reproductive biology, genetic variation and conservation of the rare endemic dysploid delphinium...

ELSEVIER Biological Conservation 86 (1998) 57--66

BIOLOGICAL CONSERVATION

Reproductive biology, genetic variation and conservation of the rare endemic dysploid Delphinium bolosii (Ranunculaceae)

Maria Bosch, Joan Simon, Juli Molero, C sar Blanch * Laboratori de Botdnica, Facultat de Farmdcia, Universitat de Barcelona, Avda. Joan XXl l I s/n, E-08028 Barcelona, Catalonia, Spain

Received 6 August 1996; received in revised form 11 November 1997; accepted 18 November 1997

Abstract

Delphinium bolosii C. Blanch~ and Molero (Ranunculaceae), is a very rare endemic plant known from only two populations in Catalonia, having fewer than 1500 individuals. A biological survey of this species included: (a) reproductive biology, comprising pollination ecology, breeding systems, seed set and pollen viability; and (b) enzyme electrophoresis to measure genetic variation within and between two populations. A trend to increasing self-pollination rates (up to 20%) and to low levels of genetic variation (1.6-1.7 alleles per locus), together with high rates of nectar robber visits are reported. The hypothetic evolutionary relationships (chromoso- mal changes and geographic isolation) of this species with its related widespread ancestor Delphiniumfissum are also discussed.

The main threats to this species are catastrophic events (especially as soil erosion), human impact activities (both agriculture and tourism) and decreasing pollination. The small populations and low genetic variation reinforce its endangered status. Some conservation measures are suggested, including in-situ and ex-situ strategies. © 1998 Elsevier Science Ltd. All rights reserved.

Keywords: Delphinium bolosii; Rare endemic; Endangered species; Conservation; Reproductive biology; Isozyme electrophoresis

1. Introduction

Delphinium bolosii C. Blanch6 and Molero (Ranun- culaceae) is a rhizomatous, tall perennial larkspur (up to 1.5-2.0 m), producing rosettes of dissected leaves and a simple raceme of up to (15) 40 (100) purple-blue flowers. Seeds are brownish-black, 2-3 mm long (Blanch~ and Molero, 1986). It is a 2n = 18 dysploid karyotype compared with the normal 2n = 16 diploid cytotypes of the related widespread species D. fissum Waldst. and Kit. (x = 8 being the usual basic number of the whole tribe Delphiniae, cf. Blanch~ et al., 1996a).

It was known only from a single population in the Montsant Massif near Ulldemolins (Priorat, Catalonia, Spain) (Blanch~ and Molero, 1983), until Ascaso and Pedrol (1991) discovered a new population of plants in Rubi6 de Baix, La Noguera, about 100km north of Ulldemolins. They described this as a new subspecies D. fissum subsp, fontqueri Ascaso and Pedrol, but further cytogenetic data (Simon et al., 1995) confirming the dysploid condition, revealed that it should be considered as a second population of D. bolosii. Finally,

* Corresponding author. Tel.: 0034 3 401 4490; fax: 0034 3 402 1886; e-mail: [email protected]

0006-3207/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved PII : S0006-3207(97)00177-8

after examination of herbarium material from Font d'Estenalles (Bages), an old erroneous citation of D. elatum L. (Bolbs and Bol6s, 1950, p. 313) has to be referred to D. bolosii. As it has not been seen since 1912 (Blancht, 1991, p. 260), it should be considered extinct (UICN, 1994) from this locality. The species is included as 'E', i.e. endangered, in the Red Book of Spanish plants (Gtmez-Campo, 1987), and is listed in the Cata- logue of Spanish Threatened Plants (BOE, 1990).

D. bolosii was regarded as a living relict (Blancht, 1991) of a focus of plant diversity following the arrival of an eastern steppe flora to the Iberian Peninsula, probably during the Messinian period (Boequet et al., 1978). During the following periods, the eastern moun- tains around the Ebro Basin conserved such ancient forms, as demonstrated by cytobiogeographic evidence (Favarger and Kiipfer, 1988). Following the classification of Favarger and Siljak-Jakovlek (1986), D. bolosii must be considered an apoendemic.

For effective conservation of such rare and endangered species, it is important to distinguish between cause and consequence of rarity (Fiedler and Ahouse, 1992). Of particular interest are population genetic studies that suggest that derivative species will have lower levels of genetic diversity than the progenitor

58 M. Bosch et al./Biological Conservation 86 (1998) 57-66

(Crawford, 1983; Loveless and Hamrick, 1988). This phenomenon was poorly studied among Mediterranean endemics, where much data on rare plants is still needed (Iriondo et al., 1994).

