variety of habitats, from wet tropical forest, to desert, to alpine, most of the species occur in tropical and warm temperate climates. Selaginella is classified in the Lycopsida with Isoetaceae (‘‘quillwort’’) and Lycopodiaceae (‘‘clubmoss’’; Korall et al. 1999). Fossil remains with striking similarity to extant Selaginella have been reported from middle Pennsylvanian deposits (Schlanker and Leisman 1969). Like seed plants, Selaginella is eusporangiate, heterosporous, and endosporic; however, Selaginella produces only spores, not seeds. A similar configuration is found in the related genus Isoetes. Heterospory and endosporic gametophyte development are found in the leptopsorangiate water ferns (Azollaceae, Marsileaceae, and Salviniaceae). Although these are not the ancestors of the seed plants, they

are a useful system for the study of seed evolution, providing a living facsimile of a seed plant precursor. Selaginella is easy to culture (Webster 1979) and has a much smaller genome than Isoetes (176 – 694 vs. 3 413 – 23 413 Mb/2C; Hanson and Leitch 2002; Obermayer et al. 2002; Wang et al. 2005). Because of these characteristics, Selaginella has been selected as a model for understanding the character transformations that must have occurred in the evolution of seeds. Currently, the genome of Selaginella moellendorffii is being sequenced by the US Department of Energy Joint Genome Institute (JGI; http://www.jgi.doe.gov/sequencing/ cspseqplans.html). One of the key criteria for selecting S. moellendorffii over other Selaginella species was its small genome size (Wang et al. 2005). At the time S. moellendorffii was chosen, there were 3 publications detailing the genome size for a total of 6 Selaginella species (Bouchard 1976; Obermayer et al. 2002; Wang et al. 2005). The Feulgen microdensitometry estimates of Bouchard have generally been disregarded for

Received 26 July 2006. Accepted 31 October 2006. Published on the NRC Research Press Web site at genome.nrc.ca on 23 May 2007. Corresponding Editor: J.H. de Jong. D.P. Little,1 R.C. Moran, E.D. Brenner, and D.Wm. Stevenson. Lewis B. and Dorothy Cullman Program for Molecular Systematic Studies, The New York Botanical Garden, Bronx, NY 10458-5126, USA. 1Corresponding

author (e-mail: [email protected]).

Genome 50: 351–356 (2007)

doi:10.1139/G06-138

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2 reasons: (i) the paper that was to include Bouchard’s data was withdrawn prior to publication (Bennett and Leitch 2001) and (ii) the value reported by Bouchard for diploid S. kraussiana is reported to be 2.7 times lower than the corresponding flow cytometric estimate (Obermayer et al. 2002). (The actual difference between the value obtained by Obermayer et al. and Bouchard is 1.4 times.) Thus, only C values obtained by flow cytometry for S. kraussiana and S. moellendorffii were seriously considered (Wang et al. 2005). Cultivated S. kraussiana occurs in a polyploid series (2n = 20, 30, 40; Jermy et al. 1967; Obermayer et al. 2002). As a result, published, trustworthy, 2C values for S. kraussiana range from 0.32 to 0.71 pg/2C (Obermayer et al. 2002; Wang et al. 2005). In contrast, published 2C values for S. moellendorffii range from 0.18 to 0.26 pg/2C (Wang et al. 2005). Unfortunately, S. moellendorffii has several undesirable characteristics precluding its use as a model species. In particular, cultivated specimens lack sexual reproduction, as manifest by abortive megasporangia (the microsporangia are functional; Fig. 1). A more extensive survey of genome size among Selaginella species will allow for the more-informed selection of a model species.

Materials and methods Scanning electron microscopy Air-dried sporangia and megaspores were attached to aluminum stubs with double-sided tape. Air-dried microspores were suspended in absolute ethanol. The spore suspension was allowed to evaporate on asphalt-covered aluminum stubs. Samples were sputter coated with a gold–palladium mixture for 2 min using a Hummer 6.2 sputtering system (Anatech, Springfield, Va.). Observations were made with a JSM-5410LV scanning electron microscope (SEM; JEOL, Tokyo, Japan) using an accelerating voltage of 15 kV. Digital images were captured using the supplied ORION software (version 1.72.1.0; JEOL USA, Peabody, Mass.). Adjustments in contrast, brightness, and cropping were made using Photoshop (version 7.0.1; Adobe Systems, San Jose, Calif.). Flow cytometry Fresh plants were collected from the New York Botanic Garden glasshouses and stored in sealed plastic bags in the dark at 4 8C for 36 h. Herbarium specimens were rehydrated for 12 h under vacuum in PBS buffer with 0.1% v/v Triton X-100 (13 mmol/L NaCl, 7 mmol/L Na2HPO4, 3 mmol/L NaH2PO4, pH 7.0). Voucher specimens for SEM and flow cytometry are deposited at the herbarium of The New York Botanical Garden (Little and Moran 925–931, 934, 935; Little 938; Moran 5477). Individual samples were subjected to a single flow-cytometry run. Multiple individuals of S. apoda and S. moellendorffii were examined by flow cytometry. The nuclei isolation and staining procedure was modified from Arumuganathan and Earle (1991). Approximately 25 mg of fresh mature Arabidopsis thaliana (L.) Heynh. ‘Columbia’ rosette leaf tissue and approximately 25 mg of fresh or 5 mg of dried Selaginella leaf and stem tissue were vigorously chopped in 1600 mL of buffer (9.6 mmol/L

