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. 2011 Aug 10;108(5):867–876. doi: 10.1093/aob/mcr208

Flow cytometry confirms reticulate evolution and reveals triploidy in Central European Diphasiastrum taxa (Lycopodiaceae, Lycophyta)

H Wilfried Bennert 1,*, Karsten Horn 2, Marion Kauth 3, Jörg Fuchs 4, Iben Sophie Bisgaard Jakobsen 5, Benjamin Øllgaard 6, Martin Schnittler 7, Matthias Steinberg 8, Ronnie Viane 9
PMCID: PMC3177684  PMID: 21835817

Abstract

Background and Aims

Interspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing.

Methods

Flow cytometry was used first to measure the relative DNA values [with 4′,6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids.

Key Results

A considerable variation of DNA 2C values (5·26–7·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·48–10·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology.

Conclusions

Diploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing events, reticulate evolution patterns arise that are similar to those known for a number of ferns.

Keywords: Diphasiastrum, Lycopodiaceae, flow cytometry, nuclear DNA content, homoploidy, triploidy, hybridization, reticulate evolution

INTRODUCTION

The lycophytes are the oldest extant group of spore-producing vascular plants, and their earliest fossil remains are from the Devonian or possibly (the genus Baragwanathia) even from the late Silurian period (Hueber, 1983; Garratt et al., 1984; Rickards, 2000; Gensel and Berry, 2001; Hao and Gensel, 2001; Wikström, 2001; Kotyk et al., 2002).

While their extant representatives comprise a number of rather small and inconspicuous herbaceous plants, in the Carboniferous, lycophytes (Lepidodendron and Sigillaria) grew as tall trees (Stewart and Rothwell, 1993; Berry and Fairon-Demaret, 2001; Pigg, 2001; Taylor et al., 2009) and, in association with the arborescent giant horsetails (Calamitaceae and Archaeocalamitaceae), formed swampland forests for 40 million years and were important contributors to coal formation.

Reconstructions of relationships for the main lineages of vascular plants that diverged since the Devonian period have revealed a basal dichotomy, separating the lycophytes from the so-called euphyllophytes (Euphyllophytina, all other vascular plants) approx. 400 million years ago (Raubeson and Jansen, 1992; Kenrick and Crane, 1997; Duff and Nickrent, 1999; Pryer et al., 2001, 2004a, b; Tsuji et al., 2007). The latter comprise two major clades: the spermatophytes (seed plants, Soltis and Soltis, 2004), and the monilophytes (ferns, sensu Pryer et al., 2004b), including horsetails, whisk ferns and all eusporangiate and leptosporangiate ferns.

The extant lycophytes include members of the homosporous Lycopodiaceae, as well as the heterosporous Selaginellaceae and Isoëtaceae. Although being extremely diverse and of virtually cosmopolitan distribution, the family of Lycopodiaceae is considered to be monophyletic (Wikström and Kenrick, 1997; Yatsentyuk et al., 2001). While in the past all species of the Lycopodiaceae were placed generally in the single genus Lycopodium, in the last several decades the family has been variously divided into 2–16 genera (Øllgaard, 1987; Wagner and Beitel, 1993; Haines, 2003). In Europe, currently four genera are recognized: Diphasiastrum, Huperzia, Lycopodium and Lycopodiella (Valentine and Moore, 1993).

The genus Diphasiastrum comprises a relatively small group of lycopods that differ morphologically from the other genera by their leaves being usually decussate (or 4–5-ranked), di- or even trimorphic, and the aerial branchlets being quadrate to flattened. It includes about 25 species (and taxa of hybrid origin) of mainly north temperate and sub-arctic distribution (Wagner and Beitel, 1993). Only a few species occur in the tropics or sub-tropics, where they are restricted to mountain areas (e.g. D. multispicatum; see Bennert et al., 2007). The base number is generally accepted to be x = 23 (Wagner, 1992), and diploidy prevails by far (Aagaard et al., 2009c).

In Continental Europe, six diploid taxa are recognized, including three parental species (D. alpinum, D. complanatum and D. tristachyum, called ‘species’ in the following), and three intermediate taxa of putative hybrid origin (D. × issleri, D. × oellgaardii and D. × zeilleri, called ‘hybrids’ in the following).

It generally has been supposed that these diploid hybrids reproduce sexually via spores, giving rise to F2 and subsequent generations, but this never has been proven beyond doubt. Also, it is not clear whether backcrosses between hybrids and parent species occur, as would be expected regarding the high crossability of species. Judging from morphology, the parental species seem to retain their identity and no transitions between parents and hybrids have been found so far, with rare putative triploids as a possible exception (see below). A total of seven homoploid hybrids have been reported from North America, Northern Asia and Europe (Wagner et al., 1985).

Flow cytometry was applied in order to check whether (a) the known Diphasiastrum hybrids in Central Europe are invariably homoploid; (b) their genome sizes would be indicative of their origin; and (c) cytotypes other than diploid exist.

MATERIALS AND METHODS

Plants of Diphasiastrum were identified by their morphological characters. While the species are easy to differentiate, recognition of the hybrids requires advanced experience. Diagnostically useful are the dimensions of the ventral and dorsal leaves and their size in relation to the stem (see Horn, 2006, 2008; Horn and Tribsch, 2009).