Delphinium bolosii provides an excellent case-study to investigate the role of genetic/reproductive isolation and mutational changes as mechanisms responsible for speciation and/or rarity in very dosed species pairs (Kar- ton, 1991). The reproductive behaviour of D. bolosii seems to differ from the expected larkspur pollination system (described by Miiller, 1883). Because of this and its apparently eroded genotype and small population, we decided to survey the reproductive biology and genetic variation in a wider project of biosystematics of the tribe Delphiniae in the W. Mediterranean area. The main specific objectives were: (1) to contribute to the knowledge of pollination ecology and breeding systems in this rare plant; (2) to assess the inter- and intrapopu- lational genetic variation of D. bolosii to determine its vulnerability; and (3) to define the baseline for estab- lishing study plots for further monitoring as well as to propose conservation measures.

nectar production in plants that had been irrigated three times a week were also taken at the nurseries in order to estimate the maximum production without insect activity. In both cases, the nectar production was measured with a Blaubrand Intramark R micropipette. The content was transferred to a field Bellingham and Stanley LTD (England) refractometer (0-50%) and sugar concentration was recorded and expressed as mg sucrose per ml nectar.

Insect behaviour and movements, number of visited flowers and flights between plants and/or inflorescences were recorded on a tape-cassette in census periods of 15 min. These alternated with 15 min of insect capture, photography and video recording. Each study took place between 0800 and sunset, from 20 June to 9 July, the limits of flowering period (3 weeks). Corbiculae and pollen loads of Hymenoptera were dispersed in a phos- phate buffer 0.1 M and transferred to fuchsinate glycero- gelatin at 60°C. Pollen identification was carried out by comparison with a reference collection at our laboratory.

2.3. Breeding systems

2. Methods

2.1. Populations studied

The two known field populations of D. bolosii were studied during the second half of June and the begin- ning of July of 1992, 1993, 1994 and 1995. The Priorat population (at Ulldemolins) is in a Rubus ulmifolius hedge in a narrow ravine through a Quercus rotundifolia forest over schists (630m elevation). The Noguera population (at Rubi6 de Baix), consists of a few lines of plants occupying ledges on a calcareous cliff facing north over the river Segre (290m elevation), at the margin of shrub communities with Jasminum fruticans, Buxus sempervirens and Acer monspessulanus. The third (extinct) population, (at Bages; Font d'Estenalles), was in a relatively moist, open clearing in a Quercus humilis forest (Fig. 1).

2.2. Pollination ecology

The total number of stamens per flower was counted and a single stamen was randomly selected to estimate the total pollen number per stamen following Dafni (1992). To study pollen fertility, another anther from the same flower was opened over a drop of Cotton Blue. After 10min incubation, the % of stained grains was estimated under the microscope (Stanley and Linskeno, 1974); a total of 500 pollen grains randomly selected were scored in each preparation.

The 24 h nectar production was measured in the field during 1995, after a rainy spring. Measurements of

Stocks to be used as parents in experimental crosses and investigation of breeding systems were transplanted from wild colonies to the laboratory nurseries. Because of the endangered condition of D. bolosii, only 20 individuals were collected from the Noguera population, and 15 from the Priorat population (from an earlier collection). Anthers were removed carefully before opening of stigmata lobes and flowers were pollinated directly by brushing with stamens from donor flowers. Controlled crosses for investigating autogamy (by artificial self-pollination), geitonogamy (pollination with pollen from other flowers of the same individual), xenogamy (pollination with pollen from flowers of other individuals of the same population), interpopulational and interspecific crosses were carried out, as well as treatments of free pollinated open flowers (control), emasculated bagged flowers (parthenogenesis) and bagged complete flowers (spontaneous self-pollination). Differences on seed production were compared using Kruskal-Wallis tests. The population at Villarino de los Aires (Salamanca, Spain) of the related diploid D. fissum Waldst. and Kit. subsp, sordidum (Cuatrec.) was surveyed for the same reproductive characteristics for comparative purposes.

2.4. Isozyme electrophoresis

Genetic variation was assessed using standard methods for starch gel electrophoresis of aUozymes (Soltis et al., 1983; Soltis and Soltis, 1989). Thirty-six plants per population were examined. Soluble enzymes were isolated from fresh pieces of actively growing leaf tissue and stored at 4°C. These leaf fragments were collected

M. Bosch et al./Biological Conservation 86 (1998) 57-66 59

q~

0

%

Fig. 1. Geographic distribution of D. bolosii (total range). • Known Ulldemolins; LNO = La Noguera: Rubi6 de Baix; BAG = Bages: Font

the day before extraction directly from field populations causing no damage to wild plants. The extraction buffer consisted of 0.05 M Tris-citric acid, 0.1% cysteine HCI, 0.1% ascorbic acid, 8% PVP-40 and l mM 2-mercap- toethanol. Eight enzymes--aspartate aminotransferase (AAT), aconitase (ACO), alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), malic enzyme (ME), 6-phosphogluconate dehydrogenase (6-PGD), phosphoglucoisomerase (PGI) and phosphoglucomu- tase (PGM)---were resolved on 12.5% starch gels employing five buffer systems. Tris-citrate buffer was used to resolve ACO and ME (pH 7.0 and pH 8.2, respectively). PGM, MDH and ADH were resolved on histidine citrate buffer pH 5.7 (Jefries and Gottlieb, 1982), 6-PGD on morpholine buffer pH 6.1 (Odrzy- koski and Gottlieb, 1984), and AAT and PGI were resolved on Tris-citrate/Lithium borate buffer pH 8.3 (Scandalios, 1969). Staining procedures for all enzymes followed Vallejos (1983), Shields et al. (1983) and Wen- del and Weeden (1989).

