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ONTOGENETIC EVOLUTION AND SPEClATlON IN MIMULUS CARDINALIS

AND M. LEWlSll (LAMIALES)

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

THOMAS HAZLE

In partial fulfilment of requirements

for the degree of

Master of Science

May, 2001

O ~hornasHazle, 2001 National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON K1A ON4 Ottawa ON KIA ON4 Canada Canada

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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette these. thesis nor substantial extracts f?om it Ni Ia thèse ni des extraits substantiels may be printed or othekse de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ONTOGENETIC EVOLUTION AND SPECIATION IN MIMULUS CARDINALE AND M. LEWlSll (LAMIALES)

Thomas Hazle Advisor: University of Guelph, 2001 Professor J.M. Canne-Hilliker

The floral ontogenies of three populations of M cardinalis were compared, as were the floral ontogenies of both races of M. lewisii. This was done to compare the Roral ontogenies of each species as exemplified by populations from Yosemite National Park, CA, and interpret differences as evolutionary transformations that occurred during speciation of M. lewisii from an M cardinalis

- like ancestor. Between the species, differences in corolla shape were observed shortiy after corolla tube formation. Corollas in buds of M lewisii became dorso - ventrally narrow while those in buds of cardinalis became

Iaterally narrow. The shorter stamens and style in flowers of M lewisii were the result of early terminations of growth relative to the growth of those in flowers of -M. cardinalis. The changes in ontogeny that had the largest impact on floral form are hypothesized to have occurred at late stages, while a series of srnaller changes occurred throughout development.

I

Acknowledgernents

No body of work is ever solely the product of one person. This thesis, like most, has had the benefit of valuable intellectual, emotional, technical and inspirational input from rnany people. I would like to express my sincere gratitude to some of these people who have been invaluable throughout the course of this study.

Primarily, I would like to thank my supervisor Judy Hilliker. She unquestioningly put her faith in me from the very beginning. If her faith ever faltered, she did not let it show. She has tnily been a source of inspiration as a scholar, a scientist, and as a teacher.

Next, I would like to thank my advisory cornmittee, Drs. Brian Husband,

Usher Posluszny and Peter Kevan. Each, in his own way, has contributed to this work as well as to my developrnent as a scientist. I would also like to thank Dr.

Richard Reader who gave so freely his time and wisdom. I want to thank Dr.

Sandy Smith for training me in the deadly art of scanning electron microscopy, and for teaching me how to build a SEM using swiule sticks and a gum wrapper.

Many thanks to Paul Beardsley and Dr. Richard Olmstead for seeds and for lending their expertise on the phylogenetic relationships within Mimulus. Also, my gratitude goes to Drs. Doug Schemske and Robert K. Vickery Jr. for seeds.

Next, I would like to thank the members of my lab, Lara Yacob and lana

Niklova, for lending expertise on (somewhat) related organisms. Also, thanks to

Leigh Anne Swayne for her exemplary work as a research assistant. .- II

Finally, 1 would like to thank Tracy Burton for her love and support, and for not saying "1 told you son (too many times, at least). I gratefully acknowledge the contributions of the aforementioned people. However, al1 mistakes are mine. iii

TABLE OF CONTENTS

Main lntroduction

Development and organismal diversification

Heterochrony

Allornetry

Additional ontogenetic hypotheses

Ontogenetic basis of breeding mating systerns

Floral morphology and pollination in Mimulus

Significance of research

Objectives

Materials and Methods

The study organisms

Microscopy

Meiosis

Al lornetric analyses

Sample sizes

Chapter one - floral ontogeny of Mimulus cardinalis

lntroduction

Results

Discussion

Figures and tables

Chapter two - floral ontogeny of Mimulus lewisii

lntroduction Results

Discussion

Figures and tables

Chapter three - ontogenetic evolution of & cardinalis and M. lewisii:

morphology, allometry and heterochrony

Introduction

Results

Discussion

Figures and tables

Main Discussion

References v

LIST OF TABLES

Chapter one

Table 1 Slopes and Regression Coefficients of Linear

Regression Lines of Bud Length versus Time

Table 2 Floral Organ Lengths at Anthesis

Table 3 Slopes and Regression Coefficients of Linear

Regression Lines of Seiected Allometric

Relationships Arnong Populations

Table 4 Slopes and Regression Coefficients of Linear

Regression Lines of Selected Allometric

Relationship Within a FIower

Chapter two

Table 1 Slopes and Regression Coefficients of Linear

Regression Lines of Bud Length versus Time

Table 2 Floral Organ Lengths at Anthesis

Table 3 Slopes and Regression Coefficients of Linear

Regression Lines of Selected Allornetric

Relationships Among Populations

Table 4 Slopes and Regression Coefficients of Linear

Regression Lines of Selected Allometric

Relationship Within a Flower Chapter 3

Table 1 Slopes and Regression Coefficients of Linear 330

Regression Lines of Bud Length versus Time

Table 2 Floral Organ Lengths at Anthesis 33 1

Table 3 Slopes and Regression Coefficients of 332

Regression of Log Transformed Adaxial and

Abaxial Corolla Lengths vs. Time vii

LIST OF FIGURES

Chapter one

Fig. 1 Lateral views of flowers of !& cardinalis.

Fig. 2 Longitudinal section of a Rower of & cardinalis.

Fig. 3 Inflorescence apices and floral apices of cardinalis.

Fig. 4 Early floral apex development of M; cardinalis.

Fig. 5 Early floral development of && cardinalis.

Fig. 6 Floral apices of M cardinalis.

Fig. 7 Floral apices of M; cardinalis.

Fig. 8 Buds of @ cardinalis.

Fig. 9 Dissected buds of M cardinalis.

Fig.1O Buds of M cardinalis.

Fig.11 Buds of M cardinalis.

Fig.12 Buds of M cardinalis.

Fig. 13 Pistils of cardinalis.

Fig.14 Buds of !&. cardinalis at sirnilar stages of development

Fig.15 Buds of M cardinalis at similar stages of developrnent

Fig.16 Buds of !& cardinalis at sirnilar stages of development

Fig.17 Buds of M cardinalis at similar stages of development

Fig. 18 Corollas of M. cardinalis just prior to anthesis.

Fig.19 Transverse sections of flowers of cardinalis.

Fig. 20 Pistils of M cardinalis, CI (DM).

Fig. 21 Line tracings of corolla lobes of cardinalis. viii

Fig. 22 Stamen development of cardinalis.

Fig. 23 Stamen development of M. cardinalis.

Fig. 24 Starnen development of M. cardinalis.

Fig. 25 Stamen alignment of cardinalis flowers (anthesis).

Fig. 26 Regression lines showing growth rates of buds

of M. cardinalis. 108

Fig. 27 Allometric plot of abaxial corolla length versus adaxial 110

corolla length for M. cardinaiis.

Fig. 28 Allometric plot of adaxial and abaxial corolla lengths 112 versus abaxial stamen length for YO.

Fig. 29 Allometrîc plots of adaxial and abaxial corolla tube lengths 114 versus ovary length for M. cardinalis.

Fig. 30 Allornetric plot of adaxial and abaxial corolla tube lengths 116 versus abaxial stamen length for plants of CI.

Fig. 31 Allometric plot of abaxial (representative of adaxial) stamen .118

length versus adaxial corolla length for M. cardinalis.

Fig. 32 Allometric plot of abaxial and adaxial stamen lengths 120 versus adaxial corolla length in YO.

Fig. 33 Allometric plot of style length veraus adaxial corolla

length for -M. cardinalis. Chapter two

Fig. 1 Flowers of M. lewisii. ix

Fig. 2 Longitudinal section of flower of M. lewisii.

Fig. 3 Inflorescence apices and floral apices of M. lewisii.

Fig. 4 Early floral developrnent of M. lewisii.

Fig. 5 Early floral development of M- lewisii.

Fig. 6 Early development of floral apices of M. lewisii.

Fig. 7 Early development of floral apices of M. lewisii.

Fig. 8 Floral buds of M. lewisii.

Fig. 9 Floral buds of M. lewisii.

Fig. 10 Floral buds of M. lewisii.

Fig. 11 Floral buds of M. lewisii.

Fig. 12 Dissected buds of M. lewisii.

Fig. 13, Buds of M. lewisii with calyx removed.

Fig. 14 Buds of ivl. lewisii with calyx removed.

Fig. 15 Buds of M- lewisii with calyx removed.

Fig. 16 Buds of M. lewisii with caiyx removed.

Fig. 17 Longitudinal sections of buds of M. lewisii.

Fig. 18 Dissected floral buds of M. lewisii.

Fig. 19 Dissected floral buds of M. lewisii.

Fig. 20 Dissected floral buds of M. lewisii.

Fig. 21 Corollas of M. lewisii.

Fig. 22 Corollas of M. lewisii.

Fig. 23 Corollas of M. lewisii.

Fig. 24 Flowers of M lewisii. X

Fig. 25 Dissected floral buds of M. lewisii.

Fig. 26 Lateral views of pistils of M. lewisii.

Fig. 27 Lateral views of nectaries of M. lewisii.

Fig. 28 Line tracings of corolla lobes of M. lewisii.

Fig. 29 Buds and fiowers of M. lewisii.

Fig. 30 Longitudinal sections of M. lewisii.

Fig. 31 Longitudinal sections of buds and flowers of M. lewisii.

Fig. 32 Regression of bud length vs. time for M. lewisii.

Fig. 33 Allometric plot of abaxial corolla length versus adaxial corolla length for M- lewisii.

Fig. 34 Allometric plot of corolla length vs. style length

Fig. 35 Allometric plot of adaxial corolla tube length versus abaxial corolla tube length for M. lewisii.

Fig. 36 Allometric plot of adaxial and abaxial corolla tube lengths versus adaxial stamen length for M. lewisii.

Fig. 37 Allometric plot of adaxial and abaxial corolla tube lengths versus adaxial stamen length for M. lewisii.

Fig. 38 Allometric plot of abaxial stamen length versus adaxial stamen length for M. lewisii.

Fig. 39 Allometric plot of adaxial and abaxial stamen length versus style length for M. lewisii.

Fig. 40 Allometric plot of style length versus adaxial stamen length for M. lewisii. xi

Chapter three

Fig. 1 Flowers of M. cardinalis and M. lewisii.

Fig. 2 Flower of cardinalis showing dimensions used in allometn'c analyses.

Fig. 3 Early floral developrnent of M cardinalis and M- lewisii.

Fig. 4 Early floral development of fi cardinalis and M- lewisii.

Fig. 5 Floral apices of !&. cardinaiis and M. lewisii.

Fig. 6 Dissected floral buds of & cardinalis and M- lewisii.

Fig. 7 Dissected floral buds of & cardinalis and M. lewisii.

Fig. 8 Diçsected floral buds of cardinalis and M_ lewisii.

Fig. 9 Floral buds of M. cardinalis and M. lewisii.

Fig. iO Corollas of cardinalis and M. Iewisii.

Fig. II Corollas of M. cardinalis and M. lewisii.

Fig. 12 Corollas of M. cardinaiis and M. lewisii.

Fig. 13 Corollas of M. cardinalis and M. lewisii.

Fig. 14 Corollas of M. cardinalis and M. lewisii.

Fig. 15 Pistils and nectaries of M. lewisii and j& cardinalis.

Fig. 16 Dissected floral buds of cardinalis and M. lewisii.

Fig. 17 Dissected buds of !'&. cardinalis and M. lewisii.

Fig. 18 Buds of M, cardinalis and M-lewisii.

Fig. 19 Dissections of M cardinalis and M. lewisii.

Fig. 20 Dissected fiowers of !& cardinalis and M. lewisii.

Fig. 21 Dissected buds of l& cardinalis and M. lewisii. XII

Fig. 22 Corollas of M. cardinalis and M. lewisii.

Fig. 23 Corollas of M. cardinalis and M. lewisii.

Fig. 24 Corollas of M. cardinalis and M. lewisii.

Fig. 25 Line tracings of corolla lobes from M. cardinalis and M Iewisii at anthesis.

Fig. 26 Regression lines showing growth rates of buds of -M. cardinalis and M. lewisii. Fig. 27 Allometric plots of corolla lengths versus ovary length. 321

Fig. 28 Allometric plots of adaxial and abaxial 323 corolla tube lengths versus ovary length.

Fig. 29 Allometric plots of stamen lengths versus ovary length. 325

Fig. 30 Allometric plot of style length versus ovary length. 327

Fig. 31 Allometric plot of abaxial corolla length 329 versus adaxial corolla length. Introduction

Development and organismal diversification

Interest in the role of development in systernatic studies haç soared during the last half of the twentieth century. Ontogenetic studies have been used extensively to investigate animal phylogeny, which has paved the way for similar studies with plant taxa (Alberch et al. 1979; Alberch 1982; Geurrant 1982;

Kellogg 1990; Hufford 1995). Work by Gould (1977) has done much to clear up ambiguities and misconceptions surrounding studies of development, which was needed due to misuse of an over-abundant terrninology.

Comrnonly, natural selection (and, to some extent, genetic drift) has been regarded as a rnechanism through which random variation is filtered in a non- random way (Diggle 1992). Alberch (1 982) suggested that the concept of development as an integral part of evolutionary theory has been largely overlooked in favour of natural selection because of a fundamental assumption of neo-Darwinian theory that nature provides a broad range of variation upon which selection can act. Because development is the process responsible for the translation of genetic information into phenotype, and because of the confounding complexity of this translation due to epistatic effects, the importance of development as an evolutionary mechanism has been recently emphasized.

Changes in developmental pathways among related taxa have been investigated in this context (Gould 1977; Alberch 1982; Kauffman 1983; Levinton 1986; Diggle

1992; Hufford 1995; Stern 2000; Kuzoff et al. 2001). Studies of development as a means of understanding the mechanisms that generate morphological variation and diversity within versus among taxa, are becoming more freguent. By 2 studying the developmental basis of floral symmetry, organ loss, connation and adnation of structures, and homeosiç (change of position of a structure), organic fon has been more accurately interpreted and multiple origins of characters have been detected (Tucker 1984; Kellogg 1990; Ramirez-Domenech and

Tucker 1990; Diggle 1992; Posluszny and Fisher 2000; Tucker 2000).The developmental means by which diagnostic characters are manifest can be useful in revealing homology or, conversely, homoplasy.

Although it is accepted that development acts as an evolutionary constraint, the role of development as a constraint has not been tested directly

(Kauffman 1983; Levinton 1986). Presumably, the existing level of knowledge of developmental regulatory genes renders experimental manipulation irnpracticable. Inçtead, comparative studies have focused on determination of the patterns of developmental variation that create phenotypic diversity among related taxa (Alberch 1982). Many of these studies have elucidated developmental frameworks for the patterns of variation that may be selected for or against during evolution. Consequently, hypotheses of ontogeny are often concomitant with hypotheses of phylogeny. Some of these will be discussed.

Hypotheses exist that predict the patterns of developmental variation observable in specific circumstances. Tucker (1984, 1997) proposed a number of distinct yet intertwined hypotheses that involve the relationship between features that are diagnostic at various taxonornic levels and the stage of development at which these features are manifested. In the Fabaceae, she reported that features that are manifested early in ontogeny (during 3 organogenesis) are diagnostic at the sub-family level (Tucker 1984; Tucker

1997). Features that are manifested during mid-stages of ontogeny are diagnostic of genera, and features that are detemined late in ontogeny tend to characterize species. As well, the developmental differences among species tend to be more quantitative than qualitative. She proposed as an explanation that features determined in early ontogeny are more stable than those determined late in ontogeny.

Heterochrony

Heterochrony is defined as changes through time in the appearance or rate of development of ancestral characters in related taxa (Gould 1977;

McNamara 1986; McKinney 1988). Studies of heterochrony are comparative analyses of divergence in developmental pathways through time and across taxa. In the twentieth century, the role of heterochrony in evolutionary studies has been greatly overlooked (McNamara 1986). Relatively recently. however, ontogenetic studies have been used to investigate animal phylogeny (Gould

1977; Aiberch et al. 1979; McNamara 1982; Marcus and McCune 1999; Swalla and Collazo 2000). This work has paved the way for sirnilar studies with plant taxa (Geurrant 1982; Kellogg 1990; Hufford 1995; Stewart and Canne-Hilliker

1998).

Six established heterochronic evolutionary processes can be explained with respect to three developmental parameters and placed into one of two categories based on the derived morphology resulting from the process. The developmental parameters are the initiation of a developmental event. the termination of a developrnental event (often defined as the initiation of a subsequent event), and the rate of development. Progenesis, defined as precocious maturation (McNamara 1986). is the result of an early termination or completion of development. Neoteny is caosed by a reduction in the rate of development and post-displacement, a delayed onset of growth (McNamara

1986) is caused &y a late initiation of development. These three processes result in paedomorphosis (McNamara 1986), which means "earlier fom" and refers to the situation where the derived or related adult organism or mature organ system has characteristics of the juvenile ancestor or related taxon. Converseiy, peramorphosis (Alberch et al. 1979) which means "beyond form", results in a derivative or relative that has a form extended "beyond" that of its ancestor or relative and results from the remaining three processes. Hypermorphosis, or delayed maturation, is caused by a delay in temination or completion of development (Le. an extended duration of development). Acceleration is an increase in the rate of development. Pre-displacement is the early onset of a developmental event. Combinations of these six heterochronic processes can occur within the orgaoisrns or organ systems such as the four whorls of fiowers that are being compared. Thus, evaluation of heterochrony requires the cornparison of ontogenies among related taxa for which we have knowledge of the timing of ontogenies. Change in, and comparisons between, ontogenies of related taxa can be portrayed allornetrically when it is first established that size is highly representative of time (McKinney 1988). 5

McKinney (1988), in the context of heterochrony, proposed hypotheses about the developmental alterations that occur at various rates during evolution, and suggested that the influence of development on evolutionary change could be inferred from these alterations. He suggested that gradua1 (neo-Darwinian) evolution would occur as the result of selection acting on a range of phenotypes, therefore, a range of developmental variation. In this case, evolution would be primarily dirocted by environmental (external) selection given the large range of phenotypic, therefore, developmental intermediates provided. Changes in development would be smali in intensity, i.e. degree of effect (depth) and potentially small in extent, i.e. number of parts affected (breadth) as small intensity changes relax the demand for accompanying changes of proximate characters. On the other hand, rapid evolutionary change (saltation) may involve a srnall number of traits due to trait dissociation which yield a functional product.

However, extreme changes (e.g. creation of a perianth tube) may require a series of concomitant alterations in many traits to accommodate the new form.

Subsequently, rapid change may involve change over a large number of traits.

These changes of large depth would most likely be internally directed whereas the accornpanying "fine-tuning" (e.g. length of the perianth tube) rnay be exkrnally directed by selection.

McKinney (1988) cites heterochrony as an important factor in saltations.

Through heterochrony and dissociation of traits, selected cornponents of an organism or an organ cornpiex like a flower rapidly change while leaving other cornponents unalterai. If functional similitude of the altered traits is maintained 6 during evolution, the ephemeral intermediates may remain viable long enough for externally driven evolution (Le. natural selection) to provide descendants with traits that confer adaptive significance.

AIlometry

Allometric analyses are studies of size and scaling of organic fom.

Traditionally, allometry has referred to a variety of concepts related to size- correlated variations in organic form and process (Niklas 1994). Niklas (1994) suggests that one of the rnost encompassing uses of the term is any depaiture from "geometric similituden(conserved shape) among forms varying in size.

While he acknowledges that geometric similitude serves well as a nul1 hypothesis for allometic analyses, allometry primarily refers to the growth of part of an organism in relation to the whole organism or another part of it. Placed in this context, the relationship between heterochrony and allometry becomes clear.

Where allometry describes changes in growth of part of an organism in relation to another part, heterochrony describes changes in growth of part (or ail) of an organism in relation to time. Therefore, studies of allometry become convenient where age data are diffÏcult to obtain. McKinney (1988) emphasizes that, in absence of age data, heterochronic processes cannot be inferred, but when age data are available, allometric methods can be used to compare ontogenies and heterochronic relationships.

Customarily, allometric analyses involve the cornparison of growth trajectories by comparing best-fit lines obtained by regressing growth data of one variable against that of another (Y1vs. Y*). lndependent variables are rarely 7 used in scaling analyses. Niklas (1994) suggests the use of major axis regression in these cases. Major axis regression considers deviations along both axes by measuring residuals as the distance of data points perpendicular to the regression line. In recent studies, investigators have opted to use least-squares regression regardless of the absence of independent variables. Presumably, the effects of the differences in methodology become minimal when R~values are very high and index variables are used in place of independent variables (Porras and Munoz 2000). Index variables are those that remain invariant (or, at least, nearly so) among comparison groups and are used to astirnate variation solely along the Y-axis. As sire is a good indicator of time within the floral buds of

Mimulus, this is the approach taken in this study of ontogeny of flowers.

Additional ontogenetic hypotheses

Heterobathmy (sensu Hufford 1995) refers to mosaic evolution. In the context of comparative developrnent, it can be the consequence of the dissociation of ontogenetic trajectories among characters such that the resulting character states occur in various combinations in related taxa (Hufford 1995).

Through evolution, ontogenetic trajectories can be altered with respect to relative rate and timing of growth (heterochrony) and the spatial patterning of growth

(heterotopy). Consequences of these alterations to ontogenetic sequences have been reported extensively. Hufford (1995) and Friedman and Carmichael (1998) report the occurrence of developmental novelties. These (sensu Hufford 1995) are the appearances of novel character states in a derived taxon relative to that of its ancestral taxon or taxa, or as autapomorphic character states in a taxon but 8

not close relatives. Hufford (1995) suggests that the evolution of novelties can

transfomi ontogenetic sequences in ways that can be summarized as additions.

deletions and substitutions. Additions and deletions simply involve the inclusion

or exclusion of an ontogenetic state or states in an ontogenetic sequence of a

taxon relative to that of its ancestral taxon, for exarnple, trichomes are or are not

formed on anthers. Substitutions involve the replacement of an ancestral

ontogenetic state in a sequence with that of a derived state, or a change in the

order of appearance of states from an ancestral ontogenetic sequence relative to

its derivative.

Tucker (1990) refers to acceleration (not heterochronic acceleration of

GouId 1977) as the relatively early occurrence of a character state in an

ontogenetic sequence and reports this in mimosoid legumes. Diminution results

in a reduction of the developrnent of a character and has been reported in

corollas of species of Besseva (Hufford 1995). Sirnilarly, Tucker (1991, 2000)

refers to supression as a process through which organs initiate but the ultimate

structure remains vestigial. Complete losses of characters (no initiation) are

reported as well (Hufford 1995; Tucker 2000). Equalization refers to the

processes through which effects of variation in timing of organogenesis among

taxa are negated during ontogeny so that adult morphologies are similar

(Ramirez-Domenech and Tucker IWO).

Comparative studies of development have proven to be useful in resolving taxonornic and phylogenetic relationships (Kellogg 1990; Han-Xing and Tucker

1995; Stewart and Canne-Hilliker 1998). Ndonly do developmental traits 9

provide a suite of characters that can be used in phylogenetic reconstructions, but by studying the developmental basis of phenotypic variation, hypotheses of

homology of characters can be more accurately forrnulated. As a result,

hypotheses of parallel or convergent evolution cm be explained as either developmental alterations that are not parallelisms, or as recurrent developmental events that are hornoplasies in closely or more distantly related taxa (Kellogg 1990; Tucker 1997).

Ontogenetic basis of breeding systems

Several studies have elucidated the developmental basis of breeding system characteristics in plant taxa. Tucker (1988) tested a hypothesis of organ suppression to explain the evolution of dioecy in Bauhinia. Others have been done to determine the developmental basis of the evolution of self-pollinating taxa from outcrossing taxa, as well as the shift from taxa containing solely chasmogamous flowers, to those that contain cleistogarnous flowers (Stewart and Canne-Hilliker 1998; Porras and Munoz 2000; Runions and Geber 2000;

Sherry and Lord 2000). Also, studies have investigated the development of traits that comprise heterostyly (Faivre 2000) and various pollination syndromes

(Geurrant 1982).

A pollination syndrome is a suite of floral traits that is consistent among plants that share a pollinator or pollinator type (Lange et al. 2000). These traits may be consistent among non-related taxa, and similar morphologies that confer pollinator specificity may not be the result of comrnon ancestry (Lange et al.

2000; Proctor et al. 1996). Plant taxa that share pollinator syndromes may also be visited by other pollinators in addition to those traditionally associated with the syndrome.

One pollination syndrome is that associated with hurnrningbird pollination.

Traits associated with humrningbird pollination include Rowers with bright, vivid colours (often red), tubular corolla, the absence of (or recurved) a lower corolla lip, abundant nectar, and large spatial separation of nectar and the antherktigma complex (Proctor et al. 1996). Hummingbird-pollination syndromes are reported in Pensternon (Scrophulariaceae), l~omopsis(Polemoniaceae), Delphinium

(Ranunculaceae), and many other taxa (Heisey et al. 1971; Linhart et al. 1987;

Grant and Temeles 1992; Campbell et al. 1993; Proctor et al. 1996; Mitchell et al.

1998; Lange et al. 2000).

Several distinct syndromes are associated with bee-pollination. Traits associated with these syndromes include flowen with tubular or bell-shaped corollas, wide corollas, lower corolla lobes that provide landing platforms for bees. nectar guides, nectar andlor pollen rewards, and hairs on corolla lobes that can be grasped by bees. Bee-pollination syndromes have been reported in

Delphinium (Ranunculaceae), Cam~anula(Campanulaceae), Polemonium

(Polemoniaceae), a variety of legumes and many other taxa (Geurrant 1982;

Proctor et al. 1996; Kobayashi et al. 1999). Both hummingbird and bee- pollination syndromes have been reported for several species within Mimulus

(Heisey et al. 1971; Bradshaw et al. 1995; Coyne 1995; Bradshaw et al. 1998).

Floral morphology and pollination in Mimulus

For decades, two species of monkeyfiower, Mimulus lewisii Pursh and Mimulus II cardinalis Douglas (fomerly Scrophulariaceae [s-1.1, sect. Ewthranthe), have been used as model organisms in studies of functional morphology and floral adaptation (Heisey et al. 1971; Sutherland and Vickery 1988; Vickery 1990;

Vickery 1992; Sutherland and Vickery 1993; Bradshaw et al. 1995; Bradshaw et al. 1998). The floral morphologies of both specieç have been cited as typifjfing forms that attract their specific pollinators (Coyne 1995). Mimulus lewisii is regarded as having floral characteristics typical of bee-pollination, which include a pink corolla with prominent yellow nectar guides, recurved lower corolla lobes that provide a landing platforrn for the bees, and anthers and stigma included within the corolla (Sutherland and Vickery 1993). Mimulus cardinalis is regarded as having typical humrningbird-pollinator characteristics which include a red corolla with a yellow tongue guide, fully reflexed corolla lobes and separated exsert anthers and stigma (Sutherland and Vickery 1993).

60th M. lewisii and & cardinalis contain two described races that generally remain morphologically distinct in allopatric populations (Vickery and

Wullstein 1987). The races of M. lewisii have been recognized based on shape and colour characteristics of the flowers as well as shape and degree of serration of leaf margins. Populations in the U.S.A. Rocky Mountain area have deep magenta-pink flowers with more recurved corolla lobes than populations in the

California Sierra Nevada region, which have pale lavender-pink flowers with lower corolla lobes that project foiward. Hybrids between the races show some reduction in pollen fertility due to irregularities of chromosome pairing at meiosis

(Heisey et al. 1971). Although allozyme data suggest that both these races merit status as species (Vickety et al. 1989), the Rocky Mountain (magenta) race has yet to be officially recognized as a taxon. Further taxonomic study is in progress at the University of Washington, Seattle (P. Beardsley, pers. comrn.)

The races of cardinalis have been characterized by differences in leaf shape, blade thickness and leaf distribution. The southern Arizona populations have narrower leaves and a slightly different habit than the west coast

(California) populations. Hybrids resulting from artificial crosses between mernbers of these races have reduced pollen fertility and show some chromosome pairing irregularities during meiosis (Heisey et al. 1971). However. species status has not been proposed for these races.

Mimulus lewisii, the more widespread species, occurs along streams in the forests of the Hudsonian-sub-alpine zones in the northern half of western

North Arnerica (Vickery 1969). Mimulus cardinalis grows by streams and in moist habitats in the wamer forests of the Transition-Canadian zones of Oregon,

California, Arizona and Baja California (Vickery 1969). The only known areas of syrnpatry for the two species occur in northern California where the two species flower simultaneously (Beardsley and Olmstead 1999).

