More than sixty origins of pantoporate pollen in angiosperms 1

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1 RESEARCH ARTICLE AMERICAN JOURNAL OF BOTANY More than sixty origins of pantoporate pollen in angiosperms 1 Charlotte Prieu 2,3,5, Hervé Sauquet 2,4, Pierre-Henri Gouyon 3, * and Béatrice Albert 2, * PREMISE OF THE STUDY: Apertures in pollen grains are key structures of the wall, involved in pollen tube germination and exchanges with the environment. Aperture types in angiosperms are diverse, but pollen with one and three apertures (including monosulcate and tricolpate, respectively) are the two most common types. Here, we investigate the phylogenetic distribution in angiosperms of pollen with many round, scattered apertures called pantoporate pollen. METHODS: We constructed a morphological data set with species producing pantoporate pollen and representative angiosperm species with other pollen types, sampled from every angiosperm order, with a total of 1260 species distributed in 330 families. This data set was analyzed with parsimony to characterize the phylogenetic distribution of pantoporate pollen in angiosperms. KEY RESULTS: We show that pantoporate pollen is distributed throughout most of the angiosperm tree, including early diverging angiosperms, monocots, and eudicots. However, this pollen type is usually restricted to a few species in a given group, and is seldom fixed at large taxonomical scales, with a few notable exceptions. CONCLUSIONS: Pantoporate pollen evolved many times during angiosperm history, but the persistence of this morphology in the long term is infrequent. This distribution pattern could indicate conflicting short-term and long-term selective pressures, pantoporate pollen being selected in the short run, but eliminated in the long run. Biological hypotheses supporting this scenario are discussed, in the context of both theoretical and empirical data on pollen biology. KEY WORDS apertures; pollen evolution; selection; pantoporate pollen; pollen phylogeny; macroevolution Pollen grains are the male gametophytes of seed plants and form a dispersal unit involved in fertilization. They exhibit a considerable level of morphological diversity, reflected in highly variable size, shape, exine ornamentation, and many other wall features ( Erdtman, 1952 ; PalDat, 2016 ). The wall of the pollen grain consists of two distinct layers: the outer layer is the exine, primarily composed of sporopollenin, whereas the inner layer, the intine, is pecto-cellulosic. Apertures are special areas of the wall where exine is absent or much thinner, and they are involved in a key process of pollen life 1 Manuscript received 24 July 2017; revision accepted 6 October Ecologie Systématique Evolution, Univ. Paris-Sud, CNRS, AgroParisTech, Université Paris- Saclay Orsay cedex, France; 3 Institut de Systématique, Évolution, Biodiversité, ISYEB UMR 7205 CNRS, MNHN, UPMC, EPHE, Muséum national d'histoire naturelle, Sorbonne Universités, 57 rue Cuvier, CP39, F-75005, Paris, France; and 4 National Herbarium of New South Wales (NSW), Royal Botanic Gardens and Domain Trust, Sydney, Australia 5 Author for correspondence ( prieu@mnhn.fr ); ORCID id *These authors contributed equally to this work. ( Edlund et al., 2004 ): the pollen tube, after landing on a stigma, germinates through these structures. The apertures are also the sites where exchanges with the environment take place, such as dehydration of the pollen grain before release and rehydration on the stigma. Moreover, apertures have a mechanical function, because they enable accommodation of volume variations due to cytoplasmic osmosis, a process called harmomegathy ( Wodehouse, 1935 ; Payne, 1972 ). An aperture pattern is defined by the number, shape, and position of apertures ( Walker and Doyle, 1975 ). Two basic aperture patterns are found in angiosperms: the monosulcate type, with a single distal furrow, and the tricolpate type, with three longitudinal furrows. Phylogenetic reconstructions show the ancestral state in angiosperms to be monosulcate, while tricolpate pollen is an evolutionary innovation of the eudicot clade ( Doyle and Hotton, 1991 ; Doyle and Endress, 2000 ; Furness and Rudall, 2004 ; Wortley et al., 2015 ). Variation between each of these and other pollen types can nonetheless be found in many taxa at a lower taxonomic level (Erdtman, 1952 ; PalDat, 2016 ). AMERICAN JOURNAL OF BOTANY 104 (12): , 2017; Botanical Society of America 1837

