Research

Evolutionary stasis in pollen morphogenesis due to natural selection

Alexis Matamoro-Vidal1,2,4, Charlotte Prieu1,2, Carol A. Furness3,Beatrice Albert2* and Pierre-Henri Gouyon1*

1Institut de Systematique, Evolution, Biodiversite, UMR 7205 – CNRS, MNHN, UPMC, EPHE Museum national d’Histoire naturelle, Sorbonne Universites, 57 rue Cuvier, CP39 F-75005 Paris, France; 2Laboratoire Ecologie Systematique et Evolution, UMR 8079 CNRS-AgroParisTech-Universite Paris-Sud, 11, F-91405 Orsay, France; 3Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK; 4Present address: Institut Jacques Monod, Universite Paris-Diderot, 75013, Paris, France

Summary Author for correspondence:  The contribution of developmental constraints and selective forces to the determination of Alexis Matamoro-Vidal evolutionary patterns is an important and unsolved question. We test whether the long-term Tel: +33 (0) 1 57 27 80 99 evolutionary stasis observed for pollen morphogenesis (microsporogenesis) in eudicots is due Email: [email protected] to developmental constraints or to selection on a morphological trait shaped by microsporoge- Received: 27 February 2015 nesis: the equatorial aperture pattern. Most eudicots have three equatorial apertures but Accepted: 15 June 2015 several taxa have independently lost the equatorial pattern and have microsporogenesis decoupled from aperture pattern determination. If selection on the equatorial pattern limits New Phytologist (2015) variation, we expect to see increased variation in microsporogenesis in the nonequatorial doi: 10.1111/nph.13578 clades.  Variation of microsporogenesis was studied using phylogenetic comparative analyses in 83 Key words: comparative analyses, species dispersed throughout eudicots including species with and without equatorial aper- constraints, macroevolution, tures. microsporogenesis, morphological evolution,  The species that have lost the equatorial pattern have highly variable microsporogenesis at morphospace, pollen aperture pattern. the intra-individual and inter-specific levels regardless of their pollen morphology, whereas microsporogenesis remains stable in species with the equatorial pattern.  The observed burst of variation upon loss of equatorial apertures shows that there are no strong developmental constraints precluding variation in microsporogenesis, and that the sta- sis is likely to be due principally to selective pressure acting on pollen morphogenesis because of its implication in the determination of the equatorial aperture pattern.

Introduction not possible as we can neither remove selection, if it is present, nor can we wait long enough to be confident that continued stasis Evolutionary stasis is common, but its causes remain unknown is not due to that selection. The evolutionary history of eudicot (Hansen & Houle, 2004; Eldredge et al., 2005; Futuyma, 2010). pollen, however, provides a natural experiment of the type Stasis in a character of a lineage can result from a failure of any Alberch proposed. The natural experiment we exploit here is that variation (caused by developmental constraints) or from the fail- a minority of eudicots produce special pollen types, in which a ure of the variation to be fixed (caused by stabilizing selection) portion of the selective pressure acting on the pollen morpho- (Charlesworth et al., 1982; Maynard Smith, 1983; Maynard genetic system (microsporogenesis) must be absent. The role of Smith et al., 1985; Williams, 1992; Gould, 2002), but the rela- this selective pressure in maintaining pollen stasis can thus be tive importance of these two causes is unclear. Here, we investi- tested by investigating whether there is an increase of develop- gate the evolutionary forces responsible for stasis in pollen mental variation in clades of species producing these special pol- development in flowering plants. len types, as compared with clades of species producing normal Alberch (1982) proposed a simple test for whether the evolu- pollen grains. This test is greatly facilitated by the fact that the tionary stasis of a morphological character is caused by stabilizing clades with special pollen types are phylogenetically nested within selection or by developmental constraints: study the variation of clades in which other members have the normal pollen types, the character in the absence of the selective pressure. If the char- thus providing clear evolutionary transitions from one condition acter remains unchanged, then developmental constraints are to the other. likely to be acting; if the character changes, then it is likely that The wall of pollen grains exhibits impressive morphological its stability was due to stabilizing selection. Such a test is usually variation (Fig. 1). This diversity is caused by variation in pollen morphogenesis, which takes place during microsporogenesis. *These authors contributed equally to this work. Microsporogenesis is the process by which, during male meiosis,

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(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

Fig. 1 Diversity of pollen aperture pattern in angiosperms. (a–d) In the monocot clade, and in the grade of early-divergent angiosperms, most species produce pollen with a single aperture located at the distal pole of the grain, but the shape of the aperture can vary. (a) Furrow-like aperture (monosulcate), Sandersonia aurantiaca (Liliaceae). (b) Three-armed furrow-like aperture (trichotomosulcate), Dianella caerulea (Liliaceae). (c) Four-armed furrow-like aperture (polychotomosulcate), Dianella tasmanica (Liliaceae). (d) Six-armed furrow-like aperture (polychotomosulcate), Hedyosmum goudotianum (Chloranthaceae). (e–h) In the eudicot clade, apertures are in most cases located at the equator of the grain. An overwhelming majority of these species produce pollen with three apertures (triaperturate pollen); the remainder show two, four, five or six equatorial apertures. (e) Two equatorial apertures (diaperturate), Banksia integrifolia (Proteaceae). (f) Three equatorial apertures (triaperturate), Discocleidion rufescens (Euphorbiaceae). (g) Four equatorial apertures (tetraaperturate), Pulmonaria officinalis (Boraginaceae). (h) Five equatorial apertures (pentaaperturate), Viola arvensis (Violaceae). (i–l) Other atypical aperture patterns can occur at lower taxonomic levels. (i) Spiral aperture (spiraperturate), Berberis vulgaris (Berberidaceae). (j) Many apertures globally distributed (pantoaperturate), Cerastium tomentosum (Caryophyllaceae). (k, l) No visible apertures (inaperturate), (k) Jatropha podagrica (Euphorbiaceae) and (l) Phyllanthus sp. (Phyllanthaceae) Scanning electron micrographs retrieved from the PalDat database (Buchner & Weber, 2000 onwards), with permission, except (d), which was courtesy of Heidemarie Halbritter. Bars, 10 lm.

the four daughter cells that will become the pollen grains are sep- et al., 2006). Evolution of microsporogenesis seems thus to repre- arated from each other by intersporal walls made of callose. sent a case of evolutionary stasis in eudicots relative to its evolu- Microsporogenesis is variable, as its three principal determinants tion in other angiosperm groups. Whether this stasis is the result (the cytokinesis type, the mode of callose deposition between of stabilizing selection or developmental constraints restricting meiotic daughter cells and the form of the tetrad of daughter the production of variation of microsporogenesis is an open ques- cells) present several possible states (Fig. 2). By determining all tion (Walker, 1974; Furness & Rudall, 2004). possible combinations of variants of these three features, a mor- Developmental constraints acting on microsporogenesis in phospace of the theoretically possible microsporogenesis patterns eudicots could, for example, be expressed as an absence of genetic can be defined (Fig. 3). This morphospace contains 20 possibili- variance, or as robustness relative to mutations, as described in ties but they do not appear to be observed equally in nature: some other biological systems (Bradshaw, 1991; Blows & Hoffmann, have never been observed, and others dominate large clades. In 2005; Stoltzfus & Yampolsky, 2009; Wagner, 2011). Alterna- the eudicot clade (an old and speciose clade with c. 165 000 tively, from a selective point of view, variation in microsporogen- species) the huge majority of species have the same microsporoge- esis could be limited by the fitness effects due to the resulting nesis type (no. 17 on Fig. 3) (Wodehouse, 1935; Blackmore & variation in pollen morphology. In particular, a selective pressure Crane, 1998; Nadot et al., 2008; Matamoro-Vidal et al., 2012). could be acting on microsporogenesis through its implication in By contrast, in the monocot clade and in early-divergent the determination of the aperture pattern (i.e. the number and angiosperms, microsporogenesis is quite variable (Furness & the position of the apertures on the pollen wall), which is a major Rudall, 1999b; Furness et al., 2002; Penet et al., 2005; Sannier component of pollen morphology. Apertures are areas where the

