Organization, available space and organ morphology within floral buds of Iris (Iridaceae)

Flora 249 (2018) 67–76

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Organization, available space and organ morphology within floral buds of Iris (Iridaceae)

T

Jinyan Guoa, , Carol A. Wilsonb ⁎

a b

Department of Biological Sciences, State University of New York at Oswego, Oswego, New York, 13126, USA University and Jepson Herbaria, University of California, Berkeley, California, 94720, USA

ARTICLE INFO

ABSTRACT

Edited by Favio Gonzalez

Before anthesis, older floral organs enclose younger ones forming a bud, within which the shape and relative size of floral organs are largely determined. The floral diversity in Iris, including sepal ornamentation, limited development of petals or sepals in some species and presence of petaloid style branches in all but one species, provides a unique opportunity to compare organ development within the confined space of a bud. Using transverse serial sections, light microscopy and measurement software, we investigated floral packing geometry (relative size and spacial relationships of floral organs) in seven species focusing on five species where we studied changes across three developmental stages. In this study, we found that floral packing geometries are diverse even among species with similar floral organ morphologies. The “filling law” proposed for vegetative buds is not applicable during floral bud development with empty space increasing as bud size increased for each of the five species that were examined. Other key findings include the presence of space between anther thecae in some species, the enlargement of sepal outgrowths and petal margins into space between thecae, differences in the relative growth of anthers versus petaloid style branches and the curvature of connectives, petals and style branches in some species. This study clarifies the integration of floral bud growth and illustrates a largely ignored yet important aspect of flower development: coordinated growth of floral organs within the bud.

Keywords: Bud packing geometry Filling law Floral anatomy Flower development Mechanical constraint Perianth elaboration

1. Introduction In angiosperms, embryonic and developing lateral organs are typically enclosed by older lateral organs and in more mature stages enclosing organs form a bud that markedly increases in volume during development. The number, morphology, relative size and arrangement of lateral organs are largely determined before the bud opens. Botanists have long been fascinated by how beautifully and efficiently packed lateral organs are within buds (Arber, 1934; Williams, 1975; Williams and Metcalf, 1975; Williams et al., 1982). Studies have shown that physical constraint within the vegetative bud has a leading role as a global regulator in the determination of leaf shape, and that there is a correlation between how leaves are folded and packed within the bud (vernation) and their final shapes (Kobayashi et al., 1998; Couturier et al., 2009, 2011, 2012; Edwards et al., 2016). Couturier et al. (2011) suggested vegetative buds follow a “filling law” where leaves fill the entire bud volume during development. A few studies have examined floral buds (Berg, 1990; Endress, 1996, 2008) and concluded these buds are also tightly packed, often with hairs or fluids as filling materials. To our knowledge, previous studies focusing on the arrangement of floral

⁎

organs in buds (aestivation), the developmental dynamics of floral bud packing geometries, and relative sizes and spatial relationships of floral organs are lacking. Iris provides a valuable opportunity to study these aspects of floral bud development because some species have sepal outgrowths and/or reduction in the sizes of sepals or petals, and all species except one have petaloid style branches, a floral characteristic of tribe Irideae. All of these floral modifications are likely to impact spatial relationships within the bud. The genus Iris L. (Iridaceae) includes approximately 270 species (Goldblatt and Manning, 2008; Wilson, 2011) and is morphologically diverse, as shown by alternative taxonomic systems, not preferred by the authors, that split the genus into 15 (Rodionenko, 2009) and 25 (Crespo et al., 2015) genera, respectively. Compared with a typical monocot flower that has six undifferentiated perianth units (tepals), six stamens and a superior ovary (Rudall, 2002; Remizowa et al., 2010), flowers in Iris are distinct. They have differentiated perianth units comprising three sepals and three petals (except I. domestica (L.) Goldblatt & Mabb. that has six tepals) where sepals are petaloid, extend horizontally, and have markings or patterns (Wilson, 2006; Guo, 2015a) while petals are typically upright and more uniformly colored

Corresponding author. E-mail addresses: [email protected] (J. Guo), [email protected] (C.A. Wilson).

https://doi.org/10.1016/j.flora.2018.10.001 Received 8 May 2018; Received in revised form 19 September 2018; Accepted 1 October 2018 Available online 05 October 2018 0367-2530/ © 2018 Elsevier GmbH. All rights reserved.

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Fig. 1. A, Floral diagram of a typical Iris flower with one pollination unit highlighted in gray. B, Schematic diagram of the lateral view of a pollination unit of Iris with sepal ornamentation highlighted in gray. C–N, Images of flowers and sepals and outlines of one half of the adaxial sepal, ornamentation shaded in gray when present; C, Flower of I. domestica; D, Flower of I. dichotoma; E, Outline of sepals lacking structural ornamentation, distal medial and proximal portions indicated; F, Sepal of I. ensata; G, Sepal of I. setosa; H, Outline of sepals with median ridges; I, Flower of I. tectorum; J, Outline of sepal with a median crest; K, Sepal of I. cristata; L, Outline of sepal with a median crest and one pair of lateral structures; M, Sepal of, I. milesii; N, Outline of sepal with a median crest and two pairs of lateral structures. Median structures on images of flowers and sepals indicated by arrowheads and lateral structures by arrows. Dotted lines demarcate the distal, medial, and proximal regions of the sepal, labeled in E. L = lateral structure; M = median structure; Sy = style; Pe = petal; pS = petaloid style; Se = sepal; St = stamen; Te = tepal.