2 1838 AMERICAN JOURNAL OF BOTANY Here we focus on a particular aperture pattern the pantoporate type ( Fig. 1 ). Pantoporate pollen (also called periporate or polyforate) possesses round apertures distributed more or less regularly over the surface of the grain ( Erdtman, 1952 ; Hesse et al., 2009 ). The number of apertures in pantoporate pollen may vary from six (as in Fumaria officinalis, Papaveraceae; Fig. 1A ) to more than 100 (as in Alcea rosea, Malvaceae, in Erdtman, 1952 ). The relative size of the apertures (compared to the size of the grain) is also variable ( Fig. 1 ). The pantoporate type is quite different from the tricolpate and monosulcate types, because apertures are both numerous and distributed evenly on the surface of the grain. These features might give special properties to the pollen grains. Pantoporate pollen is known to occur in many angiosperm species ( Erdtman, 1952 ; Pal- Dat, 2016 ), but its phylogenetic distribution and number of origins have not been characterized. The phylogenetic distribution of pantoporate pollen in angiosperms was investigated to evaluate how many times this particular aperture pattern originated. We also wanted to know if this morphology was restricted to some particular clades, or if it was widespread across flowering plants. We show that most main clades of angiosperms contain species with pantoporate pollen, and that this aperture pattern originated many times independently during flowering plant history. We also point out that in all but a few clades, only a few species or a few genera produce this particular pollen morphology. FIGURE 1 Diversity of pantoporate pollen in angiosperms. (A) Fumaria officinalis, Papaveraceae ( Oberschneider and Halbritter, 2005 ); (B) Ribes aureum, Grossulariaceae ( Halbritter, 2005b ); (C) Costus barbatus, Costaceae ( Halbritter, 2010 ); (D) Arenaria pungens, Caryophyllaceae ( Halbritter, 2005a ); (E) Portea alatisepala, Bromeliaceae ( Till and Halbritter, 2007 ); (F) Alisma lanceolatum, Alismataceae ( Svojtka and Halbritter, 2010 ); (G) Dorstenia contrajerva, Moraceae ( Halbritter and Buchner, 2011 ); (H) Daphne genkwa, Thymelaeaceae ( Buchner and Halbritter, 2009 ); (I) Alcea ficifolia, Malvaceae ( Halbritter, 2012 ).

3 DECEMBER 2017, VOLUME 104 PRIEU ET AL. PANTOPORATE POLLEN IN ANGIOSPERMS 1839 MATERIALS AND METHODS The objective of this study was to obtain a global view of pantoporate pollen evolution at the angiosperm level. In particular, we aimed at estimating the minimum number of origins of this pollen type in flowering plants. We thus built a morphological data set with representative species of both pantoporate and nonpantoporate angiosperm taxa. Taxonomic sampling We define pantoporate pollen as pollen with six or more round apertures, distributed all over the surface of the grain ( Fig. 1 ), following Erdtman (1952) who used the term polyforate and Hesse et al. (2009). All other aperture patterns are treated here as nonpantoporate (including monosulcate and tricolpate pollen). We used different sources of information to record as many species producing pantoporate pollen as possible (Appendix S1, see Supplemental Data with this article). The main sources are the reference book of Erdtman (1952) on pollen morphology, and the Palynological Database ( PalDat, 2016 ). Erdtman described pollen morphology for most angiosperm families recognized at the time, and wrote down his numerous observations. His book on pollen morphology in angiosperms remains a primary reference in palynology. The Palynological Database provides descriptions and pictures for more than 2270 species. These two main sources are likely to present some taxonomic bias (for example the European flora is overrepresented, compared to taxa from other parts of the world), but we supplemented the data set to correct this bias by adding representative angiosperm species (see below). We also used articles specifically describing species producing pantoporate pollen (Appendix S1), with a standard literature search based on keywords, to score as many taxa with pantoporate species as possible (although we are aware that we may have missed some articles, especially old ones, which are not always properly indexed in databases). With this process, we are confident that we have scored almost every family known to include at least one pantoporate species, while we acknowledge that the data set is not exhaustive at the species level. To estimate as best as we can the number of origins of pantoporate pollen, it is not necessary to be exhaustive at the species level, because one species per origin is enough. In each family with at least one pantoporate species, we also sampled one or several nonpantoporate species if these were present. These were species with