For flow cytometric studies, fresh plant material was collected (by K.H., B.Ø., I.J. and R.V.) in Denmark, France, the Czech Republic, the Central German Uplands and northern Italy (Table 1), and kept in the refrigerator prior to analysis for no longer than 2 weeks. Three to five branchlets were collected per colony; discontiguous parts of larger colonies were sampled separately. For all accessions an initial ploidy screening was performed on a Ploidy Analyser (Partec GmbH, Münster, Germany) using 4′,6-diamidino-2-phenylindole (DAPI)-stained samples (1·6 µg mL−1) isolated in 2·1 % (w/v) citric acid monohydrate, 0·5 % (w/v) Tween-20, together with Agave sisalana (5x, 2n = 150, 2C = 20·2 pg; Zonneveld et al., 2005) as an internal reference standard. Absolute 2C DNA contents were determined for selected accessions (of 2–6 phytogeographic regions with one to several localities each) using propidium iodide (PI) staining. Diphasiastrum samples and leaf sections of Pisum sativum ‘Viktoria, Kifejtö Borsó’ (Genebank Gatersleben accession number: PIS 630; 2C = 9·09 pg; Doležel et al., 2007) as an internal reference standard were simultaneously chopped and stained using the ‘CyStain PI absolute P’ nuclei extraction and staining kit (Partec GmbH, Münster) according to the manufacturer's instruction. Samples were analysed using either a Partec CyFlow SL flow cytometer (Münster, Germany) or a FACStarPLUS (BD Biosciences, San José, CA, USA). Usually 10 000 nuclei per sample were analysed in at least five replicates per accession. The absolute DNA contents of Diphasiastrum species were calculated based on the ratios of the G1 peak means of sample and reference standard.

Table 1.

