Genetic diversity and spatial genetic structure in two co-occurring ...

Does sex make a difference? Genetic diversity and spatial genetic structure in two co-occurring species of Gagea (Liliaceae) with contrasting reproductive strategies

Plant Systematics and Evolution ISSN 0378-2697 Volume 292 Combined 3-4 Plant Syst Evol (2011) 292:189-201 DOI 10.1007/ s00606-010-0404-0

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Author's personal copy Plant Syst Evol (2011) 292:189–201 DOI 10.1007/s00606-010-0404-0

ORIGINAL ARTICLE

Does sex make a difference? Genetic diversity and spatial genetic structure in two co-occurring species of Gagea (Liliaceae) with contrasting reproductive strategies Tanja Pfeiffer • Anja Klahr • Anika Heinrich Martin Schnittler

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Received: 3 May 2010 / Accepted: 20 December 2010 / Published online: 6 February 2011 Ó Springer-Verlag 2011

Abstract Gagea lutea and G. spathacea are spring geophytes naturally co-occurring in woodlands, characterised by contrasting reproductive strategies probably caused by divergent ploidy levels. The hexaploid G. lutea relies on vegetative reproduction by subterranean bulbils in young stages but completely switches to sexual reproduction once a certain bulb size is attained. The nonaploid G. spathacea seems to be sterile and reproduces only vegetatively; the plants continue to form bulbils even in the rare event of flowering. This study used AFLP genotyping to investigate the consequences of these reproductive strategies for genetic diversity. For 150 and 100 samples from three Western Pomeranian populations of G. lutea and G. spathacea, respectively, AFLP fingerprints were analysed for three different spatial scales, the patch, the transect, and the region. Applying a threshold for genotypic identity of \0.05 simple matching distance, 22–30 genets were detected in the three G. lutea populations, with all genets confined to single populations. Clonal genets consisted of 2–9 samples and extended over up to 28 m, but never occupied the whole length of a transect; 67–75% of all patches had different genets. Genetic distances between genets within populations were similar to those recorded between populations. Genotyping of G. spathacea revealed a single clonal genet for all three populations sampled within a distance of 30 km. The absent genetic diversity confirms the suspected sexual sterility. Gagea spathacea seems to be one of the few non-apomictic, fully clonal

T. Pfeiffer (&) A. Klahr A. Heinrich M. Schnittler Institute of Botany and Landscape Ecology, Ernst-Moritz-Arndt-University Greifswald, Grimmer Straße 88, 17487 Greifswald, Germany e-mail: [email protected]

vascular plants able to occupy a significant range solely by dispersal of vegetative diaspores. Keywords AFLP fingerprinting Gagea lutea Gagea spathacea Genotyping

Introduction Reproduction is one central characteristic of life: genetic diversity is mainly generated through recombination processes in sexual (generative) reproduction, which is, hence, a process of fundamental importance for population and species biology (Maynard Smith 1978). However, many higher plants also have elaborate means of vegetative reproduction. Genetic individuals can disintegrate or fragment into ramets (clonal reproduction) and/or form more or less specialised vegetative diaspores, for example bulbils or tubers (vegetative reproduction s.str.; Urbanska 1992; Frey and Lo¨sch 2004), to supplement or substitute sexual reproduction. As a consequence, each genetic individual (genet) can consist of numerous independent dividuals which are genetically identical (if somatic mutations, which may accumulate in long-lived clonal plants are disregarded; Douhovnikoff and Dodd 2003). For populations of plant taxa relying (more or less exclusively) on vegetative reproduction, reduced levels of genotypic diversity can be expected. However, studies employing molecular techniques have revealed divergent diversity patterns for such taxa. Ellstrand and Roose (1987) provided a first survey of genetic diversity in ‘‘clonal’’ species (including apomictic taxa). For 25 of the 27 studies analysed (mainly using isozyme data, involving 21 species), they calculated considerable variation within and between populations: multiclonal populations were the rule

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rather than the exception, despite reports that sexual recruitment was rare to absent. In most cases, individual clones showed local rather than regional dominance. Using other molecular techniques, similar results were obtained later. However, in a few species little to no genotypic diversity was detected, at least in taxa with very limited ranges (e.g. Lomatia tasmanica, Lynch et al. 1998; Wollemia nobilis, Peakall et al. 2003), or in isolated populations, often near the border of the distribution area (but see Tsujimura and Ishida (2008) for Sedum bulbiferum). In recent years, microsatellite and AFLP analyses have, preferentially, been applied to such studies. Both techniques generate highly polymorphic ‘‘fingerprints’’, which are ideally suited to genotype individuals and hence identify genets (Mueller and Wolfenbarger 1999; Selkoe and Toonen 2006). However, even these techniques failed to detect genotypic diversity in isolated populations, e.g. in the endemic relict W. nobilis (Peakall et al. 2003) or the Spanish Populus euphratica population (Fay et al. 1999). But in most predominantly clonal taxa these techniques revealed at least some genetic divergence, for example in sterile Grevillea infecunda (interpreted as persistence of initial diversity; Kimpton et al. 2002), or even proved rather strong genotypic differentiation (at least locally in Posidonia oceanica, Rozenfeld et al. 2007). However, especially in studies revealing low diversity, the resolving power of the applied molecular marker should be carefully examined (compare Arnaud-Haond et al. 2005). Stabilised and entirely vegetatively reproducing taxa are very rare in vascular plants. More often, basically sexual taxa have highly sterile populations, for example sterile Alpine Saxifraga cernua versus fertile Arctic populations (Bauert et al. 1998; Gabrielsen and Brochmann 1998), northern populations of Decodon verticillatus (Dorken and Eckert 2001), some morphologically monomorphic populations of Gagea bohemica (Peterson et al. 2010), or do not reproduce sexually in invasion areas, e.g. Elodea canadensis (Barrat-Segretain 1996) and Fallopia japonica (Hollingsworth and Bailey 2000) in Europe. Special cases are apomictic (agamospermous or agamosporous) lineages, which still use seeds as diaspores. Those occupying a somewhat larger than local range are often described as taxa and are usually repeatedly recreated by crosses involving at least one fully sexual parent species. Prominent examples are Taraxacum spp. (Kirschner and Stepańek 1996) or Rubus fruticosus agg. (Weber 1996). In virtually all these cases, the asexual population or lineage has closely related sexual populations or lineages as putative ancestors. Generally, variation in reproductive strategies can be expected for taxa with reticulate evolution including hybridisations and various ploidy levels. For hybrids with reduced to absent sexual fertility (e.g. hybrids with odd-