the typical aperture pattern of the family when the main aperture pattern was not pantoporate (e.g., tricolporate for Fabaceae), or, less frequently, species producing pollen with another aperture pattern when the family was mostly pantoporate (e.g., for the predominantly pantoporate Thymelaeaceae, we also sampled the genus Octolepis, which is tri- and tetraporate). When all species of a family produced pantoporate pollen, a nonpantoporate species of the most closely related family (or families) was also recorded (e.g., Saxifragaceae, nonpantoporate, sister to Grossulariaceae, pantoporate). We expanded our dataset of pantoporate species to include representative species from all major angiosperm taxa. All angiosperm orders are represented in our data set by at least one species per order. In addition to families including pantoporate species, each family of more than 100 species was also represented by at least one species. In total, 330 families (ca. 80% of all families recognized by APG III ( Angiosperm Phylogeny Group, 2009 ), 825 genera, and 1260 species were sampled in our data set. Character reconstruction All data were recorded in the PROTEUS database ( Sauquet, 2016 ), from which a NEXUS matrix was then output and analyzed with Mesquite v3.03 ( Maddison and Maddison, 2011 ) using parsimony. We acknowledge that model-based ancestral state reconstruction would also be interesting to apply to this character. However, not all pantoporate species have been sequenced yet and therefore, a complete molecular tree with branch lengths including all of the species sampled in our data set was not an option. Because our main objective was to highlight the phylogenetic distribution of this character in angiosperms, we believe that parsimony is sufficient and appropriate for this study. Phylogeny Phylogenetic relationships among families follow APG III ( Angiosperm Phylogeny Group, 2009 ) and updates are available on the Angiosperm Phylogeny Website ( Stevens, 2012 ). Clade names above order follow Cantino et al. (2007) and Soltis et al. (2011). In families with a high level of polymorphism, published phylogenies were used to refine the relationships among genera (Appendix S2), thus reducing the number of ambiguities. We assume genera to be monophyletic, unless specified otherwise in the literature. The Nexus file with the matrix and the tree is available (Appendix S4). RESULTS Fifty-four angiosperm families include at least one species with pantoporate pollen (Appendix 1), and seven of these are entirely pantoporate: Altingiaceae (13 species), Amaranthaceae (ca species), Drosophyllaceae (one species), Grossulariaceae (150 species), Halophytaceae (one species), Microteaceae (nine species), and Misodendraceae (eight species; numbers of species per family are taken from Stevens, 2012 ). With the exception of Amaranthaceae and Grossulariaceae, these are all small families with a few taxa. Moreover, the family Grossulariaceae contains only one genus (the 11 Ribes species available are all described as pantoporate). In other families with at least one pantoporate species, two different situations arise. In the first case, corresponding to most families, only a few species or a few genera were found to be pantoporate ( Fig. 2A and Appendix S3). In monocots, this is the case for Araceae (ca species), where two species of Anthurium produce pantoporate pollen, whereas other species in this family produce inaperturate or monoaperturate pollen. In eudicots, Fabaceae (ca. 19,750 species) fall in this category: six species are described as pantoporate, the pollen in this family being mainly tricolporate. The second case corresponds to families where pollen is mainly pantoporate, with a minority of species producing another aperture pattern. In monocots, this is the case of Alismataceae (one diporate species described in this family of 88 species), and in eudicots this is observed in Thymelaeaceae, Polemoniaceae, and Caryophyllaceae. In early diverging angiosperms (Amborellales, Austrobaileyales, Nymphaeales, Chloranthales, Magnoliidae, and Ceratophyllales), only very few species produce pantoporate pollen. They all belong to Chloranthaceae and Trimeniaceae ( Fig. 2A and Appendix 1). In monocots, eight families contain at least one species producing pantoporate pollen ( Fig. 2A and Appendix 1). These families are distributed in five orders: Alismatales, Commelinales, Dioscoreales, Poales, and Zingiberales. No species producing pantoporate pollen were recorded in the large order Asparagales. In eudicots, 44 families possess at least one pantoporate species ( Fig. 2A and Appendix 1).