Loci were numbered consecutively and alleles at each locus were labelled alphabetically from the most anodal form. Allozymes were inferred by observing segregation of bands among individuals in the populations sampled.

Allozyme frequencies at each locus were calculated for each population. To estimate the extent of genetic

populations, t extinct population (not collected since 1912). PRI = Priorat: d'Estenalles.

variation within populations, the following statistics were computed: A, the mean number of alleles per locus; P, the proportion of polymorphic loci when the most common allele has a frequency of < 0.95; Ho, the observed heterozygosity and He, the expected panmictic heterozygosity. The partitioning of genetic diversity within and among populations was analysed using parameters proposed by Nei (1978). Genetic variability (A, P, 1to, He) and fixation indices (deviation from Hardy-Wein- berg equilibrium) were calculated using the BIOSYS program (Swofford and Selander, 1989) and F-statistics using the GDA package (Lewis and Zaykin, 1996).

3. R ~

3.1. Conservation status

From the monitoring of its populations in the last years, the following updated diagnostic of D. bolosii can be stated:

1. Number of individuals: the Noguera population maintained a census of 1000-1100 plants but the Priorat population decreased from 300 (1983) to 70 individuals (1995) partly caused by intense

60 M. Bosch et aL/Biological Conservation 86 (1998) 57-66

rainfall in 1995 which caused severe soil erosion. In July, 1997, only 11 fruiting individuals were observed.

2. Age structure of populations: individuals plants identified by albino flowers discovered in 1983, are still living in 1995. Thus, plants can live under field conditions for at least 13 yr and plants maintained in the nurseries have lived up to 7 yr. In addition, vegetative reproduction by rhizome multiplication was easily achieved in pots. In contrast, the estimation of seed bank reserve was very poor (in June, before seed dispersal, 10 soil vouchers of each population, 5 cm deep and 20cm diameter, showed only 8 and 2 non-viable seeds at the Noguera population whereas the 18 remaining samples have no seeds) and no seedlings were seen during the visits to populations. Low germination rates, seedling mortality, seed dispersal to inade- quate sites or predation should be investigated in the future but aged populations are the current assumption.

3. Competition and predation: competition for space was observed at Priorat with expanding shrubs of Rubus ulmifolius and also a competition with the same species for pollinator services is reported below. No insect attack was observed, although in other species of Delphiniae it is a common feature (caterpillars and other insects attack seeds in D. requienii (Loisiel et al., 1995) and in Aconitum species (Bosch, 1996), and herbivores eat flowering parts in D. montanum (Simon et al., in press).

4. Impact of human activities: the Priorat population is crossed by a forest track and vineyards are < 50 m away. Cereal cultures are 100 m around the Noguera population where a castle and a camping site are also very close.

3.2. Floral morphology and reproduction

Inflorescences of D. bolosii are 0.5-1.1 m long. Flow- ers measure 33 mm in length with a pollen:ovule (P:O) ratio of 3800-5400. The upper petals are partially included in the spur and produce nectar in large quan- tifies in nectaries located at the lower end of each petal. The two upper petals constitute a tube with the nectar 11-15mm inside this tube. The proximal part of these upper petals is expanded and together they act as a funnel, defining the entrance for insects sucking nectar. The distal ends of these two petals are included inside the outer spurred sepal and are not visible from the exterior in D. bolosii (although other species of Delphi- nium such as D. picture Willd. show externally bifid spurs, cf. Bosch, 1996). The so-called lateral petals pro- vide horizontal landing platforms, similar to those described by Macior (1975) for D. tricorne and in most perennial species of the genus (Tamura, 1995).

The flowers bear 35--40 stamens and the total pollen production is high, ranging from 122000 to 164000 grains per flower, with 93-96% fertility in both populations. Stamens mature sequentially-acropetally (for 3- 4days). Each flower has three free carpels containing 21-41 primordia/flower, and each carpel has a simple stigma opening by separation of two lobes. The dry mature follicles produce subpyramidal seeds with scaly sides (15-20 wide scales/side) without longitudinal wings (Blanchr, 1991).

Nectar production was 12-14 ~tl per flower containing 8-9 mg of sucrose in the nursery and 3-4 I~1 day -1 and 2-3 mg in the natural populations.