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numbered or anorthoploid levels), three main mechanisms can grant persistence and further evolution: 1. 2. 3.

vegetative reproduction; agamospermy; and polyploidisation to regain sexual fertility.

A rare case of sexual sterility and divergent reproductive strategies associated with anorthoploidy has been documented for Butomus umbellatus introduced to America (Eckert 2002; Eckert et al. 2003): diploids have a mixed strategy with sexual and vegetative reproduction by bulbils, whereas triploid plants solely (and rarely) reproduce clonally by rhizome fragmentation. The genus Gagea (Star of Bethlehem, Liliaceae) includes species of all ploidy levels between 2 and 119 (Peruzzi 2003; Henker 2005). Here, anorthoploid taxa seem to suffer from reduced seed set (Gargano et al. 2007), but are able to survive through vegetative reproduction with bulbils. Contrasting reproductive strategies, in part probably caused by different ploidy levels, have been demonstrated for the co-occurring woodland species Gagea lutea (69) and G. spathacea (99) in populations in Western Pomerania (Schnittler et al. 2009). Both species are typical spring geophytes, with vegetation periods from February to early June. The plants consist of a subterranean parent bulb (renewed every year), which initiates new above-ground parts each year: one (G. lutea) or two basal leaves (adult G. spathacea), and, in the fertile stage, a stalk with a fewflowered inflorescence. In juvenile stages, both species reproduce vegetatively through subterranean bulbils. Once a certain parent bulb size (corresponding with resource status) is attained, the hexaploid G. lutea completely switches to sexual reproduction, and forms myrmecochorous seeds in capsular fruits. In contrast, the nonaploid G. spathacea flowers only rarely and seeds are unknown in most populations (Westerga˚rd 1936; personal observation). Because of irregular gamete formation, capability of normal sexual reproduction was doubted (Westerga˚rd 1936). Flowering plants, however, still develop bulbils, even at the cost of investment in individual growth and performance (Schnittler et al. 2009). In this species with a more restricted (mainly northern) European range, bulbils seem hence to be the sole mode of reproduction and dispersal. The objective of this AFLP fingerprinting study was to analyse and compare patterns of genetic diversity within populations of these co-occurring taxa, fully fertile G. lutea versus putative sterile G. spathacea. We tested whether genetic diversity and genet distribution correlated with the observed reproductive and dispersal strategies: in G. lutea, with a mixed sexual and vegetative strategy, genotypic diversity is expected to be much higher than in G. spathacea with at least predominant (to even exclusive) vegetative reproduction. Furthermore, the dispersal modes of

Author's personal copy Genetic diversity and spatial genetic structure in two co-occurring species of Gagea

the diaspore types, i.e. boleochorous and myrmecochorous seeds versus (more or less achorous) subterranean bulbils, should result in different spatial clustering of genets in both species. Another objective was to elucidate the habitat colonisation strategies of both species by combining data from two spatial scales (patch, transect).

Materials and methods Study species The Liliaceae G. lutea (L.) Ker Gawl. (Sect. Gagea Davliadnize) and G. spathacea (Hayne) Salisb. (monotypic sect. Spathaceae Levichev; Peterson et al. 2008) are two of ca. 7–9 species of this genus occurring in Germany. The former species has a rather wide but disjunct Eurasian range, extending through most of Central Europe and Eastern Asia, but with only limited occurrences in Western Asia. G. spathacea has a much more restricted European distribution, with a main centre in northern Germany and adjacent regions (southern Scandinavia, Poland) and a few records from western and southern parts of the continent. Germany (and especially the federal state Mecklenburg-Western Pomerania) has a large responsibility for conservation of the species, because large proportions of the world populations of G. spathacea are located here (compare Welk 2002; Ludwig et al. 2007). In the study region, both species are rather common and often co-occur in ±nutrient-rich deciduous woodlands on moderately moist soils. G. lutea also extends into more open vegetation, and often forms large populations in parks, manor-yards, and churchyards. Study region and sampling Plant material was collected from three populations of G. lutea and G. spathacea in Western Pomerania, Germany, in spring of the years 2007–2009 (Table 1). All populations occurred in a radius of ca. 30 km around the city of Greifswald. G. lutea, population A: old-growth beech forest, NW part of the ‘‘Elisenhain’’ forest SE of Greifswald (N54°040 5800 E13°260 3900 ), Fagus sylvatica L. mixed with Fraxinus excelsior L., Quercus robur L. and Acer pseudoplatanus L., spring aspect of the herb layer dominated by geophytes such as Anemone nemorosa L., some A. ranunculoides L. and Ranunculus ficaria L. Population B: Park of Griebenow estate (N54°040 4800 E13°150 0300 ) 13 km W of population A; edge of wooded part of the park, under old Tilia cordata L. undisturbed for at least 100 years. Population C: Park of Ludwigsburg castle (N54°060 1900 E13°300 2100 ) ca. 5.5 km NE of population A, under old beech and linden trees along the driveway forming the main axis of the park.