4 1840 AMERICAN JOURNAL OF BOTANY These families are distributed throughout the phylogeny of eudicots. There are nonetheless, heterogeneities in the distribution of these families: some orders, such as Caryophyllales and Malpighiales, have a particularly high number of families with pantoporate species (12 and six families respectively), whereas other clades such as Campanulidae are quite poor in pantoporate species. Very few pantoporate species were found in the most species-rich families of angiosperms: none in Orchidaceae (27,800 species) and Poaceae (11,300 species), and only six in Fabaceae (19,560 species), three in Rubiaceae (13,150 species), and 10 in Asteraceae (23,600 species). Our data ( Fig. 2B and Appendix S3) show that pantoporate pollen appeared at least 66 times independently in angiosperms and therefore is a highly convergent trait. Pantoporate pollen is almost always derived, and reversions to nonpantoporate pollen are quite rare. Th e pantoporate state is unambiguously ancestral in 11 families ( Fig. 2A and Appendix 2): the seven pantoporate families listed above (Altingiaceae, Amarathaceae, Drosophyllaceae, Grossulariaceae, Halophytaceae, Microteaceae, and Misodendraceae), where no reversion has been inferred, and in four additional families (Alismataceae, Buxaceae, Polemoniaceae, and Nyctaginaceae), where we have inferred a few reversions. In total, we found at least 12 unambiguous reversions from pantoporate to nonpantoporate pollen (Appendix 2). In eudicots, these reversions lead to species usually (Continued)

5 D E C E M B E R 2017, V O LU M E 104 P R I E U E T A L. PA N TO P O R AT E P O L L E N I N A N G I O S P E R M S 1841 FIGURE 2 Distribution and origins of pantoporate pollen in angiosperm. (A) Phylogenetic tree of pantoporate and nonpantoporate angiosperm species. Reconstruction of characters states was done using parsimony. Each terminal taxon corresponds to a species, with a total of 1260 species distributed in 330 families, representing all orders of angiosperms. Species producing pantoporate pollen are tagged in red, species with another aperture pattern in black. Branches are colored in orange when parsimony yields equivocal results. The numbers in black correspond to the different origins of pantoporate pollen. Abbreviations: Lil. stands for Liliales, Asp. for Asparagales, Zing. for Zingiberales, Sax. for Saxifragales, Malp. for Malpighiales. (B) List of origins of pantoporate pollen, corresponding to the numbers in the tree. For a more detailed tree, see Appendix S3 where all species names are given.

6 1842 AMERICAN JOURNAL OF BOTANY producing pollen with three apertures (e.g., tricolpate), although other types with intermediate aperture numbers are also found (Appendix 2). There is only one unambiguous example in monocots: Caldesia oligococca in Alismataceae, with a transition from pantoporate to diporate pollen. DISCUSSION This study demonstrates that pantoporate pollen is widespread and has evolved at least 66 times independently in angiosperms. Pantoporate pollen grains are found in many different clades, although distribution of this character state is heterogeneous in the phylogenetic tree ( Fig. 2A ): some parts of the tree include a quite high number of pantoporate species and clades, whereas other clades completely lack this pollen type. Pantoporate pollen is rare in early diverging angiosperms (rare in the ANA grade and Chloranthales, absent in Magnoliidae and Ceratophyllales) compared to monocots and eudicots. The monocot clade includes several families with pantoporate species, but only Alismataceae are predominantly pantoporate (with one transition to diporate pollen; Appendix 2). In the eudicot clade, the situation is more complex. Thirty-nine families have fewer than 10 pantoporate species, but in some cases, large clades are characterized by this aperture pattern: Amaranthaceae, core Caryophyllaceae, and the majority of Malvoideae, Thymelaeaceae, and Polemoniaceae. Thus, pantoporate pollen is more often fixed (or nearly so) in large clades in eudicots than in monocots. Moreover, we detected more unambiguous reversions in eudicots than in monocots. Most inferred reversions lead to tricolpate pollen, but other aperture patterns are produced also (tri- and tetraporate pollen in Malvaceae, pentaporate pollen in Picrodendraceae, or pollen with several longitudinal colpi in Polemoniaceae; Appendix 2). Therefore, pantoporate