3.3. Phenology

The two populations surveyed maintained open flowers for a period of 21-28 days in the 3 yr of observation. The Noguera population flowered a week earlier than the Priorat population in 1992, 1993 and 1994. The maturation pace of the inflorescence implied that, at any given time, buds and flowers in male phase and female phase were present in descending order, thus allowing self-pollination depending on insect activity.


3.4.1. Pollinators Eight species of insects regularly visited D. bolosii

inflorescences but only five Hymenoptera, and one Diptera were clearly effective pollinators (Tables 1 and 2). The overall percentage of effective pollinators visitors was 7.2% at Priorat and 17.3% at Noguera.

Analysis of pollen loads and corbiculae of captured individuals, showed that Bombus spp. carried one to four additional types of pollen types, although D. bolosii was the predominant load in all cases analysed. Field observations also showed a competition of Rubus ulmifolius for Bombus pollination services at Priorat (Rubus pollen was also found on the body of Lassioglossum sp. and Alastor atropos)

3.4.2. Nectar robbers Many flowers have perforated spurs caused by nectar

robbers (Priorat 50%, 55% and 15%, Noguera 70%, 80% and 15% in 1993-1995, respectively). Bombus terrestris was seen to be responsible for most of this (4.2% of total visits at Priorat and 15.4% at Noguera, Table 2). Secondary robbers making use of the holes produced by B. terrestris were mainly the Eumenidae Alastor atropos representing 31.2% of visits at Priorat and 8.2% at Noguera (Table 2). Primary and secondary robbers together represented 35.4°/0 of total visits at Priorat and 23.6% at Noguera, thus leading to a noticeable decrease in the nectar content without any pollination service. As some B. terrestris individuals captured were observed

M. Bosch et aL/Biological Conservation 86 (1998) 57-66

Table 1 Pollen production (mean + SE and range in parentheses), pollen viability (%) and polien/ovule ratio in D. bolosii

61

Population Stamens Pollen/anther Pollen/flower Viability Primordia Pollen/ovule ratio

Priorat 36.9 4- 0.7 4 453 4- 188 164 052 4- 22,753 96.4 30.9 4-1.8 5 427.8 4- 267.4 (35--40) (3 700-5 478) ( 134 964-208 1 64) (21-38) (3 908.3-6 452.9)

Noguera 30.3 4- 0.7 4 001 4- 294 121923 4- 10 221 93.6 31.4 4- 2.2 3,889.3 4-177.8 (27-34) (2812-5 804) (75 924-179 792) (21-41) (3 180.8-4 859.2)

carrying Delphinium pollen, a certain amount of Table2

pollination efficiency could be expected from this Insect visitorstoD, bolosii species.


The general reproductive behaviour of D. bolosii was the same in the two populations analysed (Table 3). The parthenogenesis tests did not give any seed. Despite the general flower architecture and insect rewards available, bagged flowers which excluded the presence of pollinators were able to produce a mean of 4.3-4.5 seeds per flower, showing that about 20% of the total seed set can be achieved by autogamy. When plants were actively selfed, a significant (p < 0.05) increase in seed production was observed, up to a maximum of 54%. The best results in seed production (p<0.05) were obtained through geitonogamy crosses on the same raceme (up to 16-18 seeds/flower, 59-63%), and xenogamy gave similar results (Table 3).

As a comparative external reference, the population of D. fissum at Villarino showed a similar pattern of absence of parthenogenesis, self-compatibility (up to 60%), and success of xenogamy higher than geitonogamy (Table 3). Surprisingly, however, bagged flowers did not produce any mature seed and thus spontaneous self-pollination was zero, although artificial self-pollinating demonstrated autocompatibility.

Crosses between Priorat and Noguera populations gave similar results to within-population crosses (57- 73%). Hybridizations between D. bolosii (2n= 18) and D. fissum (2n = 16) gave high percentage of viable seeds (56.3-63.7%, Table 4), in spite of the different chromosome number of the parents.

3.6. lsozyme electrophoresis

Twelve isozyme loci were resolved and interpreted (AAT-1, ACe-l , Ace-2 , ADH, MDH-1, MDH-3, ME, 6-PGD-1, 6PGD-3, PGI-1, PGI-2, PGM-2). Genetic variation was calculated at each locus from allelic frequencies and are summarised in Tables 5 and 6 for each population. A total of 20 alleles were identified in the Noguera population, compared with 19 in the Priorat population; thus, 87.5% of the recorded genetic variation is shared by the two existing populations. Alleles PGM-2b and MDH-2a were detected only in the

Insects PrioraP La Pollination Noguera b (rewards) c

Hymenoptera 66.1 42.6 Apidae

Bombus terrestris Kriiger 4.2 15.4 R (Pollen + Nectar) Bombuspasquorum L. 1.5 1.0 + + (Nectar)