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G. spathacea, population A: Elisenhain, as for G. lutea, both species growing together. Population B: Drosedower forest near Woldeforst (N53°570 0700 E13°020 1100 ) ca. 6 km N of Demmin, ca. 30 km SW of population A, closedcanopy beech forest. Population C: old-growth beech forest E of Wrangelsburg castle (N54°010 0300 E13°360 1200 ), ca. 12 km SE of population A. To assess patterns of genetic diversity, we used a grid sampling strategy on the patch and the transect scale. In all populations, pairs of plants located at a maximum distance of 0.1 m (patch scale) were sampled over a distance of at least 2 m between two such pairs, resulting in transects of at least 50 m length (transect scale). All samples were numbered consecutively, with specimens of the same patch indicated by the letters ‘‘a’’ and ‘‘b’’. A total of 25 patches per transect was sampled. All 3 9 25 patch pairs of G. lutea; 25 patch pairs of population A of G. spathacea and one plant per patch (populations B and C of G. spathacea) were subjected to the AFLP analysis, resulting in 150 samples for G. lutea and 100 for G. spathacea (Table 1). Fresh leaves (G. lutea) or whole plants with the parent bulb freed from outer sheaths (G. spathacea) were transferred to 2 ml tubes and stored at –80°C until DNA extraction. AFLP fingerprinting and profile analyses Total genomic DNA was isolated from 300 to 400 mg frozen plant material ground in liquid N2 in pre-cooled tubes. DNA extraction was carried out according to the procedure of Stein et al. (2001) with the following exceptions: after the chloroform–isoamyl alcohol step, the supernatant was transferred to new tubes, 7 M potassium acetate was added to a final concentration of 1.3 M, and the sample was then incubated for 30 min at -20°C. Another 800 ll chloroform-isoamyl alcohol (24:1) was added and the sample was inverted for at least 15 min at 4°C before centrifugation for 13 min at 17,000 rcf. Subsequent steps of RNA digestion, DNA precipitation with isopropanol, and cleaning of the pellet were carried out as in Stein et al. (2001). The pellet was dried at 37°C in 10 min using a speed-vac (RVC 2-18, Christ). It was resuspended in up to 100 ll TE-buffer (volume depending on pellet size). DNA concentrations were estimated on 0.8% agarose gels by comparison with the kHindIII length standard (Fermentas). Approximately 500 ng DNA template was used in the AFLP fingerprinting. Digestion volumes also contained 29 Tango buffer, 5 U of each EcoRI (NEB) and Tru1I (Fermentas), and H2O ad 50 ll; and were incubated for 4 h at 37°C (optimum for EcoRI-cutting) and then an additional 3 h at 65°C (optimum for Tru1I). To 20 ll restriction mix the ligation cocktail was added, with final concentrations of 0.59 T4-ligase buffer, 0.5 mM ATP (NEB), 1 lM EcoRI adapter, 10 lM MseI adapter, and

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Table 1 AFLP fingerprinting and genotyping data for the three analysed populations of G. lutea and G. spathacea using the primer combinations EcoRI-AGG/MseI-CTA (II) and EcoRI-ACA/MseI-CAG (III) Gagea lutea Pop A

Gagea spathacea Pop B

Pop C

All

Pop A

Pop B

Pop C

All

No. of samples

43

49

49

141

49

25

25

99

Fragments scoreda

415

420

422

424

314

313

313

314

194 221

196 224

197 225

197 227

145 169

144 169

144 169

145 169

188 (45.3%)

207 (49.3%)

201 (47.6%)

231 (54.5%)

10 (3.2%)

7 (2.2%)

7 (2.2%)

15 (4.8%)

Primer II

81 (41.8%)

90 (45.9%)

84 (42.6%)

97 (49.2%)

1 (0.7%)

4 (2.8%)

1 (0.7%)

5 (3.5%)

Primer III

111 (50.2%)

126 (56.3%)

118 (52.4%)

134 (59%)

9 (5.3%)

3 (1.8%)

6 (3.6%)

10 (5.9%)

Maximum (mean) 65 (44.0) fragment differences

81 (51.6)

74 (46.2)

82 (53.3)

7 (1.9)

4 (1.1)

5 (1.3)

7 (2.3)

Simple matching genetic distances: maximum (mean)

0.201 (0.129) 0.188 (0.115) 0.212 (0.133) 0.022 (0.006) 0.013 (0.003) 0.016 (0.004) 0.023 (0.007)

Primer II Primer III Polymorphic fragmentsa (%)

0.16 (0.109)

Primer II

0.167 (0.098) 0.184 (0.117) 0.172 (0.097) 0.194 (0.118) 0.011

0.025

0.010

0.031

Primer III

0.189 (0.118) 0.239 (0.139) 0.230 (0.130) 0.273 (0.145) 0.033

0.010

0.027

0.033 1

No. genets

b

22

30

28

80

1

1

1

Samples per genet

1.95

1.63

1.75

1.78

All 49

All 25

All 25

All 99

No. of clones

7

12

8

27

1

1

1

1

Samples per clone

2–9 (Ø 4.0)

