The Developmental Basis of an Evolutionary Diversiﬁcation of Female Gametophyte Structure in Piper and Piperaceae

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Annals of Botany 103: 869–884, 2009 doi:10.1093/aob/mcp011, available online at www.aob.oxfordjournals.org

The developmental basis of an evolutionary diversiﬁcation of female gametophyte structure in Piper and Piperaceae

Eric N. Madrid and William E. Friedman* University of Colorado, Department of Ecology and Evolutionary Biology, Boulder, CO 80309, USA

Received: 7 November 2008 Returned for revision: 3 December 2008 Accepted: 5 December 2008 Published electronically: 6 February 2009

† Background and Aims Fritillaria-type female gametophyte development is a complex, yet homoplasious developmental pattern that is interesting from both evolutionary and developmental perspectives. Piper (Piperaceae) was chosen for this study of Fritillaria-type female gametophyte development because Piperales represent a ‘hotspot’ of female gametophyte developmental evolution and have been the subject of several recent molecular phylogenetic analyses. This wealth of phylogenetic and descriptive data make Piper an excellent candidate for inferring the evolutionary developmental basis for the origin of Fritillaria-type female gametophytes. † Methods Developing ovules of Piper peltatum were taken from greenhouse collections, embedded in glycol methacrylate, and serially sectioned. Light microscopy and laser scanning confocal microscopy were combined to produce three-dimensional computer reconstructions of developing female gametophytes. The ploidies of the developing embryos and endosperms were calculated using microspectroﬂuorometry. † Key Results The data describe female gametophyte development in Piper with highly detailed three- dimensional models, and document two previously unknown arrangements of megaspore nuclei during early development. Also collected were microspectroﬂuorometric data that indicate that Fritillaria-type female gametophyte development in Piper results in pentaploid endosperm. † Conclusions The three-dimensional models resolve previous ambiguities in developmental interpretations of Fritillaria-type female gametophytes in Piper. The newly discovered arrangements of megaspore nuclei that are described allow for the construction of explicit hypotheses of female gametophyte developmental evolution within Piperaceae, and more broadly throughout Piperales. These detailed hypotheses indicate that the common ancestor of Piperaceae minus Verhuellia had a Drusa-type female gametophyte, and that evolutionary transitions to derived tetrasporic female gametophyte ontogenies in Piperaceae, including Fritillaria-type female gametophyte development, are the consequence of key nuclear migration and patterning events at the end of megasporogenesis.

Key words: Piper, Piperaceae, Piperales, magnoliids, female gametophyte, development, evolution, three- dimensional reconstruction.

INTRODUCTION Asteraceae (Fagerlind, 1939; Maheshwari and Srinivasan, 1944; Howe, 1964). A modiﬁed form of this ontogeny, Perhaps one of the most complex developmental sequences Plumbagella-type female gametophyte development, is reported expressed among angiosperm female gametophytes is the in Plumbagella (Dahlgren, 1915; Haupt, 1934; Fagerlind, 1938; Fritillaria-type ontogeny. This developmental pattern, which Boyes, 1939). begins with a tetrasporic coenomegaspore, involves a free- The repeated origin of such a complex ontogenetic sequence nuclear division in which three haploid megaspore nuclei initiate makes Fritillaria-type female gametophyte development an mitosis and produce two triploid restitution nuclei (instead of six ideal subject for an evolutionary developmental investigation. haploid nuclei; Bambacioni, 1928; Maheshwari, 1950; Johri Yet, basic knowledge of this ontogeny in angiosperms is et al., 1992; Batygina, 2006). As far as is known there is no limited, and the developmental basis for the origin of this example of mitosis in any group of eukaryotes in which female gametophyte type has never been explored. Although fewer nuclei are present after a mitotic division is initiated several lineages of angiosperms contain members with than before. In spite of the peculiarity of this division, Fritillaria-type female gametophyte development (see above), Fritillaria-type female gametophyte development appears to it was decided to study this ontogeny within Piperales. There have independently evolved in angiosperms at least seven are reports in Piperales of transitions from monospory to times: in Liliaceae (Bambacioni, 1928; Romanov, 1957), bispory (Johnson, 1900c; Quibell, 1941; Raju, 1961) and tetra- Piperaceae (Kanta, 1962; Nikiticheva, 1981; Prakash and Kin, spory (Johnson, 1900a, b, 1914; Swamy, 1944; Lei et al., 1982; Prakash et al., 1994), Plumbaginaceae (Dahlgren, 1916; 2002) and at least six of the ten standard female gametophyte Haupt, 1934), Euphorbiaceae (Carano, 1925, 1926; Tateishi, ontogenies recognized by Maheshwari (1950) are known to 1927; Kapil, 1961), Cornaceae (D’Amato, 1946; Chopra and exist in Piperales (Fig. 1) (Maheshwari, 1950; Davis, 1966; Kaur, 1965), Tamaricaceae (Mauritzon, 1936; Sharma, 1939, Johri et al., 1992; Batygina, 2006; Madrid and Friedman, 1940; Battaglia, 1941, 1943; Johri and Kak, 1954) and 2008). Piperales has also been the subject of several recent * For correspondence. E-mail [email protected] phylogenetic analyses (Nickrent et al., 2002; Zanis et al., 2003; # The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 870 Madrid & Friedman — Female gametophyte structure in Piper

Monosporic A 8-nucleate Polygonum-type

Bisporic B 8-nucleate Allium-type

Tetrasporic C 8-nucleate Adoxa-type

Tetrasporic D 16-nucleate Peperomia-type

Tetrasporic E 16-nucleate Penaea-type

Tetrasporic F 16-nucleate Drusa-type

Tetrasporic G 8-nucleate Fritillaria-type

F IG. 1. Selected monosporic (A), bisporic (B) and tetrasporic (C–G) patterns of female gametophyte development from Maheshwari (1950). Each of these ontogenies is found in Piperales.