Anthophoridae Xylocopa violacea 12.1 + + (Nectar) Anthophora dispar Lep. 1.8 + + (Nectar) Ceratina dentiventris Gerst. 0.2 + (Pollen)

Megaehilidae Osmia submicans Morawitz 1.2 + + (Nectar)

Scoliidae Scoliaflavifrons Fabricius 0.4 + (Pollen)

Eumenidae Alastor atropos Lep. 31.2 8.2 r (Nectar)

Other 29.2 2.9 Lepidoptera 21.2 45.6

Diptera 10.4 11.0 Bombylidae

Bombylius sp. 5.7 1.0 + + (Nectar)

Heteroptera 0.4 0.6

Coleoptera 1.9 0.3

Occasional pollinators: Hymenoptera: Ceratina dentiventris Gerst., Lassioglosswn sp., Ha//ctus sp. Lepidoptera: Papilio machaon L., Cynthia cardui L., Thymelicus sylvestris Poda, Polyommatus icarus Rottemburg, Pyronia bathseba Fabrieius, Brintesia circe Fabricius, Melanargia lachesis Hfibner, Gonopteryx rhamni L., Gonopteryx cleo- patra L., Pieris brassicae L., Artogeia rapae L., Artogeia napi L., Euchloe ausonia Hfibner, Colias croceus Creofroy, Macroglossum stellatarum L. Diptera: Melliscavea auricollis Meigen, Spaerophoria scripta L., Eupodes corollae Fabricius, Episyrphus balteatus De geer, Eristalis tenax L.

Hardly ever pollinating: Diptera: Anthrax sp., Brachycera sp. Het- eroptera: Graphosoma linetaum italioan Mfiller, Chorosoma schillingi Schummel, Cereus marginatus L. Coleoptera: Oxythirea funesta L., Mylabris sp.

a Priorat (n=478, 29h), 27-29 June and 9 July 1992, 30 June 1993. b La Noguera (n=487, 32h), 20-22 June and 4-5 July 1992, 27

June 1993, 22 June 1995. + + = effective pollinator; + = occasional pollinator; 0 = hardly

ever pollinating; R = primary robber; r = secondary robber.

larger population (Nognera), and ADH-b only at Priorat. The genetic distance (Nei, 1978, estimated by Fst) between populations is very low: 0.026. The comparison with other species of Delphinium previously studied isozymatically (Richter et al., 1994; Bosch,

62 M. Bosch et al./Biological Conservation 86 (1998) 57-66

Table 3 Breeding systems in two populations of D. bolosii and in D. fissum

Treatment N Developed seeds

nt Range m ± SE %

D. bolosii (Priorat) Free pollination 43 1256 7 - 44 29.21 + 1.17 79.75 (field) Free pollination 47 571 3 - 28 12.15+0.85 49.57 (greenhouse) Parthenogenesis 10 0 0 0 0 Passive autogamy 29 126 0 - 18 4.345:0.81 20.13 Active autogamy 14 225 8 - 24 16.07 5:0.98 54.35 Geitonogamy 40 745 3 - 40 18.63 5:1.37 63.35 Xenogamy 34 665 5 - 39 19.56± 1.31 70.90

D. bolosii (La Noguera) Free pollination 63 1508 10 - 42 23.94 ± 1.09 69.56 (field) Free pollination 50 691 2 - 27 13.82 ± 0.87 54.71 (greenhouse) Parthenogenesis 15 0 0 0 0 Passive autogamy 53 237 0 - 18 4.475:0.63 19.30 Active autogamy 37 429 0 - 24 11.595:1.15 40.70 Geitonogamy 73 1200 1 - 32 16.44±0.96 59.23 Xenogamy 38 593 5 - 38 15.61 ± 1.25 57.52

"D. fissure (ViUarino) Free pollination 18 296 8 - 25 16.44 5:1.20 50.51 (greenhouse) Parthenogenesis 5 0 0 0 0 Passive autogamy 4 0 0 0 0 Active autogamy 7 135 7 - 35 19.29±3.41 60.27 Geitonogamy 8 196 17 - 30 24.50 ± 1.26 65.55 Xenogamy 11 240 16-27 21.825:1.11 72.73

Table 4 Interpopulation and interspecific crosses between D. bolosii and D. fissure, n = sample size and % of developed seeds in each case

2-'* D. bolosii D. bolosii D. fissure ~ (Priorat) (La Noguera) (Villarino)

D. bolosii n = 46 n = 98 n = 20 (Priorat) 73.84 60.37 57.28 D. bolosii n = 34 n = 38 n = 10 (La Noguera) 70.90 62.99 56.30 D. fissum n = 34 n = 26 n = 11 (Villarino) 63.69 57.64 72.73