2–5 (Ø 2.6)

2–8 (Ø 3.6)

–

All 49

All 25

All 25

All 99

Maximal extension of genets (m)

22

22

28

28

C48

C48

C48

[30 km

Clonality index Rc

0.50

0.60

0.56

0.56

0

0

0

0

a

Totals of 424 (G. lutea) and 314 (G. spathacea) fragments were scored for all samples; the following lower numbers refer to fragments present in the respective populations (fragments absent in the whole population are excluded); fragments scored with ‘‘x’’ are disregarded

b

For respective thresholds see text

c

Clonality index (or genotypic richness), R; calculated as R = (no. of detected genets - 1)/(no. genotyped samples - 1); ranging from R = 1 (in nonclonal) to R = 0 (in fully clonal populations or taxa)

2.4 U T4-ligase (NEB). The mix was incubated for 9 h at 16°C. The pre-selective amplification was carried out with 3.5 ll ligation product (diluted 1:5), 19 NH4Cl buffer, 2.2 mM MgSO4, 0.25 mM dNTPs (Peqlab), 0.3 lM of each primer EcoRI ? A and MseI ? C, 0.35 U Taq polymerase, and H2O ad 10 ll and the cycler profile (Eppendorf Mastercycler): 10 min at 60°C (to optimise adapterbinding), initial denaturation for 2 min at 94°C followed by cycles of 30 s at 94°C, 30 s annealing, 2 min at 72°C. The annealing temperature started at 66°C, with a reduction of 1°C in each cycle. After nine cycles, the annealing temperature was kept constant at 56°C for another 20 cycles. This was followed by 60 min at 60°C and 2 h at 21°C before cooling to 4°C. PCR products from this pre-selective amplification were diluted 20-fold for the main amplification, which was carried out with PCR mixes similar to those used in the pre-selective amplification step, but with primers carrying two more selective base pairs, i.e. EcoRI ? ANN and MseI ? CNN (with N = selected

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specific bases, see below), respectively, and subjected to the cycler profile given above but leaving out the adapter binding step. Several primer combinations (primers with three or four additional bases compared with adapter sequences; EcoRIprimer with fluorescent label) were tested on a selection of samples. After this initial screening, population A of G. lutea (50 samples) was analysed with four primer combinations: I EcoRI ? AGG (VIC)/MseI ? CGT; II EcoRI ? AGG (VIC)/MseI ? CTA; III EcoRI ? ACA (6-FAM)/MseI ? CAG; and IV EcoRI ? ACC (NED)/MseI ? CAG. For further genotyping, primer combinations II and III were selected, because they produced banding patterns with the best quality and resolution; and the results obtained from these two primer combinations were similar to those obtained for all four combinations. 1.5 ll PCR product from the main amplification was mixed with 8.41 ll Hi-Di formamide and 0.09 ll Liz 500


length standard (both from Applied Biosystems) and denatured for 5 min at 95°C. The analyses were run with POP4-polymer (4% performance optimized polymer; Applied Biosystems) in 47 cm capillaries on an ABI 310 capillary sequencer. The AFLP fragments were scored with GeneMapper v3.7 (Applied Biosystems) in a semi-automated fashion, using a defined bin set but checking all peaks and evaluating their presence manually: Fragment peaks with C50 RFU were always scored as ‘‘1’’, lower peaks were either scored as 1 (if clearly defined), 0 (missing) or ‘‘x’’ (ambiguous). The resulting AFLP profiles were exported as binary matrices for further analyses. A few samples which failed to produce reliable peak patterns were omitted from the respective datasets. A limited number of peaks that could not be scored unambiguously for most samples in all three populations were omitted from the analyses. Genotyping Fragment scoring resulted in a binary matrix for each sample and fragment. From those binary AFLP profiles, pairwise distances were calculated for the absolute number of different fragments, and for simple matching and Jaccard coefficients (cf. Sneath and Sokal 1973). For identification of genets based on pairwise distances between samples, an algorithm was programmed in Excel that allowed to set a threshold for genotype identity to compensate for genotyping (biological, experimental, and scoring) errors (Douhovnikoff and Dodd 2003; Schnittler and Eusemann 2010). As an identical genotype (i.e. genet) we regard a group of samples where each sample is connected by at least one combination below the threshold with another sample (most often, all combinations were below the chosen threshold). This threshold was determined for G. lutea from the minimum in the histogram of pairwise differences between all samples and checked to show a minimum number of contradictions with dendrograms constructed using the NJ (neighbour joining) and UPGMA (unweighted pair group method with arithmetic mean) methods of winPAUP4.0b10 (Swofford 2002). Bootstrap values were calculated with PAUP, implying NJ and UPGMA (each 10,000 bootstrap replicates) searches. Such contradictions were noticed as samples of clonal genets separated by long branches from their clonal relatives. Spatial genetic structure For each population of G. lutea, a test for isolation by distance (Slatkin 1987) was carried out, applying Mantel test statistics to test for significant correlation between the genetic distance matrix (pairwise simple matching coefficients) and the geographic distance matrix (Euclidean

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square distances between sampling positions). The obtained empirical correlation coefficients r were tested for statistical significance, assuming that spatial and genetic distances are matched at random (null hypothesis). For 9,999 iterations of the original datasets, simulated correlation coefficients were computed using the PopTools Excel plugin (Hood 2009) and their 95% confidence intervals compared with the empirical r. To reveal spatial autocorrelation, four distance classes each comprising six metres, i.e. circumscribing four neighbouring patches (class 1: 0 to\8 m, 2: 8 to\16 m, 3: 16 to \24 m, 4: 24 to \32 m), and a fifth class with all distances C32 m were defined. In addition, the first class (0 m to\8 m) was subdivided into two equal classes (1a: 0 to \4 m and 1b: 4 to \8 m) to put a special focus on the smallest spatial scale investigated. For all these classes in all three populations, a Mantel permutation test was carried out and the empirical r compared with the confidence intervals of the computed r from 999 iterations.