Neinhuis et al., 2005; Wanke et al.,2007a, b). This wealth of up to 3 m in height, and has stems with distinct nodes that may developmental diversity and robust phylogenetic sampling become woody at the base. The leaves are peltate and about make Piperales an excellent candidate in which to study female 40 cm across. Developing infloresences of P. peltatum were gametophyte developmental evolution, and Piper an informative collected from greenhouse collections at the University of clade in which to study the evolution of Fritillaria-type female Colorado in the springs and summers of 2006 and 2007. gametophyte development. Each inflorescence was immersed in a modified PIPES All members of the genus Piper reported until now show the buffer (60 mM PIPES, 25 mM HEPES, 5.4mM CaCl2, Fritillaria-type (Joshi, 1944; Swamy, 1944; Maugini, 1953; 1.97 mM MgCl2) where it was dissected into smaller pieces Murty, 1959; Yoshida, 1960; Kanta, 1962; Nikiticheva, that each contained about 12 flowers. These infloresence por- 1981; Prakash and Kin, 1982; Prakash et al., 1994) and tions were then chemically fixed. female gametophyte development in Piper peltatum was investigated. Nuclear positioning, nuclear migration and nuclear division throughout the syncytial stages of female Histology gametophyte development were documented. The ploidy levels of several cell types within the mature female gameto- Developing flowers were placed in a fixative solution con- phyte and the seed were also measured. The goals were to taining either 3:1 95 % ethanol:acetic acid or 4 % paraformal- . characterize better the specific developmental processes that dehyde, 2 5 % gluteraldehyde, and 4 % acrolein in a modified are associated with female gametophyte development in PIPES buffer (above) for 12 h. Specimens were dehydrated in Piper, and use these detailed ontogenetic insights to generate a graded ethanol series, infiltrated, embedded with glycol hypotheses about how diverse patterns of female gametophyte methacrylate (JB-4 embedding kit; Polysciences, Warrington, development may have evolved in Piperaceae. PA, USA), and serially sectioned into 5-mm-thick ribbons using a Microm HM330 rotary microtome and glass knives. Sections from flowers that were fixed in the 3:1 sol- MATERIALS AND METHODS ution were stained with a solution of 0.2 mgmL21 of 40,6-diamidino-2-phenylindole (DAPI) and 0.1mgmL21 phy- Plant collection lenediamine in 0.05 M Tris (pH 7.2). Sections from flowers that Piper peltatum is a herbaceous shrub that is native to Mexico, were fixed with paraformaldehyde, gluteraldehyde and ethanol Central America and South America. Piper peltatum may grow were stained with 0.1 % toluidine blue. All slides were viewed Madrid & Friedman — Female gametophyte structure in Piper 871 with a Zeiss Axiophot microscope using brightfield, differen- template to identify equivalent female gametophyte nuclear tial interference contrast, and epifluoresence optics. Digital positioning events in other previously studied species of micrographs were taken with a Zeiss Axiocam digital Piperaceae. Character distribution and estimation of ancestral camera. Post-processing of all images was done with the and derived states were inferred using the programs Adobe Creative Suite 2 software package (San Jose, CA, MacClade (Maddison and Maddison, 1992) and Mesquite USA). Digital image manipulations were restricted to pro- (Maddison and Maddison, 2008). Characters were traced cesses that applied to the entire image unless otherwise using the most parsimonious resolving option in MacClade noted in specific figure legends. and with the maximum likelihood analysis module in Mesquite. The recent angiosperm phylogenies of Nickrent et al. (2002), Zanis et al. (2003), Neinhuis et al. (2005) and Three-dimensional computer reconstruction Wanke et al. (2007a, b) were used for character mapping. Selected 5-mm-thick serial sections of developing female Each clade was coded as either tetrasporic and the character gametophytes that had been fixed in paraformaldehyde, gluter- being present, tetrasporic and the character being absent, or aldehyde and acrolein were photographed using a Leica TCS not tetrasporic. The absence of a character was described SP2 AOBS laser scanning confocal microscope. Images were with two character states because the character could be created by exciting the toluidine blue stain at 633 nm with a absent for two distinct reasons. If a clade was tetrasporic and helium/neon laser, with detectors set to absorb within the did not have the character, this was due to patterns of 640–700 nm range. Each 5-mm-thick section was photo- nuclear migration and nuclear division. However, if a clade graphed along the z-axis to create a stack of eight to ten was not tetrasporic, then it was not capable of carrying out 0.5-mm-thick optical sections. Z-stacks from each section the actions described that involve four megaspore nuclei, and were aligned and modelled using the IMOD software this was due to preceding patterns of megasporogenesis. By package (Kremer et al., 1996). using three character states, it was possible to account for each of these explanations. Microspectrofluorometry Sections from flowers fixed in the 3:1 solution were stained with DAPI (see above) for 1 h at room temperature in a light- RESULTS free environment. Microspectrofluorometric measurements of Megasporogenesis relative DNA levels of DAPI-stained nuclei were performed within 2 h. Measurements were made with a Zeiss MSP 20 The megasporocyte of P. peltatum is densely cytoplasmic microspectrophotometer with digital microprocessor coupled and initially contains a single vacuole that is positioned on to a Zeiss Axioskop microscope equipped with epifluorescence the chalazal-side of the megasporocyte nucleus (Fig. 2A, (HBO100-W burner). An ultraviolet filter set (model number B). Smaller vacuoles accumulate throughout the cytoplasm, 48702) with excitation filter (365 nm, bandpass 12 nm), and this correlates with an increase in megasporocyte dichroic mirror (FT395) and barrier filter (LP397) was used volume (Fig. 2C, D; see Video 1 in Supplementary data, with a Zeiss Plan NeoFluar 40 objective. Before each available online). After attaining a length of approx. recording session, the photometer was standardized by 25 mm, meiosis I occurs without cytokinesis. Out of the taking a reading of fluorescence emitted from a fluorescence 272 ovules examined in this study, only one two-nucleate standard (GG 17), and this reading was taken to represent female gametophyte was observed (Fig. 2E, F; see Video 2 100 relative fluorescence units (RFU). At the completion of in Supplementary data), suggesting that meiosis II quickly each session, an additional reading was made of the fluor- follows meiosis I. escence standard to confirm that little or no deviation in the The axes of division in meiosis II are perpendicular. After this relative fluorescence value obtained from the fluorescence division, the female gametophyte is, on average, 30 mm long standard had occurred during the period of data recording. (n ¼ 9) and has four tetrahedrally arranged megaspore nuclei Relative DNA content for each nucleus was determined by (Fig. 3A–C; see Video 3 in Supplementary data, online). summing the individual fluorescence values of each of the Dense areas of cytoplasm, that may be remnants of meiotic spin- serial sections through that nucleus. A net photometric value dles, are found between megaspore pairs. However, full-fledged for each section of a nucleus was obtained by recording an phragmoplasts were never observed and wall formation did not initial reading of the nucleus and subtracting a background occur (Fig. 3A–C). As cytoplasmic connections between mega- value obtained from cytoplasm proximal to the nucleus. spore nuclei dissipate, separate vacuoles coalesce to form a Thus, background fluorescence from the glycolmethacrylate single large vacuole, and megaspore nuclei migrate from was removed from the photometric analysis of relative DNA their tetrapolar tetrahedral orientation (Fig. 3A–C) to a more content. evenly spaced tetrapolar cruciate (planar) arrangement (Fig. 3D–F; see Video 4 in Supplementary data). In this configuration, each of the four nuclei continues to occupy a separate Analyses of character evolution pole of the cell, but all four nuclei are positioned within a single The embryological literature listed in Table 1 was reviewed plane of symmetry. A small volume of cytoplasm, extending to with respect to nuclear positioning, nuclear division, and wall the wall of the syncytium, always surrounds each nucleus at this formation during megasporogenesis and megagametogenesis. stage (Fig. 3D–F) and the entire cell is, on average, 37 mm Data from the present study of P. peltatum were used as a long (n ¼ 10). 872 Madrid & Friedman — Female gametophyte structure in Piper