Diphasiastrum samples used for flow cytometric analyses

Taxon Accession number Date of collection Locality Region Type of study
D. alpinum H 08/01 20/8/2008 Gr. Sonnen-Berg N St. Andreasberg, NI, D Har b
D. alpinum H 09/01, H 09/73 11/9/2009, 18/11/2009 NNW Finsterau, BY, D BaFo a, b
D. alpinum H 09/03, H 09/75 11/9/2009, 18/11/2009 W Finsterau, BY, D BaFo a, b
D. alpinum H 09/09 11/9/2009 SW Altschönau, BY, D BaFo a
D. alpinum H 09/11 11/9/2009 SW Guglöd, BY, D BaFo a
D. alpinum H 09/27 11/9/2009 NE Frauenau, eastern locality, BY, D BaFo a
D. alpinum H 09/30 11/9/2009 NE Frauenau, western locality, BY, D BaFo a
D. alpinum H 09/36 12/9/2009 Kiesruck NE Buchenau, BY, D BaFo a
D. alpinum H 09/65 17/11/2009 Hirschenstein N Böbrach, BY, D BaFo a, b
D. alpinum H 09/68 17/11/2009 Zlíbský vrch N Strázny, C, CZ BoFo a, b
D. alpinum H 09/87b 18/11/2009 Voithenberg, western locality, BY, D ObWa a
D. alpinum H 09/57 1/10/2009 Hundseck SE Bühlertal, BW, D BlFo a
D. alpinum H 09/63 3/10/2009 Kandel N St. Peter, BW, D BlFo a
D. alpinum V 12664, V 12736 29/10/2009 Vexaincourt W Schirmeck, Vosges, F Vos a
D. alpinum V 12647 1/10/2009 SE St. Quirin, Moselle, F Vos a
D. alpinum V 12669–71, V 12681, V 12685 1/10/2009 Champ du Feu W le Hohwald, Bas-Rhin, F Vos a
D. alpinum V 12713 2/10/2009 Col du Schlucht, Haut-Rhin, F Vos a
D. alpinum V 12740 4/11/2009 Forét d'Abreschviller, Moselle, F Vos a
D. alpinum Ho 09/12 19/11/2009 Rosskopf SW Engenthal, Moselle, F Vos a
D. alpinum V ST/01–09 4/8/2010 Martalm, Ridnauntal, South Tyrol, I ST a
D. complanatum BØ 09/01, BØ 09/03 27/10/2009 Nørlund Plantage S Ikast, Midtjylland, DK Jut a
D. complanatum H 08/02 20/8/2008 Gr. Sonnen-Berg N St. Andreasberg, NI, D Har b
D. complanatum H 09/04, H 09/76 11/9/2009, 18/11/2009 W Finsterau, BY, D BaFo a, b
D. complanatum H 09/12 11/9/2009 SW Guglöd, BY, D BaFo a
D. complanatum H 09/17 11/9/2009 E Spiegelau, BY, D BaFo a,
D. complanatum H 09/31 11/9/2009 NE Frauenau, western locality, BY, D BaFo a
D. complanatum H 09/37 12/9/2009 Kiesruck NE Buchenau, BY, D BaFo a
D. complanatum H 09/43, H 09/88 12/9/2009, 18/11/2009 Voithenberg, western locality, BY, D ObWa a, b
D. complanatum H 09/69 17/11/2009 Zlíbský vrch N Strázny, C, CZ BoFo a, b
D. complanatum V ST/10–12 4/8/2010 Martalm, Ridnauntal, South Tyrol, I ST a
D. × issleri H 08/03 20/8/2008 Gr. Sonnen-Berg N St. Andreasberg, NI, D Har b
D. × issleri H 09/02, H 09/74 11/9/2009, 18/11/2009 NNW Finsterau, BY, D BaFo a, b
D. × issleri H 09/05, H 09/77 11/9/2009, 18/11/2009 W Finsterau, BY, D BaFo a, b
D. × issleri H 09/13, H 09/14 11/9/2009 SW Guglöd, BY, D BaFo a
D. × issleri H 09/18 11/9/2009 E Spiegelau, BY, D BaFo a
D. × issleri H 09/24 11/9/2009 Spiegelau, BY, D BaFo a
D. × issleri H 09/32, H 09/33 11/9/2009 NE Frauenau, western locality, BY, D BaFo a
D. × issleri H 06/07, H 09/38 12/10/2006, 12/9/2009 Kiesruck NE Buchenau, BY, D BaFo a
D. × issleri H 09/44, H 09/45, H 09/89 12/9/2009, 18/11/2009 Voithenberg, western locality, BY, D ObWa a, b
D. × issleri H 09/66 17/11/2009 Hirschenstein N Böbrach, BY, D BaFo a, b
D. × issleri H 09/67 17/11/2009 S Haidmühle, BY, D BaFo a, b
D. × issleri H 09/81 18/11/2009 Rindel-Berg E Altschönau, BY, D BaFo a, b
D. × issleri H 09/070 17/11/2009 Zlíbský vrch N Strázny, C, CZ BoFo a, b
D. × issleri H 09/59 1/10/2009 Lochrütte, Feldberg region, BW, D BlFo a, b
D. × issleri H 09/60, V 12698 2/10/2009 Le Tanet, type locality, Haut-Rhin, F Vos a, b
D. × issleri H 09/61 2/10/2009 NE Le Tanet, Haut-Rhin, F Vos a, b
D. × issleri V ST/13–16 4/8/2010 Martalm, Ridnauntal, South Tyrol, I ST a
D. × oellgaardii BØ 09/06 27/10/2009 Præstbjerg Plantage SE Vind, Midtjylland, DK Jut a
D. × oellgaardii H 09/06, H 09/78 11/9/2009, 18/11/2009 W Finsterau, BY, D BaFo a, b
D. × oellgaardii H 09/15 11/9/2009 SW Guglöd, BY, D BaFo a
D. × oellgaardii H 09/20, H 09/83 11/9/2009, 18/11/2009 E Spiegelau, BY, D BaFo a, b
D. × oellgaardii H 09/25 11/9/2009 Spiegelau, BY, D BaFo a
D. × oellgaardii H 09/34 11/9/2009 NE Frauenau, western locality, BY, D BaFo a
D. × oellgaardii H 09/39 12/9/2009 Kiesruck NE Buchenau, BY, D BaFo a
D. × oellgaardii H 09/71 17/11/2009 Zlíbský vrch N Strázny, C, CZ BoFo a, b
D. × oellgaardii H 06/19 16/11/2006 Dreiwappen ENE Althütte, BY, D ObWa a