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and patterned (Rodionenko, 1987). Species of Iris have three stamens and an inferior ovary, defining floral characteristics of Iridaceae (Goldblatt and Manning, 2008), and three free style branches that are petaloid (except I. domestica; Fig. 1A–C). The loss of the inner stamen whorl (resulting in one whorl of three stamens opposite to the sepals) is considered important to specialized pollination systems in Iridaceae (Goldblatt and Manning, 2008) and the evolution of petaloid style branches resulted in a flower that functions as three labiate flowers with each pollination unit consisting of a single sepal, stamen and petaloid style branch (Fig. 1A and B; Weberling, 1992; Westerkamp and Claßen-Bockhoff, 2007). Sepals in Iris, except I. domestica, provide the lower lip of the labiate pollination unit (Fig. 1A and B; Westerkamp and Claßen-Bockhoff, 2007) and play a crucial role in pollination (Uno, 1982; Rodionenko, 1987; Sapir et al., 2005; Morinaga and Sakai, 2006). Similar to Orchidaceae labella, Iris sepals display diversity in the degree and types of outgrowths or elaborations that add three-dimensionality to this organ (Wilson, 2006; Guo, 2015a). They can lack sepal elaborations (Fig. 1C–E) but often the sepal adaxial surface is elaborated with various structures such as beards (see Fig. 1, Wilson, 2017), ridges (a straight and smooth linear structure; Fig. 1F–H) and crests (a sinuous and/or uneven linear structure; Fig. 1I–N) along the prominent midvein of the sepal. Crested sepals in Iris are often strongly three-dimensional and can have one or two pairs of lateral structures (either ridges, crests or linear protuberances) flanking the median crest (Fig. 1K–N). These sepal elaborations and petaloid style branches do not develop in isolation but instead within the constrained space of floral buds, which lead to the following questions. How are petaloid style branches accommodated within the bud? Do gains of sepals with significant adaxial growth change the floral packing geometry compared to buds with non-elaborated sepals or sepals that are less three-dimensional? If so, do buds display different spatial arrangements that correspond to the degree and diversity of sepal elaborations? Does the reduction of size of perianth parts result in areas of empty space within the bud? What differences in packing geometry are seen among bud developmental stages? This study explores these questions by sampling three stages of floral buds from Iris species with diverse floral morphologies, with a focus on species with sepal elaborations.

stages with lengths measured from the distal end of the bud to the distal end of the floral tube (a region of perianth fusion above the ovary). Three replicates of each stage for each species were selected. Two sets of buds were sectioned by hand after infiltrating with 70% ethanol. The first set used to verify size selection and optimum orientation and the second set to identify areas within buds that were informative for each species at each stage and for comparison to fixed materials (sketches were made). The final set of buds was processed for light microscopy. Each processed bud was dehydrated through an ethanol series, infiltrated with Histo-Clear (National Diagnostics; Atlanta, Georgia, USA) followed by liquid paraffin (Paraplast; Thermo Fisher Scientific; Hampton, New Hampshire, USA) and embedded in paraffin. Transverse serial sections (at 8―10 μm thickness) were obtained from the distal end of the floral bud to the floral tube, identified as representing distal, medial, and proximal regions of the sepal (Fig. 1E, H, J, L, N, labeled in Fig. 1E) and mounted on glass slides using Sass’s adhesive (Sass, 1940). Selected slides representing each region were stained with safranin and fast green (Jensen, 1962) prior to adding coverslips. Sections were observed and imaged using a Zeiss Axiovert 135 inverted light microscope (Oberkochen, Germany) and Carl Zeiss GSZ dissecting microscope. Photos were taken using a Canon EOS Rebel T5i camera (Tokyo, Japan). Measurements of the area of a bud that was empty or filled with floral organs were obtained from images of transverse sections of five species (Iris dichotoma, I. setosa, I. tectorum, I. cristata and I. milesii) for each of the three stages (early, middle and late) using ImageJ 1.50i (Schneider et al., 2012; Fig. 2). Anther and petaloid style branch areas and the width and depth of spaces between anther thecae were also determined using ImageJ 1.50i. Measurements were obtained using sections from the medial region of the bud that most fully illustrated the organs present in each whorl. Length measurements used the imageJ ruler with the scale set to the scale bar of each image and area measurements were determined with the imageJ area tool and used the scale bar of each image to calibrate the area of pixels selected. Mean values ± SE of areas and distances are based on measurements of the three anthers and style branches from one section of each bud and differences within a bud are considered to mostly represent slight differences in the angle of the organs relative to the section. Three measurements of empty space within buds were taken to assess consistency. The area of the entire bud versus filled and empty space was determined for each bud as a final test of consistency. Measurements are considered estimates of empty space within buds and sizes of organs for each species because only one bud at each stage per species was sampled. The sections were compared to sketches of non-permanent hand sections prepared from one additional bud at each stage per species and showed similar arrangements and sizes of organs and internal space, indicating no obvious shrinkage or other artifacts during processing.