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Microsporogenesis sometimes more) equatorial apertures. This is the pollen type Three features of male meiosis more common in the eudicot clade, which is often referred as the ‘tricolpate’ clade (Judd & Olmstead, 2004), tricolpate being a Cytokinesis (a) term for pollen with three furrow-like apertures. The other microsporogenesis patterns allow less variation in pollen mor- phology, and result in pollen with lower aperture numbers on average (Fig. 3). It could be that the microsporogenesis pattern no. 17 has been Simultaneous Successive under stabilizing selection in eudicots because the resulting pollen morphologies have better performance than the morphologies (b) Callose deposition produced by other microsporogenesis patterns (Furness & * * * Rudall, 2004). In a previous study focused on the Euphorbiaceae * * family, we developed a conceptual approach allowing Alberch’s * * * * * logic to be applied to our system and thus to test whether the sta- * * sis on microsporogenesis is due to selection related to perfor- CpP CfP CfT CpT mance of pollen morphology (Matamoro-Vidal et al., 2012). Here, we extend the approach to eudicots as a whole. The (c) Tetrad form approach consists of studying the variation of microsporogenesis in a context where the putative selective pressure acting on it must be absent. For this, looking at microsporogenesis in species with nonequatorial pollen aperture patterns (e.g. lacking aper- tures (inaperturate), or with irregular or global aperture patterns) Tetragonal Rhomboidal Tetrahedral (Fig. 1i–l) is appropriate because in these, a partial or total dis- connection of aperture position from microsporogenesis occurs Fig. 2 Variation in the principal determinants of microsporogenesis. (a) Cytokinesis type (two states: successive or simultaneous). (b) Direction of (Wodehouse, 1935; Blackmore & Crane, 1998; Ressayre et al., callose deposition (four states: CpT, centripetal according to the tetrad; 2002a; Furness, 2007; Albert et al., 2009; Matamoro-Vidal et al., CpP, centripetal according to the cleavage plane; CfT, centrifugal 2012). Because of this disconnection, the effects of the selective according to the tetrad; CfP, centrifugal according to the cleavage plane). pressures, if any, acting on microsporogenesis due to aperture Asterisks show callose being deposited to separate the daughter cells. positioning should be released or even absent in these species. Arrows show the direction of callose progression. (c) Tetrad form (mainly four states: tetrahedral, rhomboidal, tetragonal and linear). Linear tetrads We thus propose the following rationale in order to discrimi- are not shown here for clarity, but see Figs 3 and 7 for illustration. These nate between two hypotheses. The first hypothesis is that the sta- three characters determine the modalities of callose deposition along the sis in microsporogenesis is maintained by selection related to wall separating the microspores. Callose is essential for the production of aperture pattern function. Under this hypothesis, microsporogen- pollen wall elements at the cell membrane level and variation in its esis in species producing pollen with typical equatorial eudicot deposition pattern can produce variation in pollen wall morphology. The images of microsporogenesis are courtesy of S. Nadot. aperture patterns (those shown in Fig. 3) is the target of a selec- tive pressure based on aperture pattern determination (Fig. 4a1). However, in species presenting atypical non-equatorial aperture external pollen wall is thin or absent (Fig. 1), providing sites at patterns (i.e. inaperturate, global or irregular aperture patterns), which pollen tube growth is initiated (Edlund et al., 2004). the aperture pattern is not determined by microsporogenesis. Changes in aperture number affect the longevity and fertilization Thus, the aperture pattern no longer imposes any selection on efficiency of pollen grains (Dajoz et al., 1991, 1993; Till-Bot- microsporogenesis, irrespective of any other selection occurring traud et al., 1999), and also the accommodation of changes in on pollen morphology in these species (Fig. 4b1). Under this pollen volume due to water gains and losses (harmomegathy) hypothesis, one would expect to observe variation in the charac- (Wodehouse, 1935; Halbritter & Hesse, 2004; Katifori et al., teristics of microsporogenesis in clades with atypical aperture pat- 2010; Firon et al., 2012). terns that are nested within clades in which other members have Microsporogenesis variation has been shown to affect the aper- equatorial apertures. In our second hypothesis, stasis in ture pattern in eudicots with equatorial apertures (e.g. Hulskamp microsporogenesis is maintained by developmental constraints. et al., 1997; Albert et al., 2010, 2011). This is achieved by modi- In this case, in species producing pollen with typical eudicot fying the modalities of callose deposition, thus altering pollen apertures patterns, microsporogenesis cannot vary because the exine wall synthesis at the level of the cell membrane (callose is characteristics of microsporogenesis are constrained (Fig. 4a2). an essential component for pollen exine wall synthesis; Dong Therefore, in species with atypical aperture patterns, even if the et al., 2005). Figure 3 shows the equatorial aperture patterns asso- phenotype of the pollen does not depend on the microsporogene- ciated with each microsporogenesis pattern, according to a model sis, the constraints should remain because disconnecting of aperture pattern ontogeny (Ressayre et al., 2002a,b). The microsporogenesis from aperture pattern determination is not microsporogenesis pattern more common in eudicots (pattern predicted to have any consequence on the constraints (i.e. on the no. 17) results in the production of pollen with three (but robustness of microsporogenesis variation relative to mutations).

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Cytokinesis type Mode of callose deposition

Tetrad form CfP CfTCpP CpT 1 2

2 1 3 Linear 3 4 56 2

Successive 1 3 Tetragonal 78910 1

2 4

3 Tetragonal 11 12 13 14 2 1 5 3 4 Rhomboidal

6 15 16 17 18 2 1 Simultaneous 5 3 4

Tetrahedral 19 20

1 2 3 Linear

Fig. 3 Morphospace for the microsporogenesis pattern. Twenty possible patterns are shown, as a function of three features of microsporogenesis: 1, cytokinesis type; 2, direction of callose deposition; 3, tetrad form. Callose progression to achieve the separation of the planes between the future pollen grains is represented in blue. White areas are the places where callose was not yet deposited at the stage represented in this scheme. Different numbersof cleavage planes are synthesized to separate the microspores during microsporogenesis, depending on the cytokinesis type and on the tetrad form (see numbering of planes on the left column). For clarity, only planes 1–3 are shown for the microsporogenesis patterns 15–18. For each microsporogenesis pattern, one or several pollen grains (grey spheres) with the corresponding aperture pattern(s) (coloured black) are illustrated, according to a model of aperture pattern ontogeny (Ressayre et al., 2002a, 2002b). CpT, centripetal according to the tetrad; CpP, centripetal according to the cleavage plane; CfT, centrifugal according to the tetrad; CfP, centrifugal according to the cleavage plane.

Consequently, microsporogenesis should remain stable, and we phylogenetically independent event suitable for testing the effect should observe continued stasis in morphogenesis in species with on microsporogenesis of the selective pressure related to aperture atypical aperture pattern (Fig. 4b2). pattern function. Previous studies (Albert et al., 2009; Mata- Inaperturate, global and irregular aperture patterns have moro-Vidal et al., 2012) have shown that microsporogenesis is evolved independently numerous times throughout the eudicot highly diverse in eudicot species producing inaperturate pollen; lineage (Furness, 2007). Each of these occurrences provides a however, these works were based on the study of a single clade of

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Hyp. 1: stasis is maintained by selection. Hyp. 2: stasis is maintained by constraints.

(a1) (a2) Constraints ?

Microsporogenesis Pollen with equatorial Microsporogenesis Pollen with equatorial aperture pattern aperture pattern ? Selection Constraints (b1) (b2) ? Pollen with Pollen with Microsporogenesis nonequatorial Microsporogenesis nonequatorial aperture pattern aperture pattern Microsporogenesis is free ? Variation of microsporogenesis to vary Selection remains constrained

Fig. 4 Rationale followed in this study, with emphasis on hypothetical selective pressures and constraints on microsporogenesis and on aperture pattern. (a) In species with equatorial aperture pattern, the aperture pattern is the result of a combination of microsporogenesis traits (black arrow), and evolutionary forces acting on pollen morphology act also on microsporogenesis and vice-versa. On the one hand, selective pressures could be acting on pollen morphology and thus these selective pressures could be preventing variation of microsporogenesis (a1; orange arrow). On the other hand, microsporogenesis stasis could be explained by constraints (e.g. mutational robustness) (a2; blue arrow). (b) In species that have lost the equatorial pattern, the aperture pattern determination is disconnected from microsporogenesis traits. As a result, selective pressures acting on pollen morphology (if any) do not affect microsporogenesis anymore (b1). Under these conditions, microsporogenesis should vary unless internal constraints are precluding variation (b2, blue arrow). inaperturate species. Here, we considerably extend these works by several buds of an individual, a number of tetrads ranging from c. studying the variation of microsporogenesis in 18 phylogeneti- 200 to 300 (Table 1). cally independent samples of species which produce pollen with atypical pollen aperture patterns dispersed throughout the eudi- Phylogeny, character optimization and test of correlated cot clade. evolution

