Conserv Genet (2012) 13:1305–1315 DOI 10.1007/s10592-012-0373-7

RESEARCH ARTICLE

Strong inbreeding depression and local outbreeding depression in the rewarding orchid Gymnadenia conopsea Nina Sletvold • John Magne Grindeland ˚ gren Pengjuan Zu • Jon A

•

Received: 28 March 2012 / Accepted: 28 May 2012 / Published online: 13 June 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Conservation of species threatened by habitat fragmentation is a major global challenge, and determining the genetic and demographic processes associated with isolation and reductions in population size will be critical for an increasing number of species. We conducted controlled crosses and field germination experiments to quantify the effects of inbreeding and outbreeding in the declining orchid Gymnadenia conopsea in two Norwegian populations that differ in size. We further compared our results with published estimates of inbreeding depression in orchids. There was severe inbreeding depression for seed production (d = 0.41–0.67) and germination (d = 0.46–0.66) in both populations, with stronger inbreeding depression in the large population. Compared to outcrossing, selfing reduced female fitness (number of seeds per fruit 9 proportion of seeds germinating) by 76 and 54 % in the large and small

Electronic supplementary material The online version of this article (doi:10.1007/s10592-012-0373-7) contains supplementary material, which is available to authorized users. ˚ gren N. Sletvold (&)  P. Zu  J. A Plant Ecology and Evolution, Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Norbyva¨gen 18 D, SE-752 36 Uppsala, Sweden e-mail: [email protected] N. Sletvold NTNU Museum of Natural History and Archaeology, 7491 Trondheim, Norway J. M. Grindeland Sør-Trøndelag University College, 7004 Trondheim, Norway Present Address: P. Zu Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland

population, respectively. The magnitude of inbreeding depression for seed production was higher than the average reported for orchids, while for germination it was similar to earlier estimates. The large population also experienced considerable outbreeding depression for seed production (d = 0.23–0.27), germination (d = 0.33) and female fitness (d = 0.47) following crosses with a population 1.6 km away. The strong inbreeding depression indicates that both populations harbour a substantial genetic load, and suggests that fragmentation may reinforce population decline in G. conopsea via increased inbreeding. Moreover, the local outbreeding depression indicates substantial genetic differentiation at a moderate spatial scale. This has important implications for the use of crosses between populations or plant translocations as conservation approaches. Keywords Conservation genetics  Genetic structure  Habitat fragmentation  Inbreeding depression  Mating system evolution  Outbreeding depression

Introduction At present, many plant species occur in small and isolated populations compared to earlier distributions due to human-induced habitat destruction and fragmentation or to changes in land use (Primack 2006). This ongoing fragmentation renders populations more vulnerable to demographic, environmental and genetic stochasticity (Lande 1988), and may influence population viability (Lienert 2004) and evolutionary processes (Eckert et al. 2010; Weber and Kolb 2011). To promote long-term population persistence and optimize conservation efforts in declining species, it is critical to understand the integrated effects of the various demographic and genetic processes associated

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with fragmentation (Oostermeijer et al. 2003; Aguilar et al. 2006; Ouborg 2010). A severe reduction in population size usually leads to a direct loss of genetic diversity, and also increases the likelihood of further loss through genetic drift and inbreeding (Young et al. 1996). In animal-pollinated plants, pollinators directly influence gene flow and mating patterns, and the potential disruption of plant–pollinator interactions by fragmentation has consequently received ˚ gren particular attention (Lennartsson 2002; Waites and A 2004; Kolb 2008; Klank et al. 2010). Pollinators often move less among patches and visit more flowers per plant in small or sparse patches compared to large or dense ones (Goverde et al. 2002; Grindeland et al. 2005), indicating a higher number of matings between closely related individuals (biparental inbreeding) and, in self-compatible species, also a higher rate of geitonogamous self-pollination (Dudash and Fenster 2000). In Betonica officinalis, experimental fragmentation during 6 years increased selfing rate and reduced genetic diversity among emerging seedlings (Rusterholz and Baur 2010), demonstrating that fragmentation may lead to rapid genetic erosion via effects on pollination. In the short term, loss of genetic diversity may lead to lower individual fitness and increased population extinction risk, while in the long term, it may constrain the potential for evolutionary response to changing conditions (Ellstrand and Elam 1993; Dierks et al. 2012). The magnitude of inbreeding depression is expected to evolve with the selfing rate, and the relationship between these two factors will depend on the genetic basis of inbreeding depression (Charlesworth and Charlesworth 1987). Most studies suggest that inbreeding depression primarily results from exposure of recessive harmful alleles (reviewed by Charlesworth and Willis 2009), allowing selfing populations to partially purge their genetic load (Husband and Schemske 1996). A recent meta-analysis showed that across species, the magnitude of inbreeding depression increases with plant population size, as expected if species occurring in small populations share a history of inbreeding (Angeloni et al. 2011). Fragmentation and increased isolation may also limit gene flow among populations and promote among-population genetic differentiation. Such differentiation could evolve both because of genetic drift and because of divergent selection, leading to local adaptation (Linhart and Grant 1996). If genetic divergence is substantial, crosses between populations can lead to outbreeding depression (Dudash and Fenster 2000). Such effects have been reported even within-populations over relatively short distances (Waser and Price 1989; McCall et al. 1991; Quilichini et al. 2001; Grindeland 2008). Alternatively, inter-population crosses may restore genetic diversity and increase progeny fitness (i.e. heterosis), leading to higher

