Kozurahova E, Richards AJ. Breeding Systems of Rare and Endemic Asytagalus, Oxytropis and Onobrychisspecies (Fabaceae) Tested with Alternative Methods

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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.

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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 kozhuharovii, 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 evolutionary 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, however, 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 autogamy 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 evolutionary 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 op.rn.Aa.bl.Sci., bulg. Acad. rend. Compt. 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

o1,2016 12, No , 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 inflorescences 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 experiment 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. Samples of ten buds each were taken and air dried from A. dasyanthus, Oxytropis urumovii and Ono- brychis pindicola, 23 buds from O. campestris and five buds from O. kozhuharovii. Rehydration was performed with mixture of alcohol, water and detergent. The standard protocol of Dafni [18] was followed. Our slight modification was the usage of Burker camera for the pollen counts. Descriptive statistics was used to summarize the results (average ± standard deviation). Results and discussion. Pollinator excluding experiments. In situ experiments with flowers of Astragalus dasyanthus excluded from pollinators revealed that few of them produce legumes. At the average 7% of the flowers were capable of spontaneous self-pollination and self-fertilization (Table 2). In addi- tion the legumes resulting from spontaneous self-pollination were localized in only 29% at the average (25.0–33.3%) of the tested inflorescences. The seed set of the few fruits resulting from excluded flowers was similar to the seed set of the fruits of free pollinated flowers (Table 2). Examination of a random sample of fully

1574 E. Kozurahova, A. Richards op.rn.Aa.bl.Sci., bulg. Acad. rend. Compt. T a b l e 2 Fruit and seed set of ﬂowers experimentally excluded from pollinators and free pollination seed set per legume (Legend: *Data published in our previous paper [21])

Astragalus dasyanthus Oxyropis urumovii Oxyropis campestris Onobrychis pindicola 1995* 2007* 2008 2009 1996 2001 2001 1996 2001 2002 Excluded inﬂorescences 18 25 34 104 3 13 10 6 13 6 Excluded ﬂowers 127 176 192 557 9 63 95 140 378 184 Legumes as 2 1 a result of with with

69 13 14 17 6 2 58 3 spontaneous abortive abortive o1,2016 12, No , self-pollination seeds seed Yearly percentage of fruit set 10.2% 7.9% 8.8% 1.1% 22.20% 92.10% 3.2% 0% 0.5% 0.5% in the excluded ﬂowers Average percentage of fruit set 7.02% 57.15% 3.2% 0.3% in the excluded ﬂowers Spontaneous self-pollination seed set per legume Average 4 2 3 2 8 8 3 0 abortive abortive Minimum 2 1 0 0 3 4 1 Maximum 8 4 6 5 10 12 7 Free pollination seed set per legume Average 4 5 5 4 5 5 6 1 Minimum 3 2 2 2 1 1 2 Maximum 6 9 8 8 9 10 11 Ovules set per pistil Average 13 14 17 1

1575 Minimum 10 15 20 1 Maximum 14 12 16 1 Fig. 1. Flowers of Astragalus dasyanthus excluded from pollinators

mature flowers (Nflowers = 30) revealed that 40% of them had the stigma and anthers at the same level (Fig. 2). Therefore, 40% of the flowers potentially have the possibility for spontaneous self-pollination and thus self-fertilization. The experimentally detected ability of only 1.1% to 10.2% of the flowers to spontaneously self-fertilize compared to the 40% potential self-fertilization suggests the presence of a genetically determined system for self-incompatibility. The fruit set of spontaneous-self-pollinated/self-fertilized flowers of Oxytropis urumovii represented 22.2–92.1% of all flowers, experimentally isolated form pollinators (Table 2). The seed set of the fruits resulting from excluded flowers was similar to the seed set of the fruits of free pollinated flowers (Table 2). In general apomixis is not reported for Oxytropis members. The experimental test of Oxytropis pilosa L. for apomixis is negative [22]. The considerable difference in the fruit set of the flowers excluded from pollinators at different sites during different seasons (Table 2) is difficult to be interpreted. One possible explana- tion might be the presence of two ploidy levels in the population of O. urumovii. Beside the diploid individuals hexaploids are registered as a result of endopolyploidy [10–12]. If our insect excluders during the first year accidentally were set on hexaploid individuals that would explain the lower percentage of spontaneous self- pollination as it is in the hexaploid species O. campestris. Only 3.2% of the flowers of O. campestris set fruits as a result of spontaneous self-pollinations (Table 2). Judging by the results from the tests of flowers excluded from pollinators, the poli- plodization in this group of closely related species of the complex O. campestris should go with adaptation towards avoiding spontaneous self-pollination.