Through a series of crossing experiments it was determined that few genetic barriers to gene exchange occur between M. lewisii and cardinalis

(Vickery 1969; Heisey et al. 1971). FI hybrids from artificial crosses were observed to be vigorous and have close to normal fertility, although fertility was higher in FI hybrids from the Sierra Nevada race of M. lewisii and & cardinalis than within the FZ from the latter taxon and the Rocky Mountain race of M. lewisii 13

(Vickery and Wullstein 1987). However, exhaustive searches have failed to detect any naturally occurring hybrids (Bradshaw et al. 1995).

It was shown in field experiments that pollinator preference alone does not

completely prevent interspecific visitation by bees and hummingbirds (Heisey et

al. 1971; Sutherland and Vickery 1993). The latter study did show that

hummingbirds more frequently visited red flowers with reflexed corolla lobes and that bumblebees more frequently visited the pink flowers with non-reflexed lobes.

Thus, it was previously hypothesized that aspects of floral morphology prevented

interspecific pollination events from occurring as a resuit of occasional visits from

other than the commonly observed pollinator (Vickery 1990). However, effective

pollinator fidelity seems to be the sole mechanisrn for reproductive isolation where lewisii and &l-.cardinalis occur in sympatry because hybrids do not

occur. For this reason some of the factors that promote pollinator fidelity and their roles in the maintenance of reproductive isolation between M. lewisii, &

cardinalis and related species have been studied extensively over the past few

decades (Heisey et al. 1971; Bradshaw et al. 1995; Vickery 1995; Bradshaw et

al. 1998; Schemske and Bradshaw 1999).

Given that assurance of reproductive isolation might lie in functional floral

morphology, Vickery (1990) conducted pollination experirnents and determined that reliance upon bird vs. bee pollinators is effective in reproductively isolating

sympatric populations of the two species. A later study (Sutherland and Vickery

1993) used a series of F2 hybrid recombinants whose floral morphologies varied

in intermediacy between those of M. lewisii and & cardinalis to observe pollinator preferences for the various recombinants. The experimenters found a high correlation between certain floral traits and rate of pollinator visitation. Traits judged to be responsible for pollinator discrimination were corolla colour. corolla shape (degree of lobe reflexation), nectar concentration, nectar volume and nectar sugar content. This conclusion was based on the observation that bees and hummingbirds did not visit the recombinants in a frequency proportional to that of their presence in the experimental population. The results of that study indicated that hummingbirds show a strong preference for flowers with fully reflexed corolla lobes. The authors interpreted a slight preference by hummingbirds for red flowers, but they point out that the birds rnay simply eschew the non-red flowers. Ultimately, they interpreted floral shape as being primarily responsible for hurnmingbird preference. In addition it was found that there was a positive correlation between increasing degree of lobe reflexation and increased floral reward (nectar sugar content). Bumblebees, on the other hand, were observed to discriminate more on floral colour, eschewing red flowers while preferring yellow and shades of pink. There seemed to be no indication of bee-preference for floral shape. It is worthwhile to note that correlation between bee approach rates and nectar characteristics was difficult to ascertain, probably due to the increased complexity of foraging strategy by bees involving both nectar and pollen as a reward. These studies did not evaiuate whether pollinator visitation resulted in successful pollination of the various fioral types presented to the poilinators.

More recently, Bradshaw et al. (1995) investigated the genetic basis of the 15 floral characteristics in M. lewisii and cardinalis that are responsible for reproductive isolation. The authors identified quantitative trait loci (QTLs) for eight floral traits in two syrnpatric populations of M. lewisii and M. cardinalis and used molecular markers to generate genome linkage maps containing these

QTLs. They determined that, for each trait, there existed at least one QTL

(containing one gene or a tight cluster of genes) that explained at least 25% of the phenotypic variance of the F2 plants used in the analysis. The eight traits were divided into three classes based on their likelihood of involvement in pollinator attraction (flower colour, shape and size), pollinator reward (nectar volume and concentration), and pollen transfer (pistil and stamen length). The investigators hypothesized that because there has been sufficient rnorphological divergence between the species to maintain reproductive isolation, but insufficient divergence in their genetic systems to render them incompatible, then

QTLs governing floral morphology might contain the genes responsible for reproductive isolation and hence speciation. A subsequent study (Bradshaw et al. 1998) increased the number of traits examined from eight to 12 and increased the size of mapping from 93 to 465 F2 progeny. This was done to reduce bias in estimating QTL number and magnitude of effect. The results implied that most

(9/12) of the traits judged to be involved in reproductive isolation are controlled by a small number of genes of large effect and substantiated the results of the earlier study.

Coyne (1995), in a review of the study by Bradshaw et al. (1995) pointed out that because reproductive isolation between these species is due to 16 adaptation to poliinators, a genetic analysis of floral characters is a study of speciation and adaptation. Although not specifically stated, Coyne's comment implies that M. lewisii and M. cardinalis share a common ancestor from which they are derived, or that one of the species is derived from the other. In other words, Coyne made two assumptions: 1. that reproductive isolation confers species status to the organisms in question, and 2. that the species are phylogenetically closely related because they are interfertile. If these assumptions are correct, then given the evolutionary differences expressed through the floral morphologies of these taxa, it would seem that comparative studies of floral characters of M. lewisii and M. cardinalis become studies of speciation and adaptation to pollinators. Because floral ontogeny generates the functional morphologies that are transformed in association wîth speciation, studies of comparative floral development will shed light on aspects of speciation dependent on floral diversification by docurnenting divergence of developmental pathways between taxa.

Taxa have rarely been studied well enough so that floral ontogeny can be investigated in the context of phylogeny (evolutionary history) and flower function

(Hufford 1995; Reeves and Olmstead 1998). Currently, a hypothesis of genealogical relationship exists for these species of Mimulus based, in part, on previous work on the classification of this section of the genus (Vickery and

Wullstein 1987; Vickery et al. 1989) and, in part, on an assumption based on the hypothesis that taxa with hummingbird-pollinated flowers are derived from taxa with bee-pollinated flowers within Mimulus. The phylogeny of this section of 17

Mimulus is being re-evaluated by Alan Yen, Paul Beardsley and Richard

Olmstead at the University of Washington, Seattle using molecular data

(Beardsley and Olmstead 1999). The study of ontogeny in the context of a

hypothesis of phylogenetic relationship is advocated. However, to compare

ontogenetic sequences as a means to study evolutionary changes, a hypothesis of direction of transformations from ancestral to derived fioral characters must be fomulated. Evaluation of the ontogenetic sequences as sources of floral

diversification can then be made in an evolutionary context (Hufford 1989). In

the case of M. lewisii and fi cardinalis, more specifically, differences in

ontogenetic character states can be related to speciation.

Significance of research

Ultimately, evolutionary changes in Roral morphology can only be

explained by differences in ontogeny regardless of selection, mutation or other

evolutionary factors. Through analyses of continuous floral ontogenetic states

from the sympatric populations of cardinalis and M. lewisii, it should be

possible to identify distinctive andior novel characters that are produced in each

taxon, and the heterochronic and/or allometric processes involved. Given an

independently produced hypothesis of phylogenetic relationship based on the

molecular work of Yen, Beardsley and Olmstead (P. Beardsley and R. Olmstead,

University of Washington, pers. comm.), and careful analyses of ontogenies, it

will be possible to generate hypotheses of the ontogenetic changes that likely

occurred and resulted in the derived rnorphology. Given the genetic,

phylogenetic and natural selection data for this group and related species 18

(Heisey et al. 1971; Vickery and Wullstein 1987; Vickery et al. 1989; Bradshaw et al. 1995; Bradshaw et al. 1998;Schemske and Bradshaw 1999), a comparative analysis of floral ontogeny should provide a vital link that ties these studies together and allows for a rare opportunity to form a comprehensive hypothesis of breeding systern evolution correlated to divergence of species involving alteration of floral morphology apparently suited primarily to different pollinators.

Objectives

The primary objective of this study is to evaluate the differences in ontogeny between the two sympatric populations of M cardinalis and M. lewisii, and interpret these differences as evolutionary transformations. Through this, it is possible to formulate hypotheses of how evolutionary transformation of Rowers of a bee-pollinated species to flowers of a hummingbird-pollinated species (or vice versa) may have occurred. A concomitant secondary objective is to document the differences in ontongeny among three widely geographically distributed populations of & cardinalis, and to document the differences in ontogeny among four geographically distributed populations (representing both recognized races) of M. lewisii. Determination of the patterns of ontogenetic variation among populations, races (potentially species with a conserved breeding system suited to bee-pollination), and closely related species (that in evolutionary time have rapidly shifted breeding systems), should make it possible to test Tuckef s hypothesis that differences among closely related taxa (i.e. features that are diagnostic at low taxonomic levels) are rnanifested late in ontogeny. 19

Material and methods

The study organisms

Mimulus cardinalis is a perennial herb that occurs along stream banks and in moist habitats in western North America frorn northern California to Baja

California, Mexico, east to Arizona at altitudes ranging from near sea level to

2500 m. Both the synsepalous calyx and sympetalous corofla are five-lobed.

Corolla colour ranges from scarlet red to red-orange and corollas are about 40 mm long and tubular. A corolla consists of a basal tube that extends to the point of insertion of two pairs of epipetalous stamens, and a throat that extends from the region of stamen insertion to the sinuses of the reflexed lobes. An oblong aperture where the throat meets the lobes is laterally narrowed. and the adaxial side of the corolla is longer than the abaxial side. The anthers and stigma are exserted from the adaxial region of the aperture (fig. 1). A trichome-lined groove occurs aIong the length of the abaxial surface of the throat.

Mimulus fewisii is a perennial herb found along strearn banks in western

North America at altitudes generally greater than 2500m. The species is distributed from southern California to Alaska and extends east to Colorado.

Both the synsepalous calyx and sympetalous corolla are five-lobed. Corolla colour ranges from pale lavender-pink (Sierra Nevada race) to deep magenta- pink (Rocky Mountain race), and corolla lengths are between 30-35 mm. Corolla form differs from that of M. cardinalis in that the oblong aperture where the throat meets the lobes is dorso-ventrally narrowed, and the abaxial side of the corolla is longer that the adaxial side. The anthers and stigma are inserted in the adaxial 20 region of the throat. Two trichome-lined ridges occur along the length of the abaxial surface of the throat, which are hypothesized to act as a nectar guide for bees (Heisey et al. 1971).

Plants were grown in growth rooms at the University of Guelph,

Department of Botany from seeds collected from seven populations: & cardinalis: Yosemite National Park, CA, U.S.A., D. Schemske, s.n., October

1998; Cedros Island, Baja California del Norte, Mexico, R. Vickery, 13315-1,

August 1994; Siskiyou Mountains, OR, U.S.A., R. Vickery. 13769-26b, August

1996; M. lewisii: Yosemite National Park, CA, U.S.A., D. Schemske, sen.

October 1998; Boise Co., ID, U.S.A., Alplains Sm., June 1998; Cascade

Mountains, Hillsboro, OR, U.S.A., J. and A. Lunn, 4839, February, 1998; and

Johnsville, Plumas Co., CA, U.S.A., P. Beardsley, 99-059, i999. For each population, 18-20 plants were grown in 8 - inch pots in a growth medium of 3: 1: 1 pro-mix: turface: composted manure. Plants were grown under uniform conditions of 18h days at 20-22 OC, fertilized as required with a commercial fiowering plant fertilizer, and watered with deionized water as required. Flowers and buds were collected frorn new growth of healthy plants. Vouchers were deposited in the University of Guelph Herbarium.

Microscopy

Flowers, floral buds and apices of varying developmental stages were either measured and dissected in 95% ethanol after having been fixed in FAA and dehydrated in a graded alcohol series, or, dissected live and then preserved and dehydrated. Dehydrated samples were critical-point dried with carbon 21 dioxide using a Ladd critical-point dryer. Dried samples were mounted on aluminum stubs, coated with gold-palladium using an Anatech Hummer VI1 sputter coater, and examined using a Hitachi S-570 scanning electron microscope at 10 kV. If necessary, samples were redissected and recoated for further microscopy. Samples were either photographed using 120 mm film or images were captured using Quartz PCI version 5.1 (Quartz lmaging

Corporation, Vancouver, BC). Larger samples were examined under a Zeiss dissecting microscope and micrographs were obtained using a Nikon E 800 digital canera.

Meiosis

Measured buds were fixed in 3:1 acetic acid: ethanol for 24h. Adaxial and abaxial anthers were excised, left in Snow's stain for 36-48h, mounted separately in Hoyer's solution and exarnined for stages of meiosis with a Zeiss cornpound light microscope using interference contrast optics.

Allometric analyses

One to four buds from each of 15-1 8 plants from each population were tagged and measured using needle-ncse digital calipers. Buds from about 2mm in length to anthesis were measured daily. A regression analysis was performed for each population and slopes were compared using ANCOVA (SPSS version

7.0,SPSS Inc., 1995). For analyses of scaling relationships, iive buds were dissected and organs were measured with needle-nose digital calipers or a calibrated ocular micrometer on a Zeiss dissecting microscope. Floral traits measured were calyx length, corolla length along the adaxial and abaxial surfaces (from base to sinus of lobes), corolla tube length along the adaxial and

abaxial surfaces (regions of corolla tube subtending the stamens), lengths of

adaxial and abaxial stamens (from the base of the filaments to the apex of the

connective), ovary length, and style length (including stigma) (chapter 1, fig. 2;

chapter 2, fig. 2; chapter 3, fig. 2). Slopes of regression lines of allometric

relationships were compared using ANCOVA. Allometric analyses customarily

include comparisons of elevations (y-intercepts) of regression lines. These

comparisons were excluded from this study because initiation data for the floral

organs were included. Although no independent variables were used, least

squares regression requires one of the variables used to be considered

independent. In place of an independent variable, some variables (e-g. ovary

length in both species, adaxial corolla length in j&. cardinalis, adaxial stamen

length in M. lewisii) were chosen as index variables against which the growth of other organs was compared. Criteria for choosing the index variable were

invariance of Iength at anthesis between taxa being compared, and highly correlated growth of the index variable with that of its calyc The latter was estimated based on a visual analysis of the scatter plot of the potential index variable against calyx length. As calyx length was shown to be an excellent predictor of age, by using an index variable that was well correlated with calyx length, inferences of organ growth through time could be made.

Sample sizes

Each of the three populations of j& cardinalis (Cedros Island, CI; Siskiyou

Mountains, SM; Yosemite National Park, YO) and one population from each of 23 the two described races of M. lewisii (Yosemite National Park, Y0and Boise Co,

BO) were fully represented in the study. One additional population of each race of M. lewisii were examined (Johnsville, JV and Oregon, OR) to veriv that the primary study populations were suitable to represent the races of that species.

For the Yosemite populations of the species, 36 M. lewisii and 53 M. cardinalis individuals were used. Each of the remaining populations was represented by 18 individuals.

for each population of cardinalis, ca. 180 buds were exarnined with the

SEM (which includes ca. 15 ovaries examined around the stage of ovule initiation), ca. 50 buds were measured for allometric analysis, ca. 100 buds were examined and/or rneasured for corolla aperture data, ca. 60 buds were examined for stamen architecture data, ca. 10 buds were exarnined during stages of rneiosis, ca. 15 fiowers were exarnined for corolla lobe data, and ca. 50 buds and flowers were examined for corolla, pistil and nectary data.

For each of the prirnary study populations of M. lewisii, Ca. 90 buds were examined with the SEM (which includes ca. 15 ovaries examined around the stage of ovule initiation), Ca. 50 buds were rneasured for allornetric analysis, Ca.

60 buds were exarnined andfor measured for corolla aperture data, ca. 60 buds were examined for starnen architecture data, Ca. 15 buds were examined at various stages of meiosis, m. 15 buds were exarnined for corolla lobe data, ca.

15 buds were examined for nectar initiation data, and Ca. 25 buds were examined for pistil and nectar-development data. For the remaining primary study populations (CI, SM and BO), data were derived from similar numbers of 24 buds as from the populations from Yosemite National Park (ca. 350 buds in total).

Additional populations (JV and OR) of M. lewisii were examined to verify the stability of floral characteristics observed in the primary çtudy populations.

For each of these populations, Ca. 25 buds were examined with the SEM, ca. 50 buds were observed and measured for allornetic analyses, Ca. 15 buds were examined for ovule initiation data, Ca. IObuds were examined during meiosis, ca. 15 buds were examined for stamen architecture data, and Ca. 15 buds were examined for corolla aperture data. 25

Ckapter one - Floral ontogeny of Mimulus cardinalis

Introduction

Two species of rnonkeyflower, Mimulus lewisii and cardinalis (formerly

Scrophulariaceae M,sect. Ervthranthe) have frequently been used as mode1 organisms in studies of functional morphology and floral adaptation (Heisey et al.

1971; Sutherland and Vickery 1988; Vickery 1990; Vickery 1992; Sutherland and

Vickery 1993; Bradshaw et al. 1995; Bradshaw et al. 1998; Schernske and

Bradshaw 1999). The floral morphologies of both species are regarded as those that attract their specific pollinators (Coyne 1995). This pollinator specificity effectively reproductively isolates these two species where they occur in sym patry.

Bradshaw et al. (1995) investigated the genetic basis of this reproductive isolating mechanisrn using analyses of quantitative trait loci (QTL). These authors found that a large amount of the morphological variation expressed in F2 hybrid recombinants was governed by a small number of QTLs containing one gene or a tight cluster of genes. Therefore, the drarnatic morphological divergence between these species is associated with a small amount of genetic divergence.

Given the evolutionary changes expressed throug h the floral morphologies of these taxa, and because floral ontogeny generates the functional morphologies that are transformed during evolution, a study of comparative floral development between M. lewisii and M. cardinalis will shed light on how rapid 26 breeding system evolution has occurred through diversification of floral developmental pathways.

Given the genetic, phylogenetic and natural selection data for this group of species, a comparative analysis of floral ontogeny between M- lewisii and fi cardinalis should provide a vital link that ties these studies together and allows for a rare opportunity to study the evolution of a bee-pollinated taxon from a hummingbird-poilinated ancestor.

Herein, as a subset of the analysis of floral ontogeny in M. lewisii and & cardinalis, the floral development of the hurnmingbird-pollinated M. cardinaiis is documented. Before developmental pathways can be compared among the species and the evolutionary differences in developmental pathways can be interpreted, the floral developmental trajectories of each taxon resulting from the interna1 developmental programs have to be determined.

The patterns of floral developmental variation among three geographically widespread populations of M. cardinalis are reported: Yosemite National Park,

YO; Cedros Island, CI; and Siskiyou Mountain, SM. Although fi cardinalis contains two races, both of which are represented here (fig. l),documentation of the floral rnorphological variation between the i-aces is scant. Furthermore, intermediate forms between members of the races are reported to exist (Hiesey et al. 1971). Because of this, interpopulational rather than interracial variation was addressed here. Documentation of the range and degree of variation in developmental characters within a species is desirable. For example, alterations in developmental trajectories may reflect evolutionary changes that are not observable in adult morphologies among populations or, alternatively, may explain the ontogenetic origins of morphological variation seen in adult morphologies among populations. Therefore, the inclusion of multiple populations that show variable developrnental character states rnay be useful in resolving phylogenetic relationships. Also, by examining the range and degree of developmental variation within a species, we may gain an understanding of how development acts as an evolutionary process that generates diversity of form

(McKinney 1988). 28

Results

Initiation of fioral apex and floral prirnordia

Buds and flowers of al1 populations are used in figures to represent the floral developrnent of the species as a whole. Developmental stages were observed to be simiiar among the populations except where noted.

At each node, two leaves each subtend a flower. Leaf, and thus floral arrangement, is decussate (fig. 3A. 38). The developrnent of the apices at a node may be synchronous or offset to varying degrees (fig. 3A, 3B). The hemispherical floral apices emerge in the axil of the subtending ieaf primordium.

Floral apices enlarge radially and vertically prior to initiation of floral organs (fig.

4A-4C). The adaxial sepal primordium appears first (fig. 40), followed by a rim meristem that surrounds the remainder of the floral apex (fig. 4E4G). Pedicel formation occurs as a result of vertical growth below the floral apex and is distinguished from the floral apex by the relatively greater radial growth of the floral apex (fig. 4G, 41). The abaxial and lateral sepal primordia emerge outward from the rim meristem apparently simultaneously (fig. 4H, 41). As the sepal primordia appear and enlarge radially, the interior of tne fioral apex takes on a pentagonal shape and becornes distinct from the calyx due to increase in height

(figs. 4H, 41, 5A). Formation of a cleft occurs between the sepals and inner apex as sepals enlarge radially and vertically (figs. 41, 5A-5F). As the sepals grow vertically, the adaxial sepal projects radially more than the lateral and abaxial sepals (fig. SB, 5D-5F). 29

After sepal initiation, five petal primordia simultaneously initiate radially on the flanks of the inner apex, and alternate to the sepal primordia (figs. 41, 5A-5F).

At initiation, the two adaxial petals are spaced more closely together than the remaining petals (fig. 5A, 5C, 5E).

Four stamen primordia arise simultaneously opposite the sepals with no primordium appearing in the adaxial median position (figs. 41, 5B-5F). In some buds, a vestigial stamen initiates in the median adaxiai position and does not enlarge further (fig. 6A).

The adaxial stamen prirnordia are hemispherical while the abaxial stamen primordia are distinctly ovoid (figs. 5A-5F, 6A, 66). Starnen primordia are taller and more massive than petals (figs. 5A-5F, 6A-6E). As the stamen primordia become more globular and taller on the floral apex, the petals enlarge radially and the calyx tube becomes evident (fig. 6B-6E). The gynoecium initiates as an oval ring primordium (fig. 68,6D). Vertical and radial growth of this prirnordium at its periphery yields an oval, then pentagonal, ovary (fig. 6D, 6F. 6H,61). A transverse septum initiates later and separates the interior of the ovary into two incipient locules (figs. 61, 7A-7H).

Calyx organogenesis

Vertical growth in calyx and petals occurs prior to trichorne appearance on the calyx and developing pedicel (figs. 41, 5A-5F). The adaxial sepal becomes distinctive in that as the other four sepals start to curve inwardly, it becornes more erect and is slightly taller (figs. 5F. 6C, 6D, 6G). Differential sepal growth and calyx tube formation result in greater calyx elongation on the adaxial side 30

(fig. 6C, 6G-61). Trichomes initiate at the base of the calyx and apex of the pedicel (fig. 66-61). As calyx lobes fold inward, trichornes increase in density and length (figs. 61, 7A-7D, 7H). Prior to calyx closure, beginning of folding of the calyx lobes is evident (figs. 7C,7H, 8A-8D). Within a calyx, trichome development is offset (fig. 8A-8D). Capitate trichornes of various lengths are present at anthesis. By the time the calyces reach ca. 5rnm in lecgth, al1 lobes cuwe toward the abaxial side (fig. 8B). The adaxial lobe and tube are longer than the abaxial side of the calyx (fig. 8A-8D). At anthesis, the calyx tube is longitudinally ribbed by folded wing-like extensions of the folded calyx lobes.

Each calyx lobe has a slender acute apex (fig. 1A-16). This shape remains constant in the three populations through development until the corolla exceeds the calyx in length.

Coroila organogenesis

After initiation, vertical and radial growth of the rounded petals continues

(figs. 6A, 6B, 6D-61, 7A-7E, 7G).At initiation of the corolla throat, petals, stamens and the gynoecium are roughly the same height (fig. 7H). As the corolla throat grows vertically and radially the lobes enlarge and curve inwardly and obscure the inner organs (fig. 9A, 9C-9G). The corolla lobes fold over the stamens and gynoecium starting with the abaxial lobe, followed by the lateral lobes and, lastly, the adaxial lobes (figs. 9H, 10A-1 OF, 1 1A-? 1 C). The corolla lobes continue expanding radially (fig. llA-11C) until the adaxial lobes (the last to fold over) obscure the abaxial and lateral lobes (figs. 15A, 16A, 16B, 17A, 176,

18A). 31

In the young bud, the sinus of the two adaxial petals is narrower than the sinuses adjacent to the lateral and abaxial petals (fig. 9D, 9G). An adaxial peak forms basal to this narrower sinus (fig. 1OG). After the corolla lobes finish folding over, and stamens and ovary are well fomed, trichomes initiate on the interior corolla surface at the junctions of the lateral and abaxial lobes and continue initiating basally along the corolla throat and are yellow in colour at anthesis (figs.

101, 12E). These trichomes increase in length and density until anthesis (figs.

14C, 15C, 16C, 16D, 17D-17G, 18B-E, 19C-19F).

After corolla closure, elongation of the corolla throat changes the corolla from globular to oblong (figs. 1IA-11 C, 12F, 12G. 14A). As the corolla throat elongates and enlarges in circumference, zona1 growth below insertion of the filaments foms the corolla tube, which is narrower than the throat (figs. 12G,

12H, 15A). The tube elongates and vertical grooves forrn along its length (fig.

16A). The troughs of these grooves or indentations represent areas subtending the points of filament attachment on the interior corolla wall (figs. 168, 17A. 18A).

Subsequently, the distal boundary of the tube is at the level of filament insertion.

As the corolla tube becomes distinct, radial growth of the corolla lobes results in radial enlargement at the apex of the corolla (figs. 16A, 17A, 176, 18A). Prior to anthesis, cilia form along the margins of the corolla lobes (figs. 16A, 17A, 178,

18A).

At anthesis, yellow trichomes are evident along the interior lateral walls of the corolla throat (figs. 19E, 19F). As the lobes begin to unfold, they remain curved inward. As the bases of the lobes reflex, the style is kept under tension 32

by the curved tips of the lobes until they reflex back. The lobes eventually fully

reflex (fig. 1). Additional shape changes in the corolla occur during enlargement

and are discussed below.

Stamen organogenesis

Prior to calyx closure, differential growth causes the starnen prirnordia to

become transversely flattened and the pairs of primordia appear more similar in

size and shape (fig. 7A-7G). Microsporangia initiate as lobes on the developing

stamens, and connectives and filaments become distinct (figs. 7F, 7H, 9A-9E,

9H, 1OA, 1OB). After the filaments become distinct (fig. 9C,9E), growth

subjacent to corolla and stamens moves the point of filament insertion ont0 the

corolla away from the floral apex (fig. 10A-1OF). Although not evident from the exterior of the corolla, this stage represents initiation of the corolla tube.

Differential development at the bases of the adaxial pair of filaments causes the recently differentiated anthers to tilt towards the median plane (fig.

AOC, IOG, IOH). About the time the adaxial corolla lobes have folded and trichomes appear on the interior of the corolla throat, trichomes appear on the surfaces of the microsporangia on both pairs of anthers (fig. 1OG, IOH). The anther trichomes elongate and become long, straight and acute (fig. 12A-12H).

Growth of filaments at angles that differ between the adaxial and abaxial pairs of stamens causes the pairs of anthers to sit in the tube at different elevations (fig.

12D, 12F, 12G). Prophase I of meiosis initiates in the abaxial microsporangia

14-15 days before anthesis (bud length = ca. 6mm) and is followed by prophase in the adaxial microsporangia. Termination of meiosis (presence of tetrads/young pollen grains) occurs when bud length is ca. 10mm (fig. 12G).

Differential growth rates between the adaxial and abaxial pairs of filaments result in abaxial stamens of greater length than adaxial stamens (fig. 12H).

At the time of their initiation on the anthers, microsporangia are adjacent along their lengths (figs. SC,9D, 106, 10G. 10H). During developrnent, the thecae diverge (fig. 17F, 17G), and eventually become arranged in tandem (fig.

25H).

Pistil organogenesis

As the primordium of the gynoecium grows, its shape is constrained by the androecial whorl, and the developing ovary changes shape from oval to pentagonal (figs. 6D-6F, 6H, 61, 7A, 78).As microsporangia and connectives fom on the stamens, a transverse cleft bisects the rim of the gynoecium aperture and separates incipient stigrna lobes (figs. 7E-7H, 9A, 9B, 9D, 9H). As the gynoecium overtakes the stamens in height, stigma lobes initiate as a flared lip around the apex of the gynoecium (figs. 9H, IOA, 10D-IOH). These lobes enlarged to fom hemispherical lobes (fig. 12A-12D). A compitum appears as a narrow channel that bisects both enlarging placentas along the median plane (fig.

10G). As trichomes on the anthers increase in density and length, a style initiates as a region of attenuation between the flared stigma lobes and the ovary

(fig. 12A-12C). The developing placentas and stigmatic surfaces are still smooth

(fig. 13A). As the hollow style elongates, it curves abaxially and the stigma lobes become completely appressed to each other (figs. 12A-12D, 12F-12H, 13A, 158,

168, 20A). After style initiation (bud length = Ca. 7rnm), round ovule primordia appear on the middle apex of the two placentas and primordia develop basally and radially (fig. 13B). Papillae start to appear around the inner apex of the developing stigma lobes (fig. 13C). Ovules initiate over the entire placentas and an integument envelops the nucellus of each ovule (fig. 13D, 13F). Stigmatic papillae continue to elongate (fig. 13E).