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population viability through ‘‘genetic rescue’’ (cf. Ingvarsson 2001). The spatial scale at which these processes operate is of great concern for conservation planning and potential reintroduction programs (Vergeer et al. 2004). One group of plants that is strongly affected by fragmentation is the orchids (Kull and Hutchings 2006; Jacquemyn et al. 2007). Many orchids have elaborate mechanisms that promote cross-pollination (Darwin 1862), and are expected to have high outcrossing rates (Tremblay et al. 2005). This should allow the accumulation of a large genetic load that will be expressed upon inbreeding (Husband and Schemske 1996). Many orchids also have dust-like seeds, potentially widely dispersed by wind, which should promote gene flow (Arditti and Ghani 2000). In line with this, several studies have documented high levels of within-population genetic diversity, and weak genetic differentiation among populations at marker loci (Alexanders˚ gren 2000; Gustafsson 2000; Machon et al. 2003). son and A If genetic structuring is weak, crosses between populations could be an efficient way of restoring genetic diversity and increasing population fitness after fragmentation. In general, previous studies have documented significant inbreeding depression in the Orchidaceae, with early stage inbreeding depression increasing from seed to protocorm production (Smithson 2006). Due to the minute seed size and slow growth of orchids, most studies of inbreeding depression estimate fitness as fruit set, the proportion of seeds with embryos, or seed viability (cf. Tremblay et al. 2005). Some have studied the germination and growth of seeds in laboratory culture (e.g., Ferdy et al. 2001; Bellusci et al. 2009), and a recent greenhouse study followed individuals to the juvenile stage (Juillet et al. 2007). However, we are aware of only one study that has used field growth techniques to estimate inbreeding depression in the source population (Smithson 2006). Inbreeding depression has repeatedly been shown to depend on environmental factors, and is in general lower when estimated under benign greenhouse conditions compared to in the wild (Cheptou and Donohue 2011). To reliably estimate the consequences of inbreeding and outbreeding, it is critical to document effects on demographic rates under field conditions. In the present study, we quantify the effects of inbreeding and outbreeding on seed production and field germination rates in 2 years in two Norwegian populations of the nectar-producing orchid Gymnadenia conopsea. In Norway, G. conopsea has been one of the more common orchid species, but is currently experiencing substantial population declines in lowland areas due to changes in land use (Ka˚la˚s et al. 2010). G. conopsea is self-compatible, but depends on pollinators for successful fruit set (no autogamy; Sletvold, unpublished data). It is visited by diurnal and nocturnal hawkmoths, butterflies and flies (Sletvold ˚ gren 2010; Sletvold et al. 2012), and is expected to be and A

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predominantly outcrossing. The two study populations differ in size and pollinator assembly (Sletvold et al. 2012). Here, we specifically ask whether the magnitude of inbreeding depression varies among populations and years, and whether there is evidence of local outbreeding depression.

Materials and methods Study species and field sites Gymnadenia conopsea (L.) Br. R. is a terrestrial orchid, distributed across Eurasia. It is associated with agricultural landscapes, growing on calcareous soils in traditional haymeadows, grazed meadows and margins of marshes and fens. Considerable genetic variation is documented within European populations at both allozyme (Soliva and Widmer 1999) and microsatellite loci (Gustafsson 2000; Gustafsson and Sjo¨gren-Gulve 2002; Campbell et al. 2007). G. conopsea is long-lived, tuberous and non-clonal. At the study sites, individuals emerge aboveground in late May to early June, and flowering individuals produce a single inflorescence with ca. 10–70 fragrant, pink flowers 3–4 weeks later. Each flower contains two pollinia, which are picked up singly or in pairs by insect visitors. The pollinium usually disintegrates and massulae from a single pollinium is likely to be deposited on multiple flowers (sectile pollinium). In the study populations, most pollinia are in the correct position to touch the stigmatic surface immediately after withdrawal from the anther (no evident pollinium reconfiguration; cf. Darwin 1862). Fruits mature 3–6 weeks after pollination, and the minute seeds are winddispersed during autumn after capsule dehiscence. High levels of fruit set are common in the study populations (60–80 %), but fruit production and fruit mass are pollen ˚ gren 2010). limited (Sletvold and A The study populations are located within two nature reserves in Central Norway, Sølendet (62°400 N, 11°500 E) and Ta˚gdalen (63°030 N, 9°050 E), separated by 145 km. At Sølendet, multiple subpopulations of G. conopsea are found scattered throughout an area of ca. 300 ha, usually with thousands of flowering individuals in total. At Ta˚gdalen, G. conopsea is restricted to a ca. 500 9 200 m area, typically containing 100–600 flowering individuals. A few other small patches with G. conopsea can be found within *10 km distance of the Ta˚gdalen population. Crossing treatments To quantify inbreeding depression we conducted controlled crosses at Sølendet in 2008, and at both sites in 2009. Sampling at Sølendet was independent between years. At

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Sølendet, we also quantified the effects of between-subpopulation crosses in both years. In 2008, we randomly selected 50 plants in the focal subpopulation at Sølendet, and in 2009 we included 40 plants at Sølendet and 35 plants at Ta˚gdalen. Plants were marked at the onset of flowering. To allow comparison of our crossing treatments with natural pollination, plants were left open for pollinator access during the first 3–7 days of flowering (O treatment). To prevent further pollinator visitation we caged all plants. On each plant, consecutive focal flowers (buds and newly opened) were marked, emasculated and randomly assigned to the pollination treatments. Newly opened flowers were determined to be virgin by the presence of two pollinia and the absence of massulae on the stigma. In both populations, treatments included self-pollination (S) and within-population outcrossing (W; distance to pollen donors 5–20 m). At Sølendet, we also crossed the experimental plants with pollen from another subpopulation 1.6 km away (betweensubpopulation cross, B). Because pollen transfer between subpopulations implied a 20 min delay from pollinia removal to pollination, and because pollen viability may deteriorate with time since removal (Kearns and Inouye 1993), we tested for a potential negative effect of delay per se by including a fourth treatment at Sølendet in 2009: within-population outcrossing with time-delay. We used cocktail sticks to collect pollen, and pollinations were performed by rubbing 1–2 pollinia across each stigma, saturating the surface with pollen. We used a minimum of two pollen donors for each flower in outcrossing treatments. Plants were caged until fruit maturation. The mature, intact capsules were collected, brought to the laboratory and stored at room temperature. In 2008, we lost six plants due to trampling by reindeer in the Sølendet population, and in 2009, four plants were grazed by sheep in the Ta˚gdalen population.

Fitness estimates Fruits were dried at room temperature for 2–3 weeks. In the laboratory, we determined fruit mass to the nearest lg and dissected the seeds from the fruits and obtained total seed mass for one fruit per plant 9 treatment combination. In 2008, we counted the seeds from all dissected fruits to obtain total seed number (n = 176 fruits). We also determined the mass of 100 seeds with embryos to the nearest lg, and estimated total number of seeds from these measurements. The counted and estimated number of seeds per fruit were highly correlated within all cross treatments (rs [ 0.88). In 2009, we used the latter method (determining total seed mass and counting and weighing 100 seeds) to estimate total number of seeds for all fruits.