TABLE 1. Megasporogenesis and female gametophyte development in magnoliids (as far as is known, female gametophyte development in Gymnotheca and Verhuellia has not been examined)

Taxon Megasporogenesis Female gametophyte development Relevant citations

Magnoliales Monosporic Polygonum-type Davis, 1966 Laurales Monosporic Polygonum-type Davis, 1966 Cannelales Monosporic Polygonum-type Davis, 1966 Piperales Aristolochiaceae Aristolochia Monosporic Polygonum-type Jacobsson-Stiasny, 1918; Johri and Bhatnagar, 1955; Gonzalez and Rudall, 2003; Madrid and Friedman, 2008 Thottea Monosporic Polygonum-type Nair and Narayanan, 1961; Gonzalez and Rudall, 2003

Asarum Monosporic Polygonum-type Hofmeister, 1858; Jacobsson-Stiasny, 1918; Wyatt, 1955; Gonzalez and Rudall, 2003 Saruma Monosporic Polygonum-type Gonzalez and Rudall, 2003 Lactoridaceae Lactoris Monosporic Polygonum-type Tobe et al., 1993; Gonzalez and Rudall, 2003 Hydnoraceae Prosopanche Bisporic Allium-type Chodat, 1916; Coccuci, 1976 Hydnora Uncertain* Uncertain* Dastur and Bombay, 1921 Saururaceae Houttuynia Monosporic Polygonum-type Murty, 1960; Raju, 1961 Anemopsis Bisporic Allium-type Quibell, 1941; Raju, 1961 Gymnotheca Unknown Unknown Saururus Bisporic Allium-type Raju, 1961 Piperaceae Verhuellia Unknown Unknown Peperomia Tetrasporic Peperomia-type Johnson, 1900a, 1914; Martinoli, 1948 Piper Tetrasporic Fritillaria-type Maheshwari and Gangulee, 1942; Joshi, 1944; Swamy, 1944; Maugini, 1953; Murty, 1959; Yoshida, 1960; Kanta, 1962 Manekia Tetrasporic Drusa-type and Penaea-type Arias and Williams, 2008 Zippelia Tetrasporic Drusa-type Lei et al., 2002

* Dastur and Bombay (1921) describe female gametophyte development in Hydnora as tetrasporic, but these data could easily be interpreted as showing a bisporic ontogeny. For the parsimony analyses, female gametophyte development in Hydnora was regarded as unknown.

When the female gametophyte is about 45 mm in length, the while the two nuclei at the chalazal pole typically have a two centrally placed (equatorial) megaspore nuclei migrate speckled appearance that makes it difficult to discern individ- towards the chalazal pole, while the micropylar- and chalazal- ual nucleoli (Fig. 4A–C). most nuclei remain stationary. The final orientation of the four A final round of free-nuclear mitoses produces eight nuclei megaspores is a bipolar 1 þ 3 arrangement in which a single within the female gametophyte. No eight-nucleate syncytial nucleus is situated at the micropylar pole of the female female gametophytes were observed. It is therefore reasonable gametophyte and three nuclei are clustered at the chalazal to conclude that cytokinesis immediately follows the last free- pole (Fig. 3G–I; see Video 5 in Supplementary data). nuclear mitotic division of female gametophyte development. Following cytokinesis, the female gametophyte contains seven cells: three antipodals, a three-celled egg apparatus, Megagametogenesis and a binucleate central cell (Fig. 4D–F; see Video 8 in Supplementary data). All four megaspores enter prophase of the first free-nuclear Initially, the three cells within the egg apparatus are cytologi- mitotic division of megagametogenesis simultaneously cally and morphologically similar (Fig. 4D, E, G, H) and it is dif- (Fig. 3G), but the divisions that occur at each pole of the ficult to distinguish between the egg and synergid cells. Polar female gametophyte are very different. At the micropylar nuclei within the central cell are visually identical (Fig. 4D), pole, the single megaspore nucleus (Fig. 3G–I) initiates a free- contain a single nucleolus (Fig. 4D) and fuse to yield a secondary nuclear mitotic division (Fig. 3J–L; see Video 6 in nucleus shortly after cytokinesis in the female gametophyte Supplementary data, online) and two nuclei are produced (Fig. 4G–I; see Video 9 in Supplementary data, online). Thus, (Fig. 4A–C; see Video 7 in Supplementary data). At the cha- female gametophyte development in P. peltatum is lazal pole, the three megaspore nuclei (Fig. 3G–I) contribute Fritillaria-type. to a single metaphase plate (Fig. 3J–L) which ultimately produces two presumably triploid restitution nuclei (as opposed to six haploid nuclei; Fig. 4A–C). This results in a second four- Female gametophyte maturation and fertilization nucleate stage of development in which two nuclei are situated at both the micropylar and chalazal poles (Fig. 4A–C). Each Following cellularization, synergids decrease in volume nucleus at the micropylar pole contains a single nucleolus, while the egg cell, antipodal cells and central cell become Madrid & Friedman — Female gametophyte structure in Piper 873