D. × oellgaardii H 09/46, H 09/47, H 09/90 12/9/2009, 18/11/2009 Voithenberg, western locality, BY, D ObWa a, b
D. × oellgaardii H 09/54 1/10/2009 Beerfelden, HE, D HeOd a
D. × oellgaardii H 09/58 1/10/2009 Hundseck SE Bühlertal, BW, D BlFo a
D. × oellgaardii H 09/62 3/10/2009 Weiherkopf S Münsterhalden, BW, D BlFo a
D. × oellgaardii V 12672, V 12673, V 12676, V 12678, V 12679, V 12686 1/10/2009 Champ du Feu W le Hohwald, Bas-Rhin, F Vos a
D. × oellgaardii V ST/17 4/8/2010 Martalm, Ridnauntal, South Tyrol, I ST a
D. tristachyum BØ 09/02, BØ 09/05 27/10/2009 Nørlund Plantage S Ikast, Midtjylland, DK Jut a
D. tristachyum H 09/07, H 09/79 11/9/2009, 18/11/2009 W Finsterau, BY, D BaFo a, b
D. tristachyum H 09/10 11/9/2009 SW Altschönau, BY, D BaFo a
D. tristachyum H 09/21 11/9/2009 E Spiegelau, BY, D BaFo a
D. tristachyum H 09/23, H 09/85 11/9/2009, 18/11/2009 Filz-Wald E Spiegelau, BY, D BaFo a, b
D. tristachyum H 09/26 11/9/2009 Spiegelau, BY, D BaFo a
D. tristachyum H 09/28 11/9/2009 NE Frauenau, eastern locality, BY, D BaFo a
D. tristachyum H 09/40 12/9/2009 Kiesruck NE Buchenau, BY, D BaFo a
D. tristachyum H 09/48, H 09/91 12/9/2009, 18/11/2009 Voithenberg, western locality, BY, D ObWa a, b
D. tristachyum H 09/55 1/10/2009 Beerfelden, HE, D HeOd a
D. tristachyum H 09/56 1/10/2009 Olfen, HE, D HeOd a
D. tristachyum H 09/64 3/10/2009 Kandel N St. Peter, BW, D BlFo a
D. tristachyum V 12639, V 12649 1/10/2009 Altdorfkopf WSW Dabo, Moselle, F Vos a
D. tristachyum V 12640, V 12645 1/10/2009 Le Canceley SE Abreschviller, Moselle, F Vos a
D. tristachyum V 12665–67, V 12668, V 12674, V 12677, V 12684, V 12687 1/10/2009 Champ du Feu W le Hohwald, Bas-Rhin, F Vos a
D. tristachyum V 12690 1/10/2009 ENE Moussey, Moselle, F Vos a
D. tristachyum V 1273 12/10/2009 St. Quirin (Tête du Calice), Moselle, F Vos a
D. tristachyum V 12738 27/10/2009 Singristerkopf near Reinhardsmunster, Bas-Rhin, F Vos a
D. tristachyum V 12735 29/10/2009 Vallée de la Sarre Blanche, Moselle, F Vos a
D. tristachyum V 12737 29/10/2009 Kappelbronn N Lutzelhouse, Bas-Rhin, F Vos a
D. tristachyum V 12739 4/11/2009 Forét domaniale de Walscheid, Moselle, F Vos a
D. × zeilleri BØ 09/07 27/10/2009 Tihøje ved Røddinglund Plantage WNW Vildbjerg, Midtjylland, DK Jut a
D. × zeilleri H 08/04 20/8/2008 Gr. Sonnen-Berg N St. Andreasberg, NI, D Har b
D. × zeilleri H 09/08, H 09/80 11/9/2009, 18/11/2009 W Finsterau, BY, D BaFo a, b
D. × zeilleri H 09/16 11/9/2009 SW Guglöd, BY, D BaFo a
D. × zeilleri H 09/22, H 09/84 11/9/2009, 18/11/2009 E Spiegelau, BY, D BaFo a, b
D. × zeilleri H 09/29 11/9/2009 NE Frauenau, eastern locality, BY, D BaFo a
D. × zeilleri H 09/35 11/9/2009 NE Frauenau, western locality, BY, D BaFo a
D. × zeilleri H 09/41 12/9/2009 Kiesruck NE Buchenau, BY, D BaFo a
D. × zeilleri H 09/49, H 09/50, H 09/92 12/9/2009, 18/11/2009 Voithenberg, western locality, BY, D ObWa a, b
D. × zeilleri H 09/72 17/11/2009 Zlíbský vrch N Strázny, C, CZ BoFo a
D. × zeilleri H 09/52, H 09/53 1/10/2009 Beerfelden, HE, D HeOd a, b
D. × zeilleri V 12679, V 12680, V 12682 1/10/2009 Champ du Feu W le Hohwald, Bas-Rhin, F Vos a
D. × zeilleri V 12728 9/10/2009 Baerenthal (Lindenkopf), Moselle, F Vos a
D. × zeilleri V 12729 9/10/2009 Wingen (Col du Litschhof, Wingen, Bas-Rhin, F Vos a
D. × zeilleri V 12732 9/10/2009 Jungenwald, Wingen, Bas-Rhin, F Vos a
D. × zeilleri V 12733 9/10/2009 Diebhalt, Climbach, Bas-Rhin, F Vos a
D. hybrid, triploid H 09/19, H 09/82 11/9/2009, 18/11/2009 E Spiegelau, BY, D BaFo a, b
D. hybrid, triploid H 09/51, H 09/86 12/9/2009, 18/11/2009 Voithenberg, eastern locality, BY, D ObWa a, b
D. hybrid, triploid H 09/42, H 09/87a 12/9/2009, 18/11/2009 Voithenberg, western locality, BY, D ObWa a, b
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Abbreviations used for accession numbers: H, K. Horn; V, R. Viane; Ho, P. Holveck; BØ, I.S. Bisgaard Jakobsen and B. Øllgaard. For localities: D, Germany; BW, Baden-Württemberg; BY, Bavaria; HE, Hesse; NI, Lower Saxony; ST, Sachsen-Anhalt; CZ, Czech Republic; C, Budejovicky Kraj; DK, Denmark; F. France; I, Italy. For regions: BaFo, Bavarian Forest; BlFo, Black Forest; BoFo, Bohemian Forest; Har, Harz Mountains; HeOd, Hessischer Odenwald; Jut, Jutland; ObWa, Oberpfälzer Wald; ST, South Tyrol; V, Vosges Mountains. For types of study: a, flow cytometry by DAPI; b, flow cytometry by PI.