Materials and Methods Phylogenetic relationships of the species for which we acquired data on pollen morphology and microsporogenesis were obtained by using available DNA sequences of rbcL and matK genes Species sampling and assessment of microsporogenesis (Table S1). Nucleotide sequences were aligned separately for each Data on microsporogenesis and on the pollen aperture pattern gene using MAFFT (Katoh et al., 2002) and combined in a single were obtained by collecting information from the literature for matrix. The tree was rooted with Ranunculales, which are sister 35 species and by making our own observations for 46 additional to all other eudicots (Soltis et al., 2011). The phylogenetic rela- species. The sampling includes species from 29 eudicot families tionships of species were inferred under the assumption of maxi- representing 18 orders (Table 1, Fig. 5). Microsporogenesis was mum likelihood (ML) using PhyML 3.0 (Guindon & Gascuel, assessed by dissecting flower buds at different developmental 2003; Guindon et al., 2010). A preliminary analysis resulted in a stages. For most species, the flower buds were obtained from topology presenting some slight discrepancies with previous fresh material collected from the living collections of botanic works (Soltis et al., 2011). Thus, we reran an analysis in which gardens. Buds were obtained by growing seeds obtained from the topology was constrained by the results of a large-scale botanic gardens in the laboratory glasshouse (Orsay, France) for angiosperm phylogeny (Soltis et al., 2011) to define relations the following species: Vernonia cinerea, Matthiola incana and among families, and of phylogenetic studies focusing at lower Linum strictum. For each species, the pollen morphology, tetrad taxonomic levels to define relations within the families for which form, cytokinesis type and mode of callose deposition during more than two species were sampled: Apocynaceae s.l (Potgieter intersporal wall formation were recorded according to a described & Albert, 2001; Ionta & Judd, 2007); Asteraceae (Liu et al., protocol (Ressayre, 2001). When more than one form of tetrad 2002; Goertzen et al., 2003); Berberidaceae (Wang et al., 2009); was observed for a given species, a survey of the proportion of Brassicaceae (Beilstein et al., 2008; Jaen-Molina et al., 2009); each tetrad form was conducted by counting, within one or Caryophyllaceae (Greenberg & Donoghue, 2011);

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Table 1 List of the studied species, with records of their pollen morphology, tetrad form, mode of callose deposition and cytokinesis type

Aperture Tetrad form Callose Species origin Taxa pattern (% of each state and n) deposition Cytokinesis or reference

Adoxaceae Viburnum tinus L. Triaperturate Td CpP Sim PBL Apocynaceae s.l. Acokanthera oppositifolia Triaperturate Td CpP Sim MNHN (no. 6061) (Lam.) Codd Amsonia tabernaemontana Walter Triaperturate Td CpP Sim MNHN (no. 22808) Carissa bispinosa (L.) Desf. Triaperturate Td (98.5%) - Rh (1.5%) (300) CpP Sim MNHN (no. 4355) Ex Brenan Apocynum cannabinum L. Global Td (26.6%) - Rh (29.7%) - CfP Succ MNHN (no. 228011) Tg (43.2%) - Lin (0.5%) (222) Periploca graeca L. Global Td (15%) - Rh (85%) (318) CpT Sim MNHN (no. 22829) Trachelospermum jasminoides Global Lin CfP Succ MNHN (no.15708) (Lem.) Apocynum androsaemifolium L. Inaperturate Td - Rh - Tg - Lin - T CfP Sim & Succ Frye & Blodgett (1905) Cynanchum callialata Buch.-Ham. Inaperturate Lin CfP Succ Devi (1964) ex Wight Hemidesmus indicus (L.) R. Br. Inaperturate T (majoritary) - Rh - Lin - Td CfP Sim Devi (1964) Hoya carnosa (L.) R. Br. Inaperturate Lin CfP Succ Schill & Dannenbaum (1984) Pergularia daemia (Forsk.) Chiov. Inaperturate Lin - T CfP Succ Devi (1964) Ochrosia elliptica Labill. Irregular Td (14.6%) - Rh (82.7%) - CpT Sim PBL Tg (2.7%) (295) Aquifoliaceae Ilex aquifolium L. Triaperturate Td (99%) - Rh (1%) (101) CpP Sim PBL Asteraceae Achillea millefolium L. Triaperturate Td CpP Sim PBL Catananche caerulea L. Triaperturate Td CpP Sim Blackmore et al. (2007) Felicia bergeriana O.Hoffm. Triaperturate Td ? Sim Sharma & Murty (1978) ex Zahlbr. Galinsoga parviflora Cav. Triaperturate Td (96%) - Rh (4%) (102) CpP Sim PBL Helianthus annuus L. Triaperturate Td ? Sim Gotelli et al. (2008) Solidago canadensis L. Triaperturate Td - Rh (rare) ? Sim Pullaiah (1978) Vernonia cinerea (L.) Less. Inaperturate Td (29%) - Rh (71%) (110) CpT Sim Collected seeds Berberidaceae Epimedium rubrum E.Morren Triaperturate Td (97.5%) - Rh (2%) - CpP Sim PBL Tg (0.5%) (294) Nandina domestica Thunb. Triaperturate Td ? Sim Furness (2008) Podophyllum peltatum L. Triaperturate Td (99.5%) - Rh (0.5%) (400) CpP Sim RBG (no. 1969 - 19646) Mahonia aquifolium Nutt. Irregular Tg CpT Succ PBL Mahonia japonica (Thunb.) Irregular Tg - Irregular CpT Succ Furness (2008) Brassicaceae Arabidopsis thaliana (L.) Heynh. Triaperturate Td CpP Sim Nadot et al. (2008) Capsella bursa-pastoris (L.) Medik. Triaperturate Td CpP Sim PBL Matthiola incana (L.) R. Br. Inaperturate Td (97.3%) - Rh (2.7%) (300) CpP & CpT Sim RBG (no. 310) subsp. glabra Caryophyllaceae Cerastium glomeratum Thuill. Global Td CpP Sim MNHN (no. 23327) Silene latifolia Poir. Global Td (81.5%) - Rh (17.9%) - CpP Sim PBL Tg (0.5%) (189) Silene pendula L. Global Td (81%) - Rh (19%) ? Sim MNHN Stellaria media (L.) Vill. Global Td (98%) - Rh (2%) (222) CpP Sim PBL Convolvulaceae Calystegia sepium (L.) R. Br. Global Td (99%) - Rh (1%) (100) CpP Sim PBL Stictocardia beraviensis Hallier f. Global Td (93.5%) -Rh (6.5%) (200) ? Sim MNHN Cucurbitaceae Bryonia dioica L. Triaperturate Td CpP Sim PBL Euphorbiaceae s.s. Dalechampia spathulata Triaperturate Td CpP Sim Matamoro-Vidal et al. (2012) (Scheidw.) Baill. Hevea brasiliensis Muell. Triaperturate Td ? Sim Rao (1964) Hura crepitans L. Triaperturate Td CpP Sim Matamoro-Vidal et al. (2012)

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Table 1 (Continued)

Aperture Tetrad form Callose Species origin Taxa pattern (% of each state and n) deposition Cytokinesis or reference

Macaranga tanarius (L.) Mull.€ Arg. Triaperturate Td CpP Sim Matamoro-Vidal et al. (2012) Mercurialis annua L. Triaperturate Td CpP Sim Matamoro-Vidal et al. (2012) Mercurialis perennis L. Triaperturate Td CpP Sim Matamoro-Vidal et al. (2012) Pedilanthus tithymaloides (L.) Triaperturate Td CpP Sim Matamoro-Vidal et al. (2012) Poit. subsp. Tithymaloides Ricinus communis (L.) Triaperturate Td CpP Sim Matamoro-Vidal et al. (2012) Manihot esculenta Crantz. Global Td (80%) - Rh (20%) (289) CpP & CpT Sim Matamoro-Vidal et al. (2012) Baloghia inophylla (G.Forst) Inaperturate Td (70%) - Rh (25%) - CpP & CpT Sim Matamoro-Vidal et al. (2012) P. Green Tg (5%) (215) Codiaeum variegatum var. pictum Inaperturate Td (72%) - Rh (20%) - CpP/CpT/CfT Sim & Succ Albert et al. (2009) (Lodd.) Mull.€ Arg. Tg (8%) (291) Eremocarpus setigerus (Hook.) Inaperturate Td (51%) - Rh (29%) - CpP & CpT Sim Matamoro-Vidal et al. (2012) Benth. Tg (19%) (228) Jatropha curcas L. Inaperturate Td - Rh ? Sim Liu et al. (2007) Jatropha gossypiifolia L. Inaperturate Td (89%) - Rh (10%) - CpP & CpT Sim Matamoro-Vidal et al. (2012) Tg (1%) (293) Jatropha integerrima Jacq. Inaperturate Td (61%) - Rh (32%) - CpP & CpT Sim (Matamoro-Vidal et al. (2012) Tg (7%) (188) Jatropha podagrica L. Inaperturate Td (83%) - Rh (15%) - CpP & CpT Sim (Matamoro-Vidal et al. (2012) Tg (2%) (218) Fabaceae Uraria crinita (L.) Desv. ex DC. Triaperturate Td CpP Sim Liu & Huang (1999) Vicia sepium L. Triaperturate Td CpP Sim PBL Juglandaceae Juglans regia L. Global Td CpP Sim PBL Lamiaceae Lamium purpureum L. Triaperturate Td CpP Sim PBL Linaceae Linum strictum L. Triaperturate Td (95.5%) - Rh (4.5%) (354) CpP Sim MNHN (no. 07-43) Reinwardtia cicanoba Global Td (20.8%) - Rh (78%) - CpP Sim RBG(no. 1973-12448) (Buch.-Ham.ex D.Don) Hara Lin (0.3%) - T (0.9%) (1277) Linderniaceae Torenia fournieri Triaperturate Td CpP Sim Vagi et al. (2004) Malvaceae Abutilon sp. ‘Red’ Triaperturate Td CpP Sim MNHN Hibiscus waimeae A. Heller Global Td (19%) - Rh (70.5%) - CpP & CpT Sim RBG (no. 1988-4056) Tg (10.5%) (278) Lavatera maritima Gouan Global Td (94%) - Rh (6%) (300) CpP Sim MNHN Nepenthaceae Nepenthes maxima Nees Inaperturate Td CpP Sim JBVP Onagraceae Chamerion angustifolium L. Triaperturate Td (98%) - Rh (1%) - CpP Sim Nadot et al. (2008) Tg (1%) (100) Orobanchaceae Orobanche hederae Duby Inaperturate Tg (91%) - Irreg (9%) (101) CfP Succ PBL Papaveraceae Chelidonium majus L. Triaperturate Td CpP Sim Nadot et al. (2008) Meconopsis cambrica (L.) Vig. Triaperturate Td (98%) - Rh (2%) (600) CpP Sim MNHN (no. 56808) Meconopsis horridula Inaperturate Td - Rh - Tg - Irreg CpP Sim & Succ MNHN (no. 47941) Hook. f. & Thomson Phyllanthaceae Phyllanthus juglandifolius Willd. Global Td (90.4%) - Rh (9.6%) (239) CpP Sim PBL Plantaginaceae Plantago lanceolata L. Global Td (69%) - Rh (18%) - CpP Sim & Succ PBL Tg (13%) (100) Veronica chamaedrys L. Triaperturate Td CpP Sim PBL Primulaceae Anagallis arvensis L. Triaperturate Td CpT Sim PBL Ranunculaceae Helleborus foetidus L. Triaperturate Td CpP Sim Ressayre et al. (2005) Ficaria verna Huds. Triaperturate Td (99%) - Rh (1%) (101) CpP Sim PBL