Results For the initial test of four primers with G. lutea population A (43 samples) we obtained the following fragment numbers (poly-/monomorphic): I 45/102, II 69/126, III 91/97, IV 49/93. All four primers showed a bimodal distribution in the histogram of pairwise distances between all samples with a broad minimum (4–8 differing fragments) between the first (near-identical samples) and the second peak (different samples). Because most of the clonal, multisample genets retained their identity with all four primer combinations, the most polymorphic primers II and III were chosen for further genotyping. Of the 150 samples of G. lutea and the 100 of G. spathacea analysed by AFLP fingerprinting, 141 and 99 samples produced consistent peak patterns and were subjected to further analyses, combining profiles produced by primers II and III. For all analyses, the scored AFLP fragments ranged in size from 100 to 500 bp. The following figures refer to all fragments with unambiguous scores (scores ‘‘x’’ are disregarded for calculations, but included in the analyses with the program PAUP). Gagea lutea In G. lutea 97/100 (poly-/monomorphic, primer II) and 134/93 fragments (primer III) were scored for the two primer combinations used in all populations. Thus, 231 fragments were polymorphic in the whole dataset of 424 peaks (54.5%). In the separate populations, the fraction of polymorphic fragments was only slightly lower (Table 1; see also for data on individual primer combinations).

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The samples of the three populations were readily differentiated by mean simple matching distances of 0.135–0.145 (maximum 80–82 different fragments). Population A shows one private fragment, population C three private fragments (all in primer combination III), but those are usually rare. Also, three and four fragments (primers II and III, respectively) in population A, one and two (population B) and one (primer III, population C) are completely missing from the respective populations but present in the other two. The histogram of pairwise differences between all 141 samples from the three populations showed a clearly bimodal distribution with a minimum at 18 fragment differences (for individual populations between 16 and 20, data not shown), corresponding to a simple matching distance of 0.05 (Fig. 1a). Applying a threshold of 18 fragment differences for the combined dataset, a total of 80 genets was identified. Each genet was confined to a single population. In population A (Elisenhain), 15 of the detected 22 genets were unique genets occurring only once, the others formed clones of up to nine analysed samples extending over distances up to 22 m. In population B (Griebenow), 18 singular and 12 clonal genets (with 2–5 samples), the latter also reaching a maximum of 22 m, were encountered. In population C (Ludwigsburg) 28 genets were detected, including 20 singletons and eight clones consisting of up to eight samples spreading over distances of 28 m (Fig. 2). For some samples, repeat runs were conducted to calculate experimental error rates. These values are much lower than the threshold, reaching a maximum of two fragments in primer combination II (18 repeat comparisons) and three fragments in combination III (18 repeats),

corresponding to mean error rates between repetitions of 0.005 and 0.004, respectively. From the originally intended 25 patch comparisons per population (comparisons between samples a and b at a specific transect position), because of failure of DNA isolation for some samples only 18, 24, and 24 were carried out for populations A, B, and C, respectively. Between 25 and 37.5% of the samples from one patch were assigned to the same genet (Table 2; Fig. 2). In all populations, most clonal genets exceeded the patch scale, i.e. were detected at distances of C2 m from the first occurrence (Figs. 2, 3). However, all clones occupied only parts of the transects. No clone extended over more than 28 m (the maximum detectable distance in our transects would be 48 m), and no clone was detected in more than one population. On average, 70.4% of the clonal genets (23.8% of all if counting singletons also) were able to disperse beyond the patch scale. Mean genetic distances within patches were slightly lower than distances between patches. In most cases, the genotypic differentiation was unequivocal—25 of 27 clonal genets and all 52 unique genets detected in the Excel analysis were retrieved in the NJ dendrogram (Fig. 4). Exceptions are clones A7 and C8, for which singular sample comparisons below the threshold lumped the respective samples in the genotyping analysis, but which occupy somewhat more distant positions in Fig. 4 (samples marked by asterisks). Within these clones, the divergent samples also have the highest numbers of ambiguous scorings, therefore their real affinities remain unresolved. (If they are excluded, this would result in one further unique genet for population A and a further clonal genet with two samples in population C.) All other clonal

Fig. 1 Histogram for pairwise comparisons (note logarithmic scale to highlight the bimodal distribution) of simple matching distances of 141 samples from three populations (43, 49, and 49 samples, respectively) of Gagea lutea (a) and 99 (49, 25, and 25 samples) of G.

spathacea (b) (class width: 0.01 for all but the first class (with absolutely identical samples, i.e. distance 0)). The arrow marks the applied threshold for genotype identity in G. lutea (0.05; see text)

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Fig. 2 Spatial distribution of genets in the three investigated populations of Gagea lutea. White boxes indicate clonal (multi-sample) genets numbered consecutively for each population, grey boxes depict singular genets