pollen is characterized by very different phylogenetic distributions and histories in early diverging angiosperms, monocots, and eudicots. These differences could be due, in part, to the specific diversity of each group (the more species rich a clade is, the more pantoporate pollen there is). One part of the tree is particularly rich in pantoporate species: the order Caryophyllales presents many origins of pantoporate pollen with 12 families, out of the 31 families sampled, possessing at least one pantoporate species. Moreover, this order includes many more large clades with pantoporate species compared to the rest of the tree: for example, pantoporate pollen is found in all species of Amaranthaceae and core Caryophyllaceae, and in most Nyctaginaceae and Polygonaceae ( Fig. 2A and Appendix S3). It is interesting to see that in the least inclusive clade with Montiaceae and Cactaceae (numbers 58 to 61 in Fig. 2A ), many species are described as pantocolpate (in Anacampserotaceae, Talinaceae, Basellaceae, and Portulacaeae, according to Erdtman (1952 ) and other references, listed in Appendix S1). This clade is thus mainly pantoaperturate, with either round or elongated apertures (although some tricolpate species can be found, for example in Montiaceae), which is quite unusual in angiosperms. All these observations suggest that the order Caryophyllales is an exception compared to the rest of angiosperms, with a high concentration of pantoaperturate species. Despite its repeated origin, except in a few cases discussed above, pantoporate pollen was seldom fixed at large taxonomic scales: in most cases, only a few species or a few genera show this aperture pattern in any given family ( Fig. 2A ). Pantoporate pollen is almost always derived, in a terminal position, and reversions to another state are scarce (Appendix 2). This recurrent evolution but limited diversification of pantoporate pollen might indicate that this pollen type is usually not selected in the long term, even if it may be selected in the short term. Thus, we hypothesize that the distribution of pantoporate pollen in angiosperms results from the combination of short-term selective advantages and long-term selective disadvantages, which could influence speciation and extinction rates. Some procedures have been suggested to test for this kind of evolutionary scenario, where a trait is favorable in the short term but deleterious in the long run, a process called macroevolutionary self-destruction in Bromham et al. (2016). This scenario produces phylogenetic trees with the focal trait mainly present in the terminal branches of the tree, or tips. Bromham et al. (2016) discuss five macroevolutionary models (plus a null model) that could generate such tippy patterns. The parameters varying among models are trait-specific speciation and extinction rates ( Maddison et al., 2007 ) and the transition rate between the two states of the trait. Maddison et al. (2007) show, using simulations, that two of these models actually produce phylogenies with tippy distribution of the focal trait. The first model is the labile model, in which diversification rates (speciation and extinction rates) are not affected by the value of the trait, but transition rates are higher than in the null model, implying that the trait is easily gained, but also easily lost. The tippy pattern of labile traits is thus due to the fact that we can only see recent origins in the reconstructed phylogenies, the older acquisitions having likely reversed to the original state of the trait. The second model producing tippy patterns is the suicide model, in which the gain rate of the focal state is higher than the loss rate, and the focal state increases the extinction rate. For suicide traits, only recent origins are seen on phylogenies, because old lineages with this trait have become extinct. Although our data set is not suited to test these models because of its taxonomic scale and sampling strategy, the study by Bromham et al. (2016) may help us understand what causes the tippy pattern of pantoporate pollen in angiosperms. We think the labile model is unlikely to explain the distribution of pantoporate pollen for developmental reasons. Aperture number and position depend on several developmental steps. Pollen grains are produced during male meiosis or microsporogenesis, and the last points of contact between the four haploid microspores generally