2. Material and methods The relative size and spatial relationships of floral organs before anthesis for five Iris species were studied using three bud stages we categorize as early, middle and late. The bud stage we identify as early occurs after floral organ initiation and differentiation when sepals have enclosed inner whorls. Two additional species were sampled but buds representing each of the three stages were not available. Studied species have sepals with no structural elaboration (I. domestica, Fig. 1C; I. dichotoma Pall., Fig. 1D), a median ridge (I. ensata Thunb., Fig. 1F; I. setosa Pall. ex Link, Fig. 1G), a median crest (I. tectorum Maxim., Fig. 1I), a median crest and a pair of lateral structures (I. cristata Aiton, Fig. 1K), and a median crest and two pairs of lateral structures (I. milesii Baker ex Foster, Fig. 1M). Iris setosa (Fig. 1G) has extremely reduced petals and I. domestica (Fig. 1C) has short, linear style branches that slightly widen at their tips and six tepals. The taxonomic placement of species included in the study and their collection information are given in Table 1. Two classification schemes, one based on morphology (Mathew, 1989) and a more recent one based on molecular phylogenetics (Wilson, 2011) are given in Table 1. Guo and Wilson (2013) provide additional information on phylogenetic relationships of these species. Floral buds for each species were collected in their native habitats or at Beijing Botanical Garden of the Chinese Academy of Sciences, and immediately fixed in a formalin-propionic acid-alcohol solution for at least two weeks. Floral buds of each species were categorized to represent early (0.5–0.8 cm), middle (1.2–1.6 cm) and late (1.8–2.5 cm)

3. Results Changes within the bud during development are most obvious at the medial region (Fig. 1E) where anthers and style branches are visible at each developmental stage (Fig. 2). For proximal regions a solid style instead of style branches is seen at early stages (Fig. 3A) while at late stages filaments have elongated with anthers not visible (Fig. 3B). For distal regions the gynoecium has not elongated and is not visible at early stages (Fig. 3C) while for middle and late stages often only the bifid tips of style branches are visible (Fig. 3D–E). 3.1. Position and development of sepal ridges and crests At the early stage of Iris domestica (not shown) (Fig. 2A) sepal outgrowths are not present. At later growths do not develop in I. domestica (not shown) (Fig. 2B–D) and at maturity these species lack sepal (Fig. 1C–D). 69

and I. dichotoma stages sepal outand I. dichotoma ridges and crests

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Table 1 Species included in study, collection information (collector and collection #, location, herbarium) and taxonomic placement; taxonomy based on Mathew (1989) and when in disagreement in parentheses based on Wilson (2011). Species

TAXONOMY

Collection information

I. I. I. I.

Subgen. Limniris sect. Lophiris (subgen. Lophiris) Pardanthopsis (subgen. Pardanthopsis) Belamcanda (subgen. Pardanthopsis) Subgen. Limniris sect. Limniris ser. Laevigatae

Karst US09-27, USA: Missouri, UC Guo CH10-29, China: Shanxi Province, UC Guo CH08-58, China: HuBei Province, UC Guo BBG07-33, China: Jilin Province grown in Beijing Botanical Garden (Chinese Academy of Sciences), UC Guo CH10-30, China: Yunnan Province, UC Guo BBG07-34, China: Jilin Province grown in Beijing Botanical Garden (Chinese Academy of Sciences), UC Guo BBG10-18, China: Beijing Botanical Garden (Chinese Academy of Sciences), UC

cristata Aiton dichotoma Pall. domestica (L.) Goldblatt & Mabb. ensata Thunb.

I. milesii Baker ex Foster I. setosa Pall. ex Link

Subgen. Limniris sect. Lophiris (subgen. Crossiris) Subgen. Limniris sect. Limniris ser. Tripetalae

I. tectorum Maxim.

Subgen. Limniris sect. Lophiris (not included in Wilson study)

For species with median ridges the prominence of the ridge varies considerably across species (Guo, 2015a). Two species included in this study, Iris ensata (Fig. 1F) and I. setosa (Fig. 1G), illustrate some of this diversity. A low sepal ridge is present in I. setosa throughout bud development (Fig. 2E–G, at arrows; illustrated in 2 H) while I. ensata has a more prominent ridge that extends into the relatively wide opening between anther thecae with little space between the ridge and thecae at the medial region of the bud (Fig. 3F, at arrow) and more space at the distal region of the bud (Fig. 3G, at arrow). For crested species, Iris tectorum (Fig. 1I), I. cristata (Fig. 1K), and I. milesii (Fig. 1M), median crests are evident at the early stage (Fig. 2I, M, Q, at arrows) and these crests extend into the opening between anther thecae (Table 2). By the middle stage, the median crest of I. cristata has enlarged more relative to its other floral organs (Fig. 2N, at black arrow), while the ridges of I. tectorum and I. milesii have enlarged relatively less than other floral organs (Fig. 2J, R, at black arrows). There are several notable changes in sepal outgrowths for crested species between the middle and late stages. Crest height in each of these species has increased, especially crests in I. tectorum (Fig. 2K, at arrow) and I. milesii (Fig. 2S, at black arrow), species with relatively little crest development between early and middle stages (Fig. 2J, R, at black arrows). The elaborate morphology of the crest in I. tectorum (Fig. 1I) is also apparent at the late stage in medial and distal sections (Figs. 2K and 3 E, at arrows). Two of the three crested species, Iris cristata (Fig. 1K) and I. milesii (Fig. 1M) have lateral structures flanking the median crests at maturity but these are not noticeable at the early bud stage (Fig. 2M and Q). At the middle stage, lateral structures of the sepal of I. cristata are apparent (Fig. 2N, at grey arrows). They occupy a space that is generally adjacent to the area between the two microsporangia of each theca. The two lateral structures on each side of the median crest of I. milesii are less prominent at this stage but are beginning to develop (Fig. 2R, at grey arrows). By the late stage, lateral crests of I. cristata (Fig. 2O, at grey arrows,) and I. milesii (Fig. 2S, at grey arrows) are obvious.