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MgSO4, 48 mmol/L KCl, 4.8 mmol/L HEPES, 1 mmol/L dithiothreitol, 0.1% w/v propidium iodide, 0.25% v/v Triton X100, pH 8.0). The homogenate was filtered with 40 mm nylon mesh. An additional 200 mL of buffer along with 400 mg of RNase A was added to the isolation. Samples were incubated at 37 8C for 15 min and then kept in the dark at 4 8C. Measurements of 30 000 events were collected with a FACscan flow cytometer (488 nm laser; BD Biosciences, San Jose, Calif.). All flow cytometric data were collected within a 30 min time period. Data analysis Flow cytometry data were analyzed with FCSPress 1.4 (Hicks 2004). The mean value of each peak was used in further calculations. ‘Columbia’ diploid genome size was assumed to be 0.34 pg/2C—the median of the published values obtained by Krisai and Greilhuber (1997), Dolezˇel et al. (1998), Bennett et al. (2003), and Schmuths et al. (2004). The median of the published estimates was used, since no objective criterion to favor one estimate over the others was manifest. It was further assumed that 1 pg of DNA is equivalent to 9.78  108 bp (Dolezˇel et al. 2003). Statistical analyses were conducted using R version 2.3.0 (R foundation, Vienna, Austria). Owing to conflicting chromosome data, statistical tests excluded values from S. involvens, S. pallescens, and S. vogelii.

Results Estimates of Selaginella genome size obtained in this study ranged from 84 to 110 megabases per holoploid genome (sensu Greilhuber et al. 2005; Table 1). When multiple samples were analyzed, similar values were obtained (e.g., variation between S. apoda samples was approximately 4.4%). In terms of particle size and distribution, the appearance of data generated from the herbarium specimen of S. moellendorffii collected in the wild is indistinguishable from the data generated from cultivated fresh material—the C values vary by approximately 1.1% (Table 1; Figs. 2C, 2D). Within this data set, Spearman’s rank correlation test indicates that chromosome number and genome size are highly correlated (p = 0.00003725). No correlation was found for the same data when only diploid individuals were included (p = 0.1012).

Discussion The estimates of nuclear genome size reported here compare well with published values: Wang et al. (2005) reported the genome size of Selaginella moellendorffii using ‘Columbia’ as an internal standard to be 0.09 pg/1C, which is similar to the values reported here (0.088 and 0.089 pg/1C; Table 1). If the genome size for ‘Columbia’ used by Wang et al. (0.36 pg/2C) were to be adopted, our observations for S. moellendorffii would exactly match the published results. The values obtained by Wang et al. using Glycine max L. Merr. or Oryza sativa L. as internal standards are approximately 1.5 times larger (0.13 pg/1C). Since the genome sizes of G. max and O. sativa are, respectively, 12.5 and 5.6 times larger than the lowest (and most reliable) published value for S. moellendorffii (Wang et al. 2005), this discrepancy is presumably the result of nonlinearity—i.e., a #

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Fig. 1. Scanning electron micrographs of cultivated S. moellendorffii (left column; Little 938) and S. moellendorffii collected in the wild (right column; Moran 5477). (A) Aborted megasporangium. (B) Functional megasporangium containing 4 megaspores. A tetrad of microspores can be seen on the bottom right. (C) Aborted megasporangium. (D) Functional megaspore partially dissected out of the megasporangium, showing the proximal face with triradiate laesurae. (E) Functional microsporangium with microspores spilling out. (F) Functional microsporangium with loose microspores. (G) Functional microspore. (H) Functional microspore.

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Genome Vol. 50, 2007 Table 1. Genome size and chromosome complement for Selaginella.

Species S. apoda (L.) Fernald S. braunii Baker S. flabellata (L.) Spring S. involvens (Sw.) Spring S. kraussiana (Kunze) A. Braun

S. S. S. S. S. S.

moellendorffii Hieron pallescens (C. Presl) Springj pulcherrima Liebm. uncinata (Desv.) Spring vogelii Springj willdenowii (Desv.) Baker

Chromosome complement

Genome species

n 9a 10c

pg/1C (coefficient of variation) 0.086 (7.28%), 0.090 (5.85%) 0.100 (11.03%) 0.12e 0.090 (5.87%), 0.11e,g 0.11e, 0.112 (8.34%), 0.16h 0.22i, 0.24h, 0.25i 0.36h 0.088 (7.64%), 0.089 (7.62%), 0.09i, 0.13i 0.088 (7.97%), 0.11e 0.093 (7.69%) 0.090 (5.50%) 0.086 (7.02%) 0.11e