Using the 2C DNA contents, a Student's test was performed to test (a) for differences between the populations within one taxon and (b) for differences between taxa based on all populations studied.

RESULTS AND DISCUSSION

Relative DNA values (DAPI measurements)

Values for relative fluorescence intensity show a 1·38-fold variation between the parent species, with D. alpinum possessing the largest and D. tristachyum the smallest genome (Table 2). The genome sizes of the three known hybrids are not only in the same range as those of the diploid species, but also intermediate between those of the assumed parents, confirming their diploid status as well as their correct identification. Within the taxa the variation between regions is small and does not exceed the inaccuracy threshold.

Table 2.

Relative fluorescence intensity (DAPI measurements) for the Diphasiastrum taxa studied, summarized by the phytogeographic regions

Taxon Genome composition Ploidy level Region No. of sampled colonies Relative fluorescence intensity (mean ± s.d.) Overall mean (± s.d.)
D. alpinum AA 2x BaFo 7 0·321 ± 0·006
BlFo 2 0·319 ± 0·006
BoFo 1 0·313
Vos 6 0·317 ± 0·006
ST 9 0·319 ± 0·006 0·317 ± 0·006
D. complanatum CC 2x BaFo 5 0·255 ± 0·009
BoFo 1 0·243
Jut 2 0·253 ± 0·009
ObWa 1 0·260
ST 3 0·248 ± 0·002 0·249 ± 0·011
D. tristachyum TT 2x BaFo 7 0·235 ± 0·012
BlFo 1 0·226
HeOd 2 0·226 ± 0·001
Jut 2 0·241 ± 0·001
ObWa 2 0·231 ± 0·003
Vos 9 0·225 ± 0·008 0·229 ± 0·010
D. × issleri AC 2x BaFo 7 0·287 ± 0·012
BlFo 1 0·286
BoFo 1 0·281
ObWa 3 0·285 ± 0·007
Vos 2 0·284 ± 0·005
ST 4 0·280 ± 0·005 0·284 ± 0·009
D. × oellgaardii AT 2x BaFo 6 0·279 ± 0·009
BlFo 2 0·268 ± 0·010
HeOd 1 0·263
Jut 1 0·283
ObWa 3 0·281 ± 0·007
Vos 6 0·267 ± 0·005
ST 1 0·278 0·273 ± 0·009
D. × zeilleri CT 2x BaFo 6 0·246 ± 0·007
BoFo 1 0·230
HeOd 2 0·234 ± 0·001
Jut 1 0·243
ObWa 3 0·250 ± 0·002
Vos 5 0·231 ± 0·006 0·238 ± 0·009
D. hybrid AAC 3x ObWa 1 0·429 ± 0·003 0·429 ± 0·003
D. hybrid AAT 3x ObWa 1 0·416 ± 0·004 0·416 ± 0·004
D. hybrid ACC 3x BaFo 1 0·402 ± 0·003 0·402 ± 0·003
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For abbreviations used, see Table 1; letters used for genome composition: A, D. alpinum; C, D. complanatum; T, D. tristachyum.

Considerably higher values were found in only three accessions, one from the Bavarian Forest and two from the Oberpfälzer Wald. These exceed those of the species by a factor of 1·30–1·80 (average 1·59). Therefore, these three colonies represent plants with a ploidy level other than diploid; they are very probably triploids (see below).

Nuclear DNA C values of the species (PI measurements)

Analogous to the results of the DAPI measurements, a considerable variation (1·42-fold) in nuclear DNA C amounts was found (Table 3), with D. alpinum having the largest 2C content (7·49 pg), D. tristachyum the smallest (5·26 pg) and D. complanatum being intermediate (5·72 pg), but closer to D. tristachyum. The intraspecific differences between localities and regions are small (usually <0·1 pg) and statistically not significant (P < 0·01).

Table 3.

DNA 2C values (pg) for the diploid Diphasiastrum taxa studied

Taxon Accession number Region Locality 2C ± s.d. mean for locality 2C ± s.d. mean for region 2C ± s.d. overall mean
D. alpinum H 09/75 BaFo W Finsterau 7·50 ± 0·04
H 09/65 BaFo Hirschenstein 7·49 ± 0·07
H 09/73 BaFo NNW Finsterau 7·45 ± 0·03 7·49 ± 0·05
H 08/01 Har Gr. Sonnenberg 7·55 ± 0·05 7·55 ± 0·05 7·52 ± 0·06
D. complanatum H 09/76 BaFo W Finsterau 5·73 ± 0·06 5·73 ± 0·06
H 09/69 BoFo Strázny 5·74 ± 0·01 5·74 ± 0·01
H 09/43 and H 09/88 ObWa Voithenberg 5·73 ± 0·07 5·73 ± 0·07
H 08/02 Har Gr. Sonnenberg 5·78 ± 0·03 5·73 ± 0·07 5·76 ± 0·05
D. tristachyum H 09/79 BaFo W Finsterau 5·26 ± 0·05
H 09/85 BaFo E Spiegelau 5·23 ± 0·07 5·25 ± 0·06
H 09/48 and H 09/91 ObWa Voithenberg 5·29 ± 0·06 5·29 ± 0·06 5·26 ± 0·06
D. × issleri H 09/77 BaFo W Finsterau 6·68 ± 0·05
H 09/66 BaFo Hirschenstein 6·59 ± 0·07
H 09/67 BaFo Haidmühle 6·59 ± 0·05
H 09/74 BaFo NNW Finsterau 6·67 ± 0·09
H 09/81 BaFo Rindel-Berg 6·52 ± 0·06 6·61 ± 0·07
H 09/70 BoFo Strázny 6·61 ± 0·06 6·61 ± 0·06
H 09/45 and H 09/89 ObWa Voithenberg 6·61 ± 0·04 6·61 ± 0·04
H 09/59 BlFo Feldberg 6·73 ± 0·08 6·73 ± 0·08
H 09/60 Vos Tanneck, type locality 6·62 ± 0·01
H 09/61 Vos Tanneck 6·62 ± 0·02 6·62 ± 0·02
H 08/03 Har Gr. Sonnenberg 6·65 ± 0·05 6·65 ± 0·05 6·64 ± 0·07
D. × oellgaardii H 09/78 BaFo W Finsterau 6·45 ± 0·06
H 09/83 BaFo E Spiegelau 6·36 ± 0·10 6·40 ± 0·09
H 09/71 BoFo Strázny 6·34 ± 0·08 6·34 ± 0·08
H 09/46 and H 09/90 ObWa Voithenberg 6·40 ± 0·06 6·40 ± 0·06 6·39 ± 0·08
D. × zeilleri H 09/80 BaFo W Finsterau 5·51 ± 0·03
H 09/84 BaFo E Spiegelau 5·46 ± 0·04 5·50 ± 0·05
H 09/49 and H 09/92 ObWa Voithenberg 5·52 ± 0·07 5·52 ± 0·07
H 09/52 HeOd Beerfelden 1 5·55 ± 0·04
H 09/53 HeOd Beerfelden 2 5·56 ± 0·02 5·55 ± 0·03
H 08/04 Har Gr. Sonnenberg 5·56 ± 0·04 5·56 ± 0·04 5·53 ± 0·05
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For abbreviations used, see Table 1.