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To quantify seed germination, we counted a maximum of three batches of 30–100 seeds with embryos from each dissected fruit, and packed each batch in a 50 9 50 mm square of fine mesh (50 lm). Seed packets were constructed by folding the mesh in half and securing the edges within a slide mounting frame (cf. Rasmussen and Whigham 1993). We compared field germination rates of seeds resulting from open-pollination and experimental crosses by conducting a seed burial experiment including 35 maternal families from the Sølendet population and 31 maternal families from the Ta˚gdalen population. In autumn, one seed packet per treatment and maternal family was planted horizontally into the soil at ca. two cm depth at three different sites in the source populations, in close proximity to established individuals. After 1 year, all seed packets were retrieved and returned to the laboratory. Recovered seeds were counted and the proportion that had germinated (seeds with fully swollen embryos with rhizoids, prior to protocorm formation) was determined. For all maternal plants and cross types we estimated female fitness as number of seeds per fruit multiplied by the proportion of seeds germinating. Statistical analyses We assessed the effect of time-delay on fruit mass and seed production by one-way ANOVA, comparing within-population outcrossing with and without time-delay (data from Sølendet 2009). Fruit mass and seed production did not differ significantly between treatments (F1,78 = 0.03, P = 0.87 and F1,78 = 0.07, P = 0.80; respectively), indicating that pollen quality did not deteriorate during the 20 min transfer period. The time-delay treatment was excluded from further analyses. We used mixed model ANOVA to determine the effects of cross type and year (fixed effects; data from the Sølendet population), and cross type and population (fixed effects; data from 2009, excluding the between-population cross at Sølendet) on number of seeds per fruit and the proportion of seeds germinating. Maternal family nested within year (Sølendet) or population (2009) was included as a random effect. If a statistically significant cross type 9 year or cross type 9 population interaction was detected, we further tested the effect of cross type separately for each year and population. We used planned contrasts to examine differences in seed production and the proportion of seeds germinating between cross types, where all other crosses were compared to the within-population cross treatment (W vs. S, B and O treatments, respectively). Significance tests were in all cases two-tailed, i.e. with no assumption on the direction of the difference. Analyses were performed with the MIXED procedure in SAS 9.2 (SAS Institute Inc., Cary, NC, USA).

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We report inbreeding depression (dID) and outbreeding depression (dOD) as relative performance for each maternal family: d = (wo - wt)/max (wt, wo), where wo is performance of the within-population cross and wt is perfor˚ gren and mance of the self or between-population cross (A Schemske 1993). Relative performance is a symmetrical function with values between -1 and 1, and positive values mean that the within-population cross outperformed the cross type to which it was compared. Population-level effects are reported as mean relative performance across families. To determine whether loci contributing to fitness reductions had similar effects on the two components of fitness examined, we calculated the correlations between maternal family inbreeding depression for seed production and germination, and between maternal family outbreeding depression for seed production and germination. To determine whether loci causing inbreeding depression also contribute to outbreeding depression, we calculated the correlation between maternal family inbreeding and outbreeding depression for each trait. To compare our results with published estimates of inbreeding depression we used three earlier reviews of inbreeding depression in orchids (Tremblay et al. 2005; Jersa´kova´ et al. 2006; Smithson 2006) and included studies that had compared the effects of self- and cross-pollination on seed production and/or germination. In addition, we searched the literature to identify recent studies providing data on inbreeding depression in orchids. For each lifehistory stage, we calculated mean inbreeding depression across species. For species with more than one estimate, we first calculated a mean based on estimates in different studies or populations.

Results There was strong inbreeding depression for both seed production and germination in both populations, and there was also strong outbreeding depression following crosses with a subpopulation 1.6 km away in the large Sølendet population. Number of seeds Compared to within-population outcrossing, all other cross types reduced the number of seeds per fruit (Table 1; Fig. 1a). In the Sølendet population, fruits produced following within-population outcrossing contained more seeds than fruits produced by selfing (Table 2; contrast F1,246 = 161.7, P \ 0.0001), between-population outcrossing (F1,246 = 30.0, P \ 0.0001), or open-pollination (F1,246 = 7.33, P = 0.0073). The effect of cross type on seed production did not differ significantly between years

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Table 1 Trait mean ± SD following within-population outcrossing (W), self-pollination (S), between-subpopulation crosses (B, only Sølendet), and open-pollination (O) in the G. conopsea populations at Sølendet (nYr1 = 44 and nYr2 = 40) and Ta˚gdalen (n = 31) Trait

W

S

B

dID

O

dOD

Fruit mass (mg) Søl Yr1

6.88 ± 0.39

4.57 ± 0.32

5.45 ± 0.33

5.97 ± 0.35

0.33 (-0.17 to 0.66)

0.18 (-0.17 to 0.66)

Søl Yr2 Ta˚g

7.93 ± 0.41

4.23 ± 0.26

5.05 ± 0.34

6.66 ± 0.39

0.37 (-0.51 to 0.89)

0.30 (-0.76 to 0.78)

11.33 ± 0.47

7.33 ± 0.38

9.60 ± 0.47

0.35 (0.10 to 0.76)

Number of seeds Søl Yr1

2143 ± 1393

810 ± 677

1509 ± 1119

1713 ± 1286

0.59 (-0.44 to 1.00)

0.27 (-0.58 to 0.74)

Søl Yr2 Ta˚g

2650 ± 1747 3481 ± 1751

657 ± 684 1972 ± 1097

1498 ± 1002

2220 ± 1580 3037 ± 1919

0.67 (-0.51 to 1.00) 0.41 (0.09 to 0.79)

0.23 (-0.91 to 0.97)

0.19 ± 0.23

0.28 ± 0.23

0.66 (-0.81 to 1.00)

0.33 (-0.85 to 1.00)

0.32 ± 0.22

0.46 (-1.00 to 1.00)

Germination Søl Ta˚g

0.30 ± 0.19

0.11 ± 0.23

0.28 ± 0.17

0.16 ± 0.24

854 ± 796

210 ± 482

1141 ± 1022

338 ± 587

Female fitness Søl Ta˚g

347 ± 492

621 ± 616

0.76 (-0.81 to 1.00)