Table 2 Comparison of genotypic divergence at patch (B0.1 m distance between samples) versus transect (C2 to 48 m distance) scales for the three investigated populations of Gagea lutea Population A

Population B

Population C

No. of samples No. of genets

43 22

49 30

49 28

No. of clonal genets (%)

7 (31.8%)

12 (40.0%)

8 (28.6%)

No. of patch comparisons

18

24

24

No. of patches with two identical plants (same genet) (%)

6 (33.3)

9 (37.5)

6 (25.0)

No. genets extending C2 m [(% of clones)/(% of all genets)]

4 (57.1/18.2)

8 (66.7/26.7)

7 (87.5/25)

Mean simple matching distance within patches

0.07

0.08

0.09

Mean simple matching distance between non-patch samples

0.11

0.13

0.12

Fig. 3 Spatial dimensions of genets in three populations of Gagea lutea (measured as pairwise Euclidean distances of positions). Clonal genets are displayed on the left scale, unique (uniq.) genets on the right-hand scale. Note separate class for patches (\0.1 m)

multi-sample genets formed clusters mostly supported by very high (C93%) to maximum bootstrap values. Genetic distances between genets from the same population are in the same range as comparisons between samples from different populations (Fig. 4). The mean genetic distance between all samples (0.132) is slightly larger than those within the populations (0.109–0.129). The

latter include numerous comparisons between clone mates; maximum distances are substantially larger (Table 1). In the constructed NJ dendrogram of the combined dataset, most of the samples belong to clusters of its respective population (Fig. 4). However, in contrast with the genet clusters the branches uniting the populations are never supported by bootstrap values [50%. Three samples from population B (Clone B8 and one unique genet) and four from population C (Clones C3, C5) form separate clusters with long branches within populations C and B, respectively. In the respective dendrograms of the two primers analysed separately (not shown), the populations are less clearly differentiated; but the above-mentioned samples also occupy distinct positions within clusters of samples from other populations. The Mantel tests yielded a slightly positive correlation for population A (empirical r = 0.352, p \ 0.001), but no large-scale correlations for populations B and C (population B: r = 0.072, p \ 0.05; population C: r = 0.208, p \ 0.001). As visible in the detailed analyses for spatial autocorrelation (Fig. 5a–c), these results can mainly be attributed to positive correlations at small scales, thus providing hints for non-random genetic structure at the smallest spatial scales investigated. In populations A and

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Fig. 4 NJ (neighbour joining) dendrogram constructed with winPAUP4.0b10 for the three investigated populations of Gagea lutea. Circles mark population A (43 samples), triangles population B (49), and rectangles population C (49), with clonal genets indicated by their respective number, singular genets by grey colour (compare

Fig. 2). Bootstrap values below 50 (and those of extremely short branches) are omitted; those characterising clonal genets are written in bold. Affinities of samples to clones A7 and C8 remain ambiguous (marked with asterisks, see text)

C, a random match of spatial and genetic distances was obvious for distances C32 m.

fragment differences. This corresponds with a maximum pairwise distance (simple matching) of 0.023 for the whole dataset. For fragment thresholds of [2, genotyping always yields a single clonal genet comprising all analysed samples from all three populations (compare Fig. 1b). Higher pairwise differences between samples were always related to others with pairwise differences not exceeding two fragments. In repeat runs, up to three (primer combination II, 40 repetitions) and five differing fragments (primer combination III, 49 repetitions) were encountered for the same sample, corresponding to error rates of 0.003 and 0.004 between runs and/or preparations of G. spathacea, respectively.

Gagea spathacea In the 99 analysed samples from G. spathacea, 145 and 169 fragments were scored for the two primer combinations, respectively (Table 1). A total of 244 fragments (77.7%) was absolutely monomorphic for the whole dataset. This number increased to 300 fragments (95.5%) if occasional ambiguous scorings (‘‘x’’) were not regarded as distinct. Between any two samples we never recorded more than seven unambiguous

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Fig. 5 Spatial autocorrelation distograms (empirical r solid line, filled diamonds) for Gagea lutea populations (A) Elisenhain, (B) Griebenow, and (C) Ludwigsburg, within six distance classes (1a: 0 to \4 m, 1b: 4 to\8 m, 2: 8 to\16 m, 3: 16 to\24 m, 4: 24 to\32 m,

5: C32 m). Note that distance classes 1a and 1b are only half the size of those following. Dashed lines represent means (open diamonds); dotted lines upper and lower margin of the 95% confidence interval for 999 permutations

Discussion

can accumulate in long-lived organisms) or contamination of samples. In our taxa, we frequently encountered the smut fungus Ustilago ornithogali (Schmidt & Kunze) Magnus, and most populations seem to be infected to some extent. Some other differences already vary between different runs of identical samples and must hence be caused by experimental and/or analytical inaccuracies. In G. spathacea, all analysed samples were assigned to one clone. However, some of the calculated error rates were only slightly smaller than the maximum divergence between all samples, i.e. experimental or genotyping errors can generate nearly the same divergence as indeed observed between clone mates. Both studied Gagea taxa are primary woodland species and naturally co-occur in Western Pomerania. Distribution patterns of the decorative, regularly flowering G. lutea have been altered by planting the species in seminatural habitats, for example church and manor yards where it multiplied and often formed large populations. In contrast, distribution patterns of the rarely flowering G. spathacea are probably mainly the result of the migration capabilities of the species. Generally, the two (sexual and vegetative) diaspore types have different dispersal strategies: Seeds as sexual diaspores are shed from the capsules by wind (or animals) and may thus be dispersed over several metres. Attracting ants, they may be further transported by myrmecochory (probably up to some tens of metres). Bulbils as vegetative diaspores are formed subterraneously in both species and are hence more or less achorous. However, they can be translocated within centimetres by solifluction (frost impact), more rarely much further by digging or wallowing activity of animals, for example wild boar (own observations), or tree falls. In general, the mean dispersal distances achieved for bulbils should be lower than those for seeds. These different dispersal strategies are obvious in the analysed datasets, with different patterns of genetic diversity on the examined spatial scales in G. lutea. Between 25.0 and 37.5% (mean 31.8%) of the samples from a patch belonged to the same genet, emphasising the relevance of