A C E V

MT D MT

D V V V

B D F

F IG. 2. Dyad formation in Piper peltatum: (A) diploid megasporocyte; (B) three-dimensional reconstruction of megasporocyte cell; (C) vacuolate megasporocyte; (D) three-dimensional reconstruction of vacuolate megasporocyte cell; (E) nuclear dyad syncytium following meiosis I; (F) three-dimensional reconstruction of dyad syncytium. Each image is oriented so that the micropyle is towards the top of the page and the chalaza is towards the bottom. The three-dimensional reconstructions in each column have been created from the light micrograph above them. In each reconstruction nuclei are blue, vacuoles are green, and the outer wall of the female gametophyte is grey. Abbreviations: D, meiotic dyad nucleus; MT, megasporocyte nucleus; V, vacuole. Scale bar ¼ 10 mm. larger (Fig. 4G–I and 5A). Just before fertilization, synergid prophase, which by definition have 4C content of DNA. cells can no longer be recognized and the egg cell has a pro- Zygote nuclei had an average RFU value of 102.71 + 9.36 minent cell wall with a large nucleus (Fig. 5A). Antipodal (n ¼ 5). Therefore, in P. peltatum, 4C content of DNA is nuclei and the secondary nucleus are also larger than at the equivalent to roughly 100 RFU, 2C content of DNA should time of female gametophyte cellularization, and two nucleoli correspond to about 50 RFU, and 1C content of DNA persist in the secondary nucleus (Fig. 5A). should be approx. 25 RFU. Four female gametophytes in which fertilization appeared to According to previously published data and the authors’ have very recently occurred were observed (Fig. 5B). The description of female gametophyte development, secondary zygote nucleus within these female gametophytes contained nuclei in mature female gametophytes of P. peltatum are two nucleoli and the primary endosperm nucleus had three derived from a presumably haploid polar nucleus from the nucleoli (Fig. 5B). Two of these ovules were used to collect micropylar pole of the female gametophyte and triploid resti- microspectrofluorometric data (see below), and both exhibited tution nucleus from the chalazal pole of the female gameto- fluorescence values indicative of fertilized female gametophyte. Thus, the secondary nucleus should be tetraploid. phytes. The central cell remains largely vacuolate through Secondary nuclei in interphase averaged 108.24 + 19.99 the time of fertilization, and the primary endosperm nucleus RFU (n ¼ 7), suggesting that these nuclei are either diploid rests against a lateral wall of the primary endosperm cell and in G2 of the cell cycle, or tetraploid and in G1.At-test (Fig. 5B). The first division of the primary endosperm cell of zygote and secondary nuclei RFU values demonstrated (Fig. 5C) produces two endosperm cells (Fig. 5D). Starch that the difference between these means was not significant accumulates in nucellus cells immediately after fertilization. (P ¼ 0.54; t-test). Mean relative fluorescence of metaphase plates in dividing nucellus cells, which should also contain 4C DNA content, was 97.04 + 11.00 RFU (n ¼ 24), and is Microspectrofluorometry also not significantly different from prophase zygote The relative fluorescence of restitution nuclei, secondary (P ¼ 0.27; t-test) and secondary nuclei (P ¼ 0.20; t-test) nuclei, zygote nuclei and endosperm nuclei stained with relative fluorescence values. DAPI, a DNA-binding fluorochrome, were quantified to deter- If the secondary nucleus is tetraploid, once fertilized, it mine DNA content. To calibrate the photometric measure- should become a pentaploid primary endosperm nucleus, and ments in P. peltatum zygote nuclei were used during contain 10C DNA during prophase. Mean relative fluorescence 874 Madrid & Friedman — Female gametophyte structure in Piper

A D G J MS MS MS V MP V

MS MS VV MS MS MS MS MP MS MS MS

B E H K

C F I L

F IG. 3. Megaspore formation and migration in Piper peltatum: (A–C) megaspores in tetrapolar tetrahedral orientation immediately following meiosis II, showing that thick areas of cytoplasm (yellow arrowheads) remain between megaspore pairs and may be remnants of meiotic spindles; (D–F) megaspores in a tetrapolar cruciate arrangement; (G–I) megaspores in prophase and in a bipolar 1 þ 3 nuclear arrangement; (J–L) ﬁrst free-nuclear division of megagametogenesis; chromosomes are distributed amongst two metaphase plates. Each image is oriented so that the micropyle is towards the top of the page and the chalaza is towards the bottom. The three-dimensional reconstructions in each column have been created from the light micrograph above them. The top-most three- dimensional reconstruction in each column is in the same orientation as the light micrograph above it, while the bottom-most three-dimensional reconstruction in each column has been rotated by 908 on the y-axis. In each reconstruction, nuclei are blue, vacuoles are green, metaphase plates are pink, and the outer wall of the female gametophyte is grey. Abbreviations: MS, megaspore nucleus; V, vacuole. Scale bar ¼ 10 mm.

of primary endosperm nuclei in prophase was 240.89 + 38.80 DISCUSSION RFU (n ¼ 4). This is almost exactly what would be expected if Fritillaria-type female gametophyte development in Piper 1C DNA content is equal to 25 RFU (10C DNA content would correspond to 250C). Therefore, primary endosperm nuclei in The ﬁrst reports of female gametophyte development in Piper Piper peltatum contain 10C content of DNA in prophase, the depict a tetrasporic Adoxa-type ontogeny (Fig. 1C) that does endosperm is pentaploid, and the secondary nuclei observed not form restitution nuclei (Johnson, 1902, 1910; Palm, in the preparations are tetraploid and situated in the G1 1915). However, the eventual discovery of restitution nuclei phase of the cell cycle. and the Fritillaria pattern of female gametophyte development Madrid & Friedman — Female gametophyte structure in Piper 875

A D G EAC EAC EAC EAC

VV V

PN PN SN

RN AA A

B E H EAC N N EAC

V V V

A A A

C F I

F IG. 4. Free-nuclear division and cytokinesis in Piper peltatum: (A–C) four-nucleate stage of development following restitution nucleus formation showing megaspore nuclei in a bipolar 2 þ 2 conﬁguration; (D–F) eight-nucleate, seven-celled female gametophyte just after cytokinesis showing that polar nuclei have not fused; (G–I) seven-nucleate, seven-celled female gametophyte with secondary nucleus. Each image is oriented so that the micropyle is towards the top of the page and the chalaza is towards the bottom. The three-dimensional reconstructions in each column have been created from the light micrographs above them. In each reconstruction, nuclei are blue, vacuoles are green, cells of the egg apparatus are purple, antipodals are dark grey, and the central cell is grey. Abbreviations: A, antipodal; EAC, egg apparatus cell; N, nucleus; PN, polar nucleus; RN, triploid restitution nucleus; SN, secondary nucleus;V, vacuole. Scale bar ¼ 10 mm. 876 Madrid & Friedman — Female gametophyte structure in Piper