9c

8k,l, 10l,m

2n 18b 20d 20d 18–20d, 20f 20d 30h 40h 20f 20d, 22n,o 20d 18d,f,p 16q, 27d 18c,d,r

Mb/1C 84–88 98 117 88–108 108–156 210–240 347 86–127 86–108 91 88 84 108

Note: Each genome size measurement reported here represents an observation from a different individual. a

Graustein (1930); serial sections. Lo¨ve (1976); method not stated. c Kuriachan (1963); squashes. d Jermy et al. (1967); squashes. e Bouchard (1976); light microscopy with Feulgen staining. f Takamiya (1993); squashes. g As S. caulescens (Wall.) Spring following the synonymy of Reed (1966). h Obermayer et al. (2002); flow cytometry with propidium iodide staining. i Wang et al. (2005); flow cytometry with propidium iodide staining. j Our sample may be misidentified. k Denke (1902); serial sections. l As S. emiliana W.H. Gibson following the synonymy of Reed (1966). m Graustein (1930); serial sections. n Tschermak-Woess and Dolezˇal-Janish (1959); squashes. o As S. cuspidate (Link) Link following the synonymy of Reed (1966). p Weng and Qiu (1988); squashes. q Heitz (1926); squashes. r Marcon et al. (2005); squashes. b

compression artifact of linear amplification that is exasperated by larger ranges of signal intensity (Bagwell et al. 1989; Dolezˇel and Bartosˇ 2005). These discrepancies cannot be attributed to the secondary chemistry of S. moellendorffii and (or) nuclear autofluorescence given the data published by Wang et al., which clearly demonstrate an increase in estimated S. moellendorffii genome size with increasing internal standard size. The magnitude and sign of the error for S. moellendorffii resulting from the use of O. sativa instead of ‘Columbia’ is similar to the discrepancy between the published flow cytometric estimates for probable diploid S. kraussiana using O. sativa as an internal standard and the value reported here—approximately a 1.4-fold difference (0.16 versus 0.112 pg/1C; Obermayer et al. 2002; Table 1). Oryza sativa has a genome approximately 6.3 times larger than the smallest (and most reliable) value reported for diploid S. kraussiana (Obermayer et al. 2002). Errors resulting from nonlinearity may also explain the approximately 1.2-fold difference between the flow cytometric estimates reported here and the results obtained using Feulgen staining of diploid S. involvens, S. kraussiana, and S. pallescens with Gallus gallus L. as a standard (Bouchard 1976). The genome size of G. gallus is approximately 22.7

times larger than the Feulgen value for these species (Bouchard 1976). Suda and Tra´vnı´cˇek (2006) tested the feasibility of gathering flow cytometric data from herbarium specimens of vascular plants that had been dried and maintained under standard conditions. After 20 months of storage most specimens still produced a detectable signal, but some displayed up to an 11% decrease in signal intensity. Although Suda and Tra´vnı´cˇek used a different sample preparation method and the sample we examined was much older (173 months; drying and storage conditions were not controlled), the normal appearance of the flow cytometric data (Fig. 2D) and the strong concordance with data gathered from fresh material (Fig. 2C) would suggest that our protocol is appropriate for herbarium specimens. Assuming that the measurement obtained from the fertile, wild-collected S. moellendorffii herbarium specimen is reliable, it appears that cultivated S. moellendorffii does not differ substantially in genome size (0.088 versus 0.089 pg/1C; Table 1). Assuming a linear relationship between the DNA content of a given chromosome and its size during metaphase, an aneuploid S. moellendorffii would have a genome size 9.8%–10.1% larger (Takamiya 1993). Instead, a 1.1% difference was observed. This difference is most likely the #

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Fig. 2. Representative flow cytometry data for Selaginella. (A) S. apoda. (B) S. involvens. (C) S. moellendorffii, sterile, cultivated specimen. (D) S. moellendorffii, fertile, wild-collected herbarium specimen.

result of measurement error. Therefore, the limited fertility observed in the cultivated plants is probably not the result of an abnormal chromosome number (e.g., induced by interspecific hybridization). This notion is further supported by the normal appearance of the microspores (Fig. 1G). Like S. moellendorffii, cultivated S. gracilis T. Moore, S. involvens, and S. viridangula Spring consistently produce abortive sporangia near the cone base (Mitchell 1910). If the production of aberrant megasporangia in these species is simply the result of culture conditions, perhaps increasing ambient ethylene concentration would result in megaspore formation—ethylene has been demonstrated to stimulate megaspore production in S. pallescens and S. wallacei Hieron (Brooks 1973). Because genome size in diploid Selaginella does not vary substantially, we propose that other criteria be used to select a model Selaginella. Considerations should include reproductive mode, generation time, strobilus arrangement, transformability, availability in cultivation, and the amount of published literature.

Acknowledgements We thank J. Hanks and C. Kellogg for protocol advice, J. Hirst for assistance with the FACscan, G. Hall for bibliographic assistance, and two anonymous reviewers for constructive comments. Funding from the National Science Foundation (DBI 0421604) to D.W.S. is gratefully acknowledged.

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