After the stigrna lobes have closed, the style elongates and the distal portion bends abaxially foming a hook (figs. 15B, 16B, 20B). In this way the style can achieve its maximum length prior to anthesis (fig. 17C). Around the stage when the stigrna lobes differentiate, the abaxial region of the base of the ovary begins to enlarge (fig. 1OH). Cell differentiation results in this region appearing dark green and swollen (figs. 17C, 20C). As the rate of corolla growth accelerates relative to that of the calyx (corolla length = ca. 15mm), the abaxial basal region of the ovary becomes nectariferous and starts to secrete nectar (fig.

20D). Nectar accumulates primarily in the abaxial region of the corolla tube below the filaments. Just prior to anthesis, the pistil is more or less at its maximum length. As the corolla opens, tension on the style is released and the style straightens to protrude from the corolla throat (fig. IA, 1B). At anthesis, the nectary is bulb-like and occupies the abaxial and part of the adaxial region of the base of the ovary (fig. 20E. 20F). Stomata are present on these surfaces (not shown). 35

Variation among Yosemite, Cedros Island and Siskiyou Mountain populations in early stages of ontogeny

Interpretation of the changes that occur during early ontogeny is facilitated by considering the development of the outmost whorl (incipient calyx) as a process decoupled from processes governing the development of the inner floral organs (see Discussion). At initiation of sepal primordia, the sizes of the floral apices at both the median and transverse planes differ among the populations.

Apices of plants from CI are significantly larger along both planes (Pc0.05) than those of Y0 and SM, which are similar in their smaller sizes. The transverse and median dimensions (mean + SE) are: CI = 228.0 f 4.1 pm X 225.6 + 6.7 pm, Y0

= 184.2 + 6.6 pm X 164.2 k6.2 Pm, and SM = 177.0 k 3.3 pm X 169.9 t 4.6 pm

(n = 8 for each population). The general circular shape of the fioral apices as seen in polar view is approximately the same arnong populations (fig. 4D-4F).

Apices on plants from SM are more straight sided in lateral view than are those of the other populations, which appear more globular (fig. 4B, 4C).

At the stage when petal and stamen primordia show enlargement, the sepal primordia have not yet begun vertical growth and remain radially projected in floral apices of plants from CI (fig. 5A, SB). At this stage, sepal primordia on plants from Y0 and SM have begun vertical growth (fig. 5C-5F). Differential vertical growth between the sepals and inner floral apex of plants from CI is less pronounced and the resulting cleft is less distinct than in plants from Y0 and SM

(fig. 5A-5F). Cornparisons of the apices at similar stages of calyx development show advanced development of the inner three whorls in apices of plants from CI 36 relative to those in plants from Y0and SM. For instance, at initiation of trichornes on the calyx, the inner organs on the apices of plants from CI (fig. 6F,

6D) are more differentiated than are those of plants from Y0and SM (fig. 6B-6E,

6H, 61). The corolla lobes of plants from CI have begun vertical growth and project farther beyond the calyx lobe sinuses than in plants from Y0 and SM populations. The stamen primordia are visually more massive and differentiated on apices of plants from Cl. The ring primordium of the gynoeciurn has become pentagonal and is starting to develop a transverse septum on apices of CI (fig.

6F). On apices of plants from Y0and SM, the ring primordium is much less discernable (fig. 6B, 6D, 6E). However, at the stage just prior to trichome initiation on the anthers, calyx trichomes on buds from SM and Y0are much elongated compared to those of CI (fig. 10D-1OF). At the time that the stigma lobes close (become appressed to each other), a much greater size difference can be seen between the adaxial and abaxial stigma lobes in plants from SM versus those on plants from Y0 and CI (fig. 12B-12D).

Rows of trichomes appear on the interior lateral walls of the corolla throat near the base of the lobes (fig. 19E,19F). These rows appear roughly midway between the adaxial and abaxial surfaces in flowers on plants from Y0 and CI

(fig. 19E) but more toward the abaxial surface of the corolla on plants from SM

(fig. 19F).

Development of corolla throat and aperture

Variation in aperture dimensions among the three populations of M. cardinalis is slight. The slopes and elevations of regression lines on allometric plots of absolute corolla height (abaxia! to adaxial surface) versus corolla length, and height: width versus corolla length at both the aperture and halfway down the corolla throat indicate that development of transverse shape and size does not differ significantly among the populations. Their development will be deçcribed collectively for the species. Populations can be assumed to be similar unless otherwise specified.

Corolla shape in transverse section at the base of the throat is approximately circular at the time the corolla starts to elongate (fig. 14A, 148).

The shape at this position does not appear to change markedly during development to anthesis (fig. 19A, 19B).

After corolla closure, transverse shape midway along the length of the throat remains circular until a groove initiates along the abaxial corolla surface

(fig. 15C). At roughly the same stage, an adaxial ridge foms along the corolla length causing an adaxial peak in transverse view (figs. 14C. 15C). Even as the groove and ridge become more pronounced, the general circular transverse form is maintained (fig. 16C). As the rate of elongation of the corolla increases relative to that of the calyx (corolla length = ca.l5mm), the relative and absolute width of the corolla increases (fig. 17D, 17E). Later, as the corolla emerges from the calyx, differential growth between the adaxial and abaxial sides of the corolla has caused the mid transverse view to appear spade-shaped and the corolla throat appears laterally compressed (fig. 186, 18C). Corollas of plants from SM have an acute peak and a distinctive external abaxial groove due to more pronounced interna1 ridges of the coroiia ihroat on either side (fig. 18C). 38

Corollas of plants from Y0 and CI have a more obtuse peak and a less distinct groove (fig. 18B). At anthesis, minor differences among the populations still exist, but the narrowly oblong-ovate transverse shape is consistent (fig. 19C.

19D).

Shape of the corolla aperture at the junction of the throat and the lobes reflects the observed shape midway along the throat (figs. 14D, 15D, 160. 17F,

17G, 18D, 18E, 19E, 19F). However, as the corolla length rapidly increases relative to calyx length (corolla length = ca.15mm). the apex of the throat does not show a relative increase in width as it does midway along the throat (fig. 17D-

17G). Change in corolla shape remains conformed to the enlarging anthers (fig.

17G). As the corolla emerges from the calyx shortly before anthesis, corollas of plants from SM are wider, causing the adaxial peak to appear acute to acuminate

(fig. 18E). Corollas of plants from Y0and CI are narrower causing the adaxial peak to appear more obtuse (fig. 18D). At anthesis, the medial elongation is achieved without a corresponding increase in width, causing the corollas to appear laterally flattened (fig. 19E, 19F). In lateral view, the corolla aperture is oblique with the adaxial tip extending 10-15 mm beyond the abaxial tip (table 3, fig. lA, 1B). Corollas of plants from SM are distinguished at anthesis by less inward curving of their lateral walls compared to thoçe on plants from Y0 and CI

(fig. 19E, 19F).

Corolla lobes at anthesis

Lobes within a corolla and among populations show variation in size and shape (fig. 21). Within a flower, and within and among populations, shape 39 variation seems to be slight but consistent. The lobes have a general obcordate shape with a straight base where they join the throat The lateral margins of the lobes range from nearly parallel in CI to convex in Y0 and SM. Parallel margins of the adaxial and lateral lobes of CI are perpendicular to the truncate base but the margins of the adaxial lobes are oblique to it. The sub-lobes of the lateral and abaxial lobes are equivalent is size and shape, but sublobes of the adaxial lobes are offset and unequal (fig. 21). Among the populations, abaxial and lateral lobes are generally bilaterally symmetrical. Lobes on plants of Y0 are widest, followed by those on plants from SM and lastly those of CI (fig. 21).

Because the lengths of the abaxial lobes Vary little among the populations, the increased widths result in a shape change that is reflected in their height: width ratios (fig. 21). Variation in the lateral lobes among the populations parallels that of the abaxial lobes. The lateral corolla lobes on plants from Y0 are wider and have more convex Iateral rnargins relative to those on plants from SM and CI.

Lateral lobes on plants from SM are intermediate in form between those of Y0 and Cl.

The adaxial corolla lobes show distinct differences in size and shape among the populations and from their lateral and abaxial counterparts. Relative to the abaxial and lateral lobes, the adaxial lobes are wider. The bilateral symmetry of these lobes is offset by relatively larger distal sub-lobes on plants frorn SM and relatively larger proximal sub-lobes on plants from Y0 and CI. At anthesis, al1 lobes are reflexed. The tateral lobes extend toward the base of the corolla, the abaxial lobes project obliquely downward and the adaxial lobes are 40 oriented upward obliquely or at right angles to the long axis of the flower (figs. 1,

2)-

Stamen architecture

Shortly after filament growth has raised the anthers off of the floral apex, both pairs of stamens are appressed to the lateral corolla walls (fig. 22A, 22B).

At this stage, differential growth in the adaxial filaments causes the adaxial stamens to bend and grow towards the adaxial surface of the corolla (fig. 22C,

220). Differential growth causes indentations of the corolla tube in the regions subtending the abaxial filaments, which directs the filaments adaxially as well

(fig . 22C,22E). Around the same stage, regions of tissue at the bases of the abaxiai filaments grow towards the median plane (fig. 22E, 22F). As the filaments continue to elongate, indentation of the corolla tube in the regions subtending the adaxial filaments occurs and may help direct stamen growth towards the median plane (fig. 23A). By this time, curvature has occurred in the abaxial filaments, causing their anthers to meet at the median plane (fig. 238).

Adaxial directional growth of the adaxial filaments causes these starnens to reach and subsequently grow along the adaxial surface of the corolla (fig. 23C,

230). Indentation of the corolla tube at the bases of the abaxial filaments continues to become more conspicuous (fig. 24A-24C). By the time the corollas have reached ca. 15mm in length, the regions of thickened tissue at the bases of the abaxial filaments have grown, and they meet at the median plane (figs. 238,

240). By anthesis, abaxial stamen growth has continued along the rnedian plane due to curvature of their filaments (fig. 25A). As well, abaxial stamen growth has occurred along the adaxial surface of the corolla due to the indentations in the corolla tube at the bases of these stamens (fig. 258, 25C, 25E). By anthesis, the indentations of the corolla tube subtending the adaxial filaments have directed the adaxial stamens along the median plane (fig. 25D, 25G, 25i-i). This directed growth of the adaxial filaments has continued to anthesis and the adaxial stamens are placed along the inner adaxial corolla surface (fig. 25E). The thickened regions at the bases of the abaxial filaments have extended ca. IOmm along the length of the filaments and remain immediately adjacent to one another

(fig. 25A, 25F).

Allometry/heterochrony

For each population, a regression analysis was performed on calyx length

(length of adaxial side of calyx) from about 2mm to anthesis. Although the relationships are best expressed by sigrnoidal equations, allometric analyses require the relationships be expressed as a straight line (fig. 26). Average growth rates of buds differ among the three populations (table 1, fig. 26). Buds on plants from SM grew at a slower rate than those on plants of YO, which grew at a slower rate than those on plants of 61 (table 1, fig. 26). Duration of bud growth was measured as the period between initiation of meiosis in the anthers, and anthesis. Initiation of meiosis is useful as a non-morphological marker of the starting point of bud growth [Geurrant, 1982 #18]. Durations of bud (calyx) growth (mean k SE) differed at the P c 0.05 level between buds of plants from

SM (16.1 + 0.3 days) and CI (18.0 i 0.3 days). Those from Y0 (16.9 + 0.4 days) 42 did not differ significantly from either of the other populations. Calyces of buds from SM have shorter lengths at anthesis than those of the other two populations

(table 2).

Without age data, changes in allometric reiationships cannot be interpreted as the result of heterochronic evolutionary processes (McKinney

1988). floral organs interna1to the calyx could not be measured throughout growth in absolute time. However, by comparing the development of organs against that of an index variable whose development remains consistent among populations, and is well correlated with calyx length (Le. time), growth relative to absolute time can be inferred. Lengths of the adaxial corolla surfaces, despite being distinct, are very similar among the populations at maturity (table 2). As well, a visual analysis of a scatter plot of adaxial corolla length versus calyx length indicates that the two variables are well correlated throughout development. For these reasons, adaxial corolla length was chosen as an index against which development of other organs was compared. Adaxial corolla length is more desirable to use as an index measurement than calyx length because data transformations were not required to obtain a straight-line relationship for regression analyses for rnany variables.

Ovary length, instead of adaxial corolla length, was used to compare corolla tube development because total corolla length is a function of corolla tube length. Ovary length meets the criteria for use as an index variable. The relationship between ovary length and corolla tube length is not best expressed as a straight line, and data transformations obscured the variation. Therefore, 43 these relationships were not analyzed statistically. To compare growth of corolla and corolla tube lengths along their abaxial and adaxial surfaces, abaxial stamen length was chosen as the index variable. To compare growth of organs within a flower, any variable can be used as the index variable.

Among the populations, on flowers of plants from SM, the abaxial cordla lengths are significantly shorter than those on plants from Y0and CI (table 2).

Average growth rate of the abaxial corolla wall on plants of CI was faster than that of SM, which was faster than that of Y0(fig. 27, table 3). As well, in buds on plants from SM, growth of the abaxial corolla wall terminates at a smaller corolla size (therefore, sooner) than on plants of the other two populations (fig. 27, table

3).

Lengths of the abaxial corolla walls significantly differ from the lengths of the adaxial corolla walls in al1 three populations (table 2). An allometric plot comparing the two corolla length measurements using adaxial stamen length as the index organ shows a reduced rate of growth of the abaxial corolla wall relative to that of the adaxial corolla wall in al1 populations (fig. 28, table 4).

Corolla tube length reflects the height at which filaments are inserted onto the corolla. Among the populations, both adaxial and abaxial corolla tube regions are significantly shorter in Y0 buds and Rowers. An allometric plot using ovary length as the index variable shows that on plants of YO. the abaxial corolla tube region terminated growth sooner relative to the growth in CI and SM (fig. 29 top). Along the adaxial surface of the corolla tube, termination times are distinct among populations (fig. 29 bottom). On plants of SM, growth time was extended 44 relative to that of CI, which was extended relative to that of Y0 (fig. 29B). These growth patterns are consistent with the observed lengths at anthesis (table 2).

Lengths of corolla tube regions subtending both adaxial and abaxial stamens are similar at anthesis for CI and SM populations (table 2). In CI flowers, the length of corolla tube subtending the adaxial stamens is significantly shorter than that subtending the abaxial stamens (table 2). An allometric plot of corolla tube lengths using abaxial stamen length as the index variable shows a reduction in growth rate of the adaxial region of the corolla tube relative to that of the abaxial corolla tube in buds of CI (fig. 30, table 4). The growth rates of the abaxial and adaxial regions of the corolla tube differ on buds of YO. However, this does not appear to be enough to make their lengths distinct at anthesis

(table 4). Growth rates of the two regions of the corolla tube do not differ in plants of SM (table 4).

Both adaxial and abaxial stamens in flowers on plants from CI have a shorter length at anthesis relative to the other populations (table 2). For both pairs of stamens in CI buds, the differences at least in part, can be attributed to a reduction in rate of growth relative to those in Y0and SM buds (fig. 31, table 3).

The data also seem to indicate that stamens in CI buds may teminate growth earlier than in the other two populations (fig. 31).

Abaxial and adaxial stamen lengths within a flower differ significantly in al1 three populations (table 2). Allometric plots of lengths of the stamen pairs against adaxial corolla length shows that adaxial stamens grow at a slower rate 45 than the abaxial stamens (table 4, fig. 32) and thus they achieve a shorter length at anthesis (table 2).

At anthesis, style length differs among al1 three populations (table 2). An allornetric plot shows that styles of buds on plants from Y0grow at a faster rate than those of CI and SM, although this is not statistically significant (table 3, fig.

33). A later termination of growth contributes to the longer length of styles on flowers on plants from Y0 (fig. 33). 46

Discussion

Ontogenetic variation among the populations of & cardinalis is slight and

reflects the morphological variation that occurs at anthesis. This may be

expected among conspecific populations; however, differences in morphologies

belie the differences in the ontogenies that generate them. Many of the

differences that occur dun'ng floral development, at least in part, explain the

divergences in floral form. Other differences during ontogeny, however, have

negligible effect at anthesis.

Variation in morphological development among populations

Differences are seen among the populations, even at very early stages

(e-g. prior to fioral organ initiation). However, these differences are of a very small degree. Prior to floral organ initiation, the lateral walls of fIoraI apices on plants of SM are straight and parallel, whereas these walls are convex on plants frorn Y0 and CI. Also, apices on plants of CI are significantly larger than those on plants of Y0and SM. Changes in early ontogeny are thought by some workers to yield morphological differences of high magnitude. Although these observed differences might contribute to some of the variation obsewed at anthesis, the morphological variation within & cardinalis was small.

The relatively early initiation of calyx growth on plants of CI (see below) resulted in floral morphological differences among the populations throughout development. Primarily, the size of sepals, and trichome development relative to the inner floral apex were different among the populations. These differences were not evident in stages following mid - ontogeny (Le. following calyx tube formation).

Slight variation was seen among the populations in corolla shape in transverse view. Corolla throats on plants of SM are relatively laterally wider, and the lateral walls of the corolla are convex. On plants of Y0 and Cl, the corolla throats are relatively more laterally narrow, and the lateral walls of the corolla curve inward. Thus, it is possible that the wider corolla throat on coroflas of SM accommodates a different species of hummingbird andlor allows for pollen deposition in a different location on the pollinator

This shape is what gives rise to the dimensions of the corolla aperture in

-M. cardinalis and, therefore, may be important in plant pollinator interactions. For example, a wide corolla aperture may alIow a visiting hummingbird to insert its face partially into the throat, thus enabling the bird to extract nectar from a longer flower than would be possible with a narrower aperture.

Heterochronic/allometricvariation among populations

Arnong the populations, calyx (bud) developrnent varied with respect to rate of growth and duration of growth. Variation in duration of growth was at least in part due to variation in timing of initiation of the calyx. Although organ growth at early stages of ontogeny could not be measured in absolute time (Le. days from anthesis), it is unlikely that variation in timing of initiation accounts for al1 of the observed variation in duration of growth. On plants of CI, the relatively late initiation, but relatively longer du ration of growth, indicates a d ifference in 48 timing of initiation and temination (anthesis) of growth from the other populations.

Calyces on plants of CI initiated Iate relative to the other populations with respect to the inner whorls. However, these calyces grew at a faster rate relative to those on plants of the other populations, and for a longer period relative to those on plants of SM (the difference in duration of growth between Y0 and CI was not significant). Calyces on plants of Y0 initiated early relative to CI, but grew at a slower rate than those of CI. Despite ontogenetic differences, at anthesis, calyx length did not differ between Y0 and CI. Calyces on plants of SM initiated similarly to those on Y0 (early relative to those of CI) but grew at a slower rate relative to the other two populations and for a shorter period relative to Y0 and CI. Thus, calyces on plants of SM were shorter at anthesis than those on plants of the other two populations.

These differences can be interpreted as a mosaic of evolutionary ontogenetic changes that work in opposition or in concert to generate the observed morphology. That is to Say, the combined changes did not necessarily work in concert to generate a longer or shorter calyx relative to other populations.

Arnong the populations, the corollas initiated as five petal primordia at the same time relative to the stamens and gynoecium. The rates of growth of the corollas along the abaxial side (measured as tube and throat) differed slightly among the three populations. Thus, these differences may not account for much of the variation in length at anthesis. Growth along the abaxial side of the corolla 49 terminated early on plants of SM relative to that of the other populations, and accounts for the shorter length in flowers from that population.

Corolla tube growth contributes to the total growth of the corolla and it may be expected that the patterns of ontogenetic variation in the dimensions of the tube reflect total corolla length. However, growth dong the abaxial side of the corolla tube terminated early in plants of Y0 relative to the other populations.

Along the adaxial side, growth terminated earlier in plants of YO, followed by those of CI, and finally, SM. Yet, it is in plants of SM that have shortened corollas, and Y0 and CI that have longer corollas. Length of the corolla tube is not indicative of total corolla length. Thus, growth in length of the tube and throat are dissociated. As with the calyx, development of corolla traits varies among populations with respect to rate and timing of termination of growth. Unlike the calyx, no differences were observed to occur in timing of initiation of corollas among the populations.

Growth of both pairs of stamens is similar among plants of Y0and SM.

Stamens on plants of Cl are shorter at anthesis due to both a lower rate of growth and early termination of growth relative to those on the other hnro populations. These developrnental variations of stamens are similar to those found in the corolla. No differences in timing of initiation of starnens were detected among populations.

Styles on plants of Y0 are longer at anthesis than those on plants of CI and SM. This is due at least in part to a relatively faster rate of growth. 50

However, temination of growth rnay have been delayed, resulting in a longer period of growth.

Ontogenetic variation within a flower

Within an organ je-g., corolla) or organ complex (e-g., androecial whorl of stamens) variation in lengths at maturity exist between abaxial and adaxial regions or pairs. In each case (total length of corolla, length of corolla tube, and length of starnens) the differences in ontogeny between the abaxial and adaxial regionslpairs were a function of differences in rate of growth.

Conclusions

Among the characters examined, alterations in ontogeny were inferred with respect to the three parameters tested in analyses of heterochrony

(comparative analyses of rates and timing of developmental events between related taxa): rate of growth, timing of initiation of growth, and termination of growth. Of the four organ complexes, only growth within the calyx showed alteration in timing of initiation (CI was Iate relative to Y0and SM). Changes in rate and timing of temination were frequent, and may be more indicative of the changes that commonly generate interpopulational variation, at least within cardinalis. As a possible explanation, early alterations in the length of calyces may be less disruptive to the growth of the rest of the bud, as these alterations occur in the outermost whorl. As well, calyx characteristics in M. cardinalis probably do not contribute to plant-pollinator interactions. Therefore, changes in them may not be as likely to be filtered out by natural selection as long as the protective function of the calyx is maintained. 51

Ontogenetic differences within a floral whorl that contributed to differences in length were limited to differences in rate of growth. Possibly, spatial constraints within the bud precluded changes in early ontogeny, i.e. ontogeny following organ initiation. Aiso, if a change was to increase the length of a structure, a delay in termination of growth could offset the timing of maturation between that structure and associated structures. For example, if the differences in length between the pairs of stamens within a flower were the result of a delayed termination of growth in the abaxial pair rather than a faster rate of growth in this pair, then pollen from the adaxial anthers may be mature and exposed within the closed bud unless maturation of these anthers was also delayed.

Ontogeny of humrningbird-pollinated traits

The flowers of M. cardinalis are regarded as having a classic hummingbird-pollinatedform (Bradshaw et al. 1995). The observed allometric relationships, in part, generate this morphology that is well suited to its pollinator.

Other developmental characteristics contribute to the general form, as well.

Overall corolla length may affect foraging behaviour of hummingbirds and may be linked io culem characteristics of the hurnrningbird visitor(s) via natural selection. In lateral view, the aperture of the corolla is oblique with the adaxial surface extending more distally than the abaxial surface. This has been reported in other taxa (e.g. Penstemon spp.) (Lange et al. 2000) and rnay serve as a mechanisrn to aid hovering hummingbirds by allowing them unhindered access to the corolla aperture because the abaxial lobes and corolla throat are recessed. 52

Within the study populations of & cardinalis it was shown that the oblique corolla aperture is achieved by differential growth rates along the abaxial and adaxial surfaces of the corolla throat, not by other mechanisms such as differential reflexation of corolla lobes.

Size and shape of the corolla aperture affect accesçibility of the hummingbird visitor to the interior of the flower and to nectar (Bradshaw et al.

1998; Bradshaw et al. 1995). Aperture characteristics (along with others such as lobe reflexation and recession of the abaxial corolla wall) determine how far a visiting hummingbird can enter into the flower. A wider aperture ailows a pollinator to insert its head deeper into the flower. Consequently, a wider corolla aperture rnay compensate for a suboptimai corolla length. Or, as rnay happen in flowers of SM, a wider aperture rnay work in conjunction with a shorter corolla to provide optimal characteristics for a hummingbird with a shorter culem.

The two rows of yellow trichomes along the length of the abaxial surface of the corolla throat, and the groove that separates these rows rnay serve as a tongue guide for hummingbird visitors. Also, in & cardinalis, these characteristics rnay be present as a result of the phylogenetic link between M. cardinalis and M. lewisii if this trait was present in the shared ancestor, Le.,the yellow rows of trichomes rnay be a sympleisiomorphy. It seems unlikely that these characteristics are functionless in M. cardinalis because the rows of trichomes lead to the opening between the abaxial filament bases where access is gained to nectar. In addition, similar characteristics occur in unrelated humrningbird-pollinated species. In Penstemon pinifolius, a yellow bearded 53 staminode lies along the abaxial surface of the corolla tube (referred to here as the corolia throat) (Lange et al. 2000). This has been suggested to serve as a nectar guide, that is, a visual andlor tactile cue that prompts a visiting pollinator to a region of the flower where nectar accumulates (Sutherland and Vickery

1993). In P. barbatus (also hummingbird-pollinated) the staminode is glabrous, however, the abaxial corolla lobe displays light-coloured Iines that resemble nectar guides of other Penstemon species (Lange et al. 2000).

Anther position relative to stigma position, and anther and stigrna position relative to corolla aperture play a role in efficiency of receipt of outcross pollen

(Lange et ai. 2000; Bradshaw et al. 1998; Bradshaw et al. 1995). Relative anther position facilitates specific placement of pollen on a pollinator. The spatial separation of anthers and stigma hinders self-pollination and also provides for efficient deposition of pollen by a pollinator ont0 the stigma of another flower.

In M. cardinalis, both pairs of stamens emerge from the adaxial side of the corolla throat in the same horizontal plane along the centre of the corolla. This requires that both pairs of stamens be directed adaxially from their points of insertion on the corolla as well as toward the median plane. Furthermore, in order for the anthers to lie in tandem and occupy the same horizontal position at anthesis, their degree of exsertion must be offset. At anthesis, the abaxial anthers sit slightly more distally from the corolla aperture than the adaxial anthen. The overall positioning of the anthers is, in part, a function of the length of the corolla tube in the regions subtending the anthers (Le. how far "up" the stamens are inserted on the corolla), length of the filaments, and the adaxial and median directional curvature of the filaments. In al1 populations, the abaxial stamens achieve a greater mature length than the adaxial stamens as a result of an increased growth rate. This is the dominant factor in determining spatial differentiation among the anther pairs, while the described stamen architecture, and points of insertion (i.e. length of corolla tube) contribute to a lesser degree.

The existence of hummingbird-pollination syndromes is well documented.

That is, it is accepted that a series of general floral traits exists that are common to a diversity of plant taxa that are pollinated by hummingbirds (Lange et al.

2000; Proctor et al. 1996). Members of the Asteridae that are reported as being visited by hummingbirds include Razisea and Hansteinia (Acanthaceae), Salvia

(Larniaceae), Penstemon (Scrophulariaceae), Ipomor>sis(Polemoniaceae),

Castilleja (Orobanchaceae), Monarda (Lamiaceae) and Mimulus (fonnerly

Scrophulariaceae) (Lange et al. 2000; Mitchell et al. 1998; Proctor et al. 1996;

Bradshaw et al. 1995; Grant and Grant 1968). Some general traits that have been identified to confer pollinator specificity in this group include long tubular corollas of bright colour (usually red), reflexed corolla lobes, anthers and stigma exserted beyond the corolla aperture, and yellow trichomes that occur along the lower interior corolla surface.

Despite consistency in general fom, variation exists within hummingbird- pollination syndromes. From this, it rnay be reasonable to assume that: 1) If taxa are not closely related, then convergence of floral form from different ancestral taxa inevitably results in some variation among descendents, regardless of the selective pressure on a given form. This may be especially true for ontogenetic variation as the unique ancestral developmental programs may constrain in different ways the number of viable alterations available to each taxon. Also, as

multiple developmental pathways cmgenerate a superficially similar

morphology, developmental variation may greatly exceed generalized

morphological variation. As a result, multiple origins of a given pollination syndrome rnay be elucidated by comparative studies of floral development in diverse taxa. 2) Whether taxa having this characteristic syndrome are closely

related or not, variation within and among hummingbird pollinated taxa throughout the plant/pollinator range is subject to varying selective pressures. thereby yielding different evolutionary responses among allopatric populations even within taxa.

Mimulus cardinalis ranges frorn southern Oregon to northern Mexico, and extends east from the Pacific coast to Arizona (Sutherland and Vickery 1993). In this range, there are an estirnated 13 species of hummingbirds with culem lengths that range from about 13 - 30 mm (Johnsgard 1997). Most of these hummingbirds with a longer culem occur farther south in Arizona and Mexico.