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Table 1 (Continued)

Aperture Tetrad form Callose Species origin Taxa pattern (% of each state and n) deposition Cytokinesis or reference

Rosaceae Chaenomeles japonica (Thunb.) Triaperturate Td CpP Sim PBL Lindl. Salicaceae Salix dasyclados Wimm. Triaperturate Td CpP & CpT Sim MNHN Populus italica Du Roi Inaperturate Td (85%) - Rh (11%) - CpT Sim PBL Tg (4%) (295) Sapindaceae Acer platanoides L. Triaperturate Td CpP Sim PBL Solanaceae Nicotiana tabacum L. Triaperturate Td CpP Sim Ressayre et al. (2003) Solanum nigrum L. Triaperturate Td CpP Sim PBL Tropaelaceae Tropaeolum majus L. Triaperturate Td CpP Sim Bolenbaugh (1928)

The last column indicates the origin of the species (with the botanical garden accession number when available) for which new data are provided or the reference for data obtained from the literature. For the tetrad form, the percentage of each form and the number of counted tetrads (n) are provided (when available). Botanical gardens: JBL, Jardin Botanique de Launay, Orsay, France; JBVP, Jardin Botanique de la Ville de Paris, France; MNHN, Museum national d’Histoire naturelle, Paris, France; RBG: Royal Botanic Gardens, Kew, UK. Tetrad form: Td, tetrahedral; Tg, tetragonal; Rh, rhomboidal; Lin, linear; T, T- shaped tetrad. Callose deposition: CpP, centripetal according to the cleavage plane; CpT, centripetal according to the tetrad; CfT, centrifugal according to the tetrad. n, number of tetrads counted to establish the percentages. Microsporogenesis: Sim, simultaneous; Succ, successive. ‘?’ denotes missing data.

Euphorbiaceae (Wurdack et al., 2005); Malvaceae (Baum et al., The two character states defined for the aperture pattern were tri- 2004; Small, 2004) and Papaveraceae (Kadereit et al., 1997). The aperturate (all the species with equatorial apertures studied were program was asked to optimize branch lengths only. The model triaperturate) or other (patterns with irregular, global or absent of nucleotide substitution was the general time reversible model apertures). Features of microsporogenesis were coded as binary (GTR). Parameter values were estimated during running. characters as follows: (1) cytokinesis, simultaneous or other (suc- Nucleotide frequencies were: (A) = 0.28995; (C) = 0.18194; cessive or heteromorphic); (2) mode of callose deposition, CpP (G) = 0.20172; (T) = 0.32640. The estimated proportion of or other (heteromorphic or CpT or CfP or CfT) and (3) tetrad invariable sites was 0.256 and a gamma distribution was assumed form, tetrahedral or other (heteromorphic or nontetrahedral for rates at invariable sites. tetrads). This coding is suitable for our aim of testing whether Pollen and microsporogenesis characters were optimized onto there is variation in microsporogenesis once aperture pattern is the phylogeny using ML, as implemented in Mesquite software not triaperturate (i.e. in any configuration where a portion of the (Maddison & Maddison, 2008). Character coding for the opti- selective pressures acting on the microsporogenesis is presumably mization analysis is shown in Table 2. Heteromorphism (the relaxed). coexistence of several states of the character within a single sta- BayesDiscrete tests for correlated evolution among pairs of men) was observed for microsporogenesis features. Callose depo- binary traits by taking into account the phylogenetic distribution sition and cytokinesis type were coded as heteromorphic when of the different states of these traits were performed. However, two or more character states were observed within a single sta- the result of the test does not rely on any reconstruction of the men. For the mode of callose deposition, heteromorphism traits’ ancestral character states. The method consists of estimat- resulted most times in the co-occurrence of the states CpP and ing the likelihood of two models of evolution: one in which the CpT. The other case of heteromorphism for the mode callose two traits evolve independently on the tree (independent model) deposition was the co-occurrence of all the states of this character. and one that assumes correlated evolution between traits (depen- These two cases were thus coded as states as such (Table 2). The dent model). The independent model has four parameters (the stages of microsporogenesis for which the mode of callose deposi- two transition rates between states 0 and 1 for each character). tion and the cytokinesis type are visible are very ephemeral, The dependent model has height parameters which are the whereas the tetrad form is relatively easy to observe. This allowed allowed transition rates (qij) between each of the four possible us to adopt a quantitative rule for heteromorphism in the case of states of the pair of traits: (0,0); (0,1); (1,0); and (1,1). Evidence the tetrad form: the tetrad form was coded as heteromorphic of correlated evolution is found when the dependent model fits when several states were observed within a single stamen and better than the independent model. We used the reversible-jump none of them was above the frequency of 0.95. Markov chain Monte Carlo (RJ MCMC) method (Pagel & We tested for a correlation between changes in pollen mor- Meade, 2006), which estimates the Bayesian posterior distribu- phology and variation in microsporogenesis features using the tions of the likelihoods of the data given the model (dependent BayesDiscrete method of the BayesTraits computer package (Pagel, or independent). The log-harmonic mean of the likelihoods is 1994). This method requires a binary coding of the characters. compared between two runs (one assuming correlated evolution

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Microsporogenesis Taxonomy Aperture pattern Tetrad shape Callose deposition Cytokinesis Family Order

Chelidonium majus TriaperturateTetrahedral CpP Sim

Meconopsis cambrica Triaperturate Tetrahedral CpP Sim Papaveraceae Meconopsis horridula Inaperturate HeteromorphicCpP Heteromorphic

Ficaria verna Triaperturate Tetrahedral CpP Sim Ranunculaceae Ranunculales Helleborus foetidus Triaperturate Tetrahedral CpP Sim Epimedium rubrum Triaperturate Tetrahedral CpP Sim Podophyllum peltatum Triaperturate Tetrahedral CpP Sim

Nandina domestica Triaperturate Tetrahedral ? Sim Berberidaceae Mahonia japonica Irregular TetragonalCpT Succ Mahonia aquifolium Irregular TetragonalCpT Succ Chamerion angustifolium Triaperturate Tetrahedral CpP Sim Onagraceae Myrtales

Acer platanoides Triaperturate Tetrahedral CpP Sim Sapindaceae Sapindales Hibiscus waimeae Global Heteromorphic CpP & CpT Sim

Abutilon sp. Triaperturate Tetrahedral CpP Sim Malvaceae Malvales Lavatera maritima Global Heteromorphic CpP Sim

Tropaeolum majus Triaperturate Tetrahedral CpP Sim Tropaelaceae

Matthiola incana glabra Inaperturate Tetrahedral CpP & CpT Sim Arabidopsis thaliana Triaperturate Tetrahedral CpP Sim Brassicaceae Brassicales Capsella bursa-pastoris Triaperturate Tetrahedral CpP Sim Chaenomeles japonica Triaperturate Tetrahedral CpP Sim Rosaceae Rosales Bryonia dioica Triaperturate Tetrahedral CpP Sim Cucurbitaceae Cucurbitales Juglans regia Global Tetrahedral CpP Sim Juglandaceae Fagales Uraria crinita Triaperturate Tetrahedral CpP Sim Fabaceae Fabales Vicia sepium Triaperturate Tetrahedral CpP Sim