1095 ± 1112

0.54 (-1.00 to 1.00)

0.47 (-0.74 to 1.00)

Mean and family range in inbreeding depression (dID) and outbreeding depression (dOD, only Sølendet) are also given

W

S

B

O

0.5

5000

(a)

(b) 0.4

4000

Prop. germination

No. seeds per fruit

Fig. 1 The effect of cross treatment on a number of seeds per fruit and b the proportion of seeds germinating in the Sølendet and Ta˚gdalen populations of G. conopsea. Bars indicate means ?1.96 SE; W within-population outcrossing, S self-pollination, B between-subpopulation cross (only Sølendet), and O openpollination

3000 2000 1000 0

SølYr1

SølYr2

0.3 0.2 0.1 0.0

Tåg

Søl

Tåg

Table 2 F ratios and P values from ANOVAs testing for differences in effects of cross type on number of seeds per fruit and proportion of seeds germinating between years in the Sølendet population, and between populations in 2009 Trait

Ta˚gdalen ? Sølendet 2009 (n = 210)

Sølendet 2008 ? 2009 (n = 336) Cross

Yr

Cross 9 Yr

Cross

Pop

Cross 9 Pop

F

P

F

P

F

P

F

P

F

P

F

P

Number of seeds

59.9

<0.0001

0.60

0.44

2.02

0.083

76.8

<0.0001

17.5

<0.0001

8.04

0.005

Germination

10.9

<0.0001

21.2

<0.0001

0.31

0.91

0.40

1.04

P \ 0.05 in bold

(Table 2). Inbreeding depression was estimated to 59 and 67 %, and outbreeding depression to 23 and 27 %, respectively. In 2009, seed production was significantly higher among plants in the Ta˚gdalen population compared to plants in the Sølendet population (Tables 1, 2; Fig. 1a). In the Ta˚gdalen population, more seeds per fruit were produced after within-population outcrossing than

following selfing (F1,58 = 56.8, P \ 0.0001) or open-pollination (F1,58 = 5.59, P = 0.022). Inbreeding depression for seed production was 41 %, which was lower than in the Sølendet population the same year (67 %; significant cross type 9 population interaction in Table 2). Number of seeds per fruit varied significantly among maternal families in both populations and years (all P \ 0.0001).

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depression estimates for seed production and germination at Sølendet (r = 0.087, P = 0.63). Estimates of inbreeding and outbreeding depression at Sølendet were positively correlated for the proportion of seeds germinating (r = 0.39, P = 0.025), but not for seed production (r = 0.099, P = 0.52).

Relative performance

1.0 0.8 0.6 0.4

Published estimates of inbreeding depression 0.2 0.0

Søl

Tåg

Fig. 2 Inbreeding depression (solid bar) and outbreeding depression (open bar, only Sølendet) for female fitness (number of seeds per fruit 9 proportion of seeds germinating) in the Sølendet and Ta˚gdalen populations of G. conopsea

Published estimates of inbreeding depression in orchids are shown in Table 3. Across species, mean inbreeding depression for seed production was 33 % (n = 49) and for germination 50 % (n = 7).

Discussion Germination The proportion of seeds germinating varied among cross treatments, but not among populations (Tables 1, 2; Fig. 1b). In the Sølendet population, 30 % of the seeds resulting from within-population outcrossing had germinated the next year, which was a significantly higher proportion than among seeds resulting from selfing (F1,102 = 27.0, P \ 0.0001) or between-population outcrossing (F1,102 = 6.39, P = 0.013), but not different from seeds resulting from open-pollination (F1,102 = 0.41, P = 0.52). There was strong inbreeding depression and outbreeding depression at the germination stage (66 % and 33 %, respectively; Table 1). In the Ta˚gdalen population, 28 % of the seeds resulting from within-population outcrossing germinated, which was a significantly higher proportion than among seeds resulting from selfing (F1,58 = 10.3, P = 0.0022), but not different from seeds resulting from open-pollination (F1,58 = 0.45, P = 0.51). Inbreeding depression for the proportion of seeds germinating was 46 % (Table 1). Germination success varied significantly among maternal families in the Sølendet population (P = 0.025), but not in the Ta˚gdalen population (P = 0.068). Female fitness and correlations among estimates There was strong inbreeding depression for female fitness (number of seeds per fruit 9 proportion of seeds germinating) in both populations (54–76 %), and also strong outbreeding depression in the Sølendet population (47 %; Table 1; Fig. 2). There were no statistically significant correlations between inbreeding depression estimates for seed production and proportion of seeds germinating at Sølendet (r = 0.13, P = 0.35, n = 33) or Ta˚gdalen (r = 0.0022, P = 0.99, n = 30), or between outbreeding

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We documented strong negative effects of inbreeding in both studied populations of G. conopsea, with a 54–76 % fitness reduction at the recruitment stage. This is consistent with a predominantly outcrossing mating system, and suggests that increased inbreeding associated with habitat fragmentation would reduce plant fitness and population viability. In addition, there was strong local outbreeding depression in the large population (47 %), indicating substantial small-scale genetic structuring. This has important implications for potential reintroductions and crossing designs to restore genetic variability. Inbreeding depression The documented inbreeding depression is among the higher estimates reported for orchid species (Table 3), indicating that both G. conopsea populations experience strong selection against selfing. The results suggest that the current trend of declining populations is likely to be accentuated if small population sizes are associated with higher levels of self-pollination (e.g., Raijmann et al. 1994; Johnson et al. 2009). Inbreeding depression was strong for both seed production and germination. The magnitude of inbreeding depression has been found to further increase from the germination to the protocorm stage (Smithson 2006), indicating that present estimates for G. conopsea may be conservative. Future studies should include field experiments that allow the quantification of effects also at later life stages. The strong inbreeding depression suggests a highly outcrossing mating system (Husband and Schemske 1996). In general, rewarding species like G. conopsea are expected to experience mixed mating, because the presence of nectar should induce pollinators to visit multiple flowers per inflorescence (Johnson et al. 2004), which could increase the rate of geitonogamous self-fertilization.

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Table 3 Published studies that have quantified inbreeding depression for seed production (seed mass or % seeds with embryo), seed germination (* field estimate), and juvenile survival in orchids. A full list of references is provided in Electronic Supplementary Material d Seeds

d Germ.

d Surv.