Multiple hummingbird species are reported to visit plants of populations of M cardinalis throughout its range (Schemske and Bradshaw 1999; Sutherland and

Vickery 1993). Given that the diversity of hummingbirds in this range probably predates the diversity within M. cardinalis, variation in culem length may have selected for a suite of (in this example) optimal corolla lengths. It should be noted that a variety of floral traits may combine to suit a hummingbird of a given 56 culem length, including degree of corolla lobe reflexation and width of the corolla aperture. 57

Fig. 1 A, Lateral views of flowen of M cardinalis, Yosemite population

(YO) (top), Siskiyou Mountain population (SM) (middle), and Cedros Island population (CI) (bottom). B. Longitudinal sections of flowen of cardinalis.

Populations are represented in the same order as A. Stamens and/or styles are displaced adaxially in sectioned flower in B. Scale = 10.0 mm; a = stamen primordiurn or anther; ab = abaxial side; ad = adaxial side; ar = anther removed; c = petal primordium, corolla or corolla lobe; ct = corolla tube; cth = corolla throat; cr = corolla or corolla lobe removed; f = filament; fa = fioral apex; g = pistil primordium; ia = inflorescence apex; k = sepal prirnordium, calyx or calyx lobe; kr

= calyx lobe removed; i = [ocule; Ip = leaf primordium; Ir = leaf removed; n = nectary; O = ovary; ov = ovule; p = pedicel; pl = placenta; se = septum; sp = stigmatic papillae; st = stigma; sy = style. Adaxial side is at the top of al1 images unless stated otherwise.

Fig. 2 Lateral view of longitudinal section of a flower of M. cardinalis (YO) showing dimensions used in allometric analyses. Not shown; adaxial filament, adaxial corolla tube, abaxial corolla. Stamen and style are adaxially displaced in this sectioned flower. Scale = 10.0 mm.

Fig. 3 A, B Polar views of inflorescence apices and floral apices of j& cardinalis, CI (SEM) showing decussate floral and leaf arrangement and offset development of floral apices at a node. Scales = 250 pm.

Fig. 4 Early floral apex development of cardinalis (SEM). A-C, Lateral views of floral apices of CI (A, 6)and SM (C) prior to floral organ initiation.

Scales = 25 Pm. D, Polar view of SM showing initiation of the adaxial sepal. E,

F, Polar views of CI (E) and SM (F) showing initiation of rim meristem of calyx

(arrowheads). G, Lateral view of SM showing early pedicel formation, and adaxial sepal and rirn meristem (arrowheads). H, Oblique polar view of Y0showing ail five sepal primordia. 1, Oblique lateral view of Y0showing sepal, petal and stamen primordia. Scales = 50 Pm.

Fig. 5 Early floral development of & cardinalis (SEM). A-F, Polar and lateral views of CI (A, B), Y0 (C, D) and SM (E, F) showing calyx development and initiation of petals and stamens. Note formation of cleft separating calyx from rernainder of floral apex. Scales = 50 Pm.

Fig. 6 Floral apices of M cardinalis (SEM). A, Polar view of Y0 showing rim meristem (arrow) and the initiation of the gynoecium (arrowhead). B-G, Polar and lateral views of Y0 (BI C), SM (D, E) and Cl (F, G) at the stage of vertical growth of calyx lobes, and calyx trichome initiation. B, Polar view showing vertical growth of sepals, petals and stamen, and initiation of gynoecium. CI

Lateral view showing taller abaxial sepals and initiation of trichomes. D-FI

Oblique polar and polar views showing curvature of sepals, enlargement of petals and stamens, and formation of a pentagonal gynoecium. Note acute petals in F. G, Lateral view showing relative heights of floral organs and trichome initiation on calyx and pedicel. H, Oblique view of Y0 with one abaxial calyx lobe removed showing start of conduplicate folding of calyx lobes and placement and size of stamen primordia relative to petal primordia. 1, Polar view of SM showing inward curvature of calyx lobes, differences in shape and distance between adaxial and abaxial starnens, and pentagonal shape of gynoecium. Scales = 50 Pm.

Fig. 7 Floral apices of cardinalis (SEM). A,B, Polar views of CI (A) and

SM (B) showing beginning of anther formation, and locule and septum formation

(arrowheads) in the gynoecium. C, Oblique lateral view of SM showing vertical growth of petals and relative heights of stamens and gynoecium. D, Oblique view of Y0 showing elongation of calyx trichomes and beginning of anther differentiation. El Oblique view of SM with distal portions of calyx lobes removed showing transverse flattening of anthers and petals, and elongation of the gynoecium. Arrowhead indicates septum. F-Hl Polar view of CI (F) and oblique views of SM (G) and Y0 (H) showing formation of rnicrosporangia and connective on anthers, and transverse clefts on rirn of gynoecium. H, Initiation of the corolla throat (arrow) and filament formation (arrowhead), and corolla, stamens and gynoecium at simiiar heights. Scales = 100 Pm.

Fig. 8 A-D, Lateral views (A, 6,D) and adaxial view (C) of buds of M. cardinalis (SEM). SM (A), CI (B) and Y0(C, D) showing elongation and radial widening of calyx tube, decurrent folds extending from lobes onto tube, enlargement of trichomes, and conduplicate folding of lobes. Scales = 300 pn.

Fig. 9 Dissected buds of cardinalis (SEM). A, Oblique view of Y0 with calyx removed, and adaxial and one lateral corolla lobe removed showing connective formation on the anthers (arrowhead), and microsporangia (arrow).

B, Lateral view of Y0with calyx, corolla and adaxial stamens removed showing vertical growth of gynoeciurn. ClLateral view of longitudinal section of SM with gynoecium removed showing inward cuwing of the abaxial corolla lobes, enlargement of microsporangia and filament formation (arrows). Dl Polar view of

CI with calyx and abaxial corolla lobe rernoved showing microsporangia (arrow), connective (arrowhead) enlargement of anthers, septum development in, and transverse clefts on rim of gynoeciurn. El Lateral view of longitudinal section of

SM with gynoecium removed showing closed calyx, incurveci abaxial petals, and fiiament formation (arrow). F, Lateral view of longitudinal section of SM with stamens and gynoecium removed showing conduplicate folded calyx lobes and inward curving of the abaxial corolla lobes. G, View of CI with calyx removed showing corolla throat and folding of the corolla lobes. H,Lateral view of Y0with calyx and adaxial corolla lobes removed showing folding of corolla lobes, and the gynoecium overtaking stamens in height. Scale = 150 Fm.

Fig. 1 O Buds of M. cardinalis (SEM). A-Cl Oblique views of longitudinal

sections of CI with the gynoecium removed (B. C)showing insertion of filaments

on the corolla, folding of corolla lobes and tilting of adaxial anthers (C). O-F,

Lateral views of longitudinal sections of Y0 (D), SM (E) and adaxial view of

longitudinal section of CI (F) showing differential elongation of trichornes among

Y0 (D), SM (€1 and CI (F), closure of corolla, placenta (arrowheads) and flaring of incipient stigma lobes. G, Polar view of Y0 showing initiation of trichomes on the tiited adaxial anthers, formation of stigrna lobes, and compitum (arrowhead) on apex of placentas. H, Oblique lateral view of Y0 showing flaring of incipient stigma lobes and formation of nectary. 1, Oblique lateral view of interior of CI corolla showing trichomes (arrowheads) initiating on the inner abaxial wall of the corolla throat. Scaies = 300 Pm.

Fig.11 Buds of cardinalis, (SEM) (YO). A-Cl Lateral view (A) and polar views (BI C) with calyx removed showing corolla closure and radial enlargement of adaxial lobes covering other lobes. Scales = 250 Fm.

Fig. 12 A-H, Buds of cardinalis (SEM). A, B. Lateral view of longitudinal sections of CI showing formation of the style, enlargement of trichomes on anthers, and adnation of filament to corolla in B. C, Pistil and adaxial stamens of SM showing unequal size of closed stigma lobes. DILateral view of (SM) showing pistil, abaxial stamen at right, tilted adaxial stamen, and closed stigma lobes. E, Laterai view of CI corolla showing region of trichorne growth (arrowhead) on the interior abaxial wall of the corolla throat. FI Lateral view of longitudinal section of CI showing abaxial cuwature of the style. G,

Lateral view of longitudinal section of CI showing elongated trichomes on anthers, appressed stigma lobesshollow style and placentas. H, Lateral view of longitudinal section of CI showing abaxially curved hollow style, ovules on the placenta, long trichomes on anthers, corolla tube below filament at right, and trichomes on inner abaxial corolla wall (arrowhead). Scales = 300 Pm.

Fig. 13 A-F, Pistils of cardinalis, CI (SEM) showing stages of initiation of ovules and stigmatic papillae. A, Adaxial view of entire pistil showing smooth placenta and smooth stigrnatic surface. B, Placenta showing initiation of ovules.

C,Stigmatic surface showing formation of stigmatic papillae. D, Placenta showing formation of integument on ovules (arrows). E, Stigmatic surface with elongating stigmatic papillae (from same pistil as D). F, Pistil at the time of integurnent formation (from sarne pistil as D). Scales = 100 Fm.

Fig. 14 A-D, Buds of cardinalis at sirnilar stages of development with calyx rernoved (adaxial corolla length = ca. 1 - 2 mm). A, Oblique lateral view of

Y0 (SEM) showing oblong shape of corolla. B-D, Transverse sections of corolias of Y0 (dissecting micrographs, DM) B. near the base of the throat showing circular shape; C,at the middle of the throat showing adaxial peak

(arrow) and two rows of trichornes aiong the interior abaxial corolla wall

(arrowheads); D, at the apex of the throat showing adaxial peak. Scales = 1.0 mm.

Fig. 15 A-D, Buds of cardinalis at similar stages of development

(adaxial corolla length = ca. 3 - 4 mm) with calyx removed in A, C, D (DM). A,

Lateral view of CI showing corolla tube formation and indentation of corolla tube

(arrowheads) at regions subtending filaments. B, Lateral view of longitudinal section of CI showing curved style and stamens of different lengths. C,

Transverse section of Y0 midway along the throat showing abaxial groove

(arrow) and rows of elongating trichomes (arrowheads). D, Transverse section of

Y0 at the apex of the throat showing circular corolla appressed to anthers.

Scales = 1.0 mm.

Fig. 16 A-D, Buds of M cardinalis at similar stages of development

(adaxial corolla length = ca. 7 - 8 mm) with calyx removed (DM). A, Lateral view of CI showing abaxial protrusion of corolla (arrow), radial enlargement of corolla at lobes, cilia initiation on corolla lobes (arrowhead with asterisk), and indentations in the exterior of the corolla tube (arrowhead). 8,Lateral oblique view of longitudinal section of CI showing bent style. Note how indentation of corolla tube (at arrow) oppoçite filament bases. C, Transverse section of CI midway along the throat showing abaxial groove, rows of trichomes along abaxial corolla wall, and rounded adaxial surface of corolla. D, Transverse section of Y0 at the apex of the throat showing abaxial groove, rows of trichomes along abaxial corolla wall (anthers removed), and adaxial peak. Scales = 1.O mm.

Fig. 17 A-G, Buds of M cardinalis at similar stages of development

(adaxial corolla length = ca. 10 - 12 mm) with calyx removed (A, B. D-G) (DM).

A, Lateral view of CI showing radial enlargement of corolla at bases of lobes, and

cilia along margins of lobes (arrowheads). Adaxial corolla wall is longer than

abaxial corolla wall. B, Oblique polar view of CI corolla showing adaxial ridge

(arrow). C,Oblique lateral view of longitudinal section of CI showing curved style

and elongating trichomes on divergent thecae of anthers. Note band of trichomes on abaxial wall of corolla (arrowheads). D, E, Transverse sections of

Y0 (D) and SM (E) midway along the throat showing increase in width (more prominent in YO). F, G, Transverse views of CI at the apex of the throat with anthers removed (F) showing general circular shape and tight packing of anthers

(G). Scales = 1.O mm.

Fig. 18 A-E, Corollas of M. cardinalis just prior to anthesis with calyces removed (DM). A, Lateral view of CI with lobes beginning to unfurl. 6,D,

Transverse section of Y0 midway along the throat (B), and at the apex of the throat (D) showing appearance of lateral compression of the corolla throat. C, E,

Transverse sections of SM midway (C) along the throat, and at the apex (E) of the throat showing wider spade-shape of the corolla throat and distinct abaxial groove. B-E, Note ridges on the abaxial interior corolla on each side of the abaxial interior groove. Scales = 1.O mm.

Fig. 19 A-F, Transverse sections of flowers of M. cardinalis with calyx rernoved (DM). Y0 (A) and SM (B) at the base of the throat showing circuiar shape. Bases of adaxial stamens are adjacent to the lateral walls of the ovary.

Bases of abaxial stamens are close together, adjacent to the abaxial wall of the ovary. Note ridges subjacent to each filament base (arrowheads), which are indentations in the external surface of the corolla tube. Y0(C) and SM (D) midway along the throat. CI (E) and SM (F) at the apex of the throat. Note the position of the filaments and style against the adaxial wall of the corolla. Note the difference in position of trichomes along the lateral walls of corollas of CI (E) and SM (F) (arrowheads) and trichomes within the abaxial groove. Scales = 1.0 mm.

Fig. 20 A-F, Pistils of cardinalis, CI (DM). A, Lateral view showing style curving abaxially. B, Lateral view showing style hooking abaxially and formation of nectary (arrowhead). ClAbaxial view of nectary showing tissue darkening.

Dl Lateral view of nectary at initiation of nectar secretion (corolla length = ca. 15 mm). El F, Abaxial views of ovary at anthesis showing fully developed nectary.

Scales = 1.O mm.

Fig. 21 Line tracings of representative adaxial, lateral and abaxial corolla lobes from each population at anthesis. Average length (vertical plane), width

(horizontal plane) and length : width ratio values are given in mm, as well as standard error. Sample sizes indicated in parentheses. Values that differ among the populations are indicated by different first superscripts (a, b, c). Values that differ among lobes within a flower are indicated by different second superscripts

(x, y, 2). Adaxial lobes are shown as the flower appears horizontally at anthesis, with the distal end of the flower to the right. Ad, Lat and Ab = adaxial, lateral and abaxial corolla lobe.

Fig. 22 Stamen developrnent of cardinalis, CI (adaxial corolla length =

ca. 3 mm). A (DM), Longitudinal section of bud showing abaxial anthers on

straight filaments. B (DM), Longitudinal section of bud in which corolla tube has

forrned below adaxial filaments. Filaments of adaxial stamens are straight. C

(SEM), Adaxial (lefi) and abaxial stamen. Filament bases showing growth of

adaxial filament toward the adaxial surface and growth of abaxial filament toward the median. D, Base of adaxial filament showing adaxial curvature. E, Base of

abaxial filament showing indentation of the corolla tube (appears as protrusion toward interior of bud) (arrow). Arrowhead indicates region of thickened tissue at the base of abaxial filament on the median side. F, Bases of abaxial filaments

showing growth of tissue toward the rnedian (arrowhead). A, 6,Scale = 1mm, C-

F, Scale = 100 Pm.

Fig. 23 Stamen development of M. cardinalis, (CI) (adaxial corolla length = ca. 10 (A) - 15 mm(f3,C)). A (DM) Longitudinal section of bud showing pistil and adaxial stamens. Indentations of the corolla tube (arrows) direct adaxial stamens toward the rnedian. B (DM), Longitudinal section of bud showing curvature of abaxial filaments toward the median. Arrowhead indicates growth of tissue at bases of abaxial filaments. C (DM), Lateral oblique view of longitudinal section showing curving of adaxial filament (at left) and indentation of corolla tube subtending abaxial filament (arrow). Note that adaxial stamen is shorter than abaxial stamen. Both are inserted ont0 corolla tube at same level. D, (SEM)

Base of abaxial (left) and adaxial (right) filament showing indentation of abaxial corolla tube (arrow), region of thickened tissue at base of abaxial filament

(arrowhead), and curved adaxial filament. A-C, Scale = 5.0 mm. D, Scaie = 1 .O mm.

Fig. 24 Stamen developrnent of M. cardinalis (adaxial corolla length = Ca.

10 - 15 mm). A (DM), Lateral view of longitudinal section of Y0 bud showing indentation of the corolla tube subtending the abaxial stamen and region of adnation of filament with corolla (arrowhead). Scale = 5.0 mm. B (DM), Close up of A, showing indentation of corolla tube (arrowhead). Scale = 1.O mm. C,

(SEM) Lateral view of CI bud showing indentation of corolla tube subtending abaxial stamen and region of adnation of filament with corolla. D, (SEM) Base of abaxial filament of CI bud showing region of thickened tissue toward the median

(arrowhead). Cl D, Scales = 100 Fm.

Fig. 25 Stamen alignment of & cardinalis Rowers (anthesis). A (DM),

Longitudinal section of base of CI showing abaxial stamen positioned afong the median, and region of thickened tissue at base of abaxial filaments (arrowhead).

B. C (DM), Lateral views of longitudinal section of Y0showing indentation of abaxial corolla tube (arrowheads) directing abaxial stamen toward the adaxial surface. D (DM), Longitudinal section of CI showing indentations of the corolla tube (arrowhead) directing adaxial stamens toward the median. E, Lateral view of longitudinal section of CI showing adaxial position of abaxial and adaxial anthers. F (DM), Transverse section of CI (calyx removed) at the junction of the corolla throat and corolla tube showing indentations of the corolla tube subtending abaxial stamens (black arrowhead) directing stamen growth towards the adaxial surface (top), and indentations subtending the adaxial stamens (white arrowhead) directing stamen growth toward the median. Regions of thickened tissue at the bases of the abaxial filaments (arrow with asterisk) meet at the median and form a tubular enclosure that terminates basally at the nectary below

(not shown). G, H, Adaxial (G) and abaxial (H) view of CI with part of calyx and corolla removed showing median positioning of both pairs of stamens. Thecae within anthers are arranged in tandem. Anthers and stigma are exsert. A, B, D,

El G, H, Scales = 5.0 mm. C,F, Scales = 1.0 mm.

Fig. 26 Regression lines showing growth rates of buds from the three populations of cardinalis. The average growth rate of buds differs among the populations at the P < 0.05 level (table 1). davs frorn anthesis Fig. 27 Allometric plot of abaxiai corolla length versus adaxial corolla

length for the three study populations of M. cardinalis. Corollas on plants from

SM teminate growth at a shorter adaxial and abaxial length (arrow) than those on plants of Y0and CI. - -

-

- Y0 - O -- Cl ..-SM - .

l 1 I I 1 1 O 10 20 30 40 50 adaxiat corolla length (mm) 111

Fig. 28 Allometric plot of adaxial and abaxial corolla lengths versus abaxial stamen length for Y0 (representative of the three study populations). The abaxial region of the corolla grows at a slower rate than the adaxial region (table

4)- .- adaxial corolla O- - abaxial corolla

O 10 20 30 40 abaxial stamen length (mm) 113

Fig. 29 Allometric plots of adaxial (top) and abaxial (bottom) corolla tube lengths versus ovary length for the three study populations. Top, The abaxial corolla tube on plants of Y0 (arrow) terminates growth before that on plants of

SM and CI. Bottom, The adaxial corolla tube on plants from Y0 tends to terminate growth before that on plants of CI, which terminates growth sooner than that on plants of SM. ovary length (mm)

ovary length (mm) Fig. 30 Allometric plot of adaxial and abaxial corolla tube lengths versus abaxial stamen length for plants of CI (representative of the three study populations). Average growth rates of the abaxial corolla tubes is sigriificantly faster at the P c 0.05 level than those of the adaxial corolla tubes (table 4). ppp O IO 20 30 40 abaxial stamen length (mm) Fig. 31 Allometric plot of abaxial (representative of adaxial) stamen length versus adaxiai corolla length for the three study populations. 60th pairs of stamens on plants of CI terminate growth at a shorter length than those of Y0 and SM, and grow at a slower rate than those on plants of Y0and SM (table 3). O 10 20 30 40 50 adaxial corolla length (mm) Fig. 32 Allometric plot of abaxial and adaxial stamen lengths versus adaxial corolla length in Y0 (representative of the three study populations).

Average growth rate of abaxial stamens is significantly higher than that of adaxial stamens at the P < 0.05 level (table 4). O 10 20 30 40 50 adaxial corolla length (mm) 121

Fig. 33 Allometric plot of style length versus adaxial corolla length for the three study populations. Although the dope obtained from Y0data appears greater, the dopes do not differ at the P < 0.05 level (table 3). Styles from CI and

SM appear to terminate growV, at a shorter length that those of YO. adaxial corolla length (mm) Table 1 Slopes (b + SE) and Regression Coefficients of Linear Regression Lines of Bud Length versus Time (as Days frorn Anthesis) cornparison group Y0 0.93 -1 -59t 0.02 205.00 0.00 2, 832 CI 0.36 -1 -65-t 0.02 SM 0.96 -1 -48 4 0.02 YO, CI 81 -47 O. 00 1, 582 YO, SM 108.10 0.00 1, 544 CI, SM 547.25 0.00 1, 537 Table 2

Floral Organ Lengths (mean k SE, (n)) in mm at Anthesis.

Population Calyx Adaxial Abaxial Ovary Style Abaxial Abaxial Adaxial Adaxial corolla corolla stamen corolla stamen corolla tube tube Y0 31.7 + 0.3 41.2$0.4 28,22 0.3 9.9f 0.1 42.3 k0.6 34.2 10.4 7.8k 0,2 31.0f 0.4 7.5 10.2 a a a ab a a a a a (23) (34) (1 7) (38) (38) (17) (17) (17) (17)

Note. Means with different letters differ at the P < 0.05 level. See fig. 2 for dimensions measured. Table 3

Abaxial vs. adaxial corolla Y0 CI SM YO, CI YO, SM CI, SM Adaxial stamen vs- adaxial corolla Y0 CI SM YO, CI YO, SM CI, SM Abaxial stamen vs. adaxial corolla Y0 CI SM YO, CI YO, SM CI, SM Style vs. Adaxial corolla Y0 CI SM YO, Cl YO, SM Cl, SM Note. See fig. 2 for dimensions measured. Table 4 Slopes (b + SE) and Regression Coefficients of Linear Regression Lines of Selected Allornetric Relationships Between Adaxial and Abaxial Dimensions of Organs Within a Flower

Population AdaxialJAbaxial corolla vs. abaxial stamen Y0 Adaxial Abaxial CI Adaxial Abaxial SM Adaxial Abaxial

AdaxiaVAbaxial corolla tube vs. abaxial stamen Y0 Adaxial Abaxial CI Adaxial Abaxial SM Adaxial Abaxiaf

AdaxiaIfAbaxiaI stamens vs. style Y0 Adaxial Abaxial CI Adaxial Abaxial SM Adaxial Abaxial

Note. See fig. 2 for dimensions measured. Chapter two - Floral ontogeny of lewisii

Introduction

In Chapter One, the development of and developmental variation among three widely geographically distributed populations of Mirnulus cardinalis were reported. This was done as part of a larger study that examined the developrnental changes that must have occurred during the evolution within the bee-pollinated M. lewisii and the hummingbird-pollinated M, cardinalis.

Given that hn. cardinalis and M. lewisii are each other's closest relatives, a study of comparative floral development between these two species will shed light on how breeding system evolution and species divergence have occurred through diversification of floral developrnental pathways. As a continuation of this study, the development of and developmental variation between both described races of M. lewisii are reported.

Mimulus lewisii occurs in the western half of North Arnerica from California to Alaska, and east to Colorado (Sutherland and Vickery 1993). Within this species, two distinct races are recognized. Plants of the Rocky Mountain race have flowers with a magenta-pink corolla with recurved lobes (figs. 1, 2). Plants of the Sierra Nevada race have a pale-pink corolla with lower lobes that project more foward (fig. 1). Allozyme data suggest that these races rnerit recognition as species (Vickery and Wullstein 1987). This hypothesis is well supported by recent molecular work done on the phylogeny of this group, and steps are being taken to forrnally recognize the Rocky Mountain (magenta) race as a separate species (P. Beardsley pers. comm.). 128

Before the ontogenies of the two species can be compared, the range and degree of developmental variation must be examined in order to detennine that each species is accurately represented. Furthemore. by examining the ontogenetic variation between the two distinct races (putative species) of M- lewisii, it is possible to gain insight as to how developrnent acts as an evolutionary process during the evolution of one bee-pollinated form into a sirnilar but distinct bee-pollinated fom, whether a speciation event is interpreted to have occurred or not.

Plants for this study were grown from field collected seeds obtained from

Yosemite National Park, CA (YO) (Sierra Nevada race); Boise Co, ID (BO)

(Rocky Mountain Race); Johnsville, Plumas Co.. CA (JV) (Sierra Nevada race);

Hillsboro, OR (OR) (Rocky Mountain race). 129

Results

Buds and flowers of al1 populations are used in figures to represent the floral developrnent of the species as a whole. Developmental stages were observed to be similar among the populations except where noted.

Initiation of floral apex and floral primordia

At each node, opposite leaves each subiend a fiower. Leaf, and thus floral arrangement, is decussate (fig. 3A. 38). The development of the floral apices at a node may be synchronous or offset to varying degrees (fig. 3A. 38).

Prior to organ initiation, the hemispherical floral apex is nearly syrnmetrical in polar view (fig. 4A. 48). After the Roral apex becomes wider transversely an unevenly thickened rim meristem initiates low around the circumference of the apex indicating incipient sepals (figs. 4B, 4C, 5A). The rim meristem is discontinuous on the abaxial side (fig. 46, 4C). Along with differential vertical growth of the apex, this causes the apex to appear asymmetrical and adaxially skewed in lateral view (fig. 4C). As the inner fioral apex increases in height, the rim widens and five rounded sepal primordia initiate on it (fig. 5A, 5D). The rim meristem forms along the abaxial surface rnaking it continuous (fig. 56-5D).

DifFerential vertical growth causes a cleft to form around the circumference of the inner floral apex that separates the calyx from the remaining floral apex. Five petal primordia initiate simultaneously as the cleft forms (fig. 5B-5D). At initiation, the two adaxial petal primordia are spaced more closely together than the other petal primordia (figs. 56, 50, 6A. 6C). Sepals begin vertical growth, and the adaxial sepal projects more radially horizontally than the other four (figs. 5C,66, 130

6D). Two pairs of stamen primordia initiate simultaneously opposite the sepak, with no starnen primordium in the median adaxial position (fig. 6A-6F). Stamen

primordia become larger than petal primordia before the latter begin vertical

growth (figs. 6D-6F, 7A-7D). A pedicel becomes distinct and lifts the floral apex

off the shoot surface (fig. 68)on plants of Y0 and JV, but somewhat later in BO

and OR (figs. 7D18G). As vertical growth of the sepals continues, they become

acute on plants from Y0and JV (fig. 6A-6C) while they remain small and

rounded on plants from BO and OR (fig. 6D-6F). As well, a leaf primordium on the node above often suppresses growth of the adaxial sepal on apices of plants from BO and OR until the apex is lifted away from the shoot apex by the

developing pedicel (figs. 6D, 7C, 7D, 8F, 8G). The two abaxial sepals project

more vertically than the lateral sepals in al1 populations (figs. 6A, 6E, 7A). The adaxial sepal continues to project more radially than the other four on plants from

Y0 and JV (fig. 7B).

A gynoecium initiates as an oval primordium in the center of the apex (fig.

7A-7D). Vertical growth causes the adaxial sepal to overtake the rernainder of the apex in height on apices of plants from Y0 (fig. 78). The other sepals on

plants from Y0are shorter than the inner floral apex (fig. 7B). The stamen

primordia become more massive. The adaxial stamens become approximately

hemispherical and the abaxial stamens become oblong (fig. 8A-81). As the gynoecium becomes more distinct on the floral apex, petal primordia begin vertical growth as well as radial growth. At this stage, trichomes initiate on the pedicels and calyces on plants from Y0 (fig. 8A-8C). Sepals exceed the height 131 of the inner organs on plants from Y0 (fig. 8B-8E), however, they have yet to achieve the same height as the inner floral organs on plants from BO and OR

(fig . 8F-8 1).

Calyx organogenesis:

After the gynoecium enlarges and a locule has formed, a calyx tube initiates on plants from Y0 and JV, calyx lobes grow in height, and trichomes elongate and increase in density (figs. 8D,10A. 1OC). On plants of BO and OR, trichornes initiate on the calyx and pedicel, and a calyx tube becomes evident

(fig. 9B-9F). Subsequently, trichomes on calyces of plants from BO elongate and increase in density in a manner similar to those on plants from Y0 (figs. 9G-91,

1OB, 10G-101). Within a calyx, trichome development is offset (figs. 1OC-1 OE,

1OH, 11A-11 D). Capitate glandular trichomes of various lengths are present at maturity. As the calyx lobes start to bend inward, longitudinal folding of the lobes occurs (figs. SG,1 OD, 1OH, 101). The lobes elongate and become narrowly lanceolate (fig. 116-1 1 D). Buds on plants from Y0 and JV are more linear than those on plants from BO and OR which have more bulbous tubes (fig. 11 C, 1l D).