Table 5 Allele frequencies in two populations of D. bolosii

Population Population

Alleles Priorat La Noguera Alleles Priorat La Noguera

n 36 36 ME-a 0.500 0.694 AAT-a 0.569 0.069 ME-b 0.375 0.194 AAT-b 0,431 0.931 ME-c 0.125 0.111 ACO-la 1.000 1.000 6PGD-la 1.000 1.000 ACO-2a 1,000 1.000 6PGD-2a 1.000 1.000 ADH-a 0.972 1.000 PGI-la 0.917 0.917 ADH-b 0.028 0.000 PGI-Ib 0,083 0.083 MDH-la 1.000 1.000 PGI-2a 0.917 0.917 MDH-2a 0,000 0.056 PGI-2b 0.083 0.083 MDH-2b 0.028 0.083 PGM-2a 1.000 0.917 MDH-2c 0.972 0.861 PGM-2b 0.000 0.083

Table 6 Genetic variability at thirteen loci in two populations of D. bolosff

Populations N A P Ho He F s

Priorat 36 1.6 33 .3 0.083 0.125 0.674 0.806 (0.2) (0.064) (0.059)

LNO 36 1.7 50.0 0.030 0.109 0.660 0.795 (0.2) (0.016) (0.041)

N = sample size; A = mean number of allels per locus; P = proportion of polymorphic loci (criterion of 0.95); Ho = heterozygosity observed; He=heterozygosity expected (in parenthesis, standard error); F = Wright's Fixation Index; s = rate of self-pollination.

Table 7 Summary of F-statistics at all polymorphic loci of D. bolosii

Locus Fls FST F~T

ACO-1 -0.710 0.075 -0.582 AAT-1 -0.435 0.287 -0.022 MDH-3 1.000 0.030 1.000 ME 0.660 0.032 0.671 PGI-1 1.000 0.000 1.000 PGI-2 1.000 0.000 1.000 PGM-2 0.273 0.043 0.304 ADH 1.000 0.014 1.000 Mean 0.226 0.298 0.092

4. Discussion

4.1. Biogeography and evolution

1996) i n d i c a t e d r e l a t ive l ow va lues o f gene t ic va r i ab i l i t y

o f D. bolosii. Values o f m e a n al leles p e r locus r a n g e d

f r o m 1.6-1 .7 a n d p o l y m o r p h i c loci r a n g e d f r o m 3 3 . 3 -

50 .0%. I n genera l , the N o g u e r a p o p u l a t i o n was s l ight ly

m o r e va r i ab le .

T h e va lues o f o b s e r v e d h e t e r o z y g o s i t y (Ho) did

n o t dev i a t e s igni f icant ly f r o m t h o s e e x p e c t e d f r o m

H a r d y - W e i n b e r g ' s e s t ima t ions (He) (Tab le 6). T h e par -

t i t ion ing o f genet ic v a r i a t i o n de r ived f r o m F-sta t is t ics

(Tab le 7) gave ev idence o f a l m o s t all the v a r i a t i o n w i th in

popu la t ions .

Species c a n be ra re in e i the r space o r t ime, o r b o t h

( R a b i n o w i t z , 1981; K r u c k e b e r g a n d R a b i n o w i t z , 1985;

F i ed l e r a n d A h o u s e , 1992). R a r e species c a n be geo -

g raph ica l ly w i d e s p r e a d b u t i n f r e q u e n t t h r o u g h o u t the i r

d i s t r ibu t ion , o r be loca l ly a b u n d a n t in a ve ry n a r r o w

g e o g r a p h i c range . Re l i c t species w h o s e p o p u l a t i o n s

h a v e b e c o m e i so la t ed a n d f r a g m e n t e d m a y also b e c o m e

t h r e a t e n e d o r e n d a n g e r e d (Fa lk , 1991).

G e n e r a l l y , species t h a t a re r a re a n d h a v e a sma l l

p o p u l a t i o n size a r e subjec t to i n b r e e d i n g d e p r e s s i o n a n d

r educed fi tness resu l t ing f r o m h o m o z y g o s i t y ( F r a n k e l


and Soule, 1981; Levin, 1983; Barret and Kohn, 1991; Lacy, 1992), in addition to being genetically homo- geneous, and therefore, more susceptible to local extinction resulting from demographic and environ- mental stochasticity (Clegg and Brown, 1983; Mitton and Grant, 1984; Guerrant, 1992). The opposite view (e.g. Lande, 1988) suggests that low genetic variation has little if any effect on abundance, and demographic factors may usually be of more immediate importance than genetic factors.

In the context of a derivative species (D. bolosii) from a widespread ancestor (D. fissure) our results could be interpreted as indicative of a certain degree of reproductive differentiation of the first species: (1) increased flower size, particularly spur length, compared with D. fissum, which critically limits the access of certain pollinators to the nectar source; (2) increased stamen number in D. bolosii, thus allowing some overlapping of the male and female phases leading to a probable increase in self-pollination rates, These morphological traits, together with the new chromosome basic number x = 9 instead of x = 8 may be related to certain reproductive features, such as the local abundance of nectar robbers which significantly decrease the nectar availability for legitimate pollinators. They may also account for the increase in selfing rates in bagged flowers (0% in D. fissure against 19-20% in D. bolosii under greenhouse conditions; Table 3).