Populus italica Inaperturate Heteromorphic CpT Sim Salicaceae Salix dasyclados Triaperturate Tetrahedral CpP & CpT Sim

Phyllanthus juglandifolius Global Heteromorphic CpP Sim Phyllanthaceae Linum strictum Triaperturate Tetrahedral CpP Sim Linaceae Reinwardtia cicanoba Global Heteromorphic CpP Sim Hura crepitans Triaperturate Tetrahedral CpP Sim Pedilanthus tithymaloides Triaperturate Tetrahedral CpP Sim Dalechampia spathulata Triaperturate Tetrahedral CpP Sim Macaranga tanarius Triaperturate Tetrahedral CpP Sim Ricinus communis Triaperturate Tetrahedral CpP Sim Mercurialis annua Triaperturate Tetrahedral CpP Sim Malpighiales Mercurialis perennis Triaperturate Tetrahedral CpP Sim

Manihot esculenta Global Heteromorphic CpP & CpT Sim Euphorbiaceae s.s. Hevea brasiliensis Triaperturate Tetrahedral ? Sim Baloghia inophylla Inaperturate Heteromorphic CpP & CpT Sim Codiaeum variegatum Inaperturate Heteromorphic Heteromorphic Heteromorphic

Eremocarpus setigerus Inaperturate Heteromorphic CpP & CpT Sim Jatropha curcas Inaperturate Heteromorphic ? Sim Jatropha integerrima Inaperturate Heteromorphic CpP & CpT Sim Jatropha gossypiifolia Inaperturate Heteromorphic CpP & CpT Sim Jatropha podagrica Inaperturate Heteromorphic CpP & CpT Sim

Nepenthes maxima Inaperturate Tetrahedral CpP Sim Nepenthaceae Cerastium glomeratum Global Tetrahedral CpP Sim Caryophyllales Stellaria media Global Tetrahedral CpP Sim Caryophyllaceae Silene latifolia Global Heteromorphic CpP Sim

Silene pendula Global Heteromorphic ? Sim Anagallis arvensis Triaperturate Tetrahedral CpT Sim Primulaceae Ericales Ilex aquifolium Triaperturate Tetrahedral CpP Sim Aquifoliaceae Aquifoliales Viburnum tinus Triaperturate Tetrahedral CpP Sim Adoxaceae Dipsacales Achillea millefolium Triaperturate Tetrahedral CpP Sim Solidago canadensis Triaperturate Tetrahedral ? Sim Vernonia cinerea Inaperturate Heteromorphic CpT Sim

Felicia bergeriana Triaperturate Tetrahedral ? Sim Asteraceae Asterales Catananche caerulea Triaperturate Tetrahedral CpP Sim Galinsoga parviflora Triaperturate Tetrahedral CpP Sim Helianthus annuus Triaperturate Tetrahedral ? Sim

Orobanche hederae Inaperturate Heteromorphic CfP Succ Orobanchaceae Lamium purpureum Triaperturate Tetrahedral CpP Sim Lamiaceae

Torenia fournieri Triaperturate Tetrahedral CpP Sim Linderniaceae Lamiales Plantago lanceolata Global HeteromorphicCpP Heteromorphic Plantaginaceae Veronica chamaedrys Triaperturate Tetrahedral CpP Sim Stictocardia beraviensis Global Heteromorphic ? Sim Convolvulaceae Calystegia sepium Global Tetrahedral CpP Sim Solanales Nicotiana tabacum Triaperturate Tetrahedral CpP Sim Solanaceae Solanum nigrum Triaperturate Tetrahedral CpP Sim

Ochrosia elliptica Irregular Heteromorphic CpT Sim Amsonia tabernaemontana Triaperturate Tetrahedral CpP Sim Acokanthera oppositifolia Triaperturate Tetrahedral CpP Sim Carissa bispinosa Triaperturate Tetrahedral CpP Sim

Hemidesmus indicus Inaperturate Heteromorphic CfP Sim Periploca graeca Global Heteromorphic CpT Sim Apocynaceae s.l. Gentianales

Trachelospermum jasminoides Global Linear CfP Succ

Apocynum androsaemifolium Inaperturate Heteromorphic CfP Heteromorphic Apocynum cannabinum Global Heteromorphic CfP Succ Hoya carnosa Inaperturate Linear CfP Succ Cynanchum callialata Inaperturate Linear CfP Succ Pergularia daemia Inaperturate Heteromorphic CfP Succ

Fig. 5 Phylogenetic mapping of changes in microsporogenesis in relation to the aperture pattern of the studied species. Clades containing species for which the selective pressure on microsporogenesis is absent (i.e. species producing atypical pollen aperture patterns) are emphasized with light grey rectangles. The taxonomic names of families and orders are according to Soltis et al. (2011). ‘?’ denotes an absence of data. CpT, centripetal according to the tetrad; CpP, centripetal according to the cleavage plane; CfT, centrifugal according to the tetrad; CfP, centrifugal according to the cleavage plane; Sim, simultaneous cytokinesis; Succ, successive cytokinesis.

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Table 2 List of characters and character states

Character states

Character State 1 State 2 State 3 State 4 State 5

Aperture pattern Triaperturate Inaperturate Global Irregular – Aperture pattern (binary) Triaperturate Inaperturate, Global or Irregular –– Tetrad form Tetrahedral Linear Tetragonal Heteromorphic – Tetrad form (binary) Tetrahedral Other (nontetrahedral or heteromorphic) –– – Mode of callose deposition CpP CpT CfP Heteromorphic 1 Heteromorphic 2 (CpP & CpT) (CpP, CpT, CfP & CfT) Mode of callose deposition CpP Other (non-CpP or heteromorphic) –– – (binary) Cytokinesis type Simultaneous Successive Heteromorphic –– Cytokinesis type (binary) Simultaneous Other (successive or heteromorphic) –– –

Alternative binary codings were defined for the purpose of the tests of correlated evolution, which can only be performed with binary characters. CpT, centripetal according to the tetrad; CpP, centripetal according to the cleavage plane; CfT, centrifugal according to the tetrad; CfP, centrifugal according to the cleavage plane.

and the other independent evolution). The relative fit of the two to the GTR model with a gamma distributed rate variation across models is tested using the Bayes factor [BF = 2(log [harmonic sites and an estimated proportion of invariable sites. Analyses were mean(dependent model)])/log[harmonic mean (independent performed as two independent runs of one million generations model)])]. Any positive value of the Bayes factor favours the each, with sampling every 100th generation. Likelihood values model assuming correlated evolution, but conventionally a ratio reached a plateau within 100 000 generations (checked with of > 2 is taken as ‘positive’ evidence, > 5 is ‘strong’ and > 10 is Tracer v.1.5; (Rambaut & Drummond, 2007)). These were ‘very strong’ evidence. Analyses were run for 10 million genera- deleted as burn-in. The tree files of the two runs were combined tions and sampled every 1000th generation. The first million in a single file from which 200 trees were randomly sampled. generations were discarded as burn-in. We used a gamma prior on the rate coefficients. Since no a priori information was avail- Results able about the mean and variance of the rate coefficients, we did not specify the mean and the variance of the gamma distribution: Data on microsporogenesis were obtained for 44 species produc- these two parameters were seeded from a uniform (0–10) hyper- ing triaperturate pollen and 37 producing pollen with atypical prior distribution (priors 1). This allows one to remain relatively aperture patterns (inaperturate, irregular or global). Reconstruc- uncommitted about the details of the prior distribution (Pagel & tion of the ancestral character state of the aperture pattern indi- Meade, 2006). Nevertheless, we measured the sensitivity of our cates that the ancestral state of our sample is triaperturate and results to the choice of priors by running analyses in which we that the species with atypical aperture patterns from 18 samples specified exponential priors seeded from a hyperprior with a uni- that are phylogenetically independent (Figs 5, 6). For the tetrad form (0–30) distribution (priors 2). form, the mode of callose deposition and cytokinesis type, the When evidence was found that a pair of traits evolves in a cor- ancestral states were tetrahedral, CpP and simultaneous, respec- – related fashion, the transition rates (qij) of the dependent model tively (Fig. 6). For the tetrad form, 14 15 transitions from the could be used to show the evolutionary sequence between the tetrahedral form to other states were observed (Fig. 6a). For mode ancestral and derived states of the two traits in a flow diagram. of callose deposition, 10 transitions from the CpP mode to other The significance of each transition rate was examined by quanti- states were found (Fig. 6b); and for cytokinesis type, there were fying the percentage of times a transition rate was assigned a value only six transitions from the ancestral simultaneous type to other of zero (Z) in the dependent model RJ MCMC chain. Transition states (Fig. 6c). rates that were rarely assigned to zero (Z < 10%) were considered The 44 triaperturate species investigated here carry out probable events (as in Higginson et al., 2012) whereas transition microsporogenesis via tetrahedral tetrads, CpP mode of callose rates often assigned to zero (Z > 90%) were considered improba- deposition and simultaneous cytokinesis (Figs 5, 6; Table 1), ble. Rates with intermediate Z values (10% ≤ Z ≤ 90%) were con- which is the typical eudicot microsporogenesis pathway. Addi- sidered as uncertain. tional modes of callose deposition were observed in two triaper- BayesTraits allows one to take into account phylogenetic uncer- turate species only. In Anagallis arvensis, the mode of callose tainty and branch length heterogeneity in the analyses. For this, deposition was CpT, and in Salix dasyclados, both CpP and CpT Bayes factors were estimated from a sample of 200 tree topologies modes of callose deposition were observed. Some slight variation obtained from a Bayesian analysis. This sample was obtained by was observed in the tetrad form for eight triaperturate species. analysing our alignment matrix with Bayesian inference using Rhomboidal tetrads were observed in addition to the typical MRBAYES version 3.1.2 (Huelsenbeck & Ronquist, 2001; tetrahedral state, but at low frequency (< 5% of the counted Ronquist & Huelsenbeck, 2003). The evolutionary model was set tetrads; Table 1).