These general shapes are maintained through to anthesis except that the apex of each lobe becomes acuminate as the calyx enlarges.

Corolla organogenesis:

As the petal primordia enlarge vertically, those on Y0 and BO are rounded but those on OR are wider and truncate when still much shorter than the stamens

(fig. 8A-8C, 8F, 8H, 81). Petals overtake the stamens and pistil in height as a corolla throat forms (figs. 9G-91, 10B). As the corolla throat enlarges, the abaxial 132 and lateral corolla lobes cuwe and subsequently fold inward (figs. 91, 1OG, 1 OH,

101, 12F). As the adaxial corolla lobes fold over, their heights are not sufficient to completely obscure the inner apex (fig. 13A-13D). As well, the lobes do not overlap at their bases (figs. 12F, 13A, 13C-13E). On plants from YO, the corolla

lobes are relatively wider than those on plants from BO and obscure more of the

inner apex (fig. 1OE, 1OG). The separation of the corolla lobes at their bases is

less distinct, and margins of adjacent lobes overlap on plants from Y0 (fig. 13A-

13C). As the adaxial lobes fold down over the lateral lobes, the apex of each

lobe has expanded radially, making the lobes oblong in shape (fig. I3B-13E). A shallow notch appears along the distal margin in the middle of each lobe (Le.the

lobes becorne retuse) which is more distinct on plants from Y0than on plants from BO (figs. 138-13E, 14B-14D, 15B, 15C). The corolla is globular in polar

and lateral views (figs. 138, 13D). The lobes continue to enlarge vertically and

radially, and completely enclose the inner floral apex (figs. 148, 14D, 15B). The order of lobe folding is occasionally disrupted, with the abaxial corolla lobe folding over one or both of the lateral lobes (figs. 158, 16B). As the corolla throat elongates, it enlarges radially but to a lesser degree basally, resulting in the formation of a narrower corolla tube. In lateral view the corolla becomes

increasingly elliptic as it elongates (figs. 14A. 14C. 15A. 15C, 2 6A, 16C. 16D.

19D, 19E, 191, 20C,20D). Cilia appear along the lateral margins of corolla lobes on plants from Y0 (fig. 15A. 158). followed by plants frorn BO (not shown) on which cilia are more sparse and not visible from the exterior of the corolla. A row of slender linear trichornes initiates on the interior abaxial wall of the

corolla throat at each of the sinuses of the abaxial and lateral lobes (fig. 18D).

These continue initiating basally along the abaxial wall of the corolla throat (figs.

191, 20A). These rows occur along the abaxial ridges that lie adjacent to the abaxial interior groove (figs. 21 H, 22F-221, 23F-231,24C-24F). During elongation, filiform trichomes at the base of the abaxial corolla lobes elongate

more rapidly than those along the throat, and become confluent with the cilia along the lateral rnargins of the corolla lobes (figs. 18G, 18H, 19D, 19E, 191,

20A). As the cilia and trichomes elongate, they protrude from the folded corolla lobes at the abaxial side on plants from Y0 (fig. 21B), but not on plants from BO.

As the corolla reaches Ca. 11-1 3 mm in length, cilia protrude from folded lobes on plants from both races (fig. 22B. 22D). At anthesis, on plants from YO, the cilia are longer and more densely arranged than on plants from BO. As well the linear trichomes on plants from Y0 that occur along the corolla throat spread over its entire abaxial surface. These are more densely arranged on the ridges. On plants from BO, the trichornes on the interior of the abaxial wall of the corolla throat are more sparse and restricted to the two rows on the ridges. Longer filiform trichomes occur on the basal regions of the abaxial lobes and distal regions of the throat (figs. IA, 1B, 2)

As the corolla becomes oblong, differential growth causes indentations to appear in the exterior of the corolla tube (figs 15A, 15C, 16A, 16C. 160, 21A-

21 Dl 22A-22D, 23A-230). The troughs of the indentations represent areas subtending the points of filament attachment on the interior corolla wall. From 134 the interior of the corolla, these merge with substaminal ridges that converge more toward the median plane at the distal region of the corolla tube as the corolla enlarges (figs. 29E,29F, 29G,30C, 30F). An abaxial groove forms along the interior abaxial surface of the throat (figs. 21 BI 21D, 21 F-211, 22B, 22D, 22F-

221, 23F-231). From the exterior of the corolla, the groove appears as a longitudinal rounded protrusion with longitudinal troughs adjacent to it on either side (figs. 15A-15C, 166-16D, 21 BI 21 DI23A, 230).

Lateral expansion of the throat continues relative to the tube, and the adaxial corolla lobes rnay remain projected siightly distally (figs. 14C, 14D, 16A,

16C, 16D). During elongation of the corolla, as the protrusion and troughs form along the exterior of the abaxial corolla throat, the throat expands radially and a ridge forrns along its exterior adaxial surface (figs. 166, 16D, ZIA-21 O, 22A-22D,

23A-230). This ridge appears as an adaxial peak in transverse section (see

"Development of corolla throat aperture" below). The radial expansion of the corolla throat is more gradua1 on plants from Y0 and increases the diameter of the throat toward the apex (figs. i5A, 16A-16D, 21A, 21B, 22A, 228,23A, 23B,

24A top). After the corolla has closed, the throat elongates more on the abaxial side than on the adaxial side (fig. 15A, 15C). Thus, the bases of the lateral lobes are oblique (fig. 15A, 15C). On plants from BO, the expansion of the throat occurs as a fiaring along the lengths of the lateral and median planes (figs. 15C,

21C, 21D, 22C, 22D, 23C, 230,24A bottom). At the distal region of the corolla, the abaxial wall of the corolla projects more distally than the adaxial side in both races (figs. 216, 21D, 228,22D, 238, 23D). At anthesis, the lengths of the 135 abaxial and adaxiai corolla throat plus tube are unequal. The abaxial region extends noticeably more distally than the adaxial region (figs. 1B. 2, 24A).

Additional shape changes occur during bud enlargement and will be discussed below.

As the abaxial side of the corolla reaches ca. 11-13 mm in length, darks spots of pigment occur along the abaxial surface of the corolla tube and throat and intensify with continued growth (figs. 22E, 22F, 22H. 238,23D. 23F-231,

248, 24C, 24E. 24F). On plants from YO, the spots are small, densely arranged and a slightly darker colour of lavender-pink than the corolla lobes. On plants from BO, the spots are larger, less densely arranged, and a dark, magenta-pink which contrasts with the paler pink colour of the corolla throat.

Stamen organogenesis:

As the corolla lobes begin vertical growth, the pairs of stamens become more distinct on the floral apex (figs. 7A-7D, 8A-81, 9A-9C). On plants from Y0 and OR, the pairs of stamens are more similar in size than those on plants from

BO in which the abaxial stamens have a greater tangential width than the adaxial stamens (figs. 8C, 8D. 8F-81, 9A). Stamens enlarge laterally so that the anthers of different pairs touch (fig. 9A, 9B). Subsequently, the abaxial anthen enlarge and their margins rneet. The space between the adaxial anthers remains throughout development (fig. 90-9H). A longitudinal cleft appears on each stamen indicating formation of incipient lobes of the microsporangia, and a connectke forms (figs. 90-91, 1OA-1 OC). At the time of their formation, two of the four microsporangia per anther are directed toward the center of the bud and two 136 are directed laterally. Four microsporangia become distinct as elongate lobes, with the outemost pair confluent at the apex of each anther (figs. 9H.91, 1OE-

IOG).

When filaments initiate, differential growth at the bases of the adaxial filaments causes their anthers to tilt adaxially and toward the median plane (figs.

12A, 12H. 17A, 17E). As the coroila lobes cuwe over the stamens, the microsporangia and connectives become more distinct (fig. 12G-121). Trichomes initiate in a continuous row along the adaxial and abaxial margins of the thecae and subsequently elongate (figs. 17A-17C, 17E, 18A-18C, 18E-181, 19A. 19D,

19E, 19H. 191). Tilting of the adaxial stamens causes their anthers to sit at a lower height than the abaxial anthers (fig. 18E-181). On plants from BO, the tilting of the adaxial stamens is more pronounced, causing a greater difference in height between the adaxial and abaxial pairs than those on plants of Y0 (fig.

18F, 18G. 181). Elongation of the filaments lifts the anthers off the floor of the bud (figs. 178, 17E, 17F, 18E-181, 19A. 19D, 19E, 19G-191). Meiosis begins, first in the abaxial anthers followed by initiation in the adaxial anthers (fig. 19A).

At prophase of meiosis, corolla length along the abaxial surface is about 1.7 mm in al1 populations. Calyx lengths on plants from Y0 are 6-7 mm, but on plants frorn BO, calyx length is 4-5 mm.

The filaments become adnate to the corolla by growth subjacent to the corolla and filaments (fig. 19G. 19N, 191). Filaments then elongate. Well-formed trichornes on the anthers on plants of BO are shorter but are more stout than on plants from Y0 (figs. 19H, 191, 20B-20D). As well, anthers on plants of BO are 137 shorter at this stage (when the style is about equal in length to the stigma lobes) than those on plants of Y0 (fig. 20C,20D). However, at anthesis size of anthers

is equivalent between races. During elongation, differential rates of growth

between the pairs of filaments contribute to the placement of abaxial anthers

sitting higher in the corolla throat than the adaxial anthers (fig. 20C,20D).

Pistil organogenesis:

Constrained by the stamens and adaxial corolla lobes, the enlarging pistil

primordium becomes pentagonal in shape (fig. 8C. 8D, 8F,8H. 81). With further

growth of the perimeter, two incipient locules fom as a cavity in the center of the

pistil primordium (fig. 8D, 8F, 8H, 81, 9A). A broad transverse septum forms frorn

the base of the gynoecium separating the cavity into two distinct locules (fig. 90).

Concurrent growth of stamens surrounding the enlarging pistil yields an oval

shaped ovary (fig. 9A, 9D, 9F).

As the septum becomes more prominent, a compiturn foms along its

rnedian length (figs. 9H, IOA, 10B, IOG, 120, 12H). As a connective forms on

the stamens, a transverse cleft bisects the rim of the gynoecium, rnarking

formation of two incipient stigma lobes (figs. SG,91, 1OA, 1OD, 1OE, 1OF, 12G,

12H). On plants from YO, the ovary is almost as tall as the stamens when the

cleft forms (fig. 10D, 1OE). In plants from BO and OR, the ovary is considerably

shorter than the stamens at this stage (figs. 9D-91, 108). As the four

microsporangia become distinct on the stamens, a vertical ridge forms along the

length of each lateral wall of the pistil. Each ridge extends slightly into the gap

between an adaxial and abaxial stamen (figs. 1OF, 101, 12A-12D). Radial 138 expansion of the ovary soon obscures the ridge (fig. 17A. 17E). On plants from

YO, the pistil overtakes the stamens in height, and stigma lobes initiate as flared lips at the apex of the pistil (fig. 1OF). On plants from BO, the pistil is shorter than the adaxial stamens, and the apex of the pistil remains constricted (figs. 101,

12A, 128). The compitum continues developrnent in the center of the septum and two placentas form (fig. 12G-12H). On plants from YO, as the stigma lobes expand radially, the abaxial stigma lobe is more massive than the adaxial lobe

(fig. 12H).

As trichomes initiate on the anthers, a nectary initiates as a disc at the

base of the pistil, and ovules initiate on the placentas (figs. 12H, I7A-l7C117E,

17F, 18A, 18F, 18H, 181). On plants from YO, a style forms as a constricted

region that separates the ovary from the laterally expanded stigma lobes (fig.

17A-17C). At this stage, on plants from BO, the stigma lobes are not as well

developed and the style is not yet present (fig. 17D-17F). As trichomes elongate

on the anthers, the nectary on the basal abaxial region of the ovary on plants

from Y0 protrudes more than at the adaxial region, and the abaxial wall of the

ovary is straight (fig. 18H, 181). On plants from BO, when the style is about as

long as the stigma lobes, the pistil is as tall as the abaxial stamens, and the

abaxial wall of the ovary is convex (fig. 18F, 1BG). At the base of the ovary. the

nectary becomes more distinct, and stomatal complexes appear on its surface

(fig. 19A-19C). On plants from YO, the nectary expands abaxially, and

invaginations occur on both the adaxial and abaxial sides that separate the

nectary from the ovary (figs. 19G, 20C). The invagination is more pronounced on 139 the abaxial side. On plants from BO, the nectary is a rnorphologically undifferentiated region of thickened tissue at the base of the ovary (fig. 19H.

20D). As ovules enlarge on the placentas, an integument forms toward the nucellus of each ovule (fig. 19F).

Around the stage where the ovary is 2-3 mm in length (adaxial corolla length = 5-6 mm), the style on plants from Y0 is about equal in length to the ovary and it curves adaxially. The ovary walls are straight sided (fig . 25A, 256).

Also, the invaginations deepen on the distal side of the nectary on both the abaxial and adaxial surfaces of the ovary (fig. 25C). Styles on plants from BO are straight and shorter than those on YO, and the invaginations do not occur above the nectary (fig. 25D-25F). Also, the ovary walls are more convex on BO

(fig. 258,25E). On plants from both populations, the nectary tissue is a darker green than the ovary tissue (fig. 25C,25F). At the time of nectar initiation (ovary length = ca. 6 mm,adaxial corolla length = Ca. 11 mm), the nectaries are fully differentiated, and their general forrn remains consistent through to anthesis (figs.

26B, 26D, 27A, 278). At nectar initiation, on plants from YO, the style is still slightly curved (fig. 26A). However, on plants from BO, the elongating style is sharply directed adaxially from its base and curves distally toward the abaxial surface of the corolla (fig. 26A. 26C). At anthesis, the styles extend along the adaxial wall beyond the anthers included within the corolla throat (figs. IA, 2,

29H, 291). Development of the corolla throat aperture:

Variation in corolla aperture dimensions arnong plants of the two races of -M. lewisii is considerable. At anthesis, average height of the apertures (abaxial wall to adaxial wall along the median of the throat at its distal region) does not

Vary significantly among the populations (ca. 7.5 mm). On plants from YO, average width of the apertures (distance between lateral walls midway between the adaxial and abaxial surfaces) is significantly less than on those from BO

(mean + SE; 10.3 k 0.2 mm, 11-7 I 0.2 mm).

Corolla shape in transverse section at the base of the throat is approximately circular as the corolla reaches 6-7 mm in length along it adaxial surface. The shape at this position does not change markedly during development to anthesis (figs. 21E, 22E, 23E. 248).

As the length of the corolla along its adaxial surface reaches 6-7 mm, the circular transverse shape midway along the length of the throat is disrupted by the groove that forms along the interior abaxial corolla surface. This groove is defined by pronounced ridges that lie on either side (fig. 21 F, 21 H). At roughly the same stage, the adaxial ridge forms along the length of the corolla throat causing an adaxial peak in transverse view (fig. 21A, 21C, 22 F, 21 H). The groove, ridges and peak are more pronounced on plants frorn YO, than on plants from BO (fig. 21 F-211). Even as the groove and ridges becorne more pronounced as growth continues, the height and width of the corolla throats are similar, and the overall transverse shape is pentagonal (fig. 21 F, 21 H). These 141 shapes are conserved along the length of the throat resulting in a similar shape in transverse section at its apex (fig. 21 G, 21 1).

Around the time of nectar initiation (corolla length = ca. ilmm), the corolla throat midway along its length on plants from Y0is still pentagonal (fig. 22F). On plants from BO, the lateral walls have expanded obscuring the pentagonal shape

(fig. 22H). The abaxial ridges and groove on plants of Y0 are not as prominent as those on plants from BO (fig. 22F, 22H). At the apex of the throat, the aperture remains pentagonal in transverse section in both races (fig. 22G,221).

On plants from YO, the adaxial peak has rounded (fig. 22G), but on plants from

BO, it has become more acuminate (fig. 221).

Later, as the corolla length rapidly increases relative to that of the calyx

(corolla length = ca. 20mm) the transverse shape of the throat rnidway along its length and at the apex is generally spade-shaped (fig. 23F. 23H). On plants from

YO, both at the midpoint and at the apex of the throat, the lateral walls are straight (fig. 23F,23G). In plants from BO, the lateral walls are convex and the transverse shape is ovate (fig. 23H, 231). At anthesis, the throat at the rnidpoint on plants from Y0 remains shade-shaped, at its apex the width has increased relative to height (fig. 24C, 24D). The lateral walls remain straight (fig. 24C,

24D). On plants from BO, the relative width has increased at the midpoint and the lateral walls slope toward the interior of the fiower (fig. 24E). At the apex, the relative width has increased as well, and the walls are convex (fig. 24F). 142

Corolla lobes at anthesis:

Lobes within a corolla and among the study populations show only slight variation in size and shape (fig. 28). Withiri a flower, lobes are emarginated to truncate, and generally about as long as wide, to slightly elongated. The bases of the adaxial and abaxial lobes are truncate where they join the throat (fig. 28).

The lateral margins of the lobes are nearly parallel, tapering only slightly toward the base. The distal margin is slightly undulate with a shallow notch in the center

(fig. 28). On plants from YO, the lobes are more emarginate with ciliate lateral margins. On plants from BO, the lobes are truncate with puberulent lateral margins (fig. 28).

Among the populations, the adaxial lobes are similar in size and shape, with lobes on plants frorn BO being slightly longer. On plants from BO, the lateral lobes are greater in length and width than on plants from YO, with no significant difference in the length: width ratio. On plants from BO, the abaxial lobe is greater in length and width than on those of YO, with only a slight difference in the length: width ratio (fig. 28). Adaxial lobes on plants from Y0 are larger than the lateral and abaxial lobes. On plants from BO, al1 lobes are similar in size and shape (fig. 28).

Stamen architecture:

In both races, shortly after filament growth has raised the anthers off of the base of the corolla, the adaxial filaments are straight, and the indentations in the exterior of the corolla tube subtending the filaments result in the stamens being directed parallel with the median plane of the bud, despite the outward cuwature 143 of the corolla throat (figs. 298,29C. 30A). As the adaxial filaments elongate, they curve toward the lateral walls. thus separating the adaxially tilted anthers

(fig. 308, 30C). As well, the adaxial filaments curve adaxially, causing the adaxial stamens to extend along the adaxial surface of the corolla throat (fig.

29D-291). When the corolla is ilmm in length (around the time of nectar secretion), the corolla throat on plants from BO has fiared outward so that the lateral walls are separated from the adaxial stamens (fig. 30C). In plants from

YO, these stamens are appressed to the lateral walls of the throat in the abaxial region of the corolla (fig. 308). On plants from BO, the thecae of the adaxial anthers are more divergent and tilt more toward the median plane than in plants frorn Y0 at this stage (fig. 308, 30C).

In buds of both races, as the abaxial filaments elongate and lift their anthers off of the base of the corolla, indentations in the exterior of the corolla tube subtending these abaxial stamens direct them foward relative to the abaxial curve of the fiaring corolla throat (fig. 29A-29C). From the interior of the corolla tube, the indentations in both the adaxial and abaxial sides of the corolla appear as substantial ridges that extend more toward the median plane at the distal region of the corolla tube than at its base (fig. 30C,30F) (see Corolla organogenesis). The abaxial filaments are directed slightly toward the lateral walls of the corolla throat (fig. 30D-30F). At the sarne time, a protrusion of tissue forms at the base of each abaxial filament and extends toward the median (fig.

30D). These protrusions grow and eventually meet at the median (fig. 30E, 30F).

During elongation the abaxial filaments cuwe toward the lateral walk of the corolla throat and the anthers lie on the trichornes on the ridges of the abaxial corolla throat (figs. 22G, 221, 30G-301, 31A, 31B). As with the adaxial stamens, in plants from BO, the pronounced lateral flaring of the corolla throat keeps its walls separated from the curved abaxial stamens (figs. 221, 30H, 301). In plants from YO, the abaxial stamens stay more appressed to the lateral walls of the corolla throat as they elongate (figs. 22G,30G). The curvature of the filaments becomes less pronounced as they elongate. At anthesis, both pairs of stamens lie along the median adaxial surface of the corolla throat where they are kept under tension by the corolla throat (fig. 31C, 310). The anthers lie subadjacent to the stigma, which is inserted within the corolla throat (fig. 31C). During development, the thecae of the anthers have become divergent and are directed abaxially. Curvature at the apex of the abaxial filaments also tilts the abaxial anthers toward the median plane. At anthesis, in plants from BO, the adaxial trajectory of al1 stamens is greater than that on plants from Y0 (fig. 29H. 291).

Allometry:

For each population, a regression analysis was performed on bud length

(length of the adaxial side of the calyx) from about 2mrn to anthesis against time as days to anthesis (fig. 32, table 1). Although relationships are best expressed by sigrnoidal equations, allometric analyses require relationships be expressed as a straight line. Average growth rates of buds differ behnreen the two primary study populations (fig. 32, table 1). Buds on plants from Y0 grew at a significantly slower rate than those on plants of BO. Duration of bud growth was measured as the number of days between initiation of prophase in meiosis (6 -7 145

mm in plants of Y0and 4 - 5 mm in plants of BO) and anthesis. Durations of

bud growth (mean + SE) differed at the P > 0.05 level between buds on plants

from Y0 (13.4 k 0.1) and BO (11.4 2 0.3). Calyx lengths did not differ

significantly at anthesis (table 2).

Without age data, changes in allometric relationships cannot be

interpreted as the result of heterochronic evolutionary processes. Also, the

phylogenetic relationships among the races and their populations of M. lewisii are

not precisely known. Thus, differences in developmental trajectories cannot be

polarized as ancestral or derived. However, comparative heterochronic

interpretations can be made among the populations, as differences in rates of

growth, and differences in timing of initiation and termination of growth. Aside from age in the growth analysis of buds, no independent variables were used.

Instead, index variables were used. The organ chosen as the index variable for

any given allometric analysis had to have a consistent length among the races at

anthesis, and had to be highly correlated with calyx length. Because lengths of

organs within a flower can be considerably different from one another between

races at anthesis, the index variable chosen from the variables that met the

criteria was done so because it had the closest straight-line relationship with the

variable being examined. The criterion of invariance did not need to be met

when comparing development of organs within a flower,

Corolla length along the adaxial surface (measured as the length of tube

plus throat) meets the criteria required for use as an index variable. Among the

races, the length at anthesis does not differ significantly (table 2). in both races, 146 corolla growth is well correlated with that of its calyx. An allometric plot of corolla length along the abaxial surface versus that along the adaxial surface. and subsequent regression anaiysis show a slightly faster average rate of growth of the abaxial surface of the corolla on plants from Y0relative to that of BO (fig. 33, table 3). The abaxial corolla lengths differ slightly among these populations at anthesis (table 2).

Within a fiower, corolla growth along the abaxial surface is faster than that along the adaxial surface (fig. 34, table 4). The abaxial lengths are greater at anthesis in both races (table 2).

In both races. the lengths of the corolla tube along the abaxiai surface are similar at anthesis and can be used to measure variation among the races in the length of the adaxial corolla tube (table 2). Differences in growth rate of the adaxiat corolla tube were observed (fig. 35, table 3). An allometric plot and regression of the length of the adaxial corolla tube versus the abaxial corolla tube throughout development indicate a significantly higher growth rate for plants from

BO (fig. 35, table 3). Within a flower, on plants from YO, the lengths of the abaxial and adaxial corolla tube differ significantly (table 2). The growth rate of the abaxial corolla tube is increased relative to that of the adaxial corolla tube

(fig. 36, table 4). Growth rates of the adaxial corolla tube and abaxial corolla tube were similar in plants of BO (fig. 37,table 4). as were their lengths at anthesis (table 2).

Abaxial stamens are significantly longer at anthesis on plants from BO relative to those of Y0 (table 2). An allometric plot using length of the adaxial 447 stamen as the index variable shows a faster rate of growth of the abaxial stamens relative to adaxial stamens on plants from BO (fig. 38, table 3).

Within a flower, abaxial stamen lengths are greater at anthesis than are those of the adaxial stamens in both races. This is due to a faster rate of growth of the abaxial starnens (fig. 39, table 4).

Among the races, style lengths differ at anthesis (table 2). However, average growth rates of the style do not differ significantly between 80 and Y0

(fig. 40, table 3). 148

Discussion

As observed in plants among populations of !& cardinalis, ontogenetic variation among populations of the races of M. lewisii is slight. However, the ontogenetic variation among these races is more substantial than in M. cardinalis- This may be intuitive based on the more divergent morphologies within M. Iewisii, nevertheless, variation arnong morphologies misrepresents the variation among the underlying ontogenies that generate them. As reported in & cardinalis, many of the differences that occur during floral developrnent, at least in part, explain the divergences in floral form. Other differences have negligible effects at anthesis.

Variation in morphological development

As observed in !&. cardinalis, calyx characteristics differed throughout development. Some of these differences (e.g. smaller, more rounded sepals of

BO versus those of Y0 at a similar stage) are accounted for by allometric relationships governing calyx length (see below). Other differences (e-g. more bulbous calyx tube of BO versus that of YO) may or may not be associated with altered rates or timing of growth between the races. The more bulbous calyx tube of BO (which would require a more rapid rate of radial growth in BO or a longer duration of growth) is likely necessary to accommodate its more laterally expansive corolla.

By the time corolla lobes of al1 populations of both races have folded over the inner floral organs, differences in lobe sizes and shapes were observed.

Lobes of Y0 were larger than those of BO at this point, yet lobes of BO were 149 generally larger at anthesis. Also, corolla lobes of Y0 had undulating distal

margins, whereas those on BO were entire. These differences were reflected at anthesis. Corolla lobe characteristics (Le. size, shape and degree of

recurvature), along with the elongated abaxial corolla wall relative to the adaxial corolla wall, provide visiting bees with a landing platform. Lobes with undulating margins may render the corolla more conspicuous to an approaching pollinator.

The laterally wide corolla throat of both races is typical of bumblebee- pollinated flowers and facilitates entry of a bee into the fiower toward the nectar.

These throats are wider in BO than they are in YO, and the walls are more rounded, perhaps suited for entry of a larger bee (which may be facilitated by the larger abaxial and lateral lobes, thus larger landing platform). During landing, a visiting bee may grasp cilia or trichomes along the bases of the abaxial corolla lobes. Once on the corolla, the insect can use the yellow hair - lined ridges as guides to nectar (Sutherland and Vickery 1993).

In both species, starnen characteristics put the anthers in a position, and facing a direction, that facilitates specific placement of pollen on the body of a bee fotaging for nectar inside the flower. Lengths of the filaments are important in placing the anther pairs in tandem inside the corolla throat (see below).

Specific stamen architecture (including features of the corolla tube) places the four anthers along the adaxial wall of the corolla at the median plane facing abaxially and obliquely toward the median plane. This orientation is achieved by a combination of the indentations in the exterior of the corolla tube (internai ridges subjacent to bases of filaments), and curvature of the filaments. The 150

outward curvature of the filaments results in these filaments putting outward pressure on the lateral walls of the corolla. Although this isn't readily evident by observing gross morphology of the flowers, this phenornenon may function during the working of a flower by the bee. If stamens are held under tension by the corolla, then movement of the corolla caused by a foraging burnblebee would cause movement of the anthers. This might increase pollen deposition on the bumblebee, or affect the location of pollen placement.

In early ontogeny (Le. prior to trichornes initiating on anthers), pistil characteristics differ between the species. Prior to initiation of a style, pistils on plants of BO are shorter than those on piants of YO. As well, walls of the ovary in BO are convex, whereas in YO, they are straight sided. These differences are not readily evident at anthesis, however.

Almost immediately following its initiation, nectary development differs between the races. On plants of Y0 and JV, the nectaries are well delineated by deep invaginations in the ovary walls. On plants of BO and OR, the nectaries are not associated with these invaginations. In plants of Y0and JV, these nectary characteristics rnay function as a hinge that allows the ovary to be defected toward the adaxial surface. This may facilitate the probing of a bee toward the base of the fiower where space is limited in flowers of these populations, which have narrower (less flared) corollas. Nectar is available only from between the bases of the abaxial filaments, which are at the same level as the apex of the ovary. Thus, a bee has to probe under the ovary where nectar collects. Aliometric/heterochronic variation among races

Among the populations, calyx (bud) development varied with respect to

rate and duration of growth. Timing of calyx initiation was late in plants of BO

(also noted in OR) relative to that in plants of Y0 (and JV). However, the

relatively shorter duration of growth can probably also be attributed to an early termination, as the effect of the altered times of initiation seem to have been

minimal. The effects of a longer duration of calyx growth in plants from Y0 seern to have been negated by the slower growth rate relative to that in plants of BO as

their average lengths at anthesis did not differ.