3.3. Development of anthers and style branches At early stages of the medial region of buds, anthers (including connective tissue) of all species occupy more space than petaloid style branches (Fig. 2A, E, I, M, Q; Table 3). Anthers and style branches of all species continue to enlarge throughout development, however, differences are seen in the relative growth of these two organs. For two species, Iris setosa and I. tectorum, the relative sizes of anthers versus style branches remains similar through bud development (Fig. 2E–G, I–K; Table 3) with anther size slightly increasing relative to style branches for I. setosa and slightly decreasing for I. tectorum. For the other three species (I. dichotoma, I. cristata and I. milesii), style branches enlarge more than anthers during development resulting in late stage size ratios for anthers versus style branches that are about 50% of ratios at early stages (Fig. 2A–C, M–O, Q–S; Table 3). 3.4. Changes in packing strategies There are changes in packing strategies through time within a species and also among species (Fig. 2). Iris dichotoma, a species without sepal elaborations, has sepals and petals with imbricate aestivation with the calyx completely enclosing inner whorls and corolla enclosing fertile whorls (Figs. 2A–D and 3 A and B). The calyx and corolla in I. dichotoma are dextrorse or overlap in a counter clockwise direction (Figs. 2A–D and 3 A and B; Schoute, 1935), an overlap pattern observed in the calyx and corolla of I. domestica (not shown) and the calyx of other species studied (Figs. 2–3). This arrangement of perianth organs and whorls is observed at the early stage in I. dichotoma (Fig. 2A) and carries through to the late stage (Fig. 2C). In general, space between thecae remains closed throughout development for I. dichotoma and I. domestica although the space between the thecae of one another in I. dichotoma is slightly open at the late stage in the bud illustrated (Fig. 2C; Table 2). In both of these species the connective is elongate and bowed bringing thecae in contact and is shown for I. dichotoma (Fig. 2A–C). Style branches in I. dichotoma enlarge through development (Table 3), mostly through marginal growth, and have relatively little thickening growth (Fig. 2B–C). Style branches in this species do not extend beyond the outer limit of the also enlarging anthers but instead curve inward (involute). This packing form is seen even at late stages (Fig. 2C) when some space is available between anthers and enclosing petals. Iris domestica lacks obvious petaloid style branches when mature and the central portion of the bud is mostly filled with developing anthers (not shown). Iris setosa and I. ensata have residual (Fig. 2E) and small petals (Fig. 3F–G), respectively, and both have sepal ridges (Figs. 1F–H, 2 E–F and 3 F–G). Both species have sepals with open aestivation that do not overlap at early flower stages (Fig. 2E). At middle and late stages of Iris setosa (Fig. 2F–G) sepal aestivation is imbricate, with counter clockwise overlap. Open aestivation continues during the middle stage in medial sections of I. ensata (Fig. 3F) while distally its sepals are wider and have imbricate aestivation, with counter clockwise overlap (Fig. 3G). Petals

3.2. Petal reduction in Iris setosa Iris setosa petals are not obvious in the mature flower because they are typically less than 2 cm in length. At the early stage, petals are visible as small structures lacking significant lateral growth that are positioned alternate and interior to sepals (Fig. 2E, illustrated in 2 H), while all other species in Fig. 2 have petals with considerable marginal growth, although petals are smaller than sepals (Fig. 2A, I, M, Q). At later stages, petals are not visible due to their limited elongation relative to the entire bud (Fig. 2F–G) while petals of other species continue to enlarge (Fig. 2B–C, J–K, N–O, R–S). Another species, I. ensata, also has petals that show limited growth in the bud (Fig. 3F–G). Mature flowers of I. ensata have upright petals that are obvious but are relatively short and narrow (∼5 × 0.5 cm) compared to their sepals (∼8 × 3 cm). 70

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Fig. 2. Medial transverse sections and schematic diagrams of Iris floral buds of A–D, I. dichotoma; E–H, I. setosa; I–L, I. tectorum; M–P, I. cristata; Q–T, I. milesii. Transverse sections are for early, middle, and late stages, left to right; sepal elaborations are shown on one of three sepals with black arrowheads for median ridges or crests and grey arrowheads for lateral ridges or crests. Diagrams on far right represent an approximation of the late stage for each species; Sepals and ridges or crests are green, petals are red, stamens are yellow, and petaloid style branches are blue.

have not elongated enough to be visible in medial sections at later stages in I. setosa (Fig. 2F–G) and remain relatively narrow and thin at the middle stage in I. enstata (Fig. 3F―G). Anthers in these two species are large and broad extending into this potential space. Of particular note is the closing of the space between anther thecae in I. setosa at middle and late stages (Fig. 2F–G; Table 2). The median sepal ridge in I. setosa (Fig. 2E–H) is adjacent to the area between thecae but does not extend into this area, even at the early stage when this space is open (Fig. 2E, at arrow) while in I. ensata the median ridge extends into the

space between thecae (Fig. 3F–G, at arrows). In I. setosa the connective does not elongate relative to anthers as the bud develops (compare Fig. 2E to Fig. 2F–G) while in I. ensata the connective is elongate and bowed (Fig. 3F–G). The margins of the style branches of I. setosa and I. ensata extend to the outer perimeter of the space between anthers where they are slightly involute (Figs. 2F–G, 3 F) but are not as curved as those in I. dichotoma (Fig. 2B and C). At the distal region of the bud in I. ensata (Fig. 3G) the bifid tips of each style branch are visible as narrow overlapping segments. 71