Species

Pollinator

Reward

Anacamptis morio

Bumblebees

0

0.713

Nilsson (1984)

Brownleea coerulea

Long-tongued flies

1

0.552

Larsen et al. (2008)

Brownleea galpinii

Long-tongued flies

1

0.982

Johnson et al. (2003)

Brownleea macroceras

NA

1

0.740

Larsen et al. (2008)

Brownleea parviflora

Bees

1

0.620

Larsen et al. (2008)

Bulbophyllum involutum

Milichiid flies

1

-0.087

Borba et al. (1999)

Bulbophyllum ipanemense

Milichiid flies

1

0.143

Borba et al. (1999)

Bulbophyllum weddellii

Milichiid flies

1

-0.414

Borba et al. (1999) 0.215

References

Caladenia tentactulata

Thynnine wasp

0

-0.004

Catasetum viridiflavum

Euglossine bees

1

0.310

Peakall and Beattie (1996)

Cleistes divaricata

Bees, bumblebees

0

0.391

Comparettia falcata

Hummingbirds

1

-0.014

Cynorkis uniflora

Hawkmoths

1

0.410

Nilsson et al. (1992)

D. incarnata

Bumblebees

0

0.100

Lammi et al. (2003)

Dactylorhiza maculata

Bumblebees, flies

0

0.146

D. praetermissa

Bumblebees

0

0.042

Tremblay et al. (2005) Gregg (1989) Salguero-Farı´a and Ackerman (1999)

Vallius (2000) 0.140

Ferdy et al. (2001), pop. A

D. praetermissa

Bumblebees

0

-0.120

D. praetermissa

Bumblebees

0

0.031

0.382

Ferdy et al. (2001), pop. B

-0.778

Ferdy et al. (2001), pop. C

Dactylorhiza sambucina

Bumblebees

0

0.427

Dactylorhiza sambucina

Bumblebees

0

Disa atricapilla

Wasps

0

Disa draconis

Long-tongued flies

0

0.299

Johnson and Steiner (1997)

Disa ferruginea

Butterflies

0

0.579

Johnson (1994)

Disa pulchra

Long-tongued flies

0

0.501

Diuris maculata

Bees

0

-0.128

Beardsell et al. (1986)

Epidendrum ciliare

Moths

0

-0.061

Ackerman and Montalvo (1990)

Goodyera oblongifolia

Bumblebees

1

0.333

Kallunki (1981)

Goodyera pubescens

Bumblebees

1

-0.162

Kallunki (1981)

Goodyera repens

Bumblebees

1

0.433

Kallunki (1981)

Goodyera tesselata

Bumblebees

1

-0.102

Gymnadenia conopsea

Hawkmoths, butterflies, flies

1

0.586

Gymnadenia conopsea

Hawkmoths, butterflies, flies

1

0.670

Gymnadenia conopsea

Hawkmoths, butterflies

1

0.413

Ionopsis utricularoides

NA

0

0.000

Montalvo and Ackerman (1987)

Leporella fimbriata

Ants

0

0.194

Peakall (1989)

Listera cordata

Fungus gnats

1

0.080

Mele´ndez-Ackerman and Ackerman (2001)

Listera ovata

Beetles, wasps

1

0.086

Nilsson (1981)

Microtis parviflora

Ants

1

0.067

Peakall and Beattie (1989)

Mystacidium venosum

Hawkmoths

1

0.625

Luyt and Johnson (2001)

Orchis mascula

Bees, bumblebees

0

0.204

Nilsson (1983a)

Orchis spitzelii

Bumblebees

0

0.379

Fritz (1990)

Platanthera bifolia

Hawkmoths

1

0.480

Nilsson (1983)

Platanthera chlorantha

Hawkmoths

1

0.675

Nilsson (1983)

Platanthera ciliaris

Butterflies

1

0.132

Gregg (1990)

Platanthera leucophaea

Hawkmoths

1

0.426

Wallace (2003)

Platanthera stricta

Bumblebees, moths, flies

1

0.451

Patt et al. (1989)

Satyrium bicorne

Moths

1

0.776

Ellis and Johnson (1999)

Satyrium coriifolium

Sunbirds

1

0.553

Ellis and Johnson (1999)

Satyrium erectum

Bees

1

0.750

Ellis and Johnson (1999)

Satyrium longicauda

Hawkmoth

1

0.749

Johnson et al. (2009)

Nilsson (1980) 0.600

0.132

0.750

Juillet et al. (2007) Steiner et al. (1994)

Johnson (2000)

Kallunki (1981) 0.655*

Sletvold et al. this study, pop. S, Yr1 Sletvold et al. this study, pop. S, Yr2

0.458*

Sletvold et al. this study, pop. T

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Conserv Genet (2012) 13:1305–1315

Table 3 continued Species

Pollinator

Reward

d Seeds

d Germ.

d Surv.

References

Serapias cordigera

Bees

0

0.711

0.929

Bellusci et al. (2009)

Serapias parviflora

Bees

0

0.242

0.376

Bellusci et al. (2009)

Serapias vomeraceae

Bees

0

0.755

0.929

Bellusci et al. (2009)

Vanilla claviculata

Euglossine bees

1

0.284

Mean ± SD

Tremblay et al. (2005)

0.327 ± 0.311

0.503 ± 0.370

0.750

Estimates of d are calculated from mean values reported in the listed reference for each species