These patterns are similar to those seen within & cardinalis and it was

hypothesized that alterations in calyx growth (especially alterations in early

ontogeny) may not be as disruptive to the growth of the rest of the bud, given the

calyx is the outermost whorl. Alternatively, in M. lewisii, calyx length probably

does not contribute to plant-pollinator interactions and, therefore, is not as

strong ly subjected to selective pressures unless the protective function of the

calyx is disrupted. Random, non-advantageous changes may not be as likely to

be filtered out by naturai seleciion.

The changes in the growth trajectories of the calyx within a flower had

opposing effects on the mature length, and were effectively nullified. That is, the

differences in morphology belied the differences in ontogeny between the

populations.

Timing in initiation of the corolla did not Vary among populations. In plants from YO, the rate of growth of the abaxial surface of the corolla (measured as 152 tube plus throat) was slightly greater than on plants from BO. This seems to be the sole factor contributing to the slightly greater length in corollas from Y0 at anthesis.

Lengths of the corolla tube along the abaxial and adaxial surfaces were generally similar among populations and within a Rower. In plants from YO, the length of the adaxial corolla tube was less than that of the abaxial tube. and less than that of both walls of the tube in plants of BO. This was observed to be the result of a decrease in growth rate in Y0relative to BO and the correspondingly shorter abaxial wall of the tube. This seems counterintuitive considering the length of the total adaxial corolla (tube plus throat) in Y0 was greater at anthesis than that in BO. This indicates a dissociation of growth within the connate corolla and was also observed in corollas of M. cardinalis.

Timing of stamen initiation was sirnilar among the populations, as was the timing of termination of elongation. The rate of growth of the abaxial starnens was lower in Y0 relative to the growth rate of these stamens on plans of BO, and can account for the shorter length at anthesis in YO.

Although differences in characteristics of the style varied among the populations, the general timings of initiation were consistent. A slightly higher rate of growth of styles in plants of BO can account for their greater length at anthesis. Position of the stigma at anthesis is crucial in determining the receipt of outcross pollen from the body of the visiting bee. Thus, differences in the position of the stigma affect the efficiency of pollen receipt from the visiting bumblebee, or, these differences accommodate a differently shaped bee. 153

Ontogenetic variation within a flower

Within an organ (e-g. corolla) or organ complex (e-g. androecial whorl of stamens) variations in lengths ai maturity exist between abaxial and adaxial regions or pairs. As observed in cardinalis, the changes in ontogeny between these regions or pairs within a flower (total length of corolla, length 3f corolla tube, and length of stamens) were limited to changes in growth rates. The differences in growth rates between the abaxial and adaxial corolla walls result in the abaxial wall extending beyond that of the adaxial wali. As indicated above, this contributes to the formation of the landing pad fomed by the abaxial wall plus the abaxial and lateral corolla lobes.

The differential rates of growth between pairs of stamens result in the tandem arrangement of the anther pairs at anthesis. The differential rates of growth are the predominant factor in achieving this arrangement, versus alterations in growth within a corolla tube (therefore. alteration in level of insertion of the filaments onto the corolla).

Conclusions

Among the characters examined, alterations in ontogeny were inferred with respect to the three parameters tested in analyses of heterchrony

(comparative analyses of changes in growth among related taxa): rate of growth, and timing of initiation and termination of growth. Among the four organ complexes, the majority of differences among the populations were differences in rates of growth. The only other differences within these trajectories were observed in calyx growth. 154

Bumblebee pollination syndromes are reported in widely diverse taxa.

Unlike some other pollination syndromes (e.g. hummingbird pollination), bee pollinated taxa seem to exhibit a variety of diverse forms that characterize bee- pollinated flowers. Some characteristics that confer bee pollinator specificity include bell-shaped corollas, lower corolla lobes that provide a landing platForm for the visiting bee, nectar guides, anthers and stigma included within the corolla, and a prominent three - dimensional form. Bees will visit flowers of various colours that include yellow, pink, blue and purple.

The flowers of M. iewisii are regarded as having a classic bee-pollinated form. The observed allometric relationships, in part, generate this morphology that is well suited to its pollinator. However, the other aspects of development described in this chapter contribute to the morphology, as well. 155

Fig. 1 A, Lateraf views of fiowers of M. lewisii, Yosemite population (YO) pale pink race (top) and Boise population (BO) magenta race (bottom). B,

Longitudinal sections of fiowers of M. lewisii showing internai fioral structure. A.

Scale = 10 mm; a = stamen primordium or anther; ab = abaxial side; ad = adaxial side; ar = anther removed; c = petal primordium, corolla or corolla lobe; ct = corolla tube; cth = corolla throat; cr = corolla or corolla lobe removed; f = filament; fa = floral apex; g = pistil primordium; ia = inflorescence apex; k = sepal primordium or calyx lobe; kr = sepal or calyx lobe removed; I = locule; Ip = leaf primordium; Ir = leaf removed; n = nectary; O = ovary; ov = ovule; p = pedicel; pl

= piacenta; se = septum; st = stigma; sy = style. Adaxial side is at the top of al1 images unless otherwise specified.

157

Fig. 2 Lateral view of longitudinal section of flower of M. lewisii (BO) showing dimensions used in allûmetric analyses. Not shown; adaxial filament, adaxial corolla tube, abaxial corolla length. Scale = 10 mm.

159

Fig. 3 A, B Polar views of inflorescence apices and floral apices of M lewisii, Y0(A) and BO (B) (SEM) showing decussate floral and leaf arrangement and offset development of floral apices at a node. Scaies = 100 Pm.

161

Fig. 4 Early floral apex development of M. lewisii (SEM). A, Abaxial view of floral apex of Y0 prior to floral organ initiation. B, Polar and C, lateral views of

BO showing initiation of discontinuous rirn meristem (arrowheads). Scales = 50

Pm.

Fig. 5 Early floral development of lewisii (SEM). A, Polar view of Y0 showing nrn meristem of incipient calyx (arrowheads). B, Polar view of Y0 showing formation of sepals and initiation of petals. C, Lateral oblique view of

Y0showing initiation of petal primordia at lower margins of inner floral apex, and adaxial sepal projecting more radially than the other sepals. D. Polar view of BO showing simultaneous initiation of al1 petals. Scales = 50 Pm.

165

Fig. 6 Early development of floral apices of M, lewisii (SEM). Polar view of

Y0(A). JV (C)and OR (E) showing simultaneous initiation of al1 stamens.

Lateral view of Y0 (B). and oblique views of BO (D) and OR (F) showing taller sepals of Y0 relative to those of BO and OR, and starnen prirnordia larger than the petal primordia. Note pedicel raising floral apex off shoot (B). Scales = 50

Pm-

167

Fig. 7 Early development of fioral apices of M, lewisii (SEM). Polar (A, C) and lateral (6,D) views of Y0 (A, B) and BO (C, D) showing gynoecium initiation

(arrowhead in A). Note suppressed adaxial calyx lobe in BO (C, D), and adaxial petai primordia closer to each other than are other petals (A, C). Scales = 100 m.

169

Fig. 8 Floral buds of M. lewisii (SEM). A-C, Polar (A, C) and lateral (B) views of Y0showing acute and talt sepals, and trichorne initiation on sepals and pedicel. D, Polar view of Y0 showing elongating trichomes, and locule fomation in the pentagonal gynoecium (arrowhead). E, Lateral view of Y0 showing calyx tube fomation (arrowhead). F, G, Polar and oblique view of BO showing shorter, rounded sepals. H, 1, Polar and oblique views of OR showing shorter sepals and truncate petals. Scales = 100 Pm.

171

Fig. 9 Floral buds of M. iewisii (SEM). Polar (A) and lateral (B, C) views of

BO showing initiation of trichomes on calyx and pedicel, vertically enlarging petal lobes, calyx tube formation (arrowhead in B), and calyx equal to the stamen in height. Note shorter adaxial calyx lobe. D, E, Polar (D) and oblique (E) views of

BO showing septum development (arrowhead in D), longitudinal cleft in anthers

(arrows) and corolla much shorter than the anthers. F, Polar oblique view of OR showing similar catyx developrnent as BO, and trichomes initiating on calyx. G,

Lateral oblique view of BO with right abaxial calyx lobe removed showing a shallow cleft (arrow) at the apex of the gynoecium. H, Polar view of BO showing elongating trichomes, and confluent microsporangia on the anthers (arrowhead).

1, Lateral oblique view of BO with abaxial and one lateral calyx lobe rernoved, showing corolfa throat, corolla taller than anthers, cleft on the apex of the gynoecium (arrow), and connective on the anthers (arrowhead). Scales =

100 Pm.

173

Fig. 10 Floral buds of M. lewisii (SEM). A, Polar view of Y0with calyx lobes removed showing cleft on anthers. B, Polar view of BO with one lateral and two abaxial calyx lobes removed showing inward curving of calyx and corolla lobes, and compitum (arrow). C, Polar view of Y0 with one abaxial calyx lobe removed showing offset lengths of trichornes and tight juxtaposition of al1 floral organs. D, Lateral view of C showing corolla lobes at about the same height as stamens and gynoecium. E, Lateral oblique view of Y0 with calyx lobes and one abaxial and lateral corolla lobe removed showing folding of truncate corolla lobes, and height of gynoecium subequal to stamens. F, Adaxial oblique view of

Y0with calyx and corolla removed showing flaring of apex of the gynoecium, and formation of longitudinal ridge along lateral walls of the ovary (arrow). Note microsporangia joined at the apex (arrowhead). G, Polar view of BO with one lateral and two abaxial calyx lobes, and one adaxial and one lateral corolla lobe removed showing inward curving of calyx and lobes and corolla lobes, trichorne elongation on the calyx, and compitum (arrowhead). H, Lateral oblique view of

BO with abaxial calyx lobes removed showing corolla throat, and conduplicate fotding of the calyx lobes (arrowhead). 1, Lateral view of longitudinal section of

BO showing longitudinal ridge along the lateral margins of the ovary (arrow) and relative heights of the floral organs. Scales = 100 Pm.

175

Fig. 11 Floral buds of M. lewisii (SEM). A, B, C,Abaxial views of BO (A,

C) and Y0(B), and lateral view of Y0 (D) showing elongating calyx lobes and tube, and elongating trichomes, and unequal trichorne lengths on calyx. Scaleç =

500 Pm.

177

Fig. 12 Dissected buds of M. lewisii (SEM). A, adaxial view of BO showing filament formation (arrowhead) and longitudinal ridge along lateral surface of the ovary (arrow). B, Lateral oblique view of BO showing slight flaring of apex of gynoecium, longitudinal ridge on gynoecium, and gynoecium shorter than stamens. C, Polar view of BO with one lateral and two abaxial calyx lobes removed, and one lateral and abaxial corolla lobes removed showing widening locuies of the gynoecium. Dl Close up view of C showing initiation of compitum

(arrowhead), and ridge along lateral walls of the gynoeciurn (arrow). E, Lateral view of C, D showing gynoecium subequal to stamens in height, and longitudinal folding of calyx lobes. F, Lateral oblique view of BO with lateral and one abaxial calyx lobe removed showing imbricate margins of folding corolla lobes. G,Polar oblique view of Y0 with calyx lobes removed and one adaxial and one lateral corolla lobe removed showing flaring of incipient stigma lobes. H, Polar view of

Y0 with calyx and corolla lobes removed showing formation of unequal stigma lobes on the ovary, enlarging compitum (arrow), and nectary at base of ovary

(arrowhead). 1, Oblique view of longitudinal section of Y0 showing formation of a placenta in the ovary. Scaies = 100 Pm.

179

Fig. 13, Buds of M. lewisii with calyx removed (SEM). A, Polar view of Y0 showing acute corolla lobes that do not overlap at their bases (arrow). B, Polar view of Y0 showing radially enlarged and retuse folded corolla lobes that do not obscure the gynoeciurn. C, Lateral view of B (YO) showing globular shape of the corolla. D, E, Polar (D) and lateral (E) views of an incompletely closed corolla of

BO showing oblong folded lobes and their non-overlapping bases (arrows).

Scales = 100 Pm.

181

Fig. 14 Buds of M. lewisii with calyx removed (SEM). A, C, Lateral views of BO showing corolla tube formation and elongating corolla throat. B, D, Polar views of BO showing obtuse corolla lobes with no notches along the distal margins. Scales = 1.O mm.

183

Fig. 15 Buds of M- lewisii with calyx removed (SEM). A, Lateral view of

Y0 showing elongating corolla throat, corolta tube with a pair of lateral indentations along its length (arrowheads), protrusion along the abaxial surface of the throat (arrow) corresponding to interior groove. Frorn the exterior, ridges adjacent to the abaxial interior groove appear as longitudinal troughs. Note cilia along the lateral margins of the adaxial corolla lobes (arrow with asterisk). 8,

Polar view of Y0showing notch in middle of distal margin of corolla lobes

(arrowhead), cilia along lateral margins of adaxial and lateral corolla lobes, and protrusion corresponding to the internal groove along abaxial surface of corolla throat (arrow) with troughs along either side. C, Lateral view of BO showing adaxial flaring of corolla throat, corolla tube with indentations (arrowhead), longitudinal protrusion corresponding to internal groove (arrow) and absence of cilia along margins of corolla lobes. Scales = 1.O mm.

185

Fig. 16 Buds of M. lewisii with calyx removed (SEM). A, Adaxial view of

Y0showing incomplete folding of adaxial corolla lobes, and radial expansion of corolla throat. Arrowheads indicate indentations forming in the exterior of the corolla tube. Arrow indicates longitudinal ridge along adaxial surface of the corolla. B, Polar view of Y0 showing elongating cilia along the lateral margins of corolla lobes. Arrowhead indicates ridge along adaxial surface of corolla. C,

Lateral view of Y0 showing protnision (interior groove) and adjacent trough along abaxial surface of corolla throat. Arrowhead indicates indentation foming in the exterior of the corolla tube. Dl Lateral oblique view of Y8 showing longitudinal ridge along the adaxial surface of corolla throat (arrowhead) and loosely folded corolla lobes. Arrowhead indicates indentation forming in the exterior of the corolla tube. Scales = 1.O mm.

187

Fig. 17 Longitudinal sections of buds of M. lewisii (SEM). A, Lateral view of Y0 with calyx and rnost of corolla removed showing stigma lobes, style initiation, nectary initiation, and initiation of trichomes on anthers, and tilted adaxial anther (at right). B, Y0showing style, and trichomes on adaxiaI anthers in closed globose corolla. C, Abaxial view of Y0 showing ovules initiating on a placenta. D, Oblique view of BO with one abaxial stamen removed showing pistil shorter than starnens. Note constricted apex of gynoeciurn. E, Lateral view of longitudinal section of BO showing trichomes initiating on anthers, style formation and initiation of nectary. Note adaxial anther tilted toward the median plane. F,

Lateral oblique view of BO showing ovules initiating on the placentas as style begins elongation. Scales = 500 Fm.

189

Fig. 18 Dissected floral buds of M. lewisii (SEM). A, Lateral oblique view of Y0with calyx, corolla, one adaxial stamen and one abaxial stamen removed showing tilting of adaxial stamen. B, Polar view of Y0 with calyx and corolla rernoved showing fiaring of unequal stigma lobes and tilted adaxial anthers. CI

Oblique view of B (YO) showing filament elongation and adnation to corolla, and anther trichomes elongating. D,Oblique view of Y0 showing trichornes initiating on the interior abaxial corolla wall at the sinus of the abaxial and lateral corolla lobe (arrowhead). Sale = 100 Pm. E, Lateral view of Y0showing short filaments and trichome initiation on one anther of each pair. F, Lateral oblique view of BO with calyx, portion of corolla and one abaxial stamen removed showing pistil at the same height as stamens, and nectary at the base of the ovary. G, Lateral view of BO with calyx, part of corolla, and one adaxial stamen removed showing extreme tilting of adaxial stamen, convex lateral walls of the ovary, and filiform trichomes at the bases of the lobes (arrow). HI Laterai view of longitudinal section of Y0 showing placenta development, straight lateral walls of the ovary, nectary expanding abaxially, and filiforrn trichomes at the bases of the abaxial corolla lobes (arrow). I, Lateral view of Y0with calyx, corolla. one abaxial stamen and one adaxial stamen removed showing different heights of abaxial and adaxial starnenç. Scales = 500 pm (except D).

Fig. 19 Dissected floral buds of M. lewisii (SEM). A, Oblique view of Y0 with calyx, corolla, and abaxial stamens rernoved showing nectary expanding abaxially, and style elongating. Note continuous row of trichomes along margins of adaxiai anthers. B, Nectary of Y0(same as A) showing stomatum. Scale =

10 Pm. C,Higher magnification of A (YO) shswing nectary differentiation. Scale

= 100 Fm. D, Lateral oblique view of longitudinal section of Y0with calyx removed showing ovule development, gradua1 lateral expansion of ovary walls toward its base, and filiform trichomes (arrowhead). E, Longitudinal section of

Y0with calyx, part of corolla, and adaxial stamens removed showing stamens appressed to lateral walls of corolla, elongated style and papillate stigma lobes.

Arrowhead indicates filiform trichomes aiong bases of abaxial corolla lobes. F,

Placenta of Y0showing nucellus and integument (arrows) formation on each ovule. Sale = 100 Pm. G, Lateral view of longitudinal section of Y0showing shallow invagination separating ovary from nectar-(arrowheads). Note that invagination is more pronounced on abaxial side. Hl Lateral view of longitudinal section of BO with calyx removed (remaining corolla and stamens displaced) showing ovule development, curvature of adaxial ovary wall, and adnation of abaxial filament to corolla. 1, Y0 showing adnation of abaxial filament to corolla, trichomes elongating along the inner abaxial wall of the corolla, and continuing to initiate toward the base of the corolla throat (arrowhead). Note corolla tube formation at region subtending filament insertion. Arrow indicates filiform trichomes. Scales = 500 prn for A, Dl E, G and H.

Fig. 20 Dissected floral buds of M. lewisii (SEM). A, Lateral view of longitudinal section of Y0 with stamens and pistil removed showing trichomes along the length of the inner abaxial wall (arrowhead), and filiforrn trichomes at bases of abaxial corolla lobe (arrows). 6,Longitudinal section of Y0with calyx and corolla removed showing elongation of trichornes on adaxial anthers, invagination separating adaxial side of ovary from adaxial side of nectary (arrow), and pistil taller than stamens. C, Lateral oblique view of Y0 showing nectary, and abaxial anther larger than adaxial anther. D, Lateral view of BO showing short, stout trichomes on anthers, pistil about equal Zn height to abaxial stamen, difference in height between abaxial and adaxial stamens, and anthers shorter than filaments. Arrow indicates nectary. Scales = 500 Pm.

Fig. 21 Corollas of M. lewisii at similar stages of development (adaxial corolla length = 6-7 mm.) (dissecting micrograph, DM). A, C, Adaxial view of Y0

(A) and BO (C) showing lateral expansion of corolla throat, indentations in exterior of corolla tube (arrows) and formation of longitudinal ridge along the exterior adaxial surface (arrowheads). B, D, Lateral view of Y0(B) and BO (D) showing dorso-ventral expansion of corolla throat, and formation of protrusion

(interior groove) along abaxial surface of corolla throat (arrowheads). Note cilia protruding from folded corolla lobes (astensk). E, Transverse section of Y0 at the base of the corolla throat showing general circular shape. F, G, Transverse section of Y0 (F) at the middle and apex (G) of the corolla throat showing groove along interior abaxial surface (arrowheads) with ridges on either side, and ridge along exterior adaxial surface (arrows). Transverse section of BO at the middle

(H) and apex (1) of the corolla throat showing pentagonal shape of corolla in transverse view, groove aiong abaxial surface (arrowheads) and ridge along adaxial surface (arrows). Scales = 1.O mm.

Fig. 22 Corollas of M. lewisii at similar stages of developrnent (adaxial corolla length = 11-13 mm.) (DM). A. C, Adaxial view of Y0 (A) and BO (C) showing lateral expansion of corolla, indentations in corolla tube (arrows) and longitudinal ridge along the adaxial surface (arrowheads). B. DlLateral view of

Y0 (B) and BO (D) showing dorso-ventral expansion of corolla, and cilia protruding from folded corolla lobes (asterisks). E, Transverse section of Y0 at the base of the corolla throat showing general circuIar shape. F, G, Transverse section of Y0 (F) at the middle and apex (G) of the corolla throat showing groove along abaxial surface (arrowhead) and ridge along adaxial surface (arrow). Note trichomes (asterisks) along abaxial ridges adjacent to groove. H, Transverse section of BO at the middle of the corolla throat showing rounded, laterally flared shape of the corolla. Arrowhead indicates abaxial groove, arrow indicates adaxiai ridge, asterisk indicates trichomes along abaxial interior ridges. 1,

Transverse section of BO at the apex of the corolla throat showing pentagonal shape of corolla in transverse view, groove along abaxial surface (arrowhead), and ridge along adaxial surface (arrow). Asterisk indicates trichomes along abaxial interior ridges. A-D, Scales = 5.0 mm, E-1, Scales = 1.O mm.

Fig. 23 Corollas of M. lewisii at similar stages of development (adaxial corolla length = ca. 20 mm). A, Adaxial oblique view of Y0showing gradual lateral expansion of the corolla throat toward the apex. BI Lateral oblique view of

Y0showing gradual dorso-ventral expansion toward the apex, and cilia protruding from folded corolla lobes (asterisk). C, DI Adaxial (C) and lateral (D) views of BO showing lateral and dorso-ventral flaring of the corolla throat, and cilia protruding from corolla lobes (asterisk in D). E, Transverse section of Y0 at the base of the corolla throat showing generai circular form. F, Transverse section of Y0 midway along the corolla throat showing shade-shape of corolla.

Arrow indicates adaxial peak of corolla throat, arrowhead indicates abaxial groove. G, Transverse section of Y0at the apex of the corolla throat showing pentagonal shape of corolla. H, Transverse section of BO midway along the corolla throat showing convex flaring of the lateral walls. 1, Transverse section of

BO at the apex of the corolla throat showing rounded spade-shape. A-DI Scales

= 5.0 mm. E-1, Scales = 1.O mm.

Fig. 24 FIowers of M. lewisii (B-FI DM). A, Lateral views of Y0 (top) and

BO (bottom) showing ridge along adaxial surface of the corolla throat

(arrowheads). Scale = i0.0 mm. 6-DI Transverse sections of corollas. B,

Section of Y0at the base of the corolla throat showing circular shape. CI

Section of Y0 midway along the corolla throat showing shade-shape. Arrow indicates adaxial peak, arrowhead indicates abaxial groove of corolla throat. D,

Section of Y0 at the apex of the corolla throat showing relative widening of corolla aperture and narrow median groove. E,Transverse section of BO midway along the corolla throat showing widening of corolla throat. FI

Transverse section of BO at the apex of the corolla throat showing rounded lateral fiaring of the corolla. 6-F, Scales = 10.0 mm.

Fig. 25 Dissected floral buds of M. lewisii (DM). A, Dl Lateral views of longitudinal sections of Y0 (A) and BO (O)showing nectary, and relative placement of anthers, style and stigma lobes. B, El Lateral views of pistils of Y0

(B) and BO (E) showing ovary and style at similar lengths. C,F, Close up views of B (YO) and D (BO) showing invaginations (arrows) that separate nectary from ovary in YO, and continuous nectary and ovary of BO. Note reflexed stigma lobes in bud stage (A, B, D, E). Scales = 1.O mm.

205

Fig. 26 Lateral views of pistils of M. lewisii (DM). A, Y0 showing curvature of style toward the abaxial surface. B, Close up view of A (YO) showing nectary at initiation of nectar production. Cl BO showing pronounced curvature of style and longer lower stigma lobe. D, Close up view of C (BO) showing nectary confluent with ovary at initiation of nectar production. Scales = 1 .O mm.

207

Fig. 27 Lateral views of nectaries of M- lewisii (DM). Y0 (A) and BO (B) showing mature nectaries at anthesis. Scales = 1 .O mm.

209

Fig. 28 Line tracings of representative adaxial, lateral and abaxial corolla lobes from Y0and BO at anthesis. Values of average length (shown as vertical plane), width (shown as horizontal plane) and length : width ratios are given in mm. as well as standard error. Sample sizes are given in parentheses. Values that differ among the populations are indicated by different first superscripts (a. b). Values that differ among lobes within a fiower are indicated by different second superscripts (x, y). Ad, Lat and Ab = adaxial, lateral and abaxial. YOS (15)

Lat 21 1

Fig. 29 Longitudinal section of buds and Rowers of M. lewisii with calyx removed (A, SEM, 8-G, DM). A, Lateral view of abaxial filament of Y0 showing growth of filament toward the adaxial surface relative to the abaxial corolla wall.

Scale = 100 Pm. B, Lateral view of Y0 with pistil removed showing horizontal projection of stamens relative to the abaxial and adaxial corolla walls. C, Lateral view of BO with calyx and pistil removed showing growth of adaxial stamen toward the adaxial surface. D, E, Lateral view of longitudinal section of Y0 (D) and BO (E) showing growth of adaxial stamen along adaxial corolla wall, and horizontal growth of the abaxial stamen relative to the flaring abaxial corolla wall.

FI G,Lateral views of Y0 (F) and BO (G) showing adaxial curvature of adaxial and abaxial filaments. F-G, Arrows indicate thickening formed by indentations in exterior of corolla tube. H, 1, Lateral views of longitudinal sections of flowers of

Y0 (H) and BO(I) showing adaxial projection of stamens and pistil when tension from corolla throat is released. Note the more extreme adaxial trajectories of organs in BO (1) than in Y0 (H). B-1, Scales = 5.0 mm.

213

Fig. 30 Longitudinal sections of M- lewisii with calyces removed. (A-C, E-1,

DM; D, SEM). A, Y0with pistil rernoved showing straight projecting adaxial stamens. B, Y0 with pistil removed showing outward lateral curving of the adaxial filaments in older bud. Note the close prcximity of the adaxial anthers to the lateral walls of the corolla. C, BO (about the same age as 8)with pistil removed showing outward lateral cuwing of the adaxial filaments, and indentation cf the exterior of the corolla tube seen as a thickening adjacent to decurrent filament (arrow). Note the distance between the adaxial anthers and the lateral walls of the corolla is greater than on plants of Y0 (B), and the thecae of the anthers are more tilted and divergent. D, Y0 with pistil removed showing growth of tissue toward the rnedian on the basal region of the abaxial filaments

(arrowheads). E, Y0 showing laterally expanding region of tissue at the base of the abaxial filaments (arrowheads). F, Y0with pistil rernoved showing bases of abaxial fitaments meeting at the median plane (arrowheads), and indentation in exterior of corolla tube seen as raised area from the interior (arrow). G, Y0with pistil removed showing lateral outward curving of the abaxial filaments. H,

Abaxial view of longitudinal section of BO showing curving of adaxial and abaxial stamens. 1, BO with pistil removed showing lateral outward curving of the abaxial filaments. Note the distance behnreen the abaxial anthers and the lateral walls of the corolla. A-C, E, G-1, Scales = 5.0 mm. D, F, Scales = 1.O mm.

Fig. 31 Longitudinal sections of buds and flowers of M. lewisii. A, Y0 with calyx and pistil removed showing outward lateral curving of abaxial filaments.

Note that the filaments extend further laterally than the lateral corolla walls when released from the corolla. B, 80 with calyx rernoved showing outward lateral curving of adaxial and abaxial filaments constrained by lateral walls of the corolla. C, D, Abaxial view of longitudinal sections of Y0 (C) and BO (D) showing longer abaxial and shorter adaxial stamens directed along the rnedian of the adaxial corolla wall. Scales = i0.0 mm.

