Kozurahova E, Richards AJ. Breeding Systems of Rare and Endemic Asytagalus, Oxytropis and Onobrychisspecies (Fabaceae) Tested with Alternative Methods
Total Page：16
File Type：pdf, Size：1020Kb
Kozurahova E, Richards AJ. Breeding Systems of Rare and Endemic Asytagalus, Oxytropis and OnobrychisSpecies (Fabaceae) Tested with Alternative Methods. Comptes Rendus de l'Academie Bulgare des Sciences 2016, 69(12), 1571-1580. Copyright: © Comptes rendus de l’Académie bulgare des Sciences. Publisher website: http://www.proceedings.bas.bg/ Date deposited: 24/03/2017 Newcastle University ePrints - eprint.ncl.ac.uk Доклади на Българската академия на науките Comptes rendus de l’Acad´emiebulgare des Sciences Tome 69, No 12, 2016 BIOLOGIE Botanique BREEDING SYSTEMS OF RARE AND ENDEMIC ASYTAGALUS, OXYTROPIS AND ONOBRYCHIS SPECIES (FABACEAE) TESTED WITH ALTERNATIVE METHODS Ekaterina Kozurahova, A. J. Richards∗ (Submitted by Academician A. Atanassov on July 14, 2016) Abstract Investigations on plant mating systems in a changing world become crucial. There are two main methods to study the rate of outcrossing (matings between unrelated genets) versus selfing (mating among close relatives) in plants. The direct method involves pollinator excluding experiments. The indirect method measures the rate of pollen-ovule ratios (P/O ratio). The aim of this study is to test the breeding systems of some rare and endemic legumes from mountain areas of the Balkans, SE Europe using both direct and indirect methods and comparatively to analyse the results. According to the results from the field experiments with flowers excluded form pollinators all studied species except Oxytropis urumovii are not capable of spontaneous self-pollination. Breeding systems data obtained by both methods are pretty well in agreement for Astra- galus dasyanthus and Onobrychis pindicola. The P/O ratio data do not fit very well with pollinator excluding data for the Oxytropis species. Although P/O ratio method is crucial in all cases when field experiments are not possible, e.g. objects with small and vulnerable populations that are hard to reach such as O. kozuharovii, the results should be interpreted with caution. Key words: Astragalus dasyanthus, Oxytropis urumovii, Oxytropis kozhu- harovii, Onobrychis pindicola, P/O ratio, excluded flowers, Bulgaria Introduction. Developing adequate conservation strategies for endemic and/or rare plant species requires the deepest possible understanding of the evo- lutionary processes that have created them. Important factor in the evolutionary processes is the reproduction. Thus local endemics with utterly restricted range 1571 such as Oxytropis kozhuharovii Pavlova, Dimitrov & Nikolova, O. urumovii Jav., Onobrychis pindicola subsp. urumovii Degen & Dren., or plants with wider but disjunctive range such as Astragalus dasyanthus Pall. and Oxytropis campestris (L.) DC., some of them endangered or critically endangered, present ideal object for comparative study on the plant breeding systems. \Plant Breeding systems" is a term used senso lato to describe the main ways of plant reproduction. Although plants can not choose their mates, they have di- verse methods by which their breeding systems manipulate and control the genetic structure of their populations and the patterns of their evolution. It is useful to think of the various plant breeding strategies in three dimensions which can, how- ever, be more simply represented in two dimensions in the form of triangle. At the apices of the triangle lie plants with a single strategy. These strategies are: panmixis (fully outcrossing, xenogamous populations); self-fertilization (often au- togamy or allogamy but geitonogamy { pollen comes from other flower of the same genet); asexuality (plants without sexual reproduction, e.g. agamospermy). The genetical and evolutionary consequences of each of these mechanisms are entirely different in each case [1]. There is increasing evidence that human disturbance can negatively impact plant-pollinator interactions such as outcross pollination and thus influence the mating systems of plants [2]. Rare and endemic plants are of particular inter- est with respect to their breeding systems and pattern of genetic variation and conservation biologists should pay special attention to the problem [3]. Summary of the data from the literature on distribution, karyology, conservation and evo- lutionary status of Astragalus dasyanthus, Oxytropis urumovii, O. kozhuharovii, O. campestris and Onobrychis pindicola are presented in Table 1 [4{17]. There are two main methods to investigate the breeding system in plants. The direct method is based on pollinator excluding experiments and analyses of the resulting fruit and seed set. The indirect method measures the ratio of the pollen grains and the ovules [18]. Pollen-ovule ratios (P/O's) of flowering plants reflect their breeding system. The evolutionary shift from obligate xenogamy towards obligate autogamy is accompanied by a significant decrease in the mean P/O. This method was developed by Cruden and his experimental emphasis is on members of family Fabaceae [19]. Additionally, a lack of correlation between P/O and the volume of pollen per flower indicates that P/O can not be used as a predictor of resource allocation to male sexual function. The species with large numbers of ovules per flower invest more in male function than plants with few ovules per flower and the greater investment is expressed as a larger number of pollen grains. There is an indication of a trade-off between pollen grain number and size [20]. The aim of this study is to test the breeding systems of some rare and endemic legumes from mountain areas of the Balkans, SE Europe using both direct and indirect methods and comparatively to analyse the results. 