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Chelidonium majus (a) Meconopsis cambrica MeconopsisMeconopsis horridulhorridulaa Ficaria verna Helleborus foetidus Epimedium rubrum Podophyllum peltatum Nandina domestica Aperture pattern MMahoniaahonia japonica Tetrade shape MMahoniaahonia aquifolium Chamerion angustifolium Triaperturate Acer platanoides Tetrahedral Inaperturate HHibiscusibiscus wawaimeaeimeae Heteromorphic Global Abutilon sp. Linear LLavateraavatera mmaritimaaritima Irregular Tropaeolum majus Tetragonal MatthiolaMatthiola incana glabra Arabidopsis thaliana Capsella bursa-pastoris Chaenomeles japonica Bryonia dioica Juglans regia Uraria crinita Vicia sepium Populus italicitalicaa Salix dasyclados PPhyllanthushyllanthus juglandifoliujuglandifoliuss Linum strictum ReReinwardtiainwardtiac cicanobaicanoba Hura crepitans Pedilanthus tithymaloides Dalechampia spathulata Macaranga tanarius Ricinus communis Mercurialis annua Mercurialis perennis MManihotanihot esculenta Hevea brasiliensis Baloghia inophinophyllaylla CCodiaeumodiaeum variegatum EEremocarpusremocarpus setsetigerusigerus JJatrophaatropha curcas JJatrophaatropha iintegerrimantegerrima Jatropha gossgossypiifoliaypiifolia JatroJatrophapha ppodagricaodagrica NNepenthesepenthes maxmaximaima CCerastiumerastium glomeratum StellariaStellaria mediamedia SileneSilene latifolilatifoliaa Silene pendulpendulaa Anagallis arvensis Ilex aquifolium Viburnum tinus Achillea millefolium Solidago canadensis VVernoniaernonia ccinereainerea Felicia bergeriana Catananche caerulea Galinsoga parviflora Helianthus annuus OOrobancherobanche hederae Lamium purpureum Torenia fournieri PlantagoPlantago lanceolatlanceolataa Veronica chamaedrys CalystegiaCalystegia sepium SStictocardiatictocardia beraviensiberaviensiss Nicotiana tabacum Solanum nigrum Ochrosia ellipticaelliptica Amsonia tabernaemontana Acokanthera oppositifolia Carissa bispinosa HeHemidesmusmidesmus inindicusdicus PPeriplocaeriploca graeca Trachelospermum jasminoides ApocynumApocynum androsaemifolium Apocynum cannabinum HHoyaoya carnoscarnosaa CCynanchumynanchum callialatacallialata Pergularia daemidaemiaa Fig. 6 Mirror trees comparing optimizations of the aperture pattern and (a) tetrad form, (b) mode of callose deposition and (c) cytokinesis type. The colours of the boxes near the species names indicate the state of the corresponding character. Pie charts on the nodes indicate the ancestral states obtained using maximum likelihood (ML). Pie chart colours represent the probability that a particular state occurred at this node. Analyses were conducted on the phylogram obtained with the PhyML analysis but are shown here on a cladogram for clarity. Clades containing species producing atypical pollen aperture patterns are emphasised with light grey rectangles. CpT, centripetal according to the tetrad; CpP, centripetal according to the cleavage plane; CfT, centrifugal according to the tetrad; CfP, centrifugal according to the cleavage plane.

With regard to the species producing atypical aperture pat- CpP (e.g. Meconopsis horridula), CpT (e.g. Vernonia), CfP (e.g. terns, pollen morphology could not be associated with any speci- Apocynum) or heteromorphic (8 species). The cytokinesis type in fic microsporogenesis pattern (Table 1, Figs 5, 6). Very diverse inaperturate species was either simultaneous (e.g. Matthiola), suc- microsporogenesis character states were found, such as successive cessive (e.g. Orobanche) or heteromorphic (e.g. Codiaeum). In the cytokinesis, rhomboidal, linear, tetragonal or irregular tetrads; same way, there was a complete lack of association between pol- and centrifugal callose deposition (Fig. 7). For inaperturate len morphology and microsporogenesis in species producing pol- species, the tetrad form could be exclusively tetrahedral (e.g. len with global aperture patterns. Regarding species with Nepenthes), linear (e.g. Hoya) or, in most of the cases (16 out of irregular aperture patterns, the tetrad form was either tetragonal 18 species), heteromorphic (i.e. several forms of tetrad were (Mahonia) or heteromorphic (Ochrosia); the mode of callose observed within the same stamen). Callose deposition in inaper- deposition was CpT, and the cytokinesis type was either succes- turate species was also very variable, as it could be exclusively sive (Mahonia) or simultaneous (Ochrosia). Overall, for the

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Chelidonium majus Meconopsis cambrica (b) MeconopsisMeconopsis horridula Ficaria verna Helleborus foetidus Epimedium rubrum Podophyllum peltatum Nandina domestica Aperture pattern MahoniaMahonia japonica Mode of callose deposition Mahonia aquifolium Chamerion angustifolium Triaperturate Acer platanoides CpP Inaperturate HiHibiscusbiscus waimeae CpT Global Abutilon sp. LLavateraavatera maritimamaritima CfP Irregular Tropaeolum majus Heteromorphic 1 (CpP & CpT) Matthiola incana glabra Heteromorphic 2 (CpP, CpT, CfP & CfT) Arabidopsis thaliana Capsella bursa-pastoris Chaenomeles japonica Bryonia dioica Juglans regia Uraria crinita Vicia sepium Populus italica Salix dasyclados Phyllanthus juglandifolius Linum strictum RReinwardtiaeinwardtiac cicanoba icanoba Hura crepitans Pedilanthus tithymaloides Dalechampia spathulata Macaranga tanarius Ricinus communis Mercurialis annua Mercurialis perennis ManihoteManihot esculenta sculenta Hevea brasiliensis Baloghia inophylla CCodiaeumodiaeum variegatum Eremocarpus setigerus JatrophaJatropha curcas Jatropha integerrima Jatropha gossypiifolia JatrophaJatropha ppodagricaodagrica NNepenthesepenthes maximmaximaa CCerastiumerastium glomeratum Stellaria medimediaa SileneSilene latifolia Silene pendula Anagallis arvensis Ilex aquifolium Viburnum tinus Achillea millefolium Solidago canadensis VVernoniaernonia ccinereainerea Felicia bergeriana Catananche caerulea Galinsoga parviflora Helianthus annuus OOrobancherobanche hederae Lamium purpureum Torenia fournieri PlantagoPlantago lanceolata Veronica chamaedrys CalystegiaCalystegia sepium StictocardiaStictocardia beraviensiberaviensiss Nicotiana tabacum Solanum nigrum Ochrosia elliptica Amsonia tabernaemontana Acokanthera oppositifolia Carissa bispinosa HHemidesmusemidesmus inindicusdicus PPeriplocaeriploca graeca TrachelospermumTrachelospermum jjasminoidesasminoides ApocynumApocynum androsaemifolium Apocynum cannabinum Hoya carnoscarnosaa CynanchumCynanchum callialata Pergularia daemia Fig. 6 Continued

species with atypical aperture patterns, microsporogenesis was and typical microsporogenesis (simultaneous cytokinesis – altered (i.e. different from the typical eudicot microsporogenesis Fig. 8b; tetrahedral tetrad – Fig. 8c; and CpP mode of callose pathway) for at least one of the three microsporogenesis features deposition – Fig. 8d) to the derived state of an atypical pollen in 32 species, whereas microsporogenesis remained identical to morphology and an atypical or variable microsporogenesis. Our the typical eudicot microsporogenesis pathway in five species only results suggest that the loss of the triaperturate morphology (Fig. 5). Intra-specific variation of microsporogenesis was always preceded alterations of any of the microsporogenesis fea- > observed in 29 out of 37 species with atypical aperture patterns. tures (q13 q12), and that this loss created conditions favourable > We detected correlated changes in the three pairs of traits to the appearance of variation of the microsporogenesis (q34 0). tested (pollen morphology/tetrad form: Bayes factor (BF) c. 49; The alternative route in which the variation of the microsporoge- pollen morphology/callose deposition: BF c. 12; pollen morphol- nesis appears first, in a context where pollen morphology is tria- ogy/cytokinesis type: BF c. 18) (Table 3), indicating strong corre- perturate, is poorly supported (q12 c. 0 for the cytokinesis type lations between the transition from triaperturate to atypical and for the tetrad form using priors 1 or 2; for the mode of cal- aperture patterns and the appearance of variation for each of the lose deposition with the priors 1 only). microsporogenesis traits. This result was not sensitive to the In the context of the loss of the triaperturate morphology, we choice of priors (Table 3). Figure 8 shows the most probable evo- found differences in the amount of variation observed between lutionary route from the ancestral state of triaperturate pollen the three microsporogenesis features: the numbers of species and