To our knowledge, there are only very few reports on 2C DNA amounts in Diphasiastrum (Table 4). Our estimates exceed both the values published by Aagaard et al. (2009c) and those that were extracted from appendix 2 in Aagaard et al. (2009b) by about 1 pg. The use of different methods, Feulgen densitometry (Aagaard et al., 2009b, c) compared with flow cytometry (present study), cannot be responsible for these differences (see Doležel et al., 1998; Vilhar et al., 2001). However, the use of frozen or dried samples (as done by Aagaard et al. 2009b, c) has to be considered with caution as, due to an altered accessibility of the stain to the DNA, an apparent nucleic acid loss cannot be excluded, especially after long-term storage (Suda and Trávníček, 2006; Aagaard et al., 2009b, c).

Table 4.

DNA 2C values (pg) for several north temperate Diphasiastrum species and hybrids

Taxon Wang et al. (2005) Aagaard et al. (2009c) Aagaard et al. (2009b) This study
D. alpinum 6·40 6·45 (6·78) [6·97] 7·52
D. complanatum 4·82 4·81 (5·12) [5·37] 5·76
D. digitatum 5·45
D. madeirense 5·63
D. tristachyum 4·45 4·28 (4·70) 5·26
D. × issleri 5·61 6·64
D. × oellgaardii 5·21 6·39
D. × zeilleri 4·53 5·53
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Data are from Wang et al. (2005), Aagaard et al. (2009b, c) and from this study; values in parentheses are mentioned by Aagaard et al. (2009b) under results and were obtained using Feulgen densitometry and those in square brackets were obtained by flow cytometric measurements using fresh material.

Aagaard et al. (2009b, appendix 2) presented a comprehensive list of Feulgen DNA image densitometry data on numerous Diphasiastrum accessions without, however, naming the taxa. The only information given is that of the origin of the chloroplast DNA (‘maternal lineage’). As a certain maternal chloroplast haplotype occurs in the corresponding species, but may also be present in the two hybrids derived from it, each accession could belong to three different taxa. For example, the chloroplast haplotype of D. alpinum is contained in D. alpinum itself, but also occurs in D. × issleri and D. × oellgaardii, provided D. alpinum was the female parent. We have attempted to identify the taxa of the accessions analysed by Aagaard et al. (2009b, appendix 2) by first selecting the three possible candidates on the basis of the maternal lineage and then comparing the reported 2C content with the values given by Aagaard et al. (2009c) for the three species. Taxa with intermediate values were interpreted as the corresponding hybrids. Some data sets could not be assigned due to incompatibility of data.

Data on genome size in other Lycopodiaceae are scarce. Omitting those of Bouchard (1976) because of large discrepancies compared with more recent studies (see Bennett and Leitch, 2001), there seem to be only two reports on members of the Lycopodiaceae. Lycopodium clavatum was determined to have a 2C DNA content of 5·72 pg (Hanson and Leitch, 2002), which is in the same range as our results for Diphasiastrum. In Huperzia lucidula a higher 2C DNA content of 11·40 pg was found (Wang et al., 2005), probably reflecting the high base number (x = 67 or 68) in this genus (Wagner and Beitel, 1993).

Nuclear DNA C values in the diploid hybrids (PI measurements)

The three diploid Diphasiastrum hybrids yielded 2C DNA values which are similar to those of the (diploid) non-hybrid species, ranging from 5·52 pg in D. × zeilleri to 6·63 pg in D. × issleri (Table 3). The values are exactly intermediate between those of their assumed parents and agree closely with those calculated from their putative parents by adding the corresponding 1C values for a single genome (Table 5). The concordance between the measured and expected values convincingly supports their hybrid origin, the assumed parentage (as inferred from morphological characters) as well as their homoploid status. Similarly to the species, the geographical variation (within and between regions) is small and statistically not significant (P < 0·01). Homoploid hybrids as confirmed by flow cytometry occur in all nine geographical regions studied (Fig. 1). In the Vosges Mountains, the Bavarian Forest, the Oberpfälzer Wald and the Bohemian Forest the occurrence of all three hybrids was confirmed, while in the remaining five areas the existence of two hybrids was established. Diphasiastrum × zeilleri appears to be the most common hybrid taxon and was shown to occur in seven out of nine regions.

Table 5.