determine aperture number and position ( Wodehouse, 1935 ; Ressayre et al., 2002 ). This mechanism is not responsible for aperture determination in pantoporate pollen, but a quite simple mechanism (involving additional callose deposits at future aperture positions) could lead to the formation of this pollen type in distant angiosperm species ( Albert et al., 2014 ), thus allowing high gain rate. However, once pollen is pantoporate, development is often variable, making it almost impossible to reconstitute the ancestral last point of contact mechanism ( Matamoro-Vidal et al., 2016 ). Reversion from pantoporate pollen to the ancestral type (tricolpate or monosulcate) thus seems highly unlikely, and we have indeed shown in our tree that reversions are very scarce. Therefore, the labile model is unlikely to apply to pantoporate pollen. In the case of the suicide model, we have to consider advantages (increasing gain rate) and disadvantages (increasing extinction rate) of pantoporate pollen. These properties may be related to two special features: the partially dehydrated state of pantoporate pollen grains, and the association of this pollen type with dry stigmas. Pantoporate pollen grains appear to be partially dehydrated

7 DECEMBER 2017, VOLUME 104 PRIEU ET AL. PANTOPORATE POLLEN IN ANGIOSPERMS 1843 after anther opening: most pollen grains are dispersed in a dehydrated state, but in some species dehydration is only moderate ( Franchi et al., 2011 ). A theoretical model of the deformation of pollen grains shows in fact that pore-shaped apertures do not allow strong deformation, in contrast to furrow-shaped apertures ( Katifori et al., 2010 ). The amount of water loss is thus likely to be limited in porate species (this characteristic increasing the risk of rupture of the exine if transport time is too long, because water loss may be inevitable during pollen dispersal). This result is supported by the distribution of partially dehydrated pollen in angiosperms, which shows that many of them have porate apertures ( Franchi et al., 2002 ). Several studies have shown that partially dehydrated pollen grains have a short life expectancy, but germinate faster when they land on a stigma ( Nepi and Pacini, 1993 ; Nepi et al., 2001 ; Franchi et al., 2002, 2011 ). Faster germination could be a competitive advantage and thus could be selected, at least in the short term: competition between different pollen grains on a stigma to produce a pollen tube and to fertilize the female gametes can be very strong, thus any factor improving competitive ability of a given pollen grain would be favored by selection. This competitive advantage could increase the transition rate from nonpantoporate to pantoporate pollen (one of the characteristics of the suicide model in Bromham et al., 2016 ). Another possibility (not exclusive of the previous assumption on competitive advantage) is simply that pantoporate pollen is easily produced, making the transition rate higher. However, pantoporate pollen grains often have a shorter life span than other pollen types, and are more sensitive to desiccation ( Nepi and Pacini, 1993 ; Nepi et al., 2001 ; Franchi et al., 2002 ). Pantoporate species are therefore more sensitive to variation in environmental conditions during dispersal, because a delay during this phase is more likely to result in the death of the pollen grain than in the case of fully dehydrated pollen (such as tricolpate/tricolporate and monosulcate pollen grains). Such a character might thus be associated with an increase of extinction rate in the long term (the other characteristic of the suicide model in Bromham et al., 2016 ), because this morphology in unlikely to persist in the long term (unless adaptations limiting desiccation appear). An association of pantoporate pollen grains with dry stigmas might also give a short-term selective advantage. This association has been suggested by several authors ( Heslop-Harrison, 1979 ; Edlund et al., 2004 ). In wet stigmas, rehydration is facilitated regardless of aperture pattern, but this step is more sensitive in the case of dry stigmas. A high number of apertures distributed all over the surface could enable rapid and efficient rehydration in this context. Pollen grains with many apertures would thus germinate faster than pollen with few apertures, and this reproductive advantage could be selected in the short term, thus increasing the