217

Fig. 32 Regression lines showing growth rates of buds from Y0 and BO populations of M- lewisii. The average growth rate of buds differs arnong the populations at the P 4 0.01 level (table 1). days from anthesis 219

Fig. 33 Allometric plot of abaxial corolla length versus adaxial corolla length for Y0 and BO populations of M. lewisii. The average growth rates of corollas along the abaxial surface differ among the populations at the P4.01 level (table 3). O 5 10 15 20 25 30 35 40 adaxial corolla length (mm) 221

Fig. 34 Allometric plot of corolla length along the abaxial and adaxial surfaces versus style tength for M. lewisii, Y0 (representative of BO). Average growth rate is faster along the abaxial side (table 4). O 5 IO 15 20 25 style length (mm) 223

Fig. 35 Allornetric plot of adaxial corolla tube length versuç abaxial corolla tube length for Y0 and BO populations of M. lewisii. The average growth rates of the corolla tube along the adaxial surface differ among the populations at the

P

Fig. 36 Allometric plot of adaxial and abaxial corolla tube lengths versus adaxial stamen length for M. lewisii, YO. The average growth rates of the corolla tube along the abaxial and adaxial side do not differ at the P<0.01 level (table 4). adaxial stamen length (mm) 227

Fig. 37 Allometric plot of adaxial and abaxial corolla tube lengths versus adaxial stamen length for M. lewisii, BO. The average growth rates of the corolla tube along the adaxial and abaxial side do not differ (table 4). adaxial stamen length (mm) 229

Fig. 38 Allometric plot of abaxial stamen length versus adaxiai stamen length for Y0 and BO populations of M- lewisii. The average growth rates of the abaxial stamen differ among populations at the Pc0.01 level (table 4). O 2 4 6 8 10 12 14 16 adaxial stamen Iength (mm) 231

Fig. 39 Allometric plot of adaxial and abaxial stamen length versus style length for M. lewisii, Y0 (representative of BO). The average growth rate of the abaxial stamen differs from that of the adaxial stamen at the P<0.01 level. Table

4 gives coefficients for Y0 and BO. style length (mm) 233

Fig. 40 Allometric plot of style length versus adaxial stamen length for Y0 and BO populations of M. lewisii. Average growth rates of styles do not differ among the populations. adaxial stamen length (mm) Table 1 Slopes + SE (b) and Regression Coefficients (?) of Regression of Adaxial Calyx Length vs. Time (as Days from Anthesis) Mean floral organ lengths and SE (in mm) at anthesis (n=30).

Population Calyx Adaxial Abaxial Ovary Style Abaxial Abaxial Adaxial Adaxial Corolta Corolla stamen tube stamen tube Y0 23.6k 0.3 34.1 k0.3 36.010.3 9,O +0,1 21,0&0,2 15.710.2 9.7kO.l 13,310.2 8.8kO.l a a a a a a a a a

Note. Means with different letters differ at the P < 0.05 level. See fig. 2 for dimensions measured. Table 3 Slopes (b f SE) and Regression Coefficients of Linear Regression Lines of Selected Allometric Relationships Among Populations

Y0 BO abaxial corolla vs. adaxial corolla b 1.02+8.17 0.99 0.11 3 0.98 0.97 adaxial corolla tube vs. abaxial corolla tube 0.90 + 0.28 1 .O2 k O. 19 f: 0.95 0.98 style vs. adaxia I stamen b 1.66 + 0.05 1.86 + 0.06 ? 0.94 0.94 abaxial stamen vs. adaxia I stamen b 1.19k0.04 1.28k0.03 8 0.94 0.98 Note. See fig. 2 for dimensions measured. Table 4 Slopes (b k SE) and Regression Coefficients of Linear Regression Lines of Selected Allometric Relationships Between Abaxial and Adaxial Dimensions of Organs Within a Flower

abaxial adaxial f P df abladaxial corolfa vs. style Y0 b 1.63 + 0.07 1.56 + 0.08 1 O. 1 3 0.00 1,127 8 0.90 O. 87 BO b 1.45 + 0.05 1.44 k 0.05 9.92 0.00 1,118 i! 0.94 O.94 a bladaxial corolla tube vs. adaxial stamen Y0 b 0.74 k 0.04 0.68 + 0.03 13.32 6 0.88 O. 88 BO b 0.77 t 0.05 0.79 + 0.05 0.1 3 ? 0.83 O.83 abladaxial stamen vs. style Y0 b 0.69 + 0.03 0.57 k 0.02 54.09 0.00 1, 127 ? 0.93 0.94 BO b 0.66 rir 0.02 0.50 f 0.02 317.56 O.00 1,118 ? 0.95 0.94 Note. See fig. 2 for dimensions rneasured. 238

Chapter three - Ontogenetic evolution of cardinalis and M. lewisii:

morphology, allometry and heterochrony

Introduction

Mimulus lewisii and l& cardinalis have been extensively studied with respect to fu nctional floral morphology that confers pollinator specificity (Heisey et al- 1971; Sutherland and Vickery 1988; Vickery 1990; Vickery 1992;

Sutherland and Vickery 1993; Bradshaw et al. 1995; Bradshaw et al. 1998;

Schemske and Bradshaw 1999). Mimulus lewisii is considered to have typical bee-pollinated flowers with a pink corolla, recurved lower corolla lobes that provide a landing platfon for bees, nectar guide, inserted anthers and stigma, and relatively low nectar volume (Sutherland and Vickery 1993) (fig. 1). Mimulus cardinalis is considered to have typid hummingbird-pollinated flowers with a long, red tubular corolla, reflexed corolla lobes, yellow tongue guide, exserted anthers and stigma, and higher nectar volume (Sutherland and Vickery 1993)

(figs. 1, 2).

Previous studies have shown that adaptation to different pollinators has provided an effective pre-mating mechanism of reproductive isolation (Vickery

1990). Natural hybrids between the species are unknown despite the fact that FI hybrids obtained from artificial crosses are shown to be vigorous and have close to normal fertility (Bradshaw et al. 1995). Because of the absence of genetic or physiological barriers to interspecific hybridization, it has been implied that lewisii and cardinalis share an ancestor-descendent relationship or that they share a common ancestor (Coyne 1995). Ongoing work at the University of Washington, Seattle on the phylogeny of this group using molecular data has confirmed this hypothesis (P. Beardsley pers. comm.). However, it remains unclear which, if either of the species is the ancestor and which is the descendent.

Recentiy, other researchers at the University of Washington, Seattle, investigated the genetic basis of the floral traits responsible for effective pollinator fidelity and, therefore, reproductive isoiation (Bradshaw et al. 1995; Bradshaw et al. 1998). Their study was conducted on sympatric populations of the species found in Yosemite National Park, CA, using analyses of quantitative trait loci

(QTLs). The authors found that for each trait examined there existed at least one

QTL (containing a gene or tight cluster of genes) that explains at least 25% of the phenotypic variation.

The extensive work done on the sympatric populations of these species provides a rare opportunity to study floral development in the context of genetic, phylogenetic and natural selection data. This comparative study of floral development has provided insight into the ontogenetic factors that may have acted during rapid evolution within this group, and has resulted in differences in floral morphology and breeding systems.

In chapter one, three widely geographically distributed populations of cardinalis were examined. In chapter two, four populations (representing both descnbed races) of M. lewisii were examined. Of the latter, only one representative population of each race was studied in greater depth. Here, the floral development of the Yosemite National Park populations of M. lewisii and M. 240 cardinalis are cornpared. The populations from Yosemite were chosen to represent each species because they have been the focus of much of the recent work on pollinator visitation and QTL analyses. Therefore, the objectives of this component of the study were to compare the ontogeny of the Yosemite population of M cardinalis against that of the Yosemite population of M. lewisii, and interpret these differences as ontogenetic evolutionary changes that must have been associated with speciation in the group. 241

Results

After initiation, in both M. cardinalis and M. lewisii the undifferentiated floral apex is globular until a rim meristern forms around its circumference. Five sepal primoridia form, more or less simultaneously on this rim, and become distinct as sepals (fig. 3A-3D). Radial growth of the rim and sepals, and verticai growth of the inner floral apex, cause a cleft to form between the calyx and inner apex (fig. 3A-3D). Thus, the inner apex is clearly demarcated from the incipient calyx.

Five petal primordia initiate simultaneously on the margins of the inner apex alternate with the sepals (fig. 3A-30). Four stamen primordia initiate on the surface of the inner apex alternate with petal primordia (fig. 3A-3D). No stamen primordium forms in the median adaxial position. Enlarging sepals are more acute on plants from M. cardinalis and have wider bases than the more rounded sepals on plants of M. tewisii which have narrower bases (fig. 4A41). After sepals and stamens begin vertical growth, an oval gynoecium initiates on the center of the floral apex (fig. 4A-4E). The globular adaxial stamen primordia, and oblong abaxial stamen primordia become distinct (fig. 4A-4E). Trichomes initiate on the bases of upright sepals and on the apical region of the pedicel (fig. 4D-

4G). Marginal growth of the gynoecium exceeds that at the center and an incipient locule forms as a cavity in the center of the pentagonal gynoecium (fig.

4F, 4H, 41). Longitudinal folding of the sepals begins at this early stage and continues as sepals and their calyx lobes elongate (figs. 41, 5A-5C). Trichomes elongate on the sepals, and increase in density as a septum initiates at the base 242 of the locule (fig. 5A-5Cl 7A-7D). A calyx tube forms, the acute calyx lobes cuwe over the inner floral whorls, and conduplicate folding of the lobes becomes more pronounced (fig. 5A-5C). On buds of M. cardinalis, the calyx lobes are more vast than those on plants of M. lewisii (fig. 5A-5C).

A longitudinal cleft forms on each stamen, marking initiation of microsporangia. A broad septum separates the two locules of the gynoecium, which has become rounded distally (fig. 5C,5D). Two transverse clefts on the apex of the gynoecium indicate formation of two incipient stigma lobes. A connective forms on each anther as four microsporangia become distinct (figs.

5D-5F, 6A, 6B). As the petals fold over the inner apex, a corolla throat forms (fig.

6A, 6B). The apex of the gynoeciurn becornes flared marking the differentiation of incipient stigma lobes on the ovary. The gynoecium now equals the stamens in height (fig. 6A, 6B). A compiturn forms on the apex of the placentas, which have arisen on the septum in each locule (fig. 7A. 7B). A nectary initiates as a radial expansion of tissue at the base of the ovary (fig. 7B-7D). The shapes of the nectaries and ovaries are similar in the species at this stage (fig. 7C,70).

After the flaring of the stigma lobes becomes more prominent, the stigma lobes on plants of cardinalis are more broadly rounded (fig. 7A). On plants of

--M. lewisii, stigma lobes are more acutely rounded. The adaxial stigma lobe is smaller than the abaxial lobe in both species (fig. 7A, 7B). A style initiates as a constricted region that separates the stigma lobes from the ovary (fig. 7C,7D).

As trichomes initiate on the anthers, filaments form, and their subsequent elongation lifts the anthers off the apex (fig. 7C-7F). In buds of cardinalis, anther trichomes are distributed over the entire surface of the rnicrosporangia but are longer along the stomia. On plants of M. lewisii, these trichornes are restricted to a continuous row that runs along the margins of the thecae (figs. 7C-

7F, 8A-8C). Differential growth within the adaxial filaments causes their anthers to tilt toward the medial plane in both species (figs. 7D-7F, 8A, 8C). As the trichomes elongate on the anthers, on plants of M. cardinalis, the style is a gradua1 narrowing above the ovary but on plants of M- lewisii the style is evident as an abruptly narrower region above the ovary (fig. 8A-8C). As the style elongates, the stigma lobes expand laterally (fig. 8A-8C). On plants from M. cardinalis, the stigma lobes are narrower and round (fig. 8B). On plants from lewisii, the stigma lobes are laterally wider, and more oblong (fig. 8A). The nectary on plants of M. cardinalis is an undifferentiated radial expansion of tissue at the base of the ovary (fig. 88, 8C). On plants of M. lewisii, at an equivalent stage the nectary is an abaxial protrusion that is separated frorn the ovary by an invagination on both the abaxial and adxial side of the ovary walls (fig. 8A).

Comparative calyx development

After the calyx lobes have enclosed the inner organs, the lobes on plants of M. cardinalis are wider, and curve inward and touch (fig. 9A). Calyx lobes on plants of M. lewisii are narrower, and project straight up or curve outward (fig.

9B). On buds of cardinalis, conduplicate folding of the calyx lobes is continued to wing-like ridges that extend basally from the lobes along the length of the calyx (fig. 9A, 9C). On buds of M. lewisii, the calyx tube has only minor ridges in areas subjacent to the conduplicate folded calyx lobes and these 244

enlarge later (fig. 9B. 9D). On plants of both species, trichome density and

lengths (which Vary within a calyx) are similar (fig. 9A, 9B). As the calyx tube and

lobes elongate, lobes on buds of M. cardinalis remain inwardly curved, and

overlap at the apex (fig. 9C). Lobes on buds of M- lewisii remain projecting

straight up or curve slightly outward (fig. 9D). As well, the calyx lobes on buds of

-M. cardinalis are broader than those of M. lewisii (fig. 9C, 9D). The broader lobes on plants of M. cardinalis cause furrowing at the apex of the calyx tube which does not occur on plants of M. lewisii (fig. 9C, 9D). Otherwise buds of both

species have a similar oblong shape, which becomes elongate tubular at anthesis (fig. 1). The calyx lobes of M. lewisii are subulate tipped at anthesis, those of M. cardinalis are acute - acuminate. The calyx of M. cardinalis is longer than that of M. lewisii at anthesis and thus encloses most of the corolla throat, whereas the distal half of the corolla in M. lewisii is exposed beyond the calyx.

Comparative corolla development

After the corolla lobes fold over the stamens and gynoecium, the overlapping lobes each develop a notch along their distal margin (figs. 1OA, IOC,

1IA, 11B). Corolla lobes on plants of both species are rounded (fig. 10A-1OD).

Lobes on plants of M. cardinalis are larger, completely overlapped, and obscure the inner organs (fig. 1OA, 1OB). Those on plants of M. lewisii are smaller and do not completely overlap at this stage (fig. 1OC, 1OD). The corolla lobes expand on plants of both species, and completely overlap on plants of M. lewisii as well as on plants of j&. cardinalis (fig. 11A, IlB). Cilia fom on the lateral margins of the adaxial and lateral lobes on buds of M. lewisii (fig. 11B), but not M cardinalis (fig. 11A). As well, on buds of M. lewisii the lobes become truncate and undulate slightly along their distal margin (fig. 116). On buds of &&. cardinalis, the larger lobes are rounded and the margins are entire except for the notch (fig. 1IA).

As the corolla throat elongates, a reduction of radial growth results in a narrow region at its base that forms an incipient corolla tube (fig. 11 C, 11 D).

Indentations fom on the exterior of the corolla tube on plants of both species

(figs. 11Dl 12A-12D, 13A-13D, 14A-14D). These indentations are protrusions in the interior of the corolla tube, and subtend the points of insertion of each filament on the interior wall of the corolla. Consequently, these indentations affect stamen architecture, and are discussed below. At this stage, the abaxial region of the corolla throat is longer than the adaxial region in buds of M. lewisii

(fig. IlD). These regions are about equal in length in corollas of M. cardinalis.

As the corollas reach 4-6 mm in length, on corollas of cardinalis, the throat is about equal in circumference along its length (fig. 124, 12B). However, the throat on corollas of M. lewisii is radially expanded toward its apex (fig. 12C,

12D). The large corolla lobes on plants of cardinalis result in a prominent radial expansion of the apex of the corolla (fig. 1ZA, 12B). On plants of M, lewisii, the corolla lobes are smaller and, therefore, the expansion of the apex is less prominent (fig. 12C, 12D). On plants of M. lewisii, cilia protrude from the corolla lobes at the abaxial side (fig. 12D). On plants of cardinalis, cilia are absent (fig. 128). In both species, a longitudinal ridge forms along the adaxial surface of the corolla throat (fig. 12A, 12C). Until this stage, in cardinalis, growth of the adaxial wall has equaled that of the abaxial corolla wall (see Allometry, fig. 31). However, with respect to these dimensions, the corolla walls of M. lewisii are unequal (fig. 12B, 120).

When the corollas reach 13-14mm in length along the adaxial surface, the walls of the throat are relatively straight on plants of M. cardinalis, and taper gradually toward the base (fig. 13A, 136). On plants of M. lewisii, the walls of the corolla throat are convex, especially on the shorter adaxial side (fig. 13C, 13D).

On plants of M; cardinalis, the prominent indentations in the exterior of the corolla tube punctuate the interface between the tube and the throat in adaxial view (fig.

13A). In lateral view, the absence of an indentation (and corresponding filament adnate to the corolla) at the adaxial position causes an undemarcated transition from tube to throat along the adaxial side (fig. 138). On plants of M. lewisii, the general characteristics of the corolla tube are similar, but the less prominent indentations in the exterior of the tube result in a more gradua1 transition from tube to throat (fig. 13C, 13D). The ridge on the adaxial exterior wall of the corolla throat becomes more pronounced in both species (fig. 13A, 13C). Cilia now protrude from within the folded corolla lobes in M. cardinalis, as well as in M. lewisii (fig. 13B, 13D). By this stage, differential growth between the adaxial and abaxial walls of the corollas of both species has occurred. In fi cardinalis, the abaxial wall is noticeably shorter than the adaxial wall (fig. 138). In M. lewisii, the abaxial wall is visibly longer than the adaxial wall (fig. 13D).

As the corollas reach 20-22 mm in Iength along the adaxial surface, the lateral walls of the corolla throat on plants from M. cardinalis are relatively straight and parallel (fig. 14A). However, the corolla throat has expanded dorso- ventrally resulting in a gradua1 increase in diameter of the corolla along the median plane (fig. 148). On plants of M. lewisii, al1 walls of the corolla throat have continued to expand laterally and thus are convex, flaring away from the median plane (fig. 14C, 14D). Continued differential growth in length between the abaxial and adaxial walls of the corolla has caused the differences in shapes between the species to become more extreme. In M. cardinalis, the apex of the adaxial corolla wall extends beyond that of the abaxial wall. In M. lewisii, the abaxial wall extends more distally than does the adaxial wall (fig. 148, 14D).

At anthesis, the aperture of the corolla is oblique in lateral view on plants of M; cardinalis (fig. lA, 18). The length along the adaxial surface of the corolla is greater than along the abaxial surface. On plants from the M. lewisii, the aperture is oblique with the abaxial length of the corolla being slightly greater than that of the adaxial length (fig. 1C, 1D). Additional differences in corolla shape occur during development, and are discussed below.

Comparative pistil development

As the style elongates, the ovary in both species is cylindrical with flattened lateral walls that taper toward the apex, and the elongating style curves abaxially. General shape and size of the pistil are similar between the species until the ovary reaches 5-6 mm in length. At this length, in buds of M. cardinalis, the style is more curved toward the abaxial surface than in buds of M. lewisii (fig.

15A, 15D). In M. lewisii the longer abaxial corolla throat accommodates the lengthening style, which will be included at anthesis (fig. AC). In buds of M, cardinalis the elongating style is accommodated in the bud by curving within the 248 corolla. When the corolla opens, the long style straightens and the stigma is positioned distal to the aperture along the adaxial side (fig. IA).

As nectar secretion begins, the nectariferous tissue becomes a darker green than that of the ovary, and the nectary is almost fully developed (fig. 158,

15E). On plants of M. cardinalis, the nectary is a bulbous region that protrudes abaxially at the base of the ovary but is not separated from the ovary by invaginations (fig.l5B, 15C). On plants of M. lewisii, the nectary is separated from the ovary by deep invaginations in the abaxial and adaxial walls of the ovary

(fig. 15E,15F).

At anthesis, the pistil is positioned along the adaxial wall of the corolla throat, adjacent to the stamens (fig. IA, 1B, 1D). The stigma occurs distally adjacent to the abaxial anthers, inserted in the corolla throat in plants of M. lewisii

(fig. 1D) and exserted in plants of & cardinalis (figs. IA. 16, 20A, 205).

Comparative development of stamen architecture

As filaments elongate, in both species, the adaxial stamen pairs grow along the adaxial wall of the corolla throat (fig. 16-16D). On plants of cardinalis, indentations in the exterior of the corolla tube subtend the adaxial filaments and the adaxial stamens are angled toward the median floral plane (not shown at this stage). On plants from M. lewisii, the indentations in the corolla tube occur at these locations, but are not as pronounced as in cardinalis (fig.

16A, 16B). Nevertheless, the adaxial filaments are slightly angled toward the median floral plane (not shown at this stage). The indentations in the corolla tube that subtend the abaxial filaments are more pronounced in M. cardinalis than in M. lewisii. Regardless, the abaxial stamens are about parallel with the median floral plane in both species (fig. 16A, 16B).

In both species, as the filaments continue to elongate (corolla length = Ca.

10 mm), each adaxial filament still occurs along the adaxial wall of the corolla

(fig. 16C, 16D). In cardinalis, the abaxial stamens are still about parallel to the median plane (fig. 16C). However, in M. lewisii, the abaxial stamens occur along the abaxial wall of the corolla and, thus, slope abaxiaily (fig. 16D).

Indentations in the exterior of the corolla tube are ridges on the interior, and are subjacent to the points of insertion of filaments onto the corolla tube. In M. cardinalis, the ridges subjacent to the abaxial filaments are augmented with a thickened region of corolla tissue (fig. 17A, 178). The corresponding abaxial filaments are directed slightly toward the adaxial surface, despite the abaxial curvature of the abaxial corolla wall (fig. 17A). The interior ridges (exterior indentations) occur in M. lewisii, but are not augmented with thickened corolla tissue (fig. 17C). In M. lewisii, the abaxial filaments are directed toward the adaxial surface of the corolla by the indentations and curvature of the filaments

(fig. 17C). However, the corollas of M. lewisii are more dorso-ventrally narrow than those of M. cardinalis. Consequently, the abaxial filaments occur closer to the adaxial wall of the corolla in M. lewisii than they do in M. cardinalis (fig. 17A, 17C).

The indentations in the regions of the corolla tube that subtend the adaxial filaments are adaxial to the transverse plane. As a result, these indentations orient the adaxial filaments toward the median plane. In buds of M. cardinalis, 250 the adaxial filaments angle toward the median plane near their bases (fig. 18A).

Further along their length, these filaments cuwe toward their adjacent lateral wall

(fig. 18A). In buds of M. lewisii, the adaxial filaments curve at their bases toward the adjacent lateral wall of the corolla and lie adjacent to the Iateral walls throughout their length (fig. 188). As the adaxial filaments elongate, in M. cardinalis, the filaments are parallel to each other (fig. 18C). In M. lewisii, the adaxial filaments are curved away from each other toward opposite lateral walls of the corolla (fig. 18D).

The indentations in the regions of the corolla tube that subtend the abaxial stamens are parallel to the median plane. Consequently, the ridges that fom subjacent to the abaxial filaments protrude both adaxially and toward the median plane (Le. toward the center of the fiower). Therefore, the indentations in the corolla tube subtending the abaxial filaments also cause the bases of the abaxial stamens to be projected toward the median plane (fig. 19A-19D). However, as the filaments elongate on plants of both species, the distal region of the abaxial filaments curve toward the adjacent lateral wall (fig. 19A-19D). On plants of M. cardinalis, the two abaxial filaments remain more adjacent to each other near the base of the corolla throat (fig. 19A, 19C). On plants of M. lewisii, the abaxial filaments curve toward the lateral wall of the corolla throat, even near their bases

(fig. 19B, 19D).

In both species at anthesis, both pairs of stamens lie along the adaxial wall of the corolla throat along the median plane (figs. IA-1D, 20A, 208). When the corolla throat is cut away, thus releasing the stamens, both pairs of stamens 25 1

spring up at a greater angle to the abaxial corolla wall in f& cardinalis than they

do in M lewisii (fig. 1B, 1D). As well, in M, cardinalis, the distal end of each

stamen crosses the median plane and the anthers are placed on the opposite

side of their origin (not shown). In M. lewisii, the stamens are directed straight,

away from the median plane (not shown). In flowers of cardinalis, the anthen

and stigma project beyond the adaxial region of the corolla throat, and al1 four

anthers are positioned with their stomia on the abaxial side (fig. 20A). In flowen

of M. lewisii, the anthers and stigma remain inserted within the corolla throat, and

the abaxial anthers are positioned so their stomia are directed toward the median

plane by curvature at the apex of each filament (fig. 208).

After the abaxial filaments lift their anthers off the floor of the floral apex, a

rounded region of tissue extends from the base of the abaxial filaments toward the median plane (fig. 21A, 21 B). As the tissue grows laterally, the two regions eventually meet at the median plane. On plants of M- lewisii, these rounded extensions remain distinct at anthesis (fig. 21 D). On plants of & cardinalis, filament elongation obscures the rounded shape of this tissue, and, at anthesis, they appear as thickened elongated strips along the interior of the abaxial filaments at their base (fig. 21 C).

Differences in corolla throat aperture

To analyze the development of the shape of the aperture of the corolla throat, transverse sections were observed at various developmental stages of the corolla throat at its base (region distally adjacent to the corolla tube), midway along its length, and at its apex (where the throat rneets the corolla lobes). Examination of the transverse shape of the corolIa throat at multiple locations throughout development was required to compare development of the shapes of

entire corollas of both species.

Transverse shape of the corolla throat at base and mid-length

At the base of the throat, shape of the transverse section in both species

is circular from the time of initiation of the tube to anthesis (not shown). As the corolla reaches 4-5 mm along its adaxial length, on plants of cardinalis the shape of the throat midway along its length is circular (fig. 224. A shallow groove has formed in the interior abaxial surface that is demarcated by two shelves on either side of it (fig. 224. A row of trichomes occurs along each of the shelves (fig. 22A). On plants from M. lewisii, the transverse corolla shape is pentagonal, and the interna1 abaxial groove is more pronounced with distinct ridges (which appear as longitudinal troughs on the exterior of the throat) on either side (fig. 228). Trichornes occur along these ridges, but are shorter and less densely arranged than those on plants of M. cardinalis (fig. 22A). In transverse view, a peak appears in both species at the adaxial surface of the corolla, which corresponds to the longitudinal adaxial ridge that forms along the adaxial surface of the throat (see "Corolla development").

Shortly before nectar initiation (adaxial corolla length = 10-11 mm) the transverse shape midway along the throat on buds from cardinalis has become pentagonal with rounded walls (fig. 22C). The abaxial groove is more pronounced, and the adjacent shelves are more distinct and ridge-like (fig. 22C).

The trichomes that occur along the shelves/ridges have elongated and their distribution has expanded laterally into the groove (fig. 22C). On plants from M. lewisii, the pentagonal shape of the corolla is more distinct with straight walls and more angled corners (fig. 22D). The trichomes that line the ridges on plants of

--M. Iewisii are much shorter than those of M. cardinalis, and their distribution extends only slightly into the abaxial groove (fig. 220).

Shortly before anthesis, transverse shape midway along the length of the corolla throat is spade-shaped in both species (fig. 22E, 22F). On plants from M. cardinalis, the throat is relatively wider, and the abaxial groove and adjacent ridges are less distinct than those of M. lewisii (fig. 22E, 22F). On plants of !&. cardinalis, the trichomes along the two ridges (referred to as shelves in earlier stages) are much taller than those that occur along the surface of the groove (fig.

22E). As well, they are much taller than those on plants of M. lewisii (fig. 22F).

At anthesis, the relative height of the corolla has increased on plants of M. cardinalis (fig. 23A) and the transverse shape is oblong with an acute adaxial peak. The transverse shape of the corolla remains spade-shaped on plants from

--M. lewisii, and the abaxial region of the throat (base of the throat in transverse view) fiares laterally (fig. 23B).

Transverse shape of the corolla at the apex of the throat

At the apex (distal region) of the throat, corolla shape in transverse section generally reflects that of the shape midway along the length (fig. 24A-24F). On plants of fi cardinalis, as the corolla reaches ca. 5-6 mm in length, the transverse shape of the throat is circular, and the peak has fomed at the adaxial surface (fig. 24A). On plants of M. lewisii, the transverse shape is pentagonal 254 with a rounded adaxial peak (fig. 24B). The adaxial peak, and abaxial ridges and groove are relatively inconspicuous (fig. 24B).

After nectar secretion begins (adaxial corolla tength = ca. 15mm), the transverse shape of the corolla in M. cardinalis is still round with an acuminate adaxial peak (fig. 24C). The abaxial groove is well defined, however, the adjacent shelves are inconspicuous (fig. 24C). Length and distribution of the trichomes are similar to those reported rnidway along the length of the throat.

The longer trichomes occur in two rows adjacent to the abaxial groove. The distribution of shorter trichomes extends into the groove (fig. 24C). In M. lewisii, the transverse shape is distinctly pentagonal with straight lateral and adaxial walls (fig. 24D). The abaxial groove and adjacent ridges are distinct, and lined with trichomes shorter than those of M. cardinalis (fig. 24D).

At anthesis, the transverse shape of corolla throats of cardinalis is laterally narrow with parallel lateral walls, an acuminate peak, and a distinct abaxial groove without adjacent ridges (fig. 24E). Trichomes have increased in length and density and are yellow (fig. 24E). In transverse view, the corollas in

--M. lewisii are laterally broad with straight walls (fig. 24F). The abaxial groove has a tall ridge on either side (fig. 24F). The trichomes that occur along the ridges are more densely arranged than they are along the groove or adjacent abaxial corolta wall (fig. 24F). The trichornes in corollas of M. cardinalis are more stout and densely arranged than they are in corollas of M. lewisii (fig. 24E, 24F).