Indeed, preliminary tests with staining of pollinia in the Sølendet population indicate that a substantial proportion of natural pollinations represents geitonogamous pollinations (Zu et al. unpublished data), but how this translates into realized outcrossing rates is not known. It may be that all plants receive sufficient cross-pollen for high levels of seed production regardless of some self-pollination. Orchids with sectile pollinia often receive a mixture of self- and cross-pollen (Johnson et al. 2005), and although rates of self-pollination was higher in small populations of the orchid Satyrium longicauda, seed production was not related to population size (Johnson et al. 2009). Outbreeding depression The documented outbreeding depression at Sølendet may be an effect of adaptive differentiation between the two subpopulations expressed at early life-history stages, or of negative epistatic gene interactions (cf. Ritland and Ganders 1987). Species that are outbreeding and disperse their seeds across large distances are not expected to express outbreeding depression after crosses between nearby populations in similar habitats. The documented 47 % fitness reduction following crosses between two subpopulations 1.6 km apart and presumably well connected by gene flow is thus surprising. In comparison, Ferdy et al. (2001) documented outbreeding depression for germination in crosses between populations of Dactylorhiza praetermissa separated by a markedly longer distance than in the present study (78 km), whereas Lammi et al. (2003) found no outbreeding depression in crosses of Dactylorhiza incarnata populations separated by 16 km. The extent and geographical scale of adaptive differentiation in G. conopsea is unknown. A wide spectrum of potential mycorrhizal partners has been documented in German populations of G. conopsea (Stark et al. 2009), suggesting that small-scale variation in habitat types and their fungal communities may contribute to local adaptation. However, in the present study, germination was scored prior to protocorm formation, and potential adaptive differentiation should involve processes influencing seed formation, seed viability and germination success in the habitats of the two subpopulations.

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Local genetic structure may also reflect more restricted seed dispersal than expected. Even though the dust-like orchid seeds can achieve long dispersal distances (Arditti and Ghani 2000), most seeds probably fall within close proximity to the maternal plant (Jersa´kova´ and Malinova´ 2007). An increasing number of studies have documented significant spatial genetic structure within orchid populations, mostly attributed to limited seed dispersal (Peakall and Beattie 1996; Chung et al. 2004; Jacquemyn et al. 2007, 2009). Moreover, pollinators of G. conopsea may forage on neighbouring plants, potentially causing biparental inbreeding and creating small-scale genetic structuring (cf. Vekemans and Hardy 2004). Reciprocal germination experiments and information on the structure of genetic variation in the Sølendet subpopulations would help clarify the mechanism behind the reduction in seed production and germination after crosses between subpopulations. Like in most previous studies exploring possible outbreeding depression, reciprocal crosses were not conducted between the two subpopulations at Sølendet. It is therefore not possible to rule out that generally lower quality of pollen in the subpopulation in which pollen donors were sampled contributed to the reduced performance observed after between-subpopulation crosses. However, the habitat in which the second subpopulation grew was very similar to that of the focal subpopulation, and plant vigour did not differ in any obvious way. The magnitude of inbreeding depression for seed production and germination was similar, but estimates were not significantly correlated. This suggests that the effects of loci responsible for fitness reductions differ between these life stages. The same was true regarding outbreeding depression at the two life stages. In contrast, estimates of inbreeding and outbreeding depression for the proportion of seeds germinating were positively correlated, indicating some overlap in loci involved. Recent theoretical models have highlighted complex evolutionary dynamics of inbreeding depression if loci that contribute to inbreeding depression also contribute to local adaptation (Epinat and Lenormand 2009), and the present results suggets that such associations deserve further attention in empirical studies.

Conserv Genet (2012) 13:1305–1315

Population-level effects The effects of inbreeding and outbreeding depression for these early life-history traits on population persistence will depend on how sensitive lifetime fitness is to decreases in seed production and germination probability. In long-lived perennials, seed production is thought to be of minor importance in determining long-term population viability (Franco and Silvertown 2004). However, recent studies suggest that seed production and seedling recruitment may limit population growth in long-lived orchids (Jacquemyn et al. 2010; Sletvold et al. 2010), indicating that processes influencing the seed stage may be critical. It is also likely that the observed effects will persist beyond the recruitment stage. Juillet et al. (2007) examined inbreeding depression beyond the protocorm stage in D. praetermissa, and found strong negative effects on juvenile survival. Costs of reproduction differ between the two studied G. conopsea populations, with significant costs in terms of reduced flower production in the Ta˚gdalen population and reduced survival in the Sølendet population (Sletvold and ˚ gren 2011). We are presently collecting demographic A data to be able to model lifetime effects of reproductive investment and inbreeding and outbreeding depression. Temporal and spatial variation in inbreeding and outbreeding depression Both gene expression and selection are likely to vary among habitats and years, and inbreeding depression has repeatedly been shown to depend on environmental conditions (reviewed by Cheptou and Donohue 2011). However, neither inbreeding nor outbreeding depression for seed production and germination varied significantly among years in the Sølendet population, suggesting weak gene-by-environment interactions for these traits in the 2 years of the study. Environmental effects on inbreeding depression for seed production may be weak compared to those on inbreeding depression for post-dispersal traits, such as seedling survival and growth. Historically, the Ta˚gdalen population is likely to have been much smaller than the Sølendet population, and it is possible that the lower inbreeding depression reflects a history of inbreeding, with partial purging of the genetic load (Husband and Schemske 1996). Direct estimates of self-pollen transfer by the use of pollen staining techniques could reveal whether the populations differ in rates of selfpollination today. Natural pollination In both populations, open-pollinated flowers produced fewer seeds compared to flowers that were cross-pollinated

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by hand, but the proportion of seeds that germinated did not differ between the two treatments. This suggests that seed production of open-pollinated flowers was limited by pollen quantity rather than pollen quality. Had the observed increase in seed production been the result of higher outcrossing (hand-pollinations were performed with pure outcross pollen, whereas natural pollination may involve a mixture of self and outcross pollen), we would have expected also a higher germination rate after cross-pollination by hand. Yet, estimates of rates of self-pollination based on pollen staining in the Sølendet population suggested a substantial fraction of geitonogamous self-pollination (Zu et al. unpublished data). The seemingly contrasting results may be due to differences between experiments in the position of focal flowers. In the present experiment, open-pollinated flowers were all situated in the lower part of the inflorescence. Hawkmoth pollinators typically forage upwards on inflorescences of G. conopsea, and it is likely that the rate of geitonogamous self-pollination increases towards the top of the inflorescence (cf. Barrett et al. 1994).

Conclusions We documented both strong inbreeding depression and local outbreeding depression in an orchid that currently experiences substantial reductions in population size and connectedness. This suggests that fragmentation may further reduce performance of G. conopsea via interactions with pollinators, and that attempts to increase genetic diversity by introducing seeds from other populations or from between-population crosses should consider the scale of genetic structuring. Habitat fragmentation is one of the major drivers of biodiversity loss today, and conservation programmes often include crossing designs or plant reintroductions to counteract negative consequences of reduced population size and increased isolation. In orchids, little is known of the scale of genetic structure and local adaptation, suggesting that such actions may represent risky options for threatened species. Acknowledgments We thank A. Moen, D.-I. Øien and A. Lyngstad for advice and support during fieldwork. Funding from the Norwegian ˚) Research Council (NS) and from the Swedish Research Council (JA is acknowledged.