transition rate to pantoporate pollen. The suicide model, in which the gain rate of the focal trait is higher than the loss rate, and in which the focal trait increases the extinction rate, is thus more likely to account for the tippy distribution of pantoporate pollen in angiosperm that the labile model: developmental arguments can account for the asymmetry of transition rates, and reproductive syndrome can explain the increase in the extinction rate. Th e suicide and labile models are likely not the only ones able to generate tippy patterns in phylogenies. Some combinations of parameters were not tested by Bromham et al. (2016), but other cases of tippy patterns have been described, with different conclusions. For example, dioecy in angiosperms has been presented as an evolutionary dead end ( Heilbuth, 2000 ), but a recent study correcting for some bias found that this trait might be associated with increased diversification rate ( Käfer et al., 2014 ; but see Sabath et al., 2016 ). More generally, differences in speciation and extinction rates depending on a trait are often mentioned as candidates to account for asymmetric distributions of traits in a phylogeny, but transition rates between the trait states can also generate this kind of pattern, with no influence on diversification rate. There are many traits like dioecy or pantoporate pollen that show a tippy distribution in flowering plants, such as extrafloral nectaries ( Weber and Keeler, 2013 ), salt tolerance in grasses ( Bennett et al., 2013 ), or selfing in Solanaceae ( Goldberg et al., 2010 ); evolutionary mechanisms responsible for these patterns may be as numerous as the examples. These hypotheses on conflicting selective pressures do not explain why pantoporate pollen is sometimes fixed in large clades (such as Alismataceae and Amaranthaceae). It is possible that certain adaptations or strategies, have been acquired in these groups, and that these adaptations or strategies would prevent elimination of this aperture pattern. With the assumption that pantoporate pollen is eliminated in the long run because it is more sensitive to desiccation during dispersal, and thus more prone to die, we may hypothesize that any adaptation reducing mortality rate during dispersal could weaken this negative long-term selective pressure. For example, many pollen grains in Caryophyllaceae and Amaranthaceae possess an operculum (see for example Chenopodium album in Amaranthaceae ( Diethart, 2016 ), or Stellaria media and Agrostemma githago in Caryophyllaceae ( Halbritter, 2016a, b ) among other examples in PalDat (2016) ), which has been hypothesized to reduce desiccation and thus mortality during dispersal ( Heslop-Harrison, 1979 ; Furness and Rudall, 2003 ). We have shown here that pantoporate pollen grains are widespread, but rare at the scale of angiosperms. Pantoporate pollen is usually restricted to a few species in any given clade, and is fixed in only a few large groups, although exceptions exist. This distribution pattern might be explained by a short-term selective advantage, balanced by long-term elimination. Some hypotheses on the relationship between morphology and reproductive biology can be made. If pantoporate pollen is indeed short-lived pollen that germinates quickly, the short-term selection could be explained by an immediate competitive advantage. However, this reproductive syndrome seems to be unfavorable in the long run, maybe due to an increased sensitivity to environmental conditions. Similar cases of multilevel selection where short-term advantage is offset by negative selection at high systematic levels have been proposed, in plants and in other organisms: sexual and asexual reproduction in Eukarya ( de Vienne et al., 2013 ), sociality in spiders ( Agnarsson et al., 2006 ), and body size in mammals ( Clauset and Erwin, 2008 ), but such patterns remain relatively unexplored. New tools are being developed to test for such a pattern ( Bromham et al., 2016 ), and we may be able to perform such tests in the future. Pantoporate pollen may thus represent an interesting new model to study multilevel selection in plants. Further studies are needed (in pantoporate, as well as in other pollen traits) to investigate the links between form and function in pollen grains. ACKNOWLEDGEMENTS The authors thank Jürg Schönenberger and the University of Vienna for funding the eflower server hosting the PROTEUS database.