Comparative rnorphology of the corolla lobes at anthesis 255

Corolla lobes differ greatly among the species. In addition to the lobes being fully reflexed on corollas of cardinalis and recurved on corollas of lewisii, shape differences occur between the species.

In general, lobes on plants of 4cardinalis are larger than lobes of lewisii along their vertical and horizontal axes, and are wider than they are long

(length : width ratio c 1) (fig. 25). Lobes on corollas of M. lewisii are smaller, and about as wide as long (length : width ratio = ca. 1) (fig. 25). The lateral rnargins of the corolla lobes of M. cardinalis are convex and the distal margins are rounded and srnooth with a notch in the center. On corollas of M. lewisii, the lateral margins of the lobes are straight, and the distal margins are truncate to rounded, and slightly undulate (fig. 25). On plants of M. lewisii, cilia occur along the lateral margins of al1 lobes, but are especially long and dense on the lateral and abaxial lobes. On plants of & cardinalis, cilia are restricted to the base of the lateral margins of the lateral and abaxial lobes, and are sparse.

Allometry

For each species, a regression analysis was performed on bud length

(length of adaxial side of calyx) against time (fig. 26). Average growth rate of buds on plants of M. lewisii is higher than that of buds on plants of M. cardinalis

(fig. 26, table 1). However, the duration of growth from initiation of meiosis to anthesis is shorter in plants of M. lewisii (13.4 + 0.1 days versus 16.9 k 0.4 days; mean +. SE). Calyx length on plants of M. lewisii is significantly less at anthesis

(table 2). 256

To interpret changes in temporai patterns of development as specific heterochronic evolutionary processes, allornetric relationships must be analyzed in the context of absolute time (age). Organs interna1 to the calyx could not be rneasured in absolute time. However, by using an index variable whose growth is well correlated with calyx growth (which is highly correlated with time), relationships of organ growth with time can be inferred. Between the species, ovary lengths are similar at anthesis (table 2), well correlated with time, and initiate at similar times relative to the other floral organs except the calyx. For this reason, ovary length was used as the index variable for allometric analyses.

Allometric plots of ovary length versus the length of each of the other variables used in the analysis (except calyx length) show that, in each case, the growth trajectories were similar between the species during rnost of the ontogeny. Divergences in ontogenies occurred as alterations in duration of growth.

The rate of growth of the adaxial side of the corolla was similar in both species (table 3). The greater length of this dimension at anthesis in !& cardinalis is due to late termination of growth relative to that in M. lewisii (fig 27 middle). The rate of growth of the abaxial corolla wall in cardinalis was significantly lower than that of M. lewisii, and accounts for its shorter length at anthesis (fig. 27 top, 27 bottorn, table 3).

Growth of the corolla tube along the adaxial and abaxial sides was abbreviated in buds of cardinalis relative to those of M-lewisii (fig. 28 top, 28 bottom). Growth of both pairs of stamens and the style were extended on plants of & cardinalis relative to that of M. lewisii (figs. 29 top, 29 bottom, 30).

An allometric plot of abaxial corolla length versus adaxial corolla length shows differential growth rates within an organ (fig. 31). Corolla shape with respect to adaxial and abaxial length were similar until corolla length = Ca. 10 mm

(fig. 31). At about this size, growth rate of the abaxial corolla wall remains consistent in M. lewisii, and decreases in &&. cardinalis relative to the growth rate of the corresponding adaxial corolla wall (fig. 31). The rate of growth of the abaxial corolla tube relative to the adaxial corolla tube did not differ between the species, nor did the growth rate of the abaxial stamens relative to that of the adaxial stamens. This indicates that, with respect to these dimensions, no differences in shape are associated with the differences in size between the species . 258

Discussion

The phylogenetic relationships of the cardinalis - M. lewisii group is

unclear. The rnost recent hypothesis supports the view that these species are

more closely related to each other than they are to any other species within their

monophyletic section of Mimulus, that is, they form a clade within the section.

However, this hypothesis includes three plausible phylogenetic scenarios. The

first scenario is that M. cardinalis is derived from a M. lewisii - like ancestor.

Because many of the most closely related extant taxa to these two species are

hummingbird-pollinated, this scenario carries an implication of multiple origins of

humrningbird-pollinated species within section Ervthranthe. The second scenario

is that M. lewisii is derived from a cardinalis - like ancestor. The two species

recognized as the outgroup to Ervthranthe are bee-pollinated. Therefore, this

scenario suggests a reversion back to a bee-pollinated form from a hummingbird-

pollinated form. In other words, if floral fom is considered a character, then in &

lewisii a floral form suited to bee - pollination is a pleisiornorphic character state.

That is, if this scenario is correct, then M. lewisii represents an independent

origin of a bee-pollinated species. The third scenario is that M cardinalis and M

lewisii are derived from a common ancestor that cannot be regarded as more simiiar to either of the descendents and couid have been pollinated by an organism other than bees or hurnmingbirds.

The hypothesis that M. lewisii (as exemplified by the Yosemite population) is derived from a hummingbird visited plant similar to & cardinalis (as exemplified by the Yosemite population) is reasonable given the current 259 phylogeny which shows M. lewisii embedded within a primarily hummingbird- pollinated clade Le., section Erythranthe. Therefore, the docurnented differences between M. lewisii and M. cardinalis can be used to formulate hypotheses of ontogeny, which are interpreted as evolutionary transformations from traits within a bird-pollinated taxon to those within a bee-pollinated taxon. The caveat is made that these hypothesized transformations might have occurred in the direction from a bee - pollinated tu a bird - pollinated taxon.

For the purposes of this study, early ontogeny is considered the period of development from floral organ initiation to organogenesis, Le., to the point of trichome initiation on the anthers. Trichorne initiation is convenient as a defining characteristic as it occurs at similar stages in both species, is easily identifiable, and roughly corresponds with initiation of meiosis in the microsporangia, and initiation of ovules.

Middle ontogeny is considered the period following initiation of trichornes on the anthers to the formation of the corolfa tube. it is during this period that all the exarnined major, floral features are formed. Subsequent differences in late ontogeny (to anthesis) are interpreted as changes (or continued changes) in existing traits.

The early floral ontogenies (floral organ initiation and organogenesis) of the two species are remarkably similar considering the rnorphotogical differences observed at anthesis. As the buds elongate, more differences accrue, yet the underlying forms are alike. Most of the differences in ontogeny that are responsible for generating the morphological variation between the species occur during late ontogeny.

Calyx

Timing of initiation of calyces is simiiar behrveeri the species, however, the duration of growth of the calyx of M. lewisii was considerably shorter than that of the calyx of M. cardinalis. Therefore, the timing of temination of growth must have occurred early in plants ancestral to extant plants of M. lewisii. In ternis of heterochrony, this cmbe attributed to progenesis. In addition, the growth rate of calyces in M. lewisii was higher than the growth rate of calyces in h?, cardinalis, indicating that acceleration may have aiso acted as an evolutionary process.

The difference in rates of calyx growth, however, was small and consistent with the type of ontogenetic variation observed among populations within each species (refer to chapters 1 and 2). Thus, progenesis probably accounts for the shorter calyx in M. lewisii.

During early ontogeny, differences in the sizes and shapes of the sepals

(incipient calyx lobes) were seen between the species. Sepals of cardinalis were larger than in M. lewisii. These differences are accentuated during mid and late ontogeny. At anthesis, the calyx lobes of M. lewisii are linear - subulate but those of M. cardinalis are triangular with short apices.

Often, the role of the calyx is that of protection of the inner bud. While the decrease in calyx size from M cardinalis to M. lewisii rnay be in response to altered protection requirements, it rnay also be in response to the decreased size of the inner organs. The aforementioned requirements rnay be correlated. The longer calyx in M cardinalis likely provides additional support when birds probe the flowers because the calyx encloses most of the length of the throat of the corolla.

Corolla

Timing of corolla initiation was consistent between the species. The rate of growth of the abaxiai surface of the corolla relative to that of the adaxial surface was similar in both species until the corolla reached ca. 6 - 7 mm in length. That is, the scaling of the corollas was the same in both species until late in ontogeny. After this point, the rate of growth of the abaxial wall of the corolla in M. lewisii increased relative to that of M. cardinalis. Therefore, acceleration is interpreted as having acted as an evolutionary process. The shorter length of the adaxial corolla wall in M. lewisii is due to progenesis or an early temination of growth relative to that of M. cardinalis. This suggests that not only has evolution resulted in differential alteration of the length of the coroila (Le., shortened the upper wall and lengthened the lower wall), but also this was done by different developmental mechanisms (Le., change in timing of temination of growth and change in rate of growth). This indicates that through dissociation of zonai growth, heterochrony can act as a mechanism of evolutionary change, even within a tubular, connate organ complex (Le., corolla). This is also the source of transverse asymmetry within the corollas of both species.

The longer corolla tube along both the adaxial and abaxial surfaces in M, lewisii relative to M. cardinalis is the result of hypermorphosis (delayed temination of growth). Since progenesis was interpreted as having acted on the 262

abaxial corolla (tube plus throat), this suggests that dissociation of zonal growth

occurred within this basal restricted region of the corolla.

The corolla tube in M. lewisii has become longer and narrower than in M.

cardinalis. As well, the indentations in the exterior of the tube are shallower in

lewisii. These characteristics likeiy affect stamen architecture and will be discussed below.

The corolla throat changed from being laterally narrow in !& cardinalis to dorso-ventrally narrow in M. lewisii. The laterally narrow corolla, especially at the aperture, is suited to hummingbird pollination as this form allows the bird to insert

its culern and face into the throat to gain access to the nectar at the base of the flower. The dorso-ventrally narrow and laterally wider form is suited to bee pollination as it accommodates a bee inserting its entire body into the flower.

The recessed abaxial wall of the corolia in fl cardinalis, along with the reflexed corolla lobes. allow a visiting hummingbird unhindered access to the corolla aperture. The slightly extended abaxial corolla wall of M. lewisii, along with the recurved lower corolla lobes, provide a landing platforrn for visiting bees. The cilia that occur on the abaxial corolla lobes, and along the lateral margins of the lateral and abaxial lobes of M. lewisii, may serve two functions. They rnay increase the three-dimensional nature of the floral form, and they may provide a holdfast for bees once they have landed on the flower. The long filiforni trichornes at the base of the abaxial corolla lobes in M. lewisii rnay be a developmental novelty that aids bees in gaining a foothold on the flower. 263

The abaxial grooves and adjacent trichorne-lined ridges/shelves occur in both species, however, there is variation of these characteristics between cardinalis and M lewisii. The groove and ridges are much more pronounced in

--M. lewisii, and the trichomes are more slender and less densely arranged than in M. cardinalis where they likely serve as a tongue guide. The yellow colour of the trichomes may initially act as a visual nectar guide for bees, and the ridges and groove may provide a tactile path that leads to the nectar.

Stamens

The relative positioning of anthers is important in determining specific placement of pollen on the pollinator. Anthers inserted in the corolla throat facilitate placement of pollen on the body of a visiting bee after it has entered the flower to forage for nectar. Anthers exserted from the corolla throat facilitate placement of pollen on the forehead or culem of a hurnmingbird as it inserts its culem toward the base of the flower where nectar collects. The change from exserted anthers in M. cardinalis to inserted anthers in M. lewisii was achieved by a shortening of the filaments through an early termination of growth

(progenesis). This is unlikely to have disrupted anther dehiscence at anthesis as anthers would have continued development dong with the other floral organs.

In addition to optimal placement of the anthers along the length of the flower, characteristics of stamen architecture determine the placement of the anthers at the adaxial surface of the corolla throat. The indentations in the exterior of the corolla tube are ridges on the interior of the tube that aid in the orientation of the filaments. In both species, the indentations appear to cause 264 the abaxial filaments to be directed toward the adaxial wall of the corolla, as well as toward the median plane. Indentations also cause the adaxial stamens to initially be oriented toward the median plane. In M cardinalis, the indentations in the corolla tube are more pronounced and, in the regions subjacent to the abaxial filaments, the internal ridges (external indentations) are reinforced with a thickening of corolla tissue. These features together are iikely responsible for the orientation of the abaxial filaments toward the adaxial wall of the corolla, and the convergence of the adaxial filaments toward the median plane. In M. lewisii, the indentations are shallower, and no reinforcement of the internal ridge occurs.

Formation of the region of thickened tissue has been lost from the ontogeny of M. lewisii. Because the corollas in M. lewisii have becorne dorso-ventrally narrow, a lesser angle of deflection of the filaments is likely required in order for the anthers to reach the adaxial wall of the corolla.

From their initiation, the adaxial filaments cuwe toward the adaxial wall of the corolla in both species. This provides a mechanism for the adaxial starnens to be positioned along the adaxial wall of the corolla. The ridges caused by the external indentations subjacent to these stamens are not oriented adaxially.

In both species, ridges caused by the exterior indentations in the corolla tube direct the abaxiai filaments toward the median plane. In M. cardinalis, the filaments are adjacent at the median plane near their bases, and distal to this region the filaments curve toward the lateral walls. In M. lewisii, the filaments curve laterally away from the median plane at their point of insertion on the corolla. In cardinalis, ridges subjacent to the adaxial filaments cause them to 265 be parallel with each other. In M. lewisii, the less pronounced ridges occur and the fiiaments cuwe toward the lateral walls.

At anthesis, in both species, the anthers are held adjacent to the adaxial wall of the corolla at the median plane. In both species, once the filaments have reached the adaxial corolla wall, they can no longer extend straight at an upward angle. Instead, the filaments lie roughly horizontally along the adaxial corolla wall, where they exert an upward force. This force is greater in & cardinalis than in M lewisii as can be seen by the greater angle of the filaments when released from the tension of the corolla when the throat is cut away. In j'& cardinalis, the filaments are directed toward the median plane where the anthers abut each other. The tight arrangement of the stamens and stigma/style within the corolla

(which is acutely narrow along the adaxial length of the wall) prevents the stamens from crossing over each other at the median. In M. lewisii, the outward, lateral cuwature of the filaments causes them to exert outward pressure on their adjacent lateral walls. These changes may be to accommodate the altered corolla shape. Alternatively, these different features rnay have a function during the working of the flower by a visiting pollinator.

In both species, a thickening of tissue occurs on the base of the abaxial filaments, and protrudes toward the median plane. In M cardinalis, this region elongates and becomes a strip along the length of the rnedian margin of adjacent filaments. In M. lewisii, this region does not elongate to the sarne extent, and remains as small rounded protrusions of the abaxial filaments. In & cardinalis, the protrusions could protect the ovary from the sharp culem of a hummingbird 266 probing for nectar. If this is true, then the structure may not be required in & lewisii, or it may be still functional but in a different way. For example, it partially encloses the region of nectar accumulation between the ovary, the abaxial filament bases and the corolla, which hold the nectar at the base of the corolla by enclosure and through capillary action. In either case, the vestigial state of this tissue thickening in M. lewisii is hypothesized to be a functional by-product of less filament elongation at this region. This hypothesis is supported by the fact that these structures are similar between the species until late ontogeny, when filament elongation continues in M cardinalis beyond that in M. lewisii.

In flowers of both species, the stigrna is positioned distally adjacent to the anthers. This facilitates transfer of pollen from the pollinator to the stigmatic surfaces pior to deposition of pollen from the current flower ont0 the pollinator.

Therefore, the stigma position had to change in concert with that of the anthers from outside the corolla at anthesis in cardinalis, to within the corolla in M.

Iewisii. As with the filaments, the styles on flowers of M. lewisii terminated growth early compared to those in M. cardinalis (progenesis). Growth of the ovary was very similar between the species. Altering growth of the style while leaving the ovary unchanged may have allowed for the timing of ovule maturation and anthesis to remain coordinated. Alterations in lengths of the style and filaments without change in sizes of anthers and ovary would also allow a change in pollinator without affecting quantity of ovules and pollen produced. 267

Nectary

Although the nectaries of both species are similar in early ontogeny, by mid - ontogeny. the nectaries in M. lewisii becorne partitioned from their ovaries by deep invaginations in the ovary walls. This does not occur in & cardinalis.

These invaginations rnay act as a hinge that allows a visiting bee to raise the ovary, and enter further into the flower as it probes for nectar. Alternatively, the hinge-like structures on ovaries of M. lewisii rnay function earlier in ontogeny and allow the ovary to flex and spatially accommodate the enlarging bases of the abaxial filaments. While the latter scenario is not necessarily supported (nor refuted, for that matter) by observations of early ontogenetic character states, it is important to consider that floral morphological traits may have adaptive significance prior to anthesis i.e., before natural selection per se has a chance to act. In other words, the structure (in this case, flower) must be viable before nature can select for or against it. 268

Fig. 1 A. Lateral views of flowers of cardinalis and M. lewisii (C).BI

Lateral view of longitudinal section of M cardinalis and M. lewisii (D) showing arrangement of starnens and pistil, which are displaced when released from the corolla throat. Scale = 10.0 mm; a = stamen primordium or anther; ab = abaxial side; ad = adaxial side; ar = anther removed; c = petal primordium, corolla or corolla lobe; ct = corolla tube; cth = corolla throat; cr = corolla or corolla lobe removed; f = filament; g = pistil or pistil primordium; k = sepal primordium. calyx or calyx lobe; kr = calyx or calyx lobe removed; I = locule; n = nectary; O = ovary; p = pedicel; se = septum; st = stigma; sy = style. Adaxial side is at the top of al1 images unless stated otherwise.

270

Fig. 2 Lateral view of longitudinal section of a flower of & cardinalis showing dimensions used in allometric analyses. Not shown; adaxial filament, adaxial corolla tube, abaxial corolla. Stamen and style are adaxially displaced in this sectioned flower. Scale = 10.0 mm.

272

Fig. 3 Early floral apex development of & cardinalis and M. lewisii (SEM)

A, Polar views of fi cardinalis and M. lewisii (C) showing sepals, petal initiation, stamen initiation, and cleft that separates sepals from the inner floral apex. 8,

Lateral oblique view of cardinalis, and D, lateral view of M. lewisii showing inner apex at a greater height than sepals, and pedicei. Scales = 50 Fm.

274

Fig. 4 Early floral apex developrnent of M. cardinalis and M. lewisii (SEM)

A, B. Polar views of M. cardinalis (A) and M. lewisii (B) showing initiation of the gynoecium (arrowheads) and enlarging petals and stamens. Note the sepals of

-M. cardinalis (A) are broader and more rounded than the narrower, more acute sepals of M. lewisii (B). C, Lateral view of M. lewisii showing vertical growth of sepals, and initiation of gynoecium (arrowhead). Note the adaxial sepal is taller than the other sepals. D, Polar view of M. cardinalis showing hemispherical stamens. E, Lateral view of cardinalis showing trichomes on sepals and apex of the elongating pedicel. F, Polar view of M. Iewisii showing locule formation in the gynoecium, upright sepals, and vertical growth of petals. G, Lateral oblique view of M. lewisii showing trichomes initiating on the sepals and apex of the pedicel. Note narrower sepals of M. lewisii (F, G) versus the wider, more rounded sepals of cardinalis (D, E). HlLateral view of M. lewisii with lateral sepal rernoved showing starnens and gynoecium taller than the petals. 1,

Oblique view of & cardinalis with abaxial sepal removed showing initiation of conduplicate folding of sepals (arrow), and vertical growth of petals. Note similar form of & cardinalis (1) and M. lewisii (H) at similar stages. Scales = 50 Fm.

276

Fig. 5 Floral apices of M. cardinalis and M. lewisii (SEM). A, Polar oblique view of cardinalis showing elongating trichomes on calyx lobes, enlarging stamens, and initiation of septum in the gynoecium. 6,Polar view of M. lewisii showing elongating trichomes on calyx lobes, enlarging stamens, and initiation of septum in the gynoecium (arrow). Note large calyx lobes and long trichomes of -M. cardinalis (A) compared to M. lewisii (B). C, D, Polar view of j&. cardinalis (C), and polar view of M. lewisii (D) with calyx lobes removed showing longitudinal cleft foming on stamens (arrowhead), broad septum, and clefts forming on the apex of the gynoecium (arrow). E, F, Oblique views of j&. cardinalis (E) and M. lewisii (F) with calyx lobes and part of corolla removed showing inward curvature of petals, microsporangia and connective (arrowheads) formation on stamens, and clefts at apex of the gynoecium (arrows). Scales = 100 Pm.

278

Fig. 6 Dissected floral buds of M. cardinalis and M. lewisii (SEM). A,

Abaxiai view of ji& cardinalis with calyx and abaxial corolla lobes removed showing folding of corolla lobes, formation of corolla throat, connective on anthers (arrowhead), and flaring at the apex of gynoecium (arrow). B, Lateral view of M. lewisii with calyx and most of corolla removed showing flaring at the apex of gynoecium. Scales = 100 prn.

280

Fig. 7 Dissected floral buds of && cardinalis and M. lewisii (SEM). A, €3,

Polar views of M. cardinalis (A) and M. iewisii (B) with calyx and corolla rernoved showing lobes of microsporangia, connective on anthers (arrowheads), unequal sizes of abaxial and adaxial stigma lobes, and formation of compitum on apex of placentas (arrows). Note initiation of nectary in B (not visible in A). C, Oblique view of cardinalis with calyx, corolla, and abaxial stamens removed showing trichomes initiating on adaxial anthers, formation of filaments, initiation of style, and nectary. Dl Lateral view of M. lewisii with calyx, corolla, one adaxial stamen and one abaxial stamen removed showing trichomes initiating on anthers, initiation of style, and nectary. Note the adaxial stamens tilt toward the median plane. E, Lateral view of one adaxial and one abaxial stamen of cardinalis showing filaments, trichomes distributed over entire anther surface, and tilting of adaxial stamen. F, Lateral view of one adaxial and one abaxial stamen of M. lewisii showing filaments, trichomes in a continuous row along margins of thecae, and tilting of adaxial stamen. Note more elongated trichomes on anthers of M. cardinalis (E) relative to those of M. lewisii (F). Scales = 300 Pm.

282

Fig. 8 Dissected floral buds of cardinalis and M. lewisii (SEM). A, M

lewisii with caiyx, corolla and abaxial stamens removed showing laterally broad stigrna lobes, narrow style, and nectary separated from ovary by invaginations in the abaxial and adaxial walts of the ovary (indicated on abaxial wall by arrow). 5,

M. cardinalis with calyx, corolla and adaxial stamens rernoved showing rounded stigma lobes, and broad style. C, Laterai view of & cardinalis with calyx, corolla, one adaxial stamen and one abaxial stamen removed showing tilting of adaxiaI stamen, and nectary not separated from the ovary by invaginations. Scales =

500 Fm.

284

Fig. 9 Floral buds of acardinalis and M. lewisii (SEM). A, Abaxial view of bud of &&cardinalis showing calyx lobes curved in, and wing-like projections extending basally from the conduplicate folded calyx lobes (arrow). 6,Abaxial view of bud of M. lewisii showing vertical conduplicate folded calyx lobes that do not extend basally into wing-like projections. C, Lateral view of older bud of M. cardinalis showing overlapping tips of calyx lobes, and furrowing at apex of calyx tube. D, Oblique view of older bud of M. lewisii showing vertical or outward curving calyx lobes, and unfurrowed calyx tube. Note that calyx lobes are broader and slightly longer in M. cardinalis than in M. lewisii. Scales = 500 pm.

286

Fig. 10 Corollas of M. cardinalis and M. lewisii at similar stages of development (SEM). A, Polar view of M. cardinalis showing overlapping of rounded lobes with a notch in the distal margins (arrow). B, Lateral view of A showing broad corolla lobes, and incornpletely folded adaxial lobes. C, Polar view of M. lewisii showing folded lobes that do not completely overiap, and notch in the distal margin of the lobes. D, Lateral view of C showing relatively narrow corolla lobes. Scales = 300 Pm.

Fig. 11 Corollas of & cardinalis and M. lewisii at similar stages of development (SEM). A, Polar view of & cardinalis showing broad, rounded corolla lobes without cilia along the margins. B. Polar view of M. lewisii showing narrower, truncate corolla lobes with undulating distal margins, and cilia (arrows).

C,Abaxiat view of & cardinalis showing oblong shape, and initiation of corolla tube (arrowhead). D, Lateral view of M. lewisii showing formation of corolla tube, indentations in exterior of tube (arrowheads), and formation of abaxial groove

(indicated by arrow, and seen on the exterior of the corolla as an abaxial protrusion with adjacent troughs). Scales = 500 Fm.

290

Fig. 12 Corollas of M. cardinalis and M. lewisii ca. 4-6 mm in length

(Dissecting micrographs, DM). A, B, Adaxiai (A) and lateral (B) views of & cardinalis showing straight walls of throat, indentations in corolla tube

(arrowheads), radially expanded apex, and ridge along the adaxial surface of the throat (arrow in A). C, O,Adaxial (C) and lateral (D) views of M. lewisii showing convex walls of the throat, indentations in the corolla tube (arrowheads), slight radial expansion of the apex, ridge along the adaxial surface of the throat

(arrow), and cilia protruding from the lobes (asterisk). Scales = 1 .O mm.

292

Fig. 13 Corollas of cardinalis and M. lewisii ca. 13-14 mm in length

(DM). A, B, Adaxial (A) and lateral (B) views of @ cardinalis showing straight walls of the throat, prominent indentations in exterior of corolla tube

(arrowheads), adaxial ndge along throat (arrow in A) and abaxial groove (arrow in 8). Note that cilia now protrude frorn the lobes on the abaxial side (asterisk in

B). C,D, Adaxial (C) and lateral (D) views of M. lewisii showing convex walls of the throat, less prominent indentations in the tube (arrowheads), adaxial ridge

(arrow in A), and abaxial groove (indicated by arrow in B and seen from the exterior as an abaxial protruçion with adjacent troughs). Asterisk indicates cilia in

B. Note the enlarged corolla apex in M cardinalis (A, B) which is truncate in lateral view (B). In M. lewisii, the apex is not enlarged, and the abaxial region extends beyond the adaxial region (C, D). Scales = 5.0 mm.

294

Fig. 14 Corollas of M. cardinalis and M. lewisii ca. 20-22 mm in length

(DM). A, B. Adaxial (A) and lateral (8)views of !& cardinalis showing parallel lateral walls of the throat, continued radial expansion of the apex, indentations in corolla tube (arrowheads), adaxial ridge (arrow in A) and abaxial groove (arrow in

B). C, D, Adaxial (C) and lateral (D) views of M. lewisii showing convex walls of the throat, adaxial ridge (arrow in A) and abaxial groove (arrow in B).

296

Fig. 15 Pistils and nectaries of M. lewisii and cardinalis (DM). A,

Lateral view of pistil of & cardinalis prior to initiation of nectar secretion showing abaxial curve of the style and dark colouration of nectary tissue. B. Lateral view of pistil of fi cardinalis around the time of initiation of nectar secretion showing more pronounced curving of the elongating style, and abaxially expanding nectary. C, Abaxial view of nectary of cardinalis at anthesis showing undifferentiated forrn and dark colouration of tissue. 0,Lateral view of pistil of lewisii showing curved style and invaginations that separate the nectary from the ovary (arrows). E, Lateral view of pistil of M. lewisii around the time of nectar initiation showing slight curve of the elongating style, and invaginations that separate the nectary from the ovary (arrows). F, Lateral view of nectary of M. lewisii at anthesis showing invaginations that separate nectary from the ovary

(arrows). A, 6,O, E, Scales = 3.0 mm; C, F, Scales = 1.O mm.

298

Fig. 16 Lateral views of dissected floral buds of cardinalis and M. lewisii

(DM). A. B. M cardinalis (A) and M. lewisii (8)showing adaxial filaments along adaxial wall of the corolla throat, abaxial filaments along the median floral plane.

Note the indentations in the corolla tube as they appear in the exterior (arrow in

A) and interior (arrow in B) of the corolla. CI DI & cardinalis (C) and M. lewisii

(D) showing adaxial filament along the adaxial wall of the corolla throat, and indentations in the corolla tube as they appear in the interior of the corolla

(arrows). Note that in & cardinalis, the abaxial filaments are parallel with the floral median plane (C), whereas in M. lewisii. they are parallel with the abaxial wall of the corolla throat (D). Scales = 1.0 mm.

300

Fig. 17 Longitudinal sections of dissected buds of cardinalis and & lewisii (DM) A, Lateral view of cardinalis showing curvature of the adaxial filament, and abaxial filament about parallel with floral median plane. Arrow indicates indentation in exterior of the corolla tube as seen in the interior of the corolla. 6,Close up of A showing ridge subjacent to abaxial filament. This ridge corresponds with an indentation in