1576 E. Kozurahova, A. Richards Fig. 2. Dissected ﬂower of Astragalus dasyanthus with stigma and anthers at the same level

At the average 0.3% of the Onobrychis pindicola flowers excluded form pollinators set fruit (Table 2). However, their seeds are abortive and never mature (Table 2). Self-pollination is possible because only 64.3% of the tested flowers (Nflowers = 50) have stigmas above the dehiscent anthers. These results indi- cate presence of self-incompatibility barrier. This self-incompatibility was experimentally confirmed because all hand self-pollinated flowers failed to set legumes (Nflowers = 20, Table 2). The excluders did not influence the fruit set because the control flowers (Nflowers = 5, excluded, hand cross-pollinated and excluded again) set fruit successfully. Pollen ovule ratio (P/O ratio). The values of P/O ratio detected for As- tragalus dasyanthus fall in the range of P/O ratio values for facultative xenogame and obligate xenogamy presented by Dafni [18] and Cruden [19] (Table 3). The values of P/O ratio of the three Oxytropis species corresponded to facultative xenogamy. Surprisingly the highest were those of O. urumovii (Table 3). Basically in this group of species probability for xenogamy is lowest compared to the other two studied legumes. P/O ratio values of Onobrychis pindicola were very high and corresponded to obligate xenogamy (Table 3). Similar strategy was detected in O. viciifolia, which demonstrated highest P/O ratio values of all studied 32 Mediterranean legume species [23]. Conclusion. According to the results from the field experiments with flowers excluded form pollinators all studied species except Oxytropis urumovii are not

4 Compt. rend. Acad. bulg. Sci., 69, No 12, 2016 1577 T a b l e 3 Number of pollen and ovules and P/O ratio

Average SD Minimum Maximum

A. dasyanthus (Nﬂowers = 10) P/O ratio 8672.4 243.3 1574.1 2345.3 Pollen 112741 2443.3 71833 155794 Ovules 13 1.1 12 14

O. urumovii (Nﬂowers = 10) P/O ratio 2532.9 493.7 1666.7 3125.0 Pollen 33950 6614 40972 21111 Ovules 14 1.2 12 15

O. kozhuharovii (Nﬂowers = 5) P/O ratio 1915.2 250.8 1574.1 2345.3 Pollen 35871 7913.6 28333 46905 Ovules 20 2.6 24 18

O. campestris (Nﬂowers = 23) P/O ratio 1975.2 976.2 888.9 5671.3 Pollen 29145 9767.3 45278 20889 Ovules 18 1.7 16 20

Onobrychis pindicola (Nﬂowers = 10) P/O ratio 23444.4 976.2 21166.7 25722.2 Pollen 21790 3204 18000 25772 Ovules 1 1 1 Obligate autogamy According to Dafni [18] 28.6 – 18.1 39.0 According Cruden [19] 27.7 3.1 Facultative autogamy According to Dafni [18] 213.9 – 31.9 396.0 According Cruden [19] 168.5 22.1 Facultative xenogamy According to Dafni [18] 1416.4 – 244.7 2588.0 According Cruden [19] 796.6 87.7 Obligate xenogamy According to Dafni [18] 98816.5 – 2108.0 195525.0 According Cruden [19] 5859.2 936.5 capable of spontaneous self-pollination. Breeding systems data obtained by both methods are in pretty good agreement for Astragalus dasyanthus and Onobrychis pindicola. The P/O ratio data do not ﬁt very well with pollinator excluding data for the Oxytropis species. The P/O ratio values of the three Oxytropis species corresponded to facultative xenogamy. Surprisingly the highest were those of

1578 E. Kozurahova, A. Richards O. urumovii and thus xenogamy would be prevailing for this species (Table 3). However, namely in this species the pollinator excluding experiments demonstrated the highest ability for self-pollination (Table 2). It has lowest number of ovules of all three 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. Our study conﬁrmed the statement of Cruden et al. [20] that in some cases P/O ratio is a complicated parameter. Since all studied species except Oxytropis urumovii are not capable to spontaneous self-pollination according to the results of this study, conservation strategies for these species would include preservation of the pollen vectors and their habitats as well. Pollinators are bumblebees [21] and this topic still requires more investigations.

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Department of Pharmacognosy and Botany Faculty of Pharmacy Medical University of Soﬁa 2, Dunav St ∗School of Biology 1000 Soﬁa, Bulgaria University of Newcastle e-mail: ina [email protected] Newcastle upon Tyne, UK

1580 E. Kozurahova, A. Richards