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Fig. 3. Transverse sections of Iris floral buds. A–B, I. dichotoma, proximal portions of bud at early and late stages, respectively. Early stage section showing solid style branches centrally (arrow) and anthers to the outside of style; late stage section showing narrow style branches and filaments (arrow) that have elongated. C–E, I. tectorum, distal region of buds from early to late stages, respectively, showing enlargement of sepal outgrowths (at arrow). At the late stage a sepal crest is evident. F, –G, I. ensata medial and distal portions of bud at the middle stage showing sepal with median ridge (at arrows) and increased space within bud distally. H, I. cristata distal portion of bud at the late stage showing a median sepal crest (black arrow), lateral crests (grey arrows) and open space within the bud.

Floral packing in species with sepal crests is more complex than in other species. Iris cristata sepals have imbricate aestivation, with counter clockwise overlap, enclosing inner whorls but petals have open aestivation (Fig. 2M–P). Petals occupy spaces between median sepal crests but are adjacent to lateral crests (at grey arrows) of the sepal (Fig. 2N–P). Style branches of I. cristata enlarge more than anthers during middle and late stages (Table 3) with style branch margins extending to the midvein region of the petal and curving with the curvature of the latter structure. The width and depth of space between anther thecae of I. cristata (Table 2) increases in conjunction with increases in median sepal crest height (Fig. 2N―P), and the crest protrudes into this space. In I. cristata the connective is less bowed than in most other species, especially at the late stage (compare Fig. 2O to Fig. 2C, K, S). The lack of curvature of the connective and relative less expansion of anthers provides considerable space between thecae that is mostly empty at the late bud stage (Fig. 2O). Floral packing in the other two crested species shares some characteristics but differ in others. Iris tectorum and I. milesii, sepals have imbricate aestivation, with counter clockwise overlap, enclosing inner whorls but petals have open aestivation (Fig. 2I–L; Q–T, respectively). Petal margins of these two species expand and by the middle stage curve into the space around the sepal median crests and between the

two thecae (Fig. 2J–L, R–T). Style branches enlarge relative to anthers during development (Table 3) with the ratio for I. milesii reduced by 50% from 1.6 to 0.8, the greatest change in relative size among the species studied. The connective is slightly bowed in these species (Fig. 3I–K, Q–S) but the space between thecae remains open and increases during development (Table 2). The style branches of I. tectorum and I. milesii widen as they expand within the bud (Fig. 2I–K, Q–S, respectively) but do not curve to the degree of I. dichotoma or I. cristata (Fig. 2C and O), respectively. At the middle stage the margins of style branches in I. milesii are slightly involute (Fig. 2R) and by the late stage are less curved (Fig. 2S) while the margins of style branches in I. tectorum show little curvature through development (Fig. 2I–K). 3.5. Space within buds For all species studied, space within the bud increases during development (Table 4). Most species have about 10% empty space at the early stage and greater than 25% by the late stage (Table 4). An exception is Iris setosa that begins with about 17% empty space at the early stage and at the late stage has about 22% empty space (Table 4). Interestingly, there is no general trend between sepal ornamentation and space within the bud. At distal regions of the bud, empty space also

Table 2 Average measured distance (μm) between thecae for three anthers from one median section across three bud stages of five Iris species. Species

I. I. I. I. I.

dichotoma setosa tectorum cristata milesii

Early

Middle

Late

Width

Depth

Width

Depth

Width

Depth

0.0 ± 0.0 58.1 ± 17.4 204.3 ± 50.9 154.7 ± 2.9 117.1 ± 6.2

0.0 ± 0.0 255.1 ± 23.2 438.6 ± 26.9 369.5 ± 8.9 331.6 ± 4.1

0.0 ± 0.0 0.0 ± 0.0 124.7 ± 14.4 138.3 ± 15.9 181.9 ± 41.9

0.0 ± 0.0 0.0 ± 0.0 668.9 ± 49.3 590.8 ± 22.2 974.6 ± 143.5

23.7 ± 23.7 42.7 ± 42.7 442.5 ± 1579.4 251.2 ± 32.8 487.9 ± 111.1

273.7 ± 273.7 173.7 ± 173.7 1148.7 ± 117.1 947.9 ± 49.3 1813.3 ± 208.2

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Table 3 Average measured area (mm2) of three anthers and style branches from one median section and ratio (anther/style branch) across three bud stages of five Iris species. Species