A B Z E

PEN SN

C D

Z EN Z

EN EN

F IG. 5. Fertilization and endosperm development in Piper peltatum: (A) mature female gametophyte prior to fertilization showing that synergids are no longer present and the secondary nucleus contains two nucleoli; (B) female gametophyte that has just been fertilized, zygote nucleus contains two nucleoli and the primary endosperm nucleus contains three nucleoli; (C) first division of the primary endosperm nucleus; (D) zygote and first two endosperm cells. Each image is oriented so that the micropyle is towards the top of the page and the chalaza is towards the bottom. All images are colour-desaturated epi-flouresence micrographs of ovules that have been stained with DAPI. Abbreviations: A, antipodal; E, egg; EN, endosperm nucleus; PEN, primary endosperm nucleus; Z, zygote. Scale bar ¼ 10 mm. in Liliaceae (Bambacioni, 1928) led some embryologists ontogenetically ambiguous. The present data indicate that (Schnarf, 1931, 1936; Maheshwari, 1937) to view previous immediately following meiosis II, megaspore nuclei in reports of Adoxa-type female gametophyte development in P. peltatum are found in a tetrapolar tetrahedral arrangement Piper with a certain degree of scepticism. The key ontogenetic (Fig. 3A–C) and, subsequently, migrate to create a tetrapolar difference between Adoxa-type (Fig. 1C) and Fritillaria-type cruciate (planar) configuration (Fig. 3D–F). This second four- (Fig. 1G) development is the presence or absence of restitution nucleate ontogenetic stage is followed by migration of the two nuclei at the chalazal pole of the female gametophyte, and all equatorially placed nuclei (midway along the micropylar– subsequent embryological investigations of Piper species have chalazal axis) to the chalazal pole of the female gametophyte consistently documented Fritillaria-type female gametophyte (Fig. 3G–I), resulting in a third four-nucleate stage, a bipolar development (Maheshwari and Gangulee, 1942; Joshi, 1944; 1 þ 3 arrangement. Swamy, 1944; Maugini, 1953; Murty, 1959; Yoshida, 1960; The tetrapolar tetrahedral and tetrapolar cruciate arrange- Kanta, 1962; Nikiticheva, 1981; Prakash and Kin, 1982; ments of nuclei observed in P. peltatum are similar to the tet- Prakash et al., 1994). The present data largely agree with rapolar arrangements described in previous investigations these reports; however, important aspects of the megasporo- (Maheshwari and Gangulee, 1942; Joshi, 1944; Swamy, genesis to megagametogenesis transition have been uncovered 1944; Murty, 1959; Yoshida, 1960). There is also reason to that serve as the basis for a new understanding of female game- believe that the linear megaspore arrangements described by tophyte developmental evolution in Piperaceae. earlier embryologists (Swamy, 1944; Murty, 1959; Kanta, Previous descriptions of nuclear positioning events between 1962) are actually tetrapolar cruciate arrangements of nuclei meiosis II and the first free-nuclear mitotic division of mega- that were observed from an angle similar to that shown in gametogenesis in Piper are both three-dimensionally and Fig. 2F. It was observed that tetrapolar cruciate and even Madrid & Friedman — Female gametophyte structure in Piper 877 tetrapolar tetrahedral megaspore nuclei could appear linear in (Saururaceae) and Verhuellia (Piperaceae) could (depending serial sections even without the benefit of three-dimensional on what is discovered) provide support for monospory at this models. node of the Piperales phylogeny. The present three-dimensional reconstructions confirm the Unweighted parsimony (Fig. 6) and maximum likelihood findings of earlier researchers, and add a degree of descriptive analyses (see Fig. S1 in Supplementary data, available structural and ontogenetic precision unavailable in previous online) also indicate that the common ancestor of Piperaceae investigations. Embryological data regarding later stage devel- minus Verhuellia did not initiate wall formation after opmental aspects of female gametophyte ontogeny in Piper meiosis I or II (Fig. 6A, B). These data indicate that a tetras- are consistent (Maheshwari and Gangulee, 1942; Joshi, poric pattern of female gametophyte development was 1944; Swamy, 1944; Maugini, 1953; Murty, 1959; Yoshida, present in the common ancestor of Piperaceae minus 1960; Kanta, 1962; Nikiticheva, 1981; Prakash and Kin, Verhuellia (Fig. 6C), and are in agreement with previous 1982; Prakash et al., 1994), and with the ambiguity in embryological investigations and comparative surveys in nuclear positioning at the four-nucleate stages resolved, it is Piperaceae (e.g. Arias and Williams, 2008). likely that the three-dimensional reconstructions for P. peltatum typify development for Piper as a whole. Nuclear positioning at the four-nucleate stage of development in Piperaceae The evolution of megasporogenesis in Piperales Peperomia-, Penaea-, Drusa- and Fritillaria-type patterns of Monosporic (Fig. 1A), bisporic (Fig. 1B) and tetrasporic female gametophyte development have been documented in (Fig. 1C–G) patterns of female gametophyte development in Piperaceae (Fig. 1 and Table 1); however, the specific develop- angiosperms have distinct patterns of meiotic wall formation mental modifications that led to evolutionary transitions that determine how many nuclei are ultimately contributed between each of these tetrasporic ontogenies have never to the functional megaspore cell. Monosporic female gameto- been examined. Both long-standing theoretical predictions phytes form walls after meiosis I and meiosis II, and produce (Schnarf, 1936; Maheshwari, 1950; Favre-Duchartre, 1977; four uninucleate megaspore cells (Fig. 1A). One of these Battaglia, 1989; Haig, 1990) and recent comparative analyses megaspores (usually the chalazal-most cell) will go on to (Friedman and Williams, 2003, 2004; Friedman et al., 2008) produce the mature female gametophyte (Fig. 1A; recognize that all angiosperm female gametophyte ontogenies Maheshwari, 1950; Davis, 1966; Johri et al., 1992). Bisporic appear to be structured around the iterative production of female gametophyte ontogenies form a wall after meiosis I nuclear ‘tetrads’, and have speculated that nuclear positioning but not after meiosis II (Fig. 1B). Lack of wall formation events early in development may be essential to the generation after meiosis II results in the placement of two nuclei within of downstream developmental and structural diversity. Yet, the the functional megaspore cell, and at maturity bisporic specific role that nuclear migration events have played in the female gametophytes contain the mitotic derivatives of two radiation of female gametophyte developmental patterns in megaspore nuclei (Fig. 1B). Tetrasporic female gametophyte Piperaceae has never been examined. development results from the absence of wall formation The present data document the presence of four distinct during meiosis I and II of megasporogenesis (Fig. 1C–G), four-nucleate ontogenetic stages associated with nuclear as is shown here in Piper (Figs 2 and 3). migration and division at the end of megasporogenesis in Previous reports of megasporogenesis in Piperales (Table 1) Piper (Figs 2–4). To understand the role these nuclear posi- were combined with published phylogenetic analyses to infer tioning events have played in female gametophyte develop- evolutionary transitions in patterns of megasporogenesis. mental evolution, the embryological literature on Piperaceae Unweighted parsimony analyses (Fig. 6) and maximum likeli- (Table 1) was surveyed and it was determined which of the hood analyses (see Fig. S1 in Supplementary data, available ontogenetic stages observed in Fritillaria-type female gameto- online) of wall formation associated with megasporogenesis phytes of P. peltatum (Figs 2–4) are present in Peperomia-, indicate that the common ancestor of Saururaceae þ Penaea- and Drusa-type female gametophyte ontogenies Piperaceae initiated wall formation after meiosis I (Fig. 6A), found in other members of Piperaceae (Fig. 7 and Table 1). but did not initiate wall formation after meiosis II (Fig. 6B). Review of the embryological literature indicates that mega- These patterns of meiotic wall formation are consistent with spore nuclei in all female gametophyte ontogenies in a bisporic pattern of megasporogenesis (Fig. 1B) in the Piperaceae conform to a tetrapolar tetrahedral arrangement common ancestor of Saururaceae þ Piperaceae (Fig. 6C). immediately after meiosis II (Fig. 7). However, only These results are interesting because they indicate that Peperomia-type female gametophytes initiate megagameto- Polygonum-type female gametophyte development in genesis directly from a tetrapolar tetrahedral nuclear configur- Houttuynia (Saururaceae) may represent an evolutionary rever- ation (Fig. 7). Penaea-, Drusa- and Fritillaria-type female sal to monospory from a bisporic ancestor (Fig. 6C). An gametophytes transition from a tetrapolar tetrahedral to a tetra- alternative, albeit less parsimonious explanation, is that the polar cruciate arrangement of megaspore nuclei (Fig. 7). common ancestor of Saururaceae þ Piperaceae was monospo- Penaea-type female gametophytes then initiate free-nuclear ric, and that bispory in Saururaceae has independently evolved divisions associated with megasporogenesis (Fig. 7), while at least twice, in Anemopsis (Saururaceae) and Saururus megaspore nuclei in Drusa- and Fritillaria-type female game- (Saururaceae). Although current empirical data indicate that tophytes undergo nuclear migration to create a bipolar 1 þ 3 the common ancestor of Saururaceae þ Piperaceae was bispo- arrangement in which a single nucleus is situated at the micro- ric (Fig. 6), future embryological studies of Gymnotheca pylar pole of the female gametophyte and three megaspore 878 Madrid & Friedman — Female gametophyte structure in Piper