1572 E. Kozurahova, A. Richards Compt. rend. Acad. bulg. Sci., T a b l e 1 Distribution, karyology evolutionary and conservation status of Astragalus dasyanthus, Oxytropis urumovii, O. kozhuharovii, O. campestris and Onobrychis pindicola Plant taxon Distribution and conservation status Evolutionary status Karyology Astragalus Pannonian{Pontian{Balkan geoelement [4]. Rather ancient origin [6] Primitive karyotype [6] dasyanthus Populations occur rather restrictedly else- Pall. where in Southern and Eastern Europe ex- tending northwards to Hungary and Central Russia [5]. Rare species for the Bulgarian flora. IUCN category is \Critically Endan- 14 69 gered" [ ]. Protected by the Biodiversity , No 12, 2016 Law [4]. Oxytropis uru- Local endemics to the marble part of North- Both morphometric and molecular analy- Diploid, 2n = 16 [10, 11]. The movii Jav. ern Pirin Mts. Localised to the north- ses confirm that O. urumovii is a very dis- karyotype is symmetrical, consist- ern marble part of the Pirin Mts, above tinct diploid species which might be an- ing of 2n = 4m + 12sm = 16 2000 m [9]. IUSN category is \Vulnera- cestral to this group (Balkan members of small and medium size chromo- 11 ble" [14]. O. campestris complex) and could be re- somes [ ]. Endopolyploidy (2n = garded as a palaeoendemic [7, 8]. 48) is also observed [12]. Oxytropis Local endemics to the marble part of North- Most closely related to O. prenja from the Tetraploid, 2n = 4x = 32 [13]. kozhurahovii ern Pirin Mts. Localised to the northern Dinaric Alps, Bosnia-Herzegovina, but is a Pavolova, marble part of the Pirin Mts, above 2000 m larger plant with a different facies and indu- Dimitrov & but it has much more restricted distribution mentum. It is possible that it has evolved as Nikolova to only one cirque IUSN category is \Criti- an allotetraploid derivative of O. urumovii cally Endangered" [14]. and O. halleri [7]. Oxytropis Much wider range in Europe and North O. urumovii and O. kozhuharovii are Hexaploid, 2n = 6x = 48, with campestris America and in Bulgaria grows on marble closely related, possibly parental to assymetric karyotype compared to (L.) DC. rocks in Northern Pirin Mts. and small area O. campestris and they all belong to the other karyotypes [11]. in Rila Mts. O. campestris complex [7]. Onobrychis Local endemic on the marbles of Pirin and Tetraploid, 2n = 4x = 28 [12]. pindicola Slavyanka Mts. SW Bulgaria. IUSN cate- subsp. gory is \Least concern" [15{17]. 1573 urumovii Degen & Dren. Material and methods. Pollinator excluding experiments were performed in the natural populations in mount Ruen, in the vicinity of Boboshevo (FM66) for Astragalus dasyanthus and in the marbleized karst region of the North Pirin Mts (FM92) for all other species. Field experiments and plant material collection were performed during the period 1995{2009. The microscope analyses were finalized in 2015. Pollinator excluding experiments. We used the standard methods sum- marized by Dafni [18] to exclude pollinators from flowers. Our slight modification was the bagging made by polyamide mesh on PVC skeleton to hold it straight with proper ventilation [21]. At least five flowers were used as control to prove the lack of negative impact of the bagging on the fruit set. Field experiments with flowers excluded from pollinators were repeated at least in two different seasons (Table 2). To test the spontaneous self-pollination ability of Astragalus dasyanthus 181 inflo- rescences or a total of 1052 flowers were in situ excluded from pollinators during four seasons (Fig. 1). Stigma level in relation to the anthers was observed in the field. The spontaneous self-pollination ability of Oxytropis urumovii was tested in situ by excluding pollinators from 16 inflorescences or a total of 72 flowers during two seasons. Experiments in situ with flowers of O. campestris excluded from pollinators were performed during two seasons (1996 and 2001) but the first ex- periment was destroyed by sheep and only the second experiment succeeded with 10 inflorescences or a total of 95 flowers. The flowers of O. kozhuharovii were not bagged to avoid disturbance of the fruit set of this critically endangered species. During a period of three seasons we excluded from pollinators 25 inflorescences or a total of 702 flowers of Onobrychis pindicola (Table 2). Additionally we excluded and hand self-pollinated 20 flowers of O. pindicola to test its self-incompatibility (respectively six flowers in 1996 and 14 flowers in 2002). Pollen ovule ratio (P/O ratio). We collected flower buds just before opening to count the total amount of pollen and ovules.