I. I. I. I. I.

dichotoma setosa tectorum cristata milesii

Early

Middle

Late

Anther

Style Branch

Ratio

Anther

Style Branch

Ratio

Anther

Style Branch

Ratio

326.0 ± 5.3 230.4 ± 2.9 478.2 ± 18.3 442.9 ± 5.4 269.5 ± 6.8

52.3 ± 2.1 76.9 ± 2.2 121.8 ± 10.3 169.4 ± 3.9 169.0 ± 6.2

6.2 3.0 3.9 2.6 1.6

794.8 ± 20.2 2308.6 ± 1035.4 872.4 ± 328.0 541.4 ± 13.4 822.0 ± 37.6

301.4 ± 6.2 8685.7 ± 377.1 211.4 ± 1.5 311.7 ± 14.0 465.3 ± 2.7

2.6 2.7 4.1 1.7 1.8

2809.7 ± 167.9 5320.6 ± 982.9 2008.9 ± 67.7 606.8 ± 21.7 1490.3 ± 14.4

829.5 ± 409.7 15388.1 ± 211.6 590.4 ± 14.7 429.1 ± 3.3 1840.0 ± 26.8

3.4 3.5 3.4 1.4 0.8

diversity when comparing sepal development to other whorls is not surprising because sepal ornamentation has evolved multiple times across Iris (Table 1; Guo and Wilson, 2013) and displays remarkable diversity (Fig. 1F–N). A study of sepal anatomy (Guo, 2015b) showed that median and lateral ridges and/or crests in Iris are created by either meristematic activities of cells on the adaxial surface of the sepal or enlargement of sepal mesophyll cells. The independent evolution of sepal median structures (ridges and crests) several times across the genus could be due to either the attainment of a genetic mechanism giving rise to an opening of the space between anther thecae first, which then provided a mechanical cue for the outgrowth of median sepal structures and the insertion of petals in response to the opening of this space; or the acquirement of a genetic program that gave rise to meristematic activities responsible for the outgrowth of median structures firstly, which then mechanically prompted the opening of the anther and changes of geometries of other floral organs, particularly petals; or both could happen at the same time. Regardless of whether the space or the sepal elaboration developed first, the resulting floral organs continued to influence and be influenced by adjacent organs and whorls of organs due to the constraint of space within the bud. Studies on physical constraint in floral organs have mostly focused on stresses within organs such as experiments on Arabidopsis (Hervieux et al., 2016) where differential growth within sepals was shown to produce tensile stresses at the sepal tip that acted as major regulators of sepal growth and shape. Hervieux et al. (2016) noted that adjacent organs might provide additional mechanical stresses restricting sepal expansion, although they did not investigate this. Endress (2008) suggested the term “imprinted shape” for modified organ shape caused by mechanical pressure of adjacent organs. Studies using manipulation to increase or decrease physical space constraints of Helianthus floral apices showed the symmetry of inflorescences and individual florets were affected by changes in physical constraints (Hernandez and Green, 1993). Hernandez and Green (1993) also showed that constraint of young Helianthus inflorescences led to development of bracts in some positions where flowers would normally form. Additional studies are warranted that investigate the roles of adjacent organs in the development of final organ morphology, including experiments incorporating manipulations of physical space. This study provides information on potential stresses due to adjacent whorls within floral buds and suggests the presence of sepal outgrowths in Iris as a promising subject for further investigation of physical constraint. Rodionenko (1987) emphasized the importance of the Iris sepal in pollination, with its distal portion serving as a landing platform and its proximal portion (with the overarching style branch) forming a pollination tunnel that also provides protection for pollinators (Fig. 1B). Uno (1982) observed pollinators on I. douglasiana, clarifying the importance of sepal coloration patterns on successful pollination while Sapir et al. (2005) and Vereecken et al. (2012) reported on the role of the tunnel-shaped Iris corolla for sheltering pollinators. While most monocots have tepals rather than morphologically distinct sepals and petals, Iris has petals that are typically smaller than sepals and upright while sepals are horizontal (perpendicular to the axis of the flower) or may be reflexed distally. This distinction between perianth whorls and

Table 4 Percent of bud that is empty from one median section across three bud stages of five Iris species. Species I. I. I. I. I.

dichotoma setosa tectorum cristata milesii

Early

% Empty Middle

Late

10.7 16.9 13.7 9.1 9.7

15.2 20.7 9.9 28.2 26.3

39.8 21.8 25.8 41.9 43.2

increases with development as shown by I. tectorum (Fig. 3C–E). We found that distal regions of buds were less filled and by the late stage had empty space associated with all whorls forming a less dense bud distally prior to anthesis as shown in sections of I. tectorum (compare Fig. 3E to Fig. 2K) and I. cristata (compare Figs. 3H–2O). 4. Discussion 4.1. Petaloid style branches A prominent feature of Iris floral buds is the presence of petaloid style branches above a shorter section of style where the three carpels are fused. Androecial development in Iris is precocious relative to the gynoecium (Fig. 2; Table 3, early stages), similar to the rapid stamen development observed in Arabidopsis during early stages of floral development (Smyth et al., 1990; Crone and Lord, 1994). In most monocots a second whorl of three stamens develops centripetally to the first whorl and opposite the petals (Remizowa et al., 2010), but in Iris this whorl is lacking. Interestingly the petaloid portion of the style is interpreted as wing-like outgrowths from the lateral edges of the abaxial side of the conduplicate carpel (Leinfellner, 1952 Fig. 1) and develops in the position where the second whorl of stamens would arise if present. After the early stage, petaloid style branch enlargement equals or outpaces anthers (Table 3) with these petaloid organs occupying the center of the bud with their margins extending into the space between anthers (Fig. 2). When the bud opens, the petaloid style branch overarches the stamen and proximal portion of the sepal contributing to the labiate pollination unit (Fig. 1A and B). 4.2. Sepal ornamentation Another prominent feature of Iris is the development of outgrowths on sepals in some species. This study finds that ornamentation of the sepal with ridges and crests is concomitant with geometrical changes of other floral whorls. The most conspicuous changes for species with sepal ornamentation are the development of an opening between thecae in some ridged (Fig. 3F–G) and all crested species (Table 2) and either reduced marginal petal growth or curvature of petal margins into the space between thecae in all ridged and crested species examined (Figs. 2–3). Other characteristics, such as the relative reduction in the size of stamens versus petaloid styles and the overall shape and position of floral organs, vary among species with sepal ornamentation. This 73