Gagea lutea and G. spathacea frequently co-occur in broad-leaved forests in the study area; they experience the same ecological conditions and are subjected to similar selective forces. Both species share major reproductive characteristics, for example features of pollination (early flowering with rather unspecific pollinators), reproduction modes (vegetatively via subterranean bulbils and sexually via seeds), and the dispersal ecology of seeds and bulbils (see below), thus enabling comparison of genetic differentiation and structure despite of their assignment to different sections of the genus (Peterson et al. 2008). In partially clonal organisms capable of vegetative reproduction, for example the Gagea species investigated, a significant portion of genets should include several individual plants. The members of each of these clones are copies of the initial, sexually derived mother plant and should be genetically identical with this plant and with each other. However, for genotyping approaches a small amount of genetic divergence is usually tolerated (ArnaudHaond et al. 2007). In this study, no context data like known clonal relationships (cf. Pfeiffer 2007; Pfeiffer et al. 2008), or sex- and distance-data (Schnittler and Eusemann 2010), were available apart from spatial distance; therefore thresholds were assessed using the histogram method (compare Rogstad et al. 2002; Douhovnikoff and Dodd 2003; Meirmans and Van Tienderen 2004). The derived threshold values of \0.05 simple matching distance in G. lutea, and even lower values in G. spathacea (Fig. 1), fit well with ranges published for other taxa (compare, e.g., Douhovnikoff and Dodd 2003). The repeat runs of identical samples (Bonin et al. 2004; Pompanon et al. 2005; Arnaud-Haond et al. 2007) also revealed small error rates and different sources of errors, either biologically and experimentally caused: Some differences between clone mates are consistent between repetitions, i.e. must be caused by ‘‘real’’ molecular divergence, either because of somatic mutations (which

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vegetative reproduction on small spatial scales. On larger scales, sexual reproduction becomes more important. This is indicated by the low percentage of neighbouring transect samples belonging to the same genet and by the rather large overall number of genets detected (up to 30 per population). The autocorrelation test reveals a slight positive correlation of spatial and genetic distances, at least for neighbouring patches (populations A, B) or up to 6 m distance (population C, Fig. 5), i.e. vegetative reproduction is mainly restricted to slightly more than the patch scale. However, in a few cases single genets can spread over larger distances (Figs. 2, 3). In these cases, bulbils must have been dispersed beyond the normal small scale. These genetic patterns reflect the combined reproduction and habitat colonisation strategy of G. lutea: By seeds, new habitats at some distance from the source population can be reached and colonised, whereas bulbils contribute to persistence and local spread of established genets (Ronsheim 1997; compare, e.g., Ziegenhagen et al. 2003; Pfeiffer et al. 2008 for mixed clonal and sexual reproduction). Vegetative reproduction is of special importance only in younger establishment phases of genets, because older plants switch to sexual reproduction and stop forming bulbils once they start flowering (Schnittler et al. 2009). This only partially clonal strategy is reflected by the comparatively high genotypic richness within populations (Table 1) and the low numbers of samples per clone. In G. spathacea, most of the analysed fragments were monomorphic in all three populations. As a result, maximum differences between any two samples never exceeded seven fragments (0.023 simple matching distance), all samples were assigned to a single clonal genet. The maximum divergence in G. spathacea is smaller than the threshold for genotypic identity applied for G. lutea. However, these thresholds are derived from highly polymorphic and usually species-specific AFLP profiles, and can hence not be generalised or directly compared between species or studies. In addition, the AFLP profiles of G. spathacea contained several very small peaks, which probably result from fragments present in low copy numbers (that may include some with somatic mutations) in the polyploid genome (marker dosage effect). Because some of these fragments could not be scored unambiguously for most samples of the populations, they were omitted from the analysis. This more restrictive scoring might have slightly diminished the already low genotypic diversity of G. spathacea. Initial tests with primer combinations I and IV revealed the same pattern like primers II and III in G. spathacea. Therefore, the extremely low genotypic diversity is not caused by inadequate resolution of the AFLP fingerprinting but is inherent in the studied populations: All