Aristolochiaceae Saururaceae Piperaceae A LactoridaceaeAristolochiaceaeHydnoraceae Saururaceae Piperaceae B Lactoridaceae Hydnoracea Magnoliales Laurales Cannelales Lactoris Thottea Aristolochia Asarum Saruma Prosopanche Hydnora Houttuynia Anemopsis Gymnotheca Saururus Verhuellia Manekia Zippelia Peperomia Piper Magnoliales Laurales Cannelales Lactoris Thottea Aristolochia Asarum Saruma Prosopanche Hydnora Houttuynia Anemopsis Gymnotheca Saururus Verhuellia Manekia Zippelia Peperomia Piper

C LactoridaceaeAristolochiaceaeHydnoraceae Saururaceae Piperaceae Magnoliales Laurales Cannelales Lactoris Thottea Aristolochia Asarum Saruma Prosopanche Hydnora Houttuynia Anemopsis Gymnotheca Saururus Verhuellia Manekia Zippelia Peperomia Piper

Present Absent Unknown / Equivocal

Monosporic Bisporic Tetrasporic

F IG. 6. Patterns of wall formation during megasporogenesis in magnoliids: (A) presence or absence of cytokinesis after meiosis I in magnoliids; (B) presence or absence of cytokinesis after meiosis II in magnoliids: (C) patterns of megasporogenesis in magnoliids that have been derived from the parsimony analyses of wall formation shown in (A) and (B). Phylogenetic relationships adapted from Nickrent et al. (2002), Zanis et al. (2003), Neinhuis et al. (2005) and Wanke et al. (2007a, b). nuclei are positioned at the chalazal pole (Fig. 7). Drusa-type gametophytes, bipolar 1 þ 3 nuclear orientation correlates female gametophytes initiate megagametogenesis from this with Drusa-type female gametophytes, and bipolar 2 þ 2 bipolar arrangement to produce 16 haploid nuclei (four at nuclear configuration correlates with Fritillaria-type female the micropylar pole and 12 at the chalazal pole), while gametophytes (Fig. 7). Fritillaria-type female gametophytes transition through All female gametophyte ontogenies in Piperaceae include a one final four-nucleate stage in which two haploid nuclei stage in which megaspore nuclei are tetrahedrally arranged, but are produced at the micropylar pole and two triploid tetrapolar cruciate and bipolar 1 þ 3 nuclear configurations restitution nuclei are produced from three haploid nuclei at arise through additional nuclear migration events linked to the chalazal pole (Fig. 7) as is shown here for P. peltatum specific female gametophyte developmental trajectories (Figs 3 and 4). (Fig. 7). Drusa- and Fritillaria-type female gametophyte onto- This comparative developmental survey makes it apparent genies transition through three distinct four-nucleate ontogen- that in Piperaceae, female gametophytes initiate megagameto- etic stages, but only Fritillaria-type female gametophytes genesis from four-nucleate tetrapolar tetrahedral, tetrapolar initiate a fourth four-nucleate stage of development as a conse- cruciate, bipolar 1 þ 3 or bipolar 2 þ 2 nuclear configurations quence of the formation of restitution nuclei. Thus, patterns of (Fig. 7). Tetrapolar tetrahedral nuclear placement correlates nuclear migration and division at the four-nucleate stage with Peperomia-type female gametophytes, tetrapolar cruciate of development in Piperaceae are predictive of the structure nuclear placement correlates with Penaea-type female of the female gametophyte at maturity. Madrid & Friedman — Female gametophyte structure in Piper 879

ration to gement ar ar ar cyte cell migrationnt to 1+3 migrationnt to 1+1 s

Differentiation of Nuclear mig Nuclear Formation of Free-nucle Nuclear Free-nucle Free-nucle megasporo Meiosis I Cytokinesis Meiosis II Cytokinesis cruciate arran arrangeme restitution nuclei mitotic division arrangeme mitotic division mitotic division Cytokinesi

Polygonum-type Absent Absent Absent

Fritillaria-type Absent Absent Absent Absent Absent

Drusa-type Absent Absent Absent Absent Absent

Peneae-type Absent Absent Absent Absent Absent Absent

Peperomia-type Absent Absent Absent Absent Absent Absent Absent

Megasporogenesis Megagametogenesis

F IG. 7. Comparative nuclear positioning in Piperaceae. Developmental modiﬁcations associated with alternate patterns of female gametophyte development are listed across the top, and female gametophyte ontogenies in Piperaceae are listed on the left. Monosporic Polygonum-type female gametophyte development is on the top-most row for comparison with the tetrasporic ontogenies below. Ontogenetic steps associated with megasporogenesis have a blue background, and developmental steps associated with megagametogenesis have an orange background.

Female gametophyte development in the common ancestor Peperomia-, Penaea- and Fritillaria-type ontogenies are evolu- of Piperaceae tionarily derived in this clade (Fig. 9). It is important to note that female gametophyte development in Data describing these ontogenetic stages in four-nucleate Manekia has been reported to be variable: female gametophyte female gametophytes of Piperaceae (Fig. 7) were combined development is Drusa-type 86 % of the time, and Penaea-type with published molecular phylogenetic analyses to reconstruct 14 % of the time. Arias and Williams (2008) conclude that ancestral female gametophyte nuclear positioning events female gametophyte development in Manekia is ancestrally (Fig. 8). These data were then used to infer the evolution of Drusa-type. If this is correct, then unweighted parsimony and female gametophyte ontogenies throughout Piperaceae maximum likelihood analyses indicate that the common ancestor (Fig. 9). Embryological data regarding female gametophyte of Piperaceae minus Verhuellia had a bipolar 1 þ 3 conﬁguration development in Verhuellia do not exist (Table 1), making it of megaspore nuclei (Fig. 7C; see Fig. S2 in Supplementary impossible to resolve character states in the common ancestor data, available online). However, if Manekia is ancestrally of Piperaceae (Fig. 8). However, ancestral state reconstruction Penaea-type and nuclear migration to a 1 þ 3 arrangement is can be resolved in the common ancestor of Piperaceae minus not plesiomorphic, parsimony and maximum likelihood analyses Verhuellia (Fig. 8). are less revealing and the evolution of nuclear migration leading Unweighted parsimony analyses (Fig. 8) and maximum to a bipolar 1 þ 3 arrangement is equivocal at all nodes of likelihood analyses (see Fig. S2 in Supplementary data, avail- Piperaceae (see Fig. S3 in Supplementary data). able online) indicate that the common ancestor of Piperaceae minus Verhuellia had a tetrasporic female gametophyte (Fig. 6C) with megaspore nuclei that were initially tetrapolar The evolution of female gametophyte development in Piperaceae tetrahedral (Fig. 8A), and then passed through tetrapolar cruciate (Fig. 8B) and bipolar 1 þ 3 nuclear conﬁgurations Drusa-type female gametophytes transition through three (Fig. 8C). Evolutionary analysis clearly indicates that the four-nucleate ontogenetic stages of development (Fig. 7). If common ancestor of Piperaceae minus Verhuellia did not this ontogeny is ancestral in the clade Piperaceae minus form restitution nuclei (Fig. 8D). In total, these developmental Verhuellia (Fig. 9), then the evolutionary loss and gain of characteristics reveal that Drusa-type female gametophyte nuclear positioning events at the four-nucleate stage of devel- development (Fig. 7) was present in the common ancestor of opment has led to the origin of derived female gametophyte Piperaceae minus Verhuellia (Fig. 8), and indicate that developmental patterns (Fig. 10). 880 Madrid & Friedman — Female gametophyte structure in Piper