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their relative roles in pollination may have contributed to further elaborations of sepals. Morinaga and Sakai (2006) manipulated the lengths of sepals and petals in I. ensata and concluded petal length influenced the number of pollinator approaches and pollen removal while sepal length influenced these two factors but also the number of pollinator landings and seed production, thus sepals influenced female and male reproductive success while petals influenced only male success in this Iris. Iris ensata has a prominent sepal ridge and was included in this present study. These studies and the occurrence of highly reduced petals in several lineages in Iris, such as I. setosa, a species included in this study, highlight the importance of sepals for sexual reproduction in Iris. Further studies on floral bud organization and spatial relations between sepals and other floral organs should include other sepal morphologies, such as sepals of diverse shapes and those with beards and/or claws. It would also be interesting to examine the effect of manipulation of sepal ornamentation on pollination, particularly pollinator landings and time spent probing flowers.

A few studies have looked at packing in floral buds but have mostly not focused on correlations between packing strategy and mature whorl or organ morphology. Berg (1990) found that floral buds in Urticales are tightly packed and often have protective hairs between floral organs. He hypothesized that tight packing and hairs provided protection to the developing flower, especially from insects (Berg, 1990). Endress (2008) also considered floral buds tightly packed and noted the presence of long hairs as filling material for irregular spaces in floral buds that are not easily packed by developing organs. Endress (2008) cited the importance of tightly packed buds for protection from herbivores, drought, or frost. Endress (2008) also suggests the shape of floral organs might be influenced by evolutionary constraints posed by fit within a tightly packed bud. Similar to our findings, he notes that in Carpinus, thecae are separated by a filamentous connective so that anthers have flexibility in their arrangement. In contrary to the “filling law” proposed by Couturier et al. (2011) for vegetative buds and studies by Berg (1990) and Endress (2008), we found that Iris floral buds are not tightly packed. Similar to patterns seen in other studies for vegetative and floral buds, early stages of Iris floral buds are approximately 80–90% filled at their median region (Table 4) indicating a greater effect of spatial constraint on developing floral organs at this stage. However, unlike vegetative buds (Couturier et al., 2009, 2011, 2012) and floral buds reported in Berg (1990) and Endress (2008), we found that late developmental stages Iris floral buds have as much as 60% empty space at their medial region (Table 4) with unfilled space found throughout the bud (Figs. 2–3). At some proximal regions (Fig. 3B) and all distal regions (Fig. 3C–E, G–H) of buds even more empty space is present. Ebar and Gülden (2011) noted the presence of space between the calyx and corolla whorls in Paulownia tomentosa (Thunb.) Steud and that this space enlarges after organ initiation. Ebar and Gülden (2011) considered the presence of this space “remarkable” and not similar to other floral buds that are tightly packed. They speculated the dome-shaped, hairy calyx provides bud protection in this species of Paulownia and that this calyx morphology might be compared to water calyces in some tropical plants (Endress, 1996) where organ packing is also not tight but young organs are protected by fluid that is secreted from glandular hairs of the calyx. Carlson and Harms (2007) studied Chrysothemis and concluded that water calyces in this plant provided protection from herbivores. Differing from the vegetative shoot apex, most floral meristems are determinate and the initiation of floral organ primordia is not continuous within a flower. This pattern of determinate growth may contribute to the presence of unoccupied space within floral buds at some stages of organ growth in some taxa. In addition, developing leaves in resting buds are enclosed in bud scales that have mechanical tissues such as sclerenchyma, collenchyma and thickened epidermal cell walls that provide protection (Foster, 1928) and a mechanical barrier restricting the growth of enclosed leaves (Hamant, 2013). The main function of the calyx or outermost whorl of sepals is also considered to be protection of the inner floral whorls (Endress, 1996). While most sepals are reduced relative to petals and often thickened or with relatively strong tissues, Iris sepals have dual roles of protecting the young bud and at later stages of expanding and serving as part of the pollination unit (Fig. 1A and B). It is interesting that Iris buds do not follow the “filling law” of vegetative buds (Couturier et al., 2011), are not tightly packed as suggested for floral buds by Berg (1990) and do not have filling hairs (Endress, 2008) or fluids (Endress, 1996; Carlson and Harms, 2007) that reduce the amount of empty space. Further studies of space in floral buds should compare buds of petaloid sepals such as those found in Iris to reduced sepals that are common in most angiosperms, including those with filling hairs and water calyces.