DNA 2C values for the species and hybrids of Diphasiastrum studied

Taxon and genome formula Genome formula Values measured (pg ± s.d.) Values predicted (pg) Difference measured – predicted (pg)
Species
D. alpinum AA 7·52 ± 0·06
D. complanatum CC 5·76 ± 0·05
D. tristachyum TT 5·26 ± 0·06
Diploid hybrids
D. × issleri AC 6·64 ± 0·07 6·64 0·00
D. × oellgaardii AT 6·39 ± 0·08 6·39 0·00
D. × zeilleri CT 5·53 ± 0·05 5·51 0·02
Triploid hybrids
D. alpinum × D. × issleri AAC 10·30 ± 0·06 10·40 –0·10
D. alpinum × D. × oellgaardii AAT 9·97 ± 0·07 10·15 –0·18
D. complanatum × D. × issleri ACC 9·48 ± 0·12 9·52 –0·04
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For the hybrids, a comparison is made between the values measured and those predicted on the basis of the parental genome sizes; the calculation for the triploids was made in accordance with the assumed origin (e.g. by summing the size of an unreduced genome of a hybrid and one half of the parental species' genome; see text); the assumed hybrid origin is indicated by genome formulae.

Fig. 1.

Fig. 1.

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Map of Central Europe showing the distribution of Diphasiastrum taxa identified; a, D. alpinum; c, D. complanatum; i, D. × issleri; o, D. × oellgaardii; t, D. tristachyum; z, D. × zeilleri. Code numbers for the regions: 1Denmark, 2Harz Mountains, 3Hessischer Odenwald, 4Vosges Mountains, 5Black Forest, 6Bavarian Forest, 7Oberpfälzer Wald, 8Bohemian Forest, 9South Tyrol.

Just like in the species, our estimates exceed the values extracted from appendix 2 in Aagaard et al. (2009b) by about 1 pg (Table 4).

Diploid backcrossing

It has been proposed that in Diphasiastrum, backcrosses between hybrids and their parent species occur at the diploid level (Aagaard et al., 2009a). Exemplified for D. × issleri, backcrossing would require this primary hybrid to produce germinable haploid meiospores (with 23 chromosomes) containing chromosomes from both parents, D. alpinum and D. complanatum. If we assume the assortment of homologues in meiosis to be random (which is likely) and to follow a Gaussian distribution, 99 % of the spores produced contain 6–17 chromosomes of each parent. During fertilization, such a mixed chromosome set would be added to a single D. alpinum genome (23 chromosomes), resulting in a sporophyte with the majority (63–87 %) of its chromosomes being derived from D. alpinum and only a minor fraction (13–37 %) from D. complanatum.

Backcrosses exhibiting such more or less intermediate genotypes should not only be morphologically striking but also detectable by flow cytometry due to their intermediate DNA contents. Our 2C DNA values, however, are virtually invariable within each species and hybrid (P < 0·01), and the differences in genome sizes between all taxa are highly significant (P < 0·0001).

A rough estimate for Central Europe, based on data from field observations, revision of herbarium specimens and regional literature, reveals that the 15–17 populations of each hybrid included in this study (see Table 1) represent 7 % of the approx. 230 known populations in D. × zeilleri, 15 % in D. × issleri with about 110 colonies, and 71 % in D. × oellgaardii with 21 records. Considering the high crossability of species and the fact that agglomerations with 4–6 taxa co-occurring are not rare, backcrosses would be expected to be not uncommon and should be detectable in our sampling.

Aagaard et al. (2009b) presented evidence that reciprocal crosses occur repeatedly in D. × issleri and × zeilleri. In such reciprocal crosses no DNA content differences would be expected.

Genetic variability was observed in neighbouring or intermixed clones in two of the diploid hybrids (D. × issleri and × zeilleri) by means of isoenzyme gel electrophoresis (see Horn, 1997), which also suggests repeated hybridization events.

Molecular studies are in preparation by one of the authors (M. Schnittler) to (a) prove the status of the hybrid taxa by using species-specific uniparental inherited markers and (b) develop markers that allow genotyping of individuals to test the occurrence of diploid backcrossing.

Nuclear DNA C values and possible parentage of three putative triploid hybrids

Considerably higher 2C DNA amounts than in the diploids were found in three accessions, with values ranging from 9·48 to 10·30 pg (Table 5). These exceed those of D. alpinum (which has the largest genome size of the diploids) by 2 to almost 3 pg, providing strong evidence that these plants are polyploids. Figure 2 shows the various genome sizes to be expected for the triploid and tetraploid genotype combinations based on the three diploid species. They range from 7·89 pg (in an autotriploid D. tristachyum, TTT) to 15·04 pg (in an autotetraploid D. alpinum, AAAA). As expected, the tetraploid cytotypes occupy the upper range, starting with 10·52 pg for an autotetraploid D. tristachyum (TTTT). This value is 0·22 pg higher than the largest 2C DNA amount measured for the new hybrids, allowing the conclusion that they represent triploids and not tetraploids. The closest concordances between measured and expected values were found assuming the following genome compositions: ACC for the accession 09/82 from the Bavarian Forest, and AAC for the accession 09/86 and AAT for the accession 09/87a, both from the Oberpfälzer Wald (Table 5). For unknown reasons, the matches are not as close as in the case of the diploid hybrids, a phenomenon observed in diploid and triploid Equisetum hybrids as well (Bennert et al., 2005). In all three Diphasiastrum hybrids the predicted values are slightly higher than those measured. Genome downsizing may be involved (see Leitch and Bennett, 2004), but, to our knowledge, has not been reported for lycophytes or ferns so far.

Fig. 2.

Fig. 2.