A LactoridaceaeAristolochiaceaeHydnoraceae Saururaceae Piperaceae C LactoridaceaeAristolochiaceaeHydnoraceae Saururaceae Piperaceae Magnoliales Laurales Cannelales Lactoris Thottea Aristolochia Asarum Saruma Prosopanche Hydnora Houttuynia Anemopsis Gymnotheca Saururus Verhuellia Manekia Zippelia Peperomia Piper Magnoliales Laurales Cannelales Lactoris Thottea Aristolochia Asarum Saruma Prosopanche Hydnora Houttuynia Anemopsis Gymnotheca Saururus Verhuellia Manekia Zippelia Peperomia Piper

Tetrapolar Biplolar Tetrahedral 1+3

ceae

B LactoridaceaeAristolochiaceaeHydnoraceae Saururaceae Piperaceae D LactoridaAristolochiaceaeHydnoraceae Saururaceae Piperaceae Magnoliales Laurales Cannelales Lactoris Thottea Aristolochia Asarum Saruma Prosopanche Hydnora Houttuynia Anemopsis Gymnotheca Saururus Verhuellia Manekia Zippelia Peperomia Piper Magnoliales Laurales Cannelales Lactoris Thottea Aristolochia Asarum Saruma Prosopanche Hydnora Houttuynia Anemopsis Gymnotheca Saururus Verhuellia Manekia Zippelia Peperomia Piper

Tetrasporic and character present Tetrapolar Tetrasporic and character absent Restitution Cruciate Not tetrasporic Nucleus Unknown / Equivocal

F IG. 8. Nuclear arrangement at the four-nucleate stage of female gametophyte development in magnoliids: (A) unweighted parsimony analysis of tetrapolar tetrahedral megaspore orientation following meiosis II; (B) unweighted parsimony analysis of tetrapolar cruciate arrangement of megaspore nuclei; (C) unweighted parsimony analysis of bipolar 1 þ 3 arrangement of megaspore nuclei; (D) unweighted parsimony analysis of restitution nucleus formation (and con- sequent bipolar 2 þ 2 nuclear conﬁguration). Phylogenetic relationships from Nickrent et al. (2002), Zanis et al. (2003), Neinhuis et al. (2005) and Wanke et al. (2007a, b).

In Peperomia the complete loss of nuclear migration events restitution nuclei appear to be the only factors that distinguish that would normally lead to bipolar 1 þ 3 and tetrapolar cruci- Fritillaria- from Drusa-type female gametophyte ontogenies ate nuclear arrangements in Drusa-type female gametophytes (Fig. 9). has given rise to Peperomia-type female gametophyte develop- Thus, Drusa-type female gametophytes in Zippelia and ment (Fig. 10). In Piper, developmental alterations associated Manekia have been inherited from the common ancestor of with the formation of restitution nuclei are appended onto the Piperaceae minus Verhuellia, while Peperomia- and ancestral Drusa-type developmental framework (Fig. 7), and Fritillaria-type female gametophytes in Peperomia and result in the formation of a fourth four-nucleate ontogenetic Piper, respectively (Table 1), have evolved through either stage and a Fritillaria-type female gametophyte. Patterns of losses or gains of four distinct four-nucleate ontogenetic cytokinesis in Fritillaria-type female gametophytes are stages that have been identiﬁed at the end of megasporogenesis similar to those of Drusa-type female gametophytes (a single (Figs 6, 8 and 9). Interestingly, this evolutionary developmen- nucleus from each pole of the syncytium becomes partitioned tal scenario predicts that the occasional production of into the common cytoplasmic space of the central cell), and Penaea-type female gametophytes in Manekia is likely to be developmental modiﬁcations leading to the formation of the consequence of the loss of the nuclear migration event Madrid & Friedman — Female gametophyte structure in Piper 881

Fritillaria-type female gametophyte development in angiosperms Fritillaria-type female gametophyte development has LactoridaceaeAristolochiaceaeHydnoraceae Saururaceae Piperaceae evolved at least six times outside of Piperaceae (Joshi, 1938; Maheshwari, 1946; Davis, 1966; Johri et al., 1992), and a brief review of this literature provides some interesting paral- lels with the conclusions presented in the present study. In Tamaricaceae, Fritillaria-type Myricaria (Schnarf, 1931; Magnoliales Laurales Cannelales Lactoris Thottea Aristolochia Asarum Saruma Prosopanche Hydnora Houttuynia Anemopsis Gymnotheca Saururus Verhuellia Manekia Zippelia Peperomia Piper Battaglia, 1943) is sister to Tamarix (Lledo et al., 1998; Cuenoud et al., 2002), which produces Adoxa-, Drusa- and Fritillaria-type female gametophytes (Joshi and Kajale, 1936; Sharma, 1939, 1940; Johri and Kak, 1954). Euphorbia (Euphorbiaceae) contains members with Fritillaria-type female gametophytes (Carano, 1925, 1926; Kapil, 1961) and is within the larger clade Euphorbioideae, which is sister to Acalyphoideae (Tokuoka, 2007). Acalyphoideae is exclusively tetrasporic and contains several members with Penaea-type female gametophytes (Tateishi, 1927; Swamy and Balakrishna, 1946; Banerji, 1949; Thathachar, 1952; Johri

Polygonum-type (monosporic) and Kapil, 1953; Kajale and Murthy, 1954; Mukherjee, Allium-type (bisporic) 1958; Kapil, 1960). Penaea-type female gametophyte develop- Peperomia-type (tetrasporic) ment is also reported within Euphorbia itself, in E. procera Drusa-type (tetrasporic) (Modilewski, 1909, 1910). Liliaceae is largely Fritillaria-type Fritillaria-type (tetrasporic) (Bambacioni, 1928; Bambacioni and Giombini, 1930;