References ˚ gren J, Schemske DW (1993) Outcrossing rate and inbreeding A depression in two annual monoecious herbs, Begonia hirsuta and B. semiovata. Evolution 47:125–135

123

1314 Aguilar R, Ashworth L, Galetto L, Aizen MA (2006) Plant reproductive susceptibility to habitat fragmentation: review and synthesis through a meta-analysis. Ecol Lett 9:968–980 ˚ gren J (2000) Genetic structure in the nonreAlexandersson R, A warding, bumblebee-pollinated orchid Calypso bulbosa. Heredity 85:401–409 Angeloni F, Ouborg NJ, Leimu R (2011) Meta-analysis on the association of population size and life history with inbreeding depression in plants. Biol Conserv 144:35–43 Arditti J, Ghani AKA (2000) Numerical and physical properties of orchid seeds and their biological implications. New Phytol 145:367–421 Barrett SCH, Harder LD, Cole WW (1994) Effects of flower number and position on self-fertilization in experimental populations of Eichhornia paniculata (Pontederiaceae). Funct Ecol 8:526–535 Bellusci F, Pellegrino G, Musacchio A (2009) Different levels of inbreeding depression between outcrossing and selfing Serapias species. Biol Plant 53:175–178 Campbell VV, Rowe G, Beebee TJC, Hutchings MJ (2007) Genetic differentiation amongst fragrant orchids (Gymnadenia conopsea s.l.) in the British Isles. Bot J Linn Soc 155:349–360 Charlesworth D, Charlesworth B (1987) Inbreeding depression and its evolutionary consequences. Annu Rev Ecol Syst 18:237–268 Charlesworth D, Willis JH (2009) The genetics of inbreeding depression. Nat Rev Genet 10:783–796 Cheptou P-O, Donohue K (2011) Environment-dependent inbreeding depression: its ecological and evolutionary significance. New Phytol 189:395–407 Chung MY, Nason JD, Chung MG (2004) Spatial genetic structure in populations of the terrestrial orchid Cephalanthera longibracteata (Orchidaceae). Am J Bot 91:52–57 Darwin C (1862) On the various contrivances by which British and foreign orchids are fertilised by insects. John Murray, London Dierks A, Baumann B, Fischer K (2012) Response to selection on cold tolerance is constrained by inbreeding. Evolution. doi: 10.1111/j.1558-5646.2012.01604.x Dudash MR, Fenster CB (2000) Inbreeding and outbreeding depression in fragmented populations. In: Young AG, Clarke GM (eds) Genetics, demography and viability of fragmented populations. Cambridge University Press, Cambridge, pp 35–53 Eckert CG et al (2010) Plant mating systems in a changing world. Trends Ecol Evol 25:35–43 Ellstrand NC, Elam DR (1993) Population genetic consequences of small population size: implications for plant conservation. Annu Rev Ecol Syst 24:217–242 Epinat G, Lenormand T (2009) The evolution of assortative mating and selfing with in- and outbreeding depression. Evolution 63:2047–2060 Ferdy JB, Loriot S, Sandmeier M, Lefranc M, Raquin C (2001) Inbreeding depression in a rare deceptive orchid. Can J Bot 79:1181–1188 Franco M, Silvertown J (2004) Comparative demography of plants based upon elasticities of vital rates. Ecology 85:531–538 Goverde M, Schweizer K, Baur B, Erhardt A (2002) Small-scale habitat fragmentation effects on pollinator behaviour: experimental evidence from the bumblebee Bombus veteranus on calcareous grasslands. Biol Conserv 104:293–299 Grindeland JM (2008) Inbreeding depression and outbreeding depression in Digitalis purpurea: optimal outcrossing distance in a tetraploid. J Evol Biol 21:716–726 Grindeland JM, Sletvold N, Ims RA (2005) Effects of floral display size and plant density on pollinator visitation rate in a natural population of Digitalis purpurea. Funct Ecol 19:383–390 Gustafsson S (2000) Patterns of genetic variation in Gymnadenia conopsea, the fragrant orchid. Mol Ecol 9:1863–1872