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Chelidonium majus (c) Meconopsis cambrica MMeconopsiseconopsis horridulhorridulaa Ficaria verna Helleborus foetidus Epimedium rubrum Podophyllum peltatum Nandina domestica Aperture pattern MMahoniaahonia japonicajaponica MMahoniaahonia aquifolium Cytokinesis type Chamerion angustifolium Triaperturate Acer platanoides Simultaneous Inaperturate HibiscusHibiscus waimeaewaimeae Successive Global Abutilon sp. LLavateraavatera mmaritimaaritima Heteromorphic Irregular Tropaeolum majus MatthiolaMatthiola incana glabra Arabidopsis thaliana Capsella bursa-pastoris Chaenomeles japonica Bryonia dioica Juglans regia Uraria crinita Vicia sepium Populus italica Salix dasyclados PPhyllanthushyllanthus juglandifoliujuglandifoliuss Linum strictum RReinwardtiaeinwardtia cicanoba cicanoba Hura crepitans Pedilanthus tithymaloides Dalechampia spathulata Macaranga tanarius Ricinus communis Mercurialis annua Mercurialis perennis MManihotanihot esculentaesculenta Hevea brasiliensis Baloghia inophinophyllaylla CCodiaeumodiaeum variegatum Eremocarpus setigerusetigeruss JJatrophaatropha curcacurcass JJatrophaatropha iintegerrimantegerrima Jatropha gossgossypiifoliaypiifolia JatroJatrophapha ppodagricaodagrica NNepenthesepenthes maxmaximaima CCerastiumerastium glomeratum SStellariatellaria medimediaa SSileneilene latifolilatifoliaa Silene pendulpendulaa Anagallis arvensis Ilex aquifolium Viburnum tinus Achillea millefolium Solidago canadensis VVernoniaernonia ccinereainerea Felicia bergeriana Catananche caerulea Galinsoga parviflora Helianthus annuus OOrobancherobanche hederae Lamium purpureum Torenia fournieri PPlantagolantago lanceolatlanceolataa Veronica chamaedrys CCalystegiaalystegia sepium SStictocardiatictocardia beraviensiberaviensiss Nicotiana tabacum Solanum nigrum Ochrosia elliptica Amsonia tabernaemontana Acokanthera oppositifolia Carissa bispinosa HeHemidesmusmidesmus inindicusdicus PPeriplocaeriploca graecgraecaa TrachelospermumTrachelospermum jasminoidesjasminoides ApocynumApocynum androsaemifolium ApocynumApocynum cannabinum HoyaHoya carnosa CynanchumCynanchum callialatcallialataa Pergularia daemia Fig. 6 Continued of clades with atypical microsporogenesis states were different for modes (Fig. 9b). In the same way, simultaneous cytokinesis was the three microsporogenesis features. Tetrad form, callose deposi- observed much more frequently than successive cytokinesis tion and cytokinesis varied in 31, 25 and 12 species, respectively, (Fig. 9c). and these alterations were distributed among 15, 10 and 6 inde- pendent phylogenetic samples, respectively (Figs 5, 6). Discussion Within each microsporogenesis trait, in the context of the loss of the triaperturate morphology, the different states of each trait The stability of pollen morphology and development in eudicots were not observed in equal proportions (Fig. 9). For the tetrad is a fact that is widely recognized among botanists. The eudicot form, within the 18 groups of species producing atypical pollen clade encompasses phenomenal variation in morphological, morphology, the tetrahedral, rhomboidal and tetragonal states anatomical, ecological and biochemical characters (Judd & were observed in 16, 14 and 10 groups, respectively, whereas Olmstead, 2004), though microsporogenesis and pollen mor- other tetrad forms (linear, irregular and T-shaped) were observed phology in this group are relatively conserved: most species at a lower frequency, in one or two groups only (Fig. 9a). For the produce pollen with three equatorial apertures through a very mode of callose deposition, we observed a higher number of stereotypic microsporogenesis pattern. According to the criterion occurrences for the centripetal modes than for the centrifugal of Williams (1992), this pattern may thus be considered as

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(a) (b) (c) (d) (e)

Fig. 7 (a) ‘Typical’ eudicot microsporogenesis pattern and (b–e) some examples of changes after the removal of selective pressure. Each example is accompanied by a diagram to aid the interpretation of the images. In (b) and (d), the images correspond to a longitudinal section of the tetrad shown in the diagrams. In the diagrams, the microspores (future pollen grains, represented as grey spheres) are being separated by the progressive deposition of callose walls (blue). (a) ‘Typical’ eudicot microsporogenesis. In this pattern, the microspores are arranged in a tetrahedral tetrad, cytokinesis is simultaneous, and callose is deposited centripetally (Lavatera maritima). (b) Microspores arranged in a rhomboidal tetrad. As in (a), cytokinesis is simultaneous, and callose is deposited centripetally (Reinwardtia cicanoba). (c) Microspores arranged in a tetragonal tetrad. Cytokinesis is successive, and callose is deposited centrifugally (Apocynum cannabinum). (d) Microspores arranged in a linear tetrad. Cytokinesis is simultaneous, and callose is deposited centripetally (Reinwardtia cicanoba). (e) Microspores arranged in a linear tetrad. Cytokinesis is successive, and callose deposition is centrifugal (Trachelospermum jasmino€ıdes).

evolutionary stasis. The nature of the evolutionary processes microsporogenesis then microsporogenesis is not constrained, underlying the conservation of pollen aperture pattern and and it is likely that the evolutionary stasis in microsporogenesis is microsporogenesis is fundamental for our understanding of, on due to selection on pollen morphology. A drawback of this the one hand, the evolutionary morphology of plants (Walker, rationale is that finding an absence of variation in species with 1974; Furness & Rudall, 2004) and, on the other hand, a com- atypical aperture pattern would be in part inconclusive because mon evolutionary pattern (evolutionary stasis) (Hunt, 2007; this result could be explained by constraints or by unidentified Kellermann et al., 2009; Piras et al., 2009; Ellegren, 2010; selective pressures acting on microsporogenesis. However, we Lavoue et al., 2011; Van Bocxlaer & Hunt, 2013). found a remarkable burst of variation affecting all of the three In this study we hypothesized that stasis in microsporogenesis microsporogenesis traits studied in the species producing atypical was due to a selective pressure related to the determination of a aperture patterns, clearly showing that there is a relationship pollen morphological trait (aperture pattern). This was tested by between the involvement of microsporogenesis in pollen aperture studying the variation of microsporogenesis in a context where pattern ontogeny and stasis. the hypothetical selective pressure must be absent. Such condi- Evolutionary transitions from the triaperturate to an atypical tions are found in species with atypical aperture patterns pollen morphology were correlated with the occurrence of intra- because in these species microsporogenesis is not the determinant individual and inter-specific variation in microsporogenesis. of the aperture pattern. The disconnection of microsporogenesis Some slight variation in microsporogenesis was observed among from aperture pattern determination requires the loss of the the triaperturate species investigated herein, but it is quite negli- equatorial aperture pattern, a transformation that could be con- gible if compared with the huge variation observed in species strained. However, Dobritsa and Coerper (2012) have shown with atypical pollen. Only a few species producing atypical pollen that a mutation in a single gene (INP1) was sufficient to lose all morphologies had microsporogenesis identical to that of the tria- three apertures in Arabidopsis thaliana, resulting in plants with an perturate species. Instead, species producing atypical pollen mor- inaperturate pollen morphology lacking any other visible mor- phology exhibited a wide range of microsporogenesis pathways. phological abnormality, and otherwise normal and fertile. More- For example, in Reinwardtia, we observed, within a single plant, over, inaperturate pollen appeared independently in distant four different microsporogenesis patterns, though pollen mor- groups both in eudicots and in monocots (Furness & Rudall, phology was always the same, namely global. 1999a; Furness, 2007). This suggests that aperture loss, and thus The finding that in most of our species with atypical aperture the disconnection of microsporogenesis from the determination patterns pollen morphology could not be associated with any of aperture pattern is not a difficult transformation to achieve. microsporogenesis pattern, and that microsporogenesis was vari- According to our rationale (see Introduction and Fig. 4), if able at the intra-individual level without any change in pollen species with atypical aperture patterns present variable morphology, is consistent with previous works (Wodehouse,