F IG. 9. Female gametophyte developmental evolution in magnoliids, in Bambacioni-Mezzetti, 1932; Cooper, 1934, 1935; accordance with the data and analyses in the present study. Westergard, 1936; Fagerlind, 1938), but contains members with Drusa- and Adoxa-type female gametophytes in the genus Tulipa (Romanov, 1936, 1938, 1957). Fritillaria-type female gametophyte development is reported in that normally leads to a bipolar 1 þ 3 arrangement of mega- Plumbaginaceae in Limonium (Dahlgren, 1916) and Armeria spore nuclei (Fig. 10), a prediction that is in agreement with (Dahlgren, 1916). Armeria and Limonium share a recent ideas initially put forth by Arias and Williams (2008). common ancestor (Lledo et al., 1998; Cuenoud et al., 2002)

Fritillaria-type

Drusa-type

Penaea-type

Peperomia-type

F IG. 10. Developmental evolution of tetrasporic female gametophytes in Piperaceae from an ancestral Drusa-type ontogeny. Crucial differences betweeneach ontogeny occur during the four-nucleate stage of development, and arrows indicate points of ontogenetic divergence. 882 Madrid & Friedman — Female gametophyte structure in Piper and Limonium also contains members that produce Battaglia E. 1941. Contributo all’embriologia delle Tamaricaceae. Nuovo Penaea-type and Adoxa-type female gametophytes Giornale Botanico Italiano 48: 572–612. Battaglia E. 1943. Alcune osservazione sullo svlluppo del gametofito femmi- (Fagerlind, 1938; Davis, 1966). neo della Myricaria germanica. Nuovo Giornale Botanico Italiano 49: Thus, five of the seven independent origins of Fritillaria-type 464–467. female gametophyte development in angiosperms have occurred Battaglia E. 1989. Embryological questions. 14. The evolution of the female in clades that are associated with Drusa- and/or Penaea-type gametophyte of angiosperms: an interpretive key. Annali di Botanica female gametophyte ontogenies. The evolutionary developmen- (Roma) 47: 7–144. Batygina TB. 2006. Embryology of flowering plants, terminology and con- tal hypotheses presented in this study predict that both of these cepts. Enfield, NH: Science Publishers. developmental patterns would be antecedent to and/or closely Boyes JW. 1939. Development of the embryo sac of Plumbagella micrantha. allied with transitions to Fritillaria-type female gametophyte American Journal of Botany 26: 539–547. development (Figs 7 and 10). The Adoxa-type ontogeny is Carano E. 1925. Sul particulare sviluppo del gametofito female di Euphorbia dulcis L. Consiglio Nazionale Delle Ricerche Accademia Nazionale dei also commonly associated with clades that include members Lincei, series 6A 1: 633–635. with Fritillaria-type female gametophytes. An interesting goal Carano E. 1926. Ulteriori osservazioni su Euphorbia dulcis L. in rapporto col for future studies would be to characterize nuclear migration suo comportamento apomittico. Annali di Botanica (Roma) 17: 50–79. and positioning events in syncytial stages of Adoxa-type Chodat R. 1916. Hydnoraceae. Bulletin de la Socie´te´ botanique de Ge`neve 8: female gametophyte development, and determine which (if 186–201. Chopra RN, Kaur H. 1965. Some aspects of the embryology of Cornus. any) of the ontogenetic modifications that are described in Phytomorphology 15: 353–359. Peperomia-, Penaea-, Drusa- and Fritillaria-type female gameto- Cocucci AE. 1976. Estudios en el genero Prosopanche (Hydnoraceae). III. phytes also occur in Adoxa-type ontogenies. Provided that Embriologia. Kurtziana 9: 19–39. future embryological investigations include detailed information Cooper DC. 1934. Development of the embryo sac of Lilium henryi. about nuclear migration and positioning events throughout Proceedings of the National Academy of Sciences of the USA 20: 163–166. female gametophyte development, the generality of these Cooper DC. 1935. Macrosporogenesis and development of the embryo sac of hypotheses can be tested in other angiosperm lineages, and Lilium henryi. Botanical Gazette 97: 346–355. used to understand the developmental basis of evolutionary Cuenoud P, Savolainen V, Chatrou LW, Powell M, Grayer RJ, Chase MW. transitions to Fritillaria-type female gametophyte development 2002. Molecular phyogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. American throughout angiosperms. Journal of Botany 89: 132–144. D’Amato F. 1939. Ricerche embryologiche e caryologiche sul genere Euphorbia. Nuovo Giornale Botanico Italiano 46: 470–509. SUPPLEMENTARY DATA D’Amato F. 1946. Osservasioni cito-embriologiche su Cornus mas L. con par- Supplementary data are available online at www.aob.oxford ticolare riguardo alla sterillita di un Biotipo triploide. Nuovo Giornale Botanico Italiano 55: 170–210. journals.org and consists of the following files. Nine videos are Dahlgren KVO. 1915. Der embryosack von Plumbagella, Ein neuer Typus provided of the three-dimensional models that are presented in unter den Angiospermen. Archiv fu¨r Botanik 14: 1–10. the text (Figs 2, 3 and 4): in each video, a single three- Dahlgren KVO. 1916. Zytologische und embryologische Studien uber die dimensional model rotates by 3608 on the y-axis, and then Reihen Primulales und Plumbaginales. Kongligen Svenska Vetenskaps Akademiens Handlingar 56: 1–80. again by 3608 on the x-axis. The colour-coding scheme used Dastur RH, Bombay BS. 1921. Notes on the development of the ovule, for cellular components within each model is identical to the cor- embryo sac and embryo of Hydnora africana, Thunb.. Transactions of responding figure in the text. A file is also available containing the the Royal Society of South Africa 9: 27–31. following three figures: maximum likelihood analyses of wall Davis GL. 1966. Systematic embryology of the angiosperms. New York, NY: formation associated with megasporogenesis (Fig. S1), nuclear John Wiley and Sons. Fagerlind F. 1938. Embryosack von Plumbagella und Plumbago. Arkiv fu¨r migration (Fig. S2) and nuclear division events (Fig. S3). Botanik 29B:1–8. Fagerlind F. 1939. Drei Beispiele des Fritillaria-Typs. Svensk Botanisk Tidskrift 33: 188–204. ACKNOWLEDGEMENTS Favre-Ducharte M. 1977. Eight interpretations of the embryo sac. Phytomorphology 27: 407–418. We thank Joe Williams for helpful discussions in preparing Friedman WE, Williams JE. 2003. Modularity of the angiosperm female this manuscript, and the very constructive comments of two gametophyte and its bearing on the early evolution of endosperm in flow- anonymous reviewers. 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