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Bar chart displaying genome sizes (DNA values in pg) to be expected for the ten triploid (seven allotriploid and three autotriploid) and six tetraploid (three allotetraploid and three autotetraploid) genotype combinations (black columns) as calculated from the values measured for the six diploid taxa. The DNA contents of the three triploids found in nature are shown by white columns; for abbreviations used for regions, see Table 1; for letters used for genome composition, see Table 2.

The morphology of the triploid hybrids corroborates our interpretation (Fig. 3). The plant of accession 09/82 is generally intermediate between D. complanatum and D. × issleri, but displays a strong influence of D. complanatum, such as the distinctly flattened upright axes and the spreading lateral leaves, which narrow abruptly toward the slender apices and reduced lanceolate ventral leaves. In accession 09/86, considered to be an intermediate between D. alpinum and D. × issleri (AAC), the D. complanatum-like characters are less distinct, and the influence of D. alpinum is more pronounced as becomes manifest by longer ventral and dorsal leaves (Fig. 3). Accession 09/87 (intermediate between D. alpinum and D. × oellgaardii, AAT) is not reminiscent of D. complanatum, but shares characters with D. × oellgaardii, such as the length and shape of the ventral leaves; the shoots, however, are terete to triangular, approaching those of D. alpinum. Additional morphological details (growth form and habit of plants, number and insertion of strobili), giving further evidence for the assumed origin of the three triploids, will be published separately.

Fig. 3.

Fig. 3.

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Photographs of shoots of the three putative triploid Diphasiastrum hybrids: (A, B) dorsal and ventral view of the hybrid interpreted as D. complanatum × D. × issleri (ACC), (C, D) dorsal and ventral view of the hybrid interpreted as D. alpinum × D. × issleri (AAC), (E, F) dorsal and ventral view of the hybrid interpreted as D. alpinum × D. × oellgaardii (AAT). Scale bar = 2·5 mm.

In total, seven allotriploid genome combinations are possible (Figs 2 and 4): six with a genome pair derived from the same parental species combined with a third chromosome set from another species (like AAC), and a unique combination composed of one genome each from all three diploid species (ACT). As shown in Fig. 4, there are three different routes by which this hybrid could be achieved. While in Diphasiastrum this three-parent hybrid is unknown, such a hybrid was detected in the genus Equisetum (Bennert et al., 2005; Lubienski et al., 2010).

Fig. 4.

Fig. 4.

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Hybridization scheme showing the origin of the three known diploid and all seven possible triploid hybrids within Diphasiastrum in Central Europe; diploid species and hybrids as well as the three new triploids are shown in the grey boxes; for the triploid genotype ACT, all three possible origins are indicated.

Out of the seven possible triploids, three apparently exist in nature and, together with the diploid hybrids, deliver an impressive example of reticulate evolution in Diphasiastrum.

How could triploids have formed in nature?

When studying spore morphology, in all three diploid hybrids, besides well-formed spores and aborted spores, diplospores were discovered, which are recognizable by their larger size and more globose shape; details of this study will be published separately. The occurrence of diplospores was reported earlier for D. × oellgaardii by Øllgaard and Tind (1993). These unreduced diplospores, sometimes called ‘giant’ or ‘basketball’ spores (Wagner et al., 1986; Øllgaard and Tind, 1993), result from incomplete meiotic divisions and are believed to be capable of germinating and producing diploid gametophytes.

Tetraploids are unknown in European Diphasiastrum species, and our screening also failed to prove the existence of tetraploids in the regions studied. Thus, the common route of yielding triploids, i.e. from a cross between a tetraploid and a diploid species or cytotype, obviously is unlikely. We propose that the triploids arose instead as crosses between diploid gametophytes (stemming from a hybrid's diplospore) and a haploid gametophyte (derived from a normal meiospore of the second parent).

For the formation of the three putative triploid hybrids (ACC, AAC and AAT), only two diplospore-producing diploid hybrids would be required, namely D. × issleri (AC) and D. × oellgaardii (AT). For establishment of the genotypes AAC and ACC, a diploid D. × issleri gametophyte would backcross with either parent (D. alpinum or D. complanatum), while to obtain AAT a diploid gametophyte of D. × oellgaardii is required, which would backcross with D. alpinum. At the locality in the Bavarian Forest where the triploid ACC is growing, all six diploid taxa are present, and the same applies for the stand of the triploid AAT in the Oberpfälzer Wald, while the colony of AAC is separated by about 750 m from the main Diphasiastrum assemblage also containing the maximum number of six diploid taxa. So far, the range of the triploid hybrids is confined to southeastern Germany (Fig. 1). This is one centre of distribution of the diploid hybrids (D. × issleri and D. × oellgaardii) that are considered as parents (Bennert, 1999).

The significance of diplospores for the formation of triploids has been shown, e.g. for Athyrium (Schneller and Rasbach, 1984; Rasbach et al., 1991) and Equisetum, where several triploid taxa exist (Bennert et al., 2005), and, as in European Diphasiastrum, no tetraploids are known.

ACKNOWLEDGEMENTS

We thank Mrs Ilse Wessel, Bochum, Germany, and Mr Guy Van der Kinderen, Ghent, Belgium, for careful help with part of the lab work, Mr Pascal Holveck, Rauhwiller, France, for collecting a number of samples in the Vosges Mountains, and Mr Ralf Wieland, Bochum, Germany, for providing the geographical map of Central Europe.

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