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Table 3 Results of the tests for correlated evolution by this microsporogenesis pattern should present some improved performance as compared with the other morphologies shown in Value Fig. 3. Trait X Trait Y Parameter Priors 1 Priors 2 One possibility is that selection comes from the direct effects of aperture number on pollen reproductive success. Dajoz et al. Aperture pattern Cytokinesis type BF 18.9 16.3 (1991, 1993) and Till-Bottraud et al. (1999) have shown the exis- Z (%) q 99.8 99.3 12 tence of a trade-off such that increased aperture number corre- q21 29.8 31.7 q13 0.0 0.0 lates with a faster pollen germination rate upon landing on the q31 1.3 1.7 stigma, but also with decreased pollen survival during dispersal. q24 26.4 27.7 Another function of apertures is to contribute to the accommoda- q42 82.2 82.1 tion of pollen wall deformations further to volume changes of the q 0.0 0.0 34 cell due to water exchanges with the environment (Wodehouse, q43 41.4 45.7 Aperture pattern Tetrad form BF 49.8 49.2 1935; Halbritter & Hesse, 2004; Katifori et al., 2010). Moreover, Z (%) q12 99.2 91.9 aperture sites are involved in the regulation of pollen hydrody- q21 13.0 12.0 namics (Heslop-Harrison, 1979; Firon et al., 2012). The aperture q13 0.4 4.9 pattern is thus linked with many functions, and it could be that q 0.1 1.1 31 aperture patterns (especially the triaperturate one) resulting from q24 21.7 18.9 q42 95.2 90.6 eudicot typical microsporogenesis do a relatively good trade-off q34 0.2 1.6 between all these functions. Future experimental work on the q43 3.4 2.1 relationships between pollen morphology and pollen perfor- Aperture pattern Mode of BF 12.7 16.3 mance will have to test this prediction. Another possibility could callose deposition Z (%) q 84.3 40.3 12 be that it is the flexibility in pollen morphology provided by the q21 14.7 16.5 q13 0.5 0.5 microsporogenesis pattern no. 17 that represents an advantage. q31 2.7 15.0 < There is growing evidence that mechanisms facilitating the gener- q24 9.5 5.6 ation of variation can be maintained in the long-term by lineage < q42 6.5 18.9 selection, despite no obvious short-term advantage and even q 8.0 28.7 < 34 strong short-term disadvantages (Goldberg et al., 2010; de q43 69.0 62.8 Vienne et al., 2013). Under this scenario, the stasis on the For each pair of traits X–Y tested, the value of the Bayes factor (BF) and of microsporogenesis would have been maintained, in the long- the percentage of times each transition rate was assigned to zero (Z) in the term, by selection of lineages having morphological diversity and reversible-jump Markov chain Monte Carlo (RJ MCMC) chain are given. rapid adaptation in a changing environment. This hypothesis is Results are given for the two sets of hyperpriors used: gamma (Priors 1) and exponentials (Priors 2). Bold numbers indicate significant values of BF consistent with the maintenance of a single microsporogenesis or transitions rates rarely assigned to zero (Z < 10%). Grey numbers depict pattern despite environmental change over long periods of time. improbable transition rates (Z ≥ 90%). The ‘<‘ marks indicate discrepancies However, this hypothesis lacks an explanation for how between the two sets of hyperpriors used. microsporogenesis has been maintained in the short-term, and thus it calls for the possibility that other evolutionary forces are involved in the maintenance of the stasis. Accordingly, our obser- 1935; Blackmore & Crane, 1998; Ressayre et al., 2002a; Furness, vations on the microsporogenesis variation in the absence of 2007) proposing that in these species microsporogenesis is dis- selection due to aperture pattern suggest that there might be con- connected from pollen aperture pattern determination. The fact straints and/or additional selective pressures acting on the possi- that under these conditions microsporogenesis was highly vari- ble variations of microsporogenesis. able also supports the view that microsporogenesis is not strongly We found that the tetrad form and callose deposition change constrained by the genotype-phenotype map or by other uniden- more often and more rapidly in response to the absence of tified selective pressures. selective pressure than does the cytokinesis type (Figs 5, 6). Sim- Our evolutionary model (Fig. 8) supports a scenario in which ilar results have been obtained in inaperturate monocot species, changes in aperture pattern occur first, allowing for a release of which have been found to be highly labile in the tetrad form selection on microsporogenesis and then, in most cases, this is but not in the cytokinesis type (Nadot et al., 2006; Pereira followed by the occurrence of variation in microsporogenesis. Nunes et al., 2009). These differences in the timing of changes Note that if the variation observed was due to a release of a devel- between characters may be interpreted in three non-exclusive opmental constraint on microsporogenesis, one would expect ways. First, spontaneous variation rates may differ among the that variation in microsporogenesis appears first and is accompa- three traits, for example, because of differences in the mutation nied by variation in aperture pattern but it is the alternative sce- rates between different parts of the genome (Tian et al., 2008) nario which is supported by the data (Fig. 8). The conservation or because of differences in the robustness to mutations of each of microsporogenesis pattern no. 17 is likely due principally to a of these traits (Wagner, 2011). A second explanation is that the selective pressure related to its implication in the determination morphogenetic system is such that certain changes must neces- of aperture pattern. It follows that pollen morphologies produced sarily occur if certain others are to become established (Alberch

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(a) Trait X = 0 q 12 Trait Y = 0 q 13

q 31 q 21 Trait X = 0 Trait X = 1 Trait Y = 1 Trait Y = 0

q 42 q 43 q Trait X = 1 24 q Trait Y = 1 34

(b)X: Pollen (triaperturate/atypical) (c)X: Pollen (triaperturate/atypical) (d) X: Pollen (triaperturate/atypical) Y: Cytokinesis type (simultaneous/other) Y: Tetrad form (tetrahedral/other) Y: Callose deposition (CpP/other)

Triaperturate Triaperturate Triaperturate Simultaneous Tetrahedral CpP

Triaperturate Atypical Triaperturate Atypical Triaperturate Atypical Other Simultaneous Other Tetrahedral Other CpP

Atypical Atypical Atypical Other Other Other

Fig. 8 Flow diagrams illustrating the most likely evolutionary sequence from the ancestral state to the derived state for the different pairs of traits analysed. Related to Table 3. (a) The flow diagram, as explained in Pagel (1994). There are eight possible transitions (qij) among the four state combinations, (0,0), (0,1), (1,0) and (1,1), resulting from two binary traits (X and Y). (b–d) Flow diagrams for the analyses between pairs of traits: (b) ‘aperture pattern’ and ‘cytokinesis type’; (c) ‘aperture pattern’ and ‘tetrad form’; and (d) ‘aperture pattern’ and ‘mode of callose deposition’. In (b–d), probable transitions (Z < 10%) are depicted by thick black arrows; uncertain transitions (10% ≤ Z ≤ 90%) are depicted by thin black arrows; and improbable transitions (Z > 90%) were removed from the figure. Dashed arrows indicate transitions that were probable in the analysis using the ‘Priors 1’ set of hyperpriors, and uncertain using the ‘Priors 2’ set of hyperpriors. CpP, centripetal according to the cleavage plane.

(a) 100 16/18 (89 %) 14/18 (78 %) 80

10/18 (56 %) % 60

40

20 2/18 (11 %) 2/18 (11 %) 1/18 (6 %) 0 Tetrahedral Rhomboidal Tetragonal Linear Irregular T-shaped Tetrad shape Fig. 9 Variation of the microsporogenesis (b) (c) 100 100 traits in the species producing pollen with 16/18 (89 %) atypical aperture patterns. For each trait (a, tetrad form; b, mode of callose deposition, c, 80 80 cytokinesis type) the number of occurrences 12/18 (67 %) of each state in the 18 groups of species 60 60 producing pollen with atypical aperture 9/18 (50 %) % % patterns, and the corresponding percentages, 40 40 6/18 (33 %) are given. For example, for the tetrad form, the tetrahedral state was observed at least in one species in 16 of the 18 groups of species 20 20 2/18 (11 %) investigated. CpT, centripetal according to 1/18 (5,5 %) the tetrad; CpP, centripetal according to the 0 0 cleavage plane; CfT, centrifugal according to CpP CpT CfP CfT Simultaneous Successive the tetrad; CfP, centrifugal according to the Mode of callose deposition Microsporogenesis cleavage plane.

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