Thus, D. bolosii appears to suffer a loss of reproductive fitness which could be identified as one of the causes of the present rarity of the species. The loss of nectar resources (both by physical limitation and by robbery), the lack of genetic barriers avoiding self-compatibility and the mod- ification of size in flower parts to a less functional architecture are significant; they have not been found in any of the perennial species of the Tribe Delphiniae reported in the literature except for some Pyrenean populations of .4conitum lycoctonum (Bosch, 1996; Bosch et al., 1997). This is an analogous case of extreme area border effect (nectar robbers, spur length higher than the available fauna proboscis and self-compatibility) but without taxonomic or cytogenetic differentiation.

Currently we cannot evaluate the long-term reproductive consequences of the ratio between pollinators (about 60%) and robbers visits (24--35%) but the energy investment in nectar production seems to be a very inefficient pattern given the apparently very specialized flowers of D. bolosii.

Some additional pollination services may be achieved by the mOth Macroglossum stellatarum, whose limited contacts with anthers or stigmata and low pollen burden is compensated for by its high number of visits, analogous to the bumblebees/hummingbird competition reported by Waser and Price (1990) for American species of Delphinium.


The partially selfing and out-crossing mating system (as suggested from the data of P/O ratio), is a common reproductive strategy in higher plants (Richards, 1986; Brown, 1990, among others). This mixed system (including autogamy to some extent) could have been acquired from its relative D. fissure (0% of seeds produced when insects are excluded; Table 3). The loss of mechanisms to avoid selfing is consistent with the morphological differentiation mentioned above.

The case is analogous to that of Limnanthes bakeri in which there is reduced protandry resulting in a much higher level of fertility than is found in the widespread related L. douglasii (Kesseli and Jain, 1984, 1986). Karron (1991) reports another similar example of Stephanomeria malheurensis, a rare species living in a very restricted area that derives from a widespread species S. exigua (Gottlieb, 1973). Although the widespread ancestor is an obligate outcrosser, S. malheurensis is highly autofertile. Many similar cases are given by the literature, although some inverse exam- ples are also known (i.e. the rare Oenothera organensis from New Mexico became an obligate outcrosser with a well developed self-incompatibility system; Karron, 1991).

4.4. Genetic variation


The spectrum of insect visitors is similar to that reported by Macior (1975) for D. tricorne in the USA, but wider than for other Delphiniae, mainly pollinated by bumblebees (or hummingbirds) (Waser and Price, 1990; Bosch, 1996). However, in spite of a large list of visitors, the effective pollinating vectors are few and the nectar robbers are more numerous. Nectar robbers were reported previously in other genera of Ranunculaceae with zygomorphic flowers and spurs, such as Aconitum (L~ken, 1949) and Aquilegia (Macior, 1966), but not in Delphinium (Bosch et al., 1997).

If causes of rarity can be inferred from the facts stated above, the consequences are less clear. Although several rare plants studied for genetic variation appear to be genetically depauperate because of small population size effects, it may be premature to assume that this is a universal feature of rare species (Stebbins, 1980; Barret and Kohn, 1991). However, at least in the case of D. bolosii, the results of the isozyme survey indicated low levels of genetic variation (if compared with other Mediterranean species reported in Bosch, 1996), thus resulting in agreement with previous reports for rare and geographically restricted plant species (Hamrick and Godt, 1989; Barret and Kohn, 1991): low number

64 M. Bosch et aL/Biological Conservation 86 (1998) 57-66

of alleles per locus, low percentage of polymorphic loci and low heterozygosity. The only available data for comparative purposes among rare endemics Delphinium spp. showed higher levels of genetic variation (D. vir- idescens, Richter et al., 1994) although the number of known populations was also much higher (13).

Factors responsible for these low levels of genetic variation in rare species included chance events (such as genetic drift) and directional selection for genetic uni- formity (such as inbreeding associated with selection against homozygous for rare alleles) (Karron, 1991). Because the only two remaining populations of D. bolosii contain fewer than hundreds of individuals (or thousands at the very best), stochastic factors have most certainly played a significant part in the reduction of genetic variation.

If we combine this low genetic diversity, the increased selfing rates and the decreasing size of populations, we might assume that the levels of inbreeding in our populations could increase over time, at rates depending on the population size per generation. Following Barret and Kohn (1991), therefore, populations become inbred more rapidly when they are of small size. The rates of success of self-pollination obtained from bagged flowers in experimental conditions (19-20%, Table 3) are infer- ior to the actual selfing rates estimated in natural populations by isozyme evidence (selfing rate 's': 0.795- 0.806, Table 6). These data suggest that an important proportion of self-fertilization must derive from insect- mediated autogamy and geitonogamy and that vegetative propagation could maintain the genotypes both across space and time, among other. These facts need further research.