123

Conserv Genet (2012) 13:1305–1315 Gustafsson S, Sjo¨gren-Gulve P (2002) Genetic diversity in the rare orchid, Gymnadenia odoratissima and a comparison with the more common congener, G. conopsea. Conserv Genet 3: 225–234 Husband BC, Schemske DW (1996) Evolution and timing of inbreeding depression in plants. Evolution 50:54–70 Ingvarsson PK (2001) Restoration of genetic variation lost—the genetic rescue hypothesis. Trends Ecol Evol 16:62–63 Jacquemyn H, Brys R, Hermy M, Willems JH (2007) Long-term dynamics and population viability in one of the last populations of the endangered Spiranthes spiralis (Orchidaceae) in The Netherlands. Biol Conserv 134:14–21 Jacquemyn H, Wiegand T, Vandepitte K, Brys R, Rolda´n-Ruiz I, Honnay O (2009) Multigenerational analysis of spatial structure in the terrestrial, food-deceptive orchid Orchis mascula. J Ecol 97:206–216 Jacquemyn H, Brys R, Jongejans E (2010) Seed limitation restricts population growth in shaded populations of a perennial woodland orchid. Ecology 91:119–121 Jersa´kova´ J, Malinova´ T (2007) Spatial aspects of seed dispersal and seedling recruitment in orchids. New Phytol 176:237–241 Jersa´kova´ J, Johnson SD, Kindlmann P (2006) Mechanisms and evolution of deceptive pollination in orchids. Biol Rev 81: 219–235 ˚ gren J (2004) The effects of nectar addition Johnson SD, Peter CI, A on pollen removal and geitonogamy in the non-rewarding orchid Anacamptis morio. Proc R Soc B 271:803–809 Johnson SD, Neal PR, Harder LD (2005) Pollen fates and the limits on male reproductive success in an orchid population. Biol J Linn Soc 86:175–190 ˚ gren J (2009) Relationships between Johnson SD, Torninger E, A population size and pollen fates in a moth-pollinated orchid. Biol Lett 5:282–285 Juillet N, Dunand-Martin S, Gigord LDB (2007) Evidence for inbreeding depression in the food-deceptive colour-dimorphic orchid Dactylorhiza sambucina (L.) Soo`. Plant Biol 9:147–151 ˚ , Henriksen S, Skjelseth S (eds) (2010) The 2010 Ka˚la˚s JA, Viken A Norwegian Red List for species. Norwegian Biodiversity Information Centre, Trondheim Kearns CA, Inouye DW (1993) Techniques for pollination biologists. University Press of Colorado, Niwot Klank C, Pleuss AR, Ghazoul J (2010) Effects of population size on plant reproduction and pollinator abundance in a specialized pollination system. J Ecol 98:1389–1397 Kolb A (2008) Habitat fragmentation reduces plant fitness by disturbing pollination and modifying response to herbivory. Biol Conserv 141:2540–2549 Kull T, Hutchings MJ (2006) A comparative analysis of decline in the distribution ranges of orchid species in Estonia and the United Kingdom. Biol Conserv 129:31–39 Lammi A, Vallius E, Vauhkonen T, Kuitunen M (2003) Outcrossing, hybridization, pollen quantity, and the evolution of deceptive pollination in Dactylorhiza incarnata. Ann Bot Fenn 40:331–338 Lande R (1988) Genetics and demography in biological conservation. Science 241:1455–1460 Lennartsson T (2002) Extinction thresholds and disrupted plant– pollinator interactions in fragmented plant populations. Ecology 83:3060–3072 Lienert J (2004) Habitat fragmentation effects on fitness of plant populations—a review. J Nat Conserv 12:53–72 Linhart YB, Grant MC (1996) Evolutionary significance of local genetic differentiation in plants. Annu Rev Ecol Syst 27: 237–277 Machon N, Bardin P, Mazer SJ, Moret J, Godelle B, Austerlitz F (2003) Relationship between genetic structure and seed and

Conserv Genet (2012) 13:1305–1315 pollen dispersal in the endangered orchid Spiranthes spiralis. New Phytol 157:677–687 McCall C, Mitchell-Olds T, Waller DM (1991) Distance between mates affects seedling characters in a population of Impatiens capensis (Balsaminaceae). Am J Bot 78:964–970 Oostermeijer JGB, Luijten SH, den Nijs JCM (2003) Integrating demographic and genetic approaches in plant conservation. Biol Conserv 113:389–398 Ouborg NJ (2010) Integrating population genetics and conservation biology in the era of genomics. Biol Lett 6:3–6 Peakall R, Beattie AJ (1996) Ecological and genetic consequences of pollination by sexual deception in the orchid Caladenia tentaculata. Evolution 50:2207–2220 Primack R (2006) Essentials of conservation biology, 4th edn. Sinauer Associates, Sunderland Quilichini A, Debussche M, Thompson JD (2001) Evidence for local outbreeding depression in the Mediterranean island endemic Anchusa crispa Viv. (Boraginaceae). Heredity 87:190–197 Raijmann LEL, van Leuwen NC, Kersten R, Oostermeijer JGB, den Nijs HCM, Menken SBJ (1994) Genetic variation and outcrossing rate in relation to population size in Gentiana pneumonanthe L. Conserv Biol 8:1014–1026 Rasmussen HN, Whigham DF (1993) Seed ecology of dust seeds insitu—a new study technique and its application in terrestrial orchids. Am J Bot 80:1374–1378 Ritland K, Ganders FR (1987) Crossability of Mimulus guttatus in relation to components of gene fixation. Evolution 41:772–786 Rusterholz H-P, Baur B (2010) Delayed response in a plant–pollinator system to experimental grassland fragmentation. Oecologia 163:141–152 ˚ gren J (2010) Pollinator-mediated selection on floral Sletvold N, A display and spur length in the orchid Gymnadenia conopsea. Int J Plant Sci 171:999–1009 ˚ gren J (2011) Among-population variation in costs of Sletvold N, A reproduction in the long-lived orchid Gymnadenia conopsea: an experimental study. Oecologia 167:461–468 Sletvold N, Øien D-I, Moen A (2010) Long-term influence of mowing on population dynamics in the rare orchid Dactylorhiza

1315 lapponica: the importance of recruitment and seed production. Biol Conserv 143:747–755 ˚ gren J (2012) Separating Sletvold N, Trunschke J, Wimmergren C, A selection by diurnal and nocturnal pollinators on floral display and spur length in Gymnadenia conopsea. Ecology. doi: 10.1890/11-2044 Smithson A (2006) Pollinator limitation and inbreeding depression in orchid species with and without nectar rewards. New Phytol 169:419–430 Soliva M, Widmer A (1999) Genetic and floral divergence among sympatric populations of Gymnadenia conopsea s.l. (Orchideaceae) with different flowering phenology. Int J Plant Sci 160:897–905 Stark C, Babik W, Durka W (2009) Fungi from the roots of the common terrestrial orchid Gymnadenia conopsea. Mycol Res 113:952–959 Tremblay RL, Ackerman JD, Zimmerman JK, Calvo RN (2005) Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversification. Biol J Linn Soc 84:1–54 Vekemans X, Hardy OJ (2004) New insights from fine-scale spatial genetic structure analyses in plant populations. Mol Ecol 13: 921–935 Vergeer P, Sonderen E, Ouborg NJ (2004) Introduction strategies put to the test: local adaptation versus heterosis. Conserv Biol 18:812–821 ˚ gren J (2004) Pollinator visitation, stigmatic pollen Waites AR, A loads, and among-population variation in seed set in Lythrum salicaria. J Ecol 92:512–526 Waser NM, Price MV (1989) Optimal outcrossing in Ipomopsis aggregata: seed set and offspring fitness. Evolution 43: 1097–1109 Weber A, Kolb A (2011) Evolutionary consequences of habitat fragmentation: population size and density affect selection on inflorescence size in a perennial herb. Evol Ecol 25:417–428 Young A, Boyle T, Brown T (1996) The population genetic consequences of habitat fragmentation for plants. Trends Ecol Evol 11:413–418

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