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analysed samples belong to the same genet, i.e. derived from a common ancestor without sexual reproduction, despite geographical distances up to 30 km between the three populations investigated. However, the occurrence of a single ‘‘private fragment’’ in about half of the samples of population A indicates slight differentiation between populations. This divergent fragment is probably the result of somatic mutations causing the gain or loss of respective restriction and/or primer binding sites. Such mutations will be passed on to all clonal descendants and may in the long run lead to a more pronounced divergence of the isolated stands, despite common clonal origin, probably even to total sexual extinction (compare Eckert 2002; Honnay and Bossuyt 2005). The present monoclonality of G. spathacea can only be explained by sexual sterility and complete reliance on vegetative reproduction, followed by slow stepwise dispersal of the bulbils over large times, perhaps accompanied by rare large-scale translocations by migrating animals. In G. spathacea, sexual reproduction is severely hampered by the high and anorthoploid ploidy level (nonaploid state), with malfunctioning meiosis resulting in irregular formation of gametes (Westerga˚rd 1936). Although some authors mention rare fruiting (Kalheber and Kalheber 1966; Seybold 1998; D. Kunzmann, personal communication) and/or describe seed characteristics (Tomovic and Niketic 2005), we have been unable to observe fruiting plants in Western Pomerania (Schnittler et al. 2009) nor to obtain detailed information about successful sexual reproduction (confirmed by I. Levichev, personal communication). This fact was also stated and commented on by Westerga˚rd (1936) for Danish populations. He doubted the capability of normal sexual reproduction in G. spathacea, but did not exclude a parthenogenetic reproduction a priori. Using molecular methods, such apomictic clones of genetically identical offspring from unreduced somatic cells replacing zygotes cannot be distinguished from those produced by bulbils. Their migration ability, however, should be higher, because of the more mobile seeds. Yet this dispersal strategy remains highly doubtful because we have no confirmed reports of seed set. However, total sexual sterility cannot be accepted a priori. At least some pollen grains seem to develop normally and might be fertile. If the same applies for egg cells, sexual reproduction is potentially possible. Over long periods of time, rare sexual events are conceivable, limiting sexual reproduction to ‘‘windows of opportunity’’ under suitable conditions. Alternatively, severely reduced fertility might not prevent cross-pollination with other Gagea species, especially with the co-occurring and fully fertile G. lutea. Generally, hybridisation is common in the genus (Peruzzi 2008; Peruzzi et al. 2008; Peterson et al. 2008,


2009 and references therein) but has not yet been documented for G. spathacea as a parent. Some authors hypothesise that G. spathacea is a hybridogeneous taxon itself (Westerga˚rd 1936; Mesıćek and Hrouda 1974). If this is true, it may further diminish chances of successful sexual reproduction in this anorthoploid (nonaploid) taxon, because it complicates pairing of homologous chromosomes during meiosis. If the species originated from multiple hybridisation events, substantial initial genetic variability must be assumed. Subsequent changes may be caused by somatic mutations, rare sexual reproduction (both increasing diversity), or selection (decreasing diversity). Generally, these mechanisms are of special importance during initial colonisation (compare Kimpton et al. 2002) as they determine not only the initial diversity but may also affect the future diversity of populations. However, as for a singular hybrid origin, locally this could result in dominance of single or few welladapted genets with good migration and/or persistence features (see also Brochmann and Ha˚pnes 2001). In this study, this question cannot be satisfactorily addressed, because the analysed populations grow within the same region, although separated by up to 30 km. To assess genetic variation at the species level and to elucidate the origin and colonisation history of G. spathacea (and its monotypic section Spathaceae), AFLP screening of populations from larger parts of the current distribution area would be desirable. Respective analyses are in progress. To answer our initial question: Yes, sex makes a fundamental difference, for both genotypic diversity and structure in the two co-occurring Gagea species! In G. lutea, the mixed vegetative and sexual reproductive strategy is reflected by rather high diversity, with the different dispersal distances of the respective diaspores (bulbils vs. seeds) resulting in spatial structuring of the detected divergence. With G. spathacea we seemingly discovered the rare case of an entirely clonal species not reproducing by agamospermy. Nevertheless, G. spathacea occupies a small yet distinct North-Central European range (compare Meusel et al. 1965; Hulteń and Fries 1986). In contrast with other German species of the genus Gagea most populations inhabit natural to near-natural forests (Henker 2005). We thus can assume that the species maintains this range mainly by natural (non-anthropogenic) dispersal via bulbils. As stated by Schnittler et al. (2009) the extremely high bulbil production (up to 50 bulbils per plant and year) seems to be the key adaptation for the survival of this sterile species. These features distinguish G. spathacea from other taxa with asexual populations or lineages (but see S. bulbiferum, Tsujimura and Ishida 2008), where monoclonality is most likely the result of ongoing extinction (as in the relictual W. nobilis, Peakall et al. 2003), recent origin (e.g. in hybridogeneous, partially

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fertile Saxifraga svalbardensis with very low (to absent) genetic diversity; Brochmann and Ha˚pnes 2001), or an ancient (singular) hybridisation event, as suggested for the narrow endemic Lomatia tasmanica (Lynch et al. 1998), but most often it is evident only in parts of species’ distribution ranges. Examples of the latter case include Alpine populations of S. cernua (Bauert et al. 1998), some North American populations of B. umbellatus (Eckert et al. 2003), and probably monomorphic populations of G. bohemica (e.g. from Czech Republic, Peterson et al. 2010), which are all able to persist by vegetative reproduction via bulbils, but—in contrast with G. spathacea—also reproduce sexually in other parts of their respective ranges. Acknowledgments We want to thank Susanne Starke and several student collaborators for their help in sampling; David Harter, Noreen Formella, and Anna Roschanski for help with DNA extraction and AFLP reactions; and Dr Igor Levichev and Dr Angela Peterson for sharing and discussion of ideas. Permission from environmental authorities (Staatliches Amt fu¨r Umwelt und Natur Ueckermu¨nde) to collect plant material in the nature reserve ‘‘Eldena’’ is gratefully acknowledged. This study was supported by a Ka¨the-Kluth scholarship of the Ernst-Moritz-Arndt-University Greifswald to T.P.

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