4.5. Conservation implications

Low levels of genetic variation and low heterozygosity, together with some disturbed pollinator activity and declining population size neatly assesses the endangered character of D. bolosii. From the data reported, the following conservation measures could be proposed (some of them have already been undertaken).

4.5.1. Ex-situ conservation program For several years, seed-collecting field work permitted

some material to be obtained for gene banks. Seeds from both populations are currently conserved in the seed bank of Professor C. G6mez-Campo (Universidad Politrcnica, Madrid) and in our laboratory at the Uni- versitat de Barcelona. The plants which were collected many years ago from wild populations are also conserved in the nurseries of the Universitat de Barcelona, and plants obtained by vegetative propagation were sent to, and are currently preserved in the Jardin Botanique de Brest (in Brittany, France) under the auspices of Dr J.Y. Lessouef.

4.5.2. In-situ conservation There are no current measures for conservation of the

wild populations. An initial need is to build a monitoring program for D. bolosii, including intensive explora- tion of favourable habitats while looking for additional natural populations; special effort has to be made at the site of the extinct population (Bages), within the boundaries of the Sant Lloren~ Natural Park (Diputa- ci6 de Barcelona).

4.5.3. Noguera population This reaches the minimum rank of MVP suggested by

Menges (1991) and Iriondo (1996), i.e. 103-106 individuals. This population should be considered as the reservoir of genetic diversity of the total pool of the species, which is maintained by the relatively high genetic diversity and demographic number. In addition to legal protection for this site (see below), measures to avoid a pollinator recruitment failure (Washitani, 1996) should be implemented.

4.5.4. Priorat population The genetic, reproductive and demographic char-

acteristics of this population suggests that management intervention for in-situ conservation may be necessary, although it is recommended only if the population size falls below a certain critical point (Bawa and Ashton, 1991). These authors proposed the use of seeds from the same population in reinforcing strategies; the results from our isozyme survey showed a certain differentiation among populations at some alleles which may indicate the existence of some adaptive genes. Thus, a hypothetical reinforcing program should avoid mixing seeds from the two existing populations.

In addition, Karron (1991) suggested that conservation programs should attempt to protect nearby pollinator populations such as bumblebees for example by reducing pesticides in the adjacent areas. In the mean- time, Catalonia in-situ hand pollination should not be disregarded. It will be useful both to increase genetic diversity (to avoid inbreeding depression) and to com- pensate losses by robbers. All these measures should be accompanied by site protection.

4.5.5. Bages population As discussed in other papers (Blanch6 et al., in press

b), the reintroduction of this species to the historical site of Font d'Estenalles (Bages population) needs further studies, but some genetic guidelines are provided by the present paper. A self-sustaining population must contain sufficient genetic variation to adapt to natural habitat changes (Kress et al., 1994). Current levels of genetic variation of extant populations should serve as the minimum level and, because of its longer size, the Noguera population should be the source (50% of loci polymorphic compared with 33.3% in Priorat).


4.5.6. Protection

Legal protect ion measures are also needed. Al though this species is listed as endangered ( G t m e z - C a m p o , 1987 and BOE, 1990), there was no managemen t plan or habi ta t conservat ion program. As in Spain the Nat - ure Conservat ion policies are transferred to the Au ton- omous Governments , the Cata lan authorities should use its legal resources (a ne twork o f protected areas called ' P E I N ' , D O G C , 1993) by including Pr iorat into the ' P E I N ' area named 'Mont san t ' , only 2-3 km f rom the populat ion. Alternative policies, by creation o f a Nat - ural Reserve, seem more suitable for Noguera , because the closest ' P E I N ' area ( 'Aiguabarre ig del Segre') is far away f rom the populat ion.

Acknowledgements

T h a n k you to J. Pedrol for his kind help in finding Nogue ra popula t ion and J. M~irquez for his helpful technical advice on pollen identification in loads analysis. Insect identification was kindly provided by L. Cas- tro (Bombinae and Eumenidae), J. Bosch, N. Vicens (Hymenoptera) , J. Dan ta r t (Lepidoptera), M.A. Marcos (Syrphidae), M. Gou la (Heteroptera) and X. Vfizquez (Coleoptera). P. Anis , M. Aguader , J. Caujap t , D. E. Soltis and P.S. Soltis helped us with their comments and ideas on isozyme work and interpretation. Finally, the editors and referees comments much improved this paper. This work was subsidized by grants PB.91-268 ( D G I C Y T , Ministerio de E d u c a c i t n y Ciencia, Spain) and AMB.97-375 (CICYT).

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