8 1844 AMERICAN JOURNAL OF BOTANY The authors also thank the Action Transversale du Muséum Formes possibles, formes réalisées (Museum National d Histoire Naturelle) for funding, and the Palynological Database PalDat for sharing pictures, especially Heidemarie Helbritter who helped us to clarify some data. The authors also thank the Associate Editor, Jim Doyle, and an anonymous reviewer for constructive comments. LITERATURE CITED Agnarsson, I., L. Avilés, J. A. Coddington, and W. P. Maddison Sociality in theridiid spiders: repeated origins of an evolutionary dead end. Evolution 60 : Albert, B., Z. Toghranegar, and S. Nadot Diversity and evolution of microsporogenesis in Bromeliaceae. Botanical Journal of the Linnean Society 176 : Angiosperm Phylogeny Group An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society 161 : Bennett, T. H., T. J. Flowers, and L. 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9 DECEMBER 2017, VOLUME 104 PRIEU ET AL. PANTOPORATE POLLEN IN ANGIOSPERMS 1845 Svojtka, M., and H. Halbritter Alisma lanceolatum. PalDat Palynological Database Available at: lanceolatum/ [Accessed July 17, 2015]. Till, W., and H. Halbritter Portea alatisepala. PalDat Palynological Database Available at: alatisepala/ [Accessed July 17, 2015]. Walker, J. W., and J. A. Doyle The bases of angiosperm phylogeny: palynology. Annals of the Missouri Botanical Garden 62 : APPENDIX 1 Families with at least one pantoporate species. Families that are entirely pantoporate are listed in bold type. Number of pantoporate species Family (order) recorded Acanthaceae (Lamiales) 2 Alismataceae (Alismatales) 28 Altingiaceae (Saxifragales) 7 Amaranthaceae (Caryophyllales) 45 Anacardiaceae (Sapindales) 4 Araceae (Alismatales) 2 Asteraceae (Asterales) 10 Balanophoraceae (Santalales) 1 Basellaceae (Caryophyllales) 3 Bromeliaceae (Poales) 6 Buxaceae (Buxales) 7 Cactaceae (Caryophyllales) 5 Campanulaceae (Asterales) 5 Caryophyllaceae (Caryophyllales) 94 Chloranthaceae (Chloranthales) 1 Convolvulaceae (Solanales) 8 Costaceae (Zingiberales) 7 Cucurbitaceae (Cucurbitales) 5 Cyperaceae (Poales) 7 Drosophyllaceae (Caryophyllales) 1 Euphorbiaceae (Malpighiales) 12 Fabaceae (Fabales) 6 Grossulariaceae (Saxifragales) 11 Haemodoraceae (Commelinales) 2 Halophytaceae (Caryophyllales) 1 Haloragaceae (Saxifragales) 1 Juglandaceae (Fagales) 2 Linaceae (Malpighiales) 1 Malpighiaceae (Malpighiales) 9 Malvaceae (Malvales) 44 Martyniaceae (Lamiales) 2 Microteaceae (Caryophyllales) 2 Misodendraceae (Santalales) 7 Montiaceae (Caryophyllales) 2 Moraceae (Rosales) 58 Nyctaginaceae (Caryophyllales) 16 Papaveraceae (Ranunculales) 10 Phyllanthaceae (Malpighiales) 7 Picrodendraceae (Malpighiales) 11 Plantaginaceae (Lamiales) 11 Podostemaceae (Malpighiales) 2 Polemoniaceae (Ericales) 33 Polygonaceae (Caryophyllales) 6 Ranunculaceae (Ranunculales) 19 Rivinaceae (Caryophyllales) 1 Rubiaceae (Gentianales) 3 Talinaceae (Caryophyllales) 2 Thismiaceae (Dioscoreales) 1 Thymelaeaceae (Malvales) 37 Trimeniaceae (Austrobaileyales) 4 Urticaceae (Rosales) 3 Vivianiaceae (Geraniales) 8 Zingiberaceae (Zingiberales) 1 Zygophyllaceae (Zygophylalles) 5 Weber, M. G., and K. H. Keeler The phylogenetic distribution of extrafloral nectaries in plants. Annals of Botany 111 : Wodehouse, R. P Pollen grains: their structure, identification and significance, in science and medicine. Hafner Publishing Co., New York. Wortley, A. H., H. Wang, L. Lu, D. Li, and S. Blackmore Evolution of angiosperm pollen. 1. Introduction. Annals of the Missouri Botanical Garden 100 : APPENDIX 2 Unambiguous transitions from pantoporate pollen to another aperture pattern. Species Corresponding origin in Fig. 2 Caldesia oligococca (Alismataceae) 4 Diporate Clematis vitalba (Ranunculaceae) 16 Tricolpate Aperture pattern Buxus obtusifolia (Buxaceae) 17 Tricolporate Abutilon (Malvaceae) 25 Tri- and tetraporate Petalostigma pubescens (Picrodendraceae) Ipomopsis, Langloisia, Eriastrum, Gilia, Collomia (Polemoniaceae) At least three independent transitions Convolvulus arvensis (Convolvulaceae) Persicaria alpina (Polygonaceae) Bougainvillea, Abronia (Nyctaginaceae) Two independent transitions 32 Pentaporate 44 Longitudinal colpi (between six and nine) 48 Tricolpate 54 Tricolpate 57 Tricolpate