Novel computed tomography-based tools reliably quantify plant reproductive investment

Journal of Experimental Botany, Vol. 69, No. 3 pp. 525–535, 2018 doi:10.1093/jxb/erx405 Advance Access publication 23 December 2017 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Novel computed tomography-based tools reliably quantify plant reproductive investment Y. M. Staedler1,†,*, T. Kreisberger1,†, S. Manafzadeh1,†, M. Chartier1,†, S. Handschuh2, S. Pamperl1, S. Sontag1, O. Paun3 and J. Schönenberger1 1 3 Department of Botany and Biodiversity Research, Division of Structural and Functional Botany, University of Vienna, Austria VetCORE – Facility for Research, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria Department of Botany and Biodiversity Research, Division of Systematic and Evolutionary Botany, University of Vienna, Austria † These authors contributed equally to this work. * Correspondence: Received 4 September 2017; Editorial decision 17 October 2017; Accepted 19 October 2017 Editor: Daphne Goring, University of Toronto, Canada Abstract The flower is a bisexual reproductive unit where both genders compete for resources. Counting pollen and ovules in flowers is essential to understand how much is invested in each gender. Classical methods to count very numerous pollen grains and ovules are inefficient when pollen grains are tightly aggregated, and when fertilization rates of ovules are unknown. In this study we have therefore developed novel counting techniques based on computed tomography. In order to demonstrate the potential of our methods in very difficult cases, we counted pollen and ovules across inflorescences of deceptive and rewarding species of European orchids, which possess both very large numbers of pollen grains (tightly aggregated) and ovules. Pollen counts did not significantly vary across inflorescences and pollination strategies, whereas deceptive flowers had significantly more ovules than rewarding flowers. The within-inflorescence variance of pollen-to-ovule ratios in rewarding flowers was four times higher than in deceptive flowers, possibly demonstrating differences in the constraints acting on both pollination strategies. We demonstrate the inaccuracies and limitations of previously established methods, and the broad applicability of our new techniques: they allow measurement of reproductive investment without restriction on object number or aggregation, and without specimen destruction. Keywords: Deceptive orchids, machine counting, micro computed tomography, ovule count, pollen count, pollination. Introduction It should be evident to most human beings that the mere existence of genders is a harbinger of conflicts for resources. Gender conflicts are nowhere more acute than in hermaphroditic organisms where both genders have to draw from the resource pool of the same organism to maximize fitness (Charnov, 1979; Lloyd, 1979). In the overwhelmingly hermaphroditic flowering plants, counting the pollen grains and ovules of flowers allows us to understand how much a plant invests in the male versus female part of its fitness. Pollen counting methods fall into three groups (Costa and Yang, 2009): counting with the naked eye, particle counters, and image-processing algorithms. Counting visually usually involves spreading samples on specialized slides with a grid and counting a sub-sample (e.g. Jorgensen, 1967; Kearns and Inouye, 1993), which is then extrapolated (Kannely, © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 526 | Staedler et al. of an inflorescence during a visit, and visit the same inflorescence repeatedly in order to harvest its rewards (Van der Cingel, 1995; Fig. 1A, B). In taxa with deceptive flowers, however, pollinators tend to learn to avoid deception and to visit only the first open flowers they encounter while they are still naive (Jersáková and Kindlmann, 1998; Van der Cingel, 1995; Fig. 1D). Consequently, in rewarding plants the fruit set is usually higher and the fruits are spread across the inflorescence (Neiland and Wilcock, 1995; Fig. 1C), whereas in deceptive plants only the first flowers to open tend to bear fruit (Nilsson, 1980; Vogel, 1993; Jersáková and Kindlmann, 1998; Fig. 1E). In orchids in general, the ratio of pollen to ovules (P:O) increases from the bottom to the top of inflorescences (Salisbury, 1942; Nazarov and Gerlach, 1997; Kopylov-Gus’kov Yu et al., 2006). Due to decreased pollinator visits to the top flowers, we hypothesise that the decrease in ovule number (increase in P:O) should be stronger across inflorescences of deceptive flowers than across inflorescences of rewarding flowers (Fig. 1F–I). The Orchidinae, to which most European orchids belong, are a good system to test this hypothesis because their pollination biology and phylogenetic relationships are very well understood (Van der Cingel, 1995; Claessens and Kleynen, 2011; Inda et al., 2012). The phylogenetic relationship between the species studied needs to be known in order to control for potential phylogenetic constraints. This study was aimed at: (1) establishing new methods for pollen and ovule counting that can be used even for flowers with many, densely aggregated pollen grains and ovules; and (2) demonstrating the potential of these methods by focussing on species of European orchids to determine whether the differences of pollinator behaviour in rewarding versus deceptive plants lead to different patterns of reproductive investment at the level of the inflorescence. Materials and methods Plant material We sampled three rewarding and five deceptive species of the subtribe Orchidinae (Inda et al., 2012; see Supplementary Table S1 at JXB online). We collected three flowers for 2–4 inflorescences per species, for a total of 76 flowers (see Supplementary Table S2). Collection method Open flowers (including pollinia, i.e. the pollen aggregates of orchids) or buds close to anthesis were collected from the bottom, middle, and top sections of inflorescences and were immediately fixed in 1% phosphotungstic acid (the contrast agent) in formalin–acetic acid– alcohol (1% PTA / FAA; Fig. 2A). The flowers were removed from the plants using razor blades and tweezers. Sample preparation The sampled flowers and buds were de-aerated for 20–30 min using a water-jet vacuum pump. Total infiltration time in 1% PTA / FAA was 4–6 weeks during which the solution was changed twice. The long infiltration time allowed saturation of the sample with the contrast agent. 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