4.3. Organ reduction, organ loss and labiate form Petal reduction occurs in several lineages of Iris (Ikinci et al., 2011; Guo and Wilson, 2013; Wheeler and Wilson, 2014). This study found that I. setosa buds have space interior to the reduced petals at the early stage (Fig. 2E). In later stages (Fig. 2F–G) petals are not visible because they have not elongated as the bud enlarged and space at the petal positions is occupied by the overlapping sepal margins, involute petaloid style branch margins and enlarged anthers. Similarly as discussed above, the petaloid style branches occupy the space where the inner stamen whorl, that is not present in Iris, would occur. In Iris the reduction and/or loss of floral whorls are associated with shifts in the position of other organs and/or growth of other organs into this space. Similarly, Couturier et al. (2012) showed that removal of one of a pair of leaf primordia in Acer vegetative buds resulted in the enlargement and growth of the remaining leaf into the space made available. The labiate pollination units, each comprised of a petaloid style branch, one stamen opposite to the sepal, and the sepal, is a unique feature of Irideae (Fig. 1A and B). We suggest the loss of the inner stamen whorl, the change from tepals with similar size and shape to obvious whorls of typically larger sepals and smaller petals, and the development of a petaloid style branch can be attributed to coordinated changes within the floral bud, allowing the development of a labiateform. Radović et al. (2017) quantified floral organ symmetry in Iris pumila and concluded that the symmetrical petaloid style branch might signal the presence of rewards for pollinators and that the shapes and sizes of sepals and style branches both contribute to a wide and symmetrical pollination tunnel that enhances pollination, especially pollination by relatively large insects, such as bumblebees. The overall shape of Iris floral buds is not circular but instead is trilobed (Figs. 2–3) with greater width at the three pollination units and less width at the position of petals, which occur alternate to pollination units. We consider the reduction of petals in some species across Iris as further development of the labiate form. 4.4. Empty space within floral buds Couturier et al. (2011) proposed that vegetative buds are always filled and follow a “filling law” where morphologies of mature leaves can be predicted by parameters of leaf packing within buds. In addition, they proposed that small changes in the packing of leaves can result in major changes to final leaf morphologies with leaf growth and lobing influenced by previous and subsequent leaves through mechanical regulation by contact within the filled bud. Edwards et al. (2016) suggested that while mechanical constraint is one hypothesis to explain lobes and teeth on leaves associated with a folded and tightly packed arrangement in buds, another possibility is that leaves with these particular shapes may produce a more compact bud.

4.5. Coordinated growth of floral organs As discussed above, mechanical forces generated by spatial constraint alone do not fully explain the coordinated growth of floral 74

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organs within Iris floral buds. Several examples of coordinated growth across organ whorls are demonstrated in this study, including the occurrence of space between anther thecae and the growth of sepal ridges and crests and petal margins into this space, the enlargement of anthers into space typically occupied by petals in species with extreme petal reduction, the elongation and curvature of stamen connectives showing flexibility in stamen position and the development of petaloid style branches in the center of the bud that is possible because of the inferior ovary and lack of an inner stamen whorl. Studies on biochemical or genetic mechanisms of coordinated growth of floral organs are scarce but illuminating. Goldschmidt and Huberman (1974) showed the wounding of a single petal changed the radial symmetry of Citrus flowers and disrupted the balance of sucrose and acetate among petals as well as among other floral whorls. They concluded there is a potential regulatory mechanism that balances the relative metabolic activities among floral organs in the same whorl as well as determines the relative metabolic activities among different floral whorls (Goldschmidt and Huberman, 1974). Genetic programs are known that control the development of each floral organ (Sablowski, 2015; Stewart et al., 2016), but there may also be molecular regulatory mechanisms across floral organ boundaries to achieve the consistent shape and relative size of all floral organs. Eriksson et al. (2010) discovered that a cytochrome P450 KLU-dependent novel mobile growth signal acts non-cell-autonomously to promote the growth of floral organs and can move beyond the boundaries of floral organs of the same flower as well as across the inflorescence to determine the final size of individual floral organs, thus providing a potential mechanism for coordinated growth of floral organs. This study illustrates the importance of studying the morphogenesis of floral organs in the context of other whorls within the bud. This study also calls for future studies of the mechanical, biochemical and genetic mechanisms of coordinated growth of floral organs. Finally, this study examines floral bud development of five Iris species with complex floral morphology and suggests areas of research that are likely to be informative through more focused study. This study relied heavily on the medial region of floral buds while a three dimensional imaging study of buds is likely to provide more insights into their development.

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Acknowledgements We thank Rancho Santa Ana Botanic Garden (RSABG), the Microscopy Laboratory at Brigham Young University and the Department of Biological Sciences at SUNY Oswego for providing facilities for this research; Jinzheng Zhang and Guofeng Sun (Beijing Botanical Garden, the Chinese Academy of Sciences), Cuifen Wu and Qianyu Zuo (Caojian Forest Institute of Yunlong County), Weibang Sun (Kunming Institute of Botany), personnel from the Shaw Nature Preserve, Lisa Karst, and Jinyan Guo’s parents (Jiang Guo and Ruili Wang) for assisting with field trips or providing plant materials. Funding was provided by the Graduate Student Research Awards Program at RSABG (JG), the American Iris Society Foundation (JG), NSF: DEB–1011731 (JG) and NSF: DEB–1020826 (CAW). The Early Start Program at SUNY Oswego allowed JG time to work on the final portions of this study. References Arber, A., 1934. The Gramineae: A Study of Cereal, Bamboo and Grass. Cambridge University Press, Cambridge, UK. Berg, C.C., 1990. Differentiation of flowers and inflorescences of Urticales in relation to their protection against breeding insects and to pollination. Sommerfeltia 11, 13–34. Carlson, J.E., Harms, K.E., 2007. The benefits of bathing buds: water calyces protect flowers from a microlepidopteran herbivore. Biol. Lett. 3, 405–407. https://doi.org/ 10.1098/rsbl.2007.0095. Couturier, E., Courrech du Pont, S., Douady, S., 2009. A global regulation inducing the shape of growing folded leaves. PLoS One 4, e7968. Couturier, E., Courrech du Pont, S., Douady, S., 2011. The filling law: a general framework for leaf folding and its consequences on leaf shape diversity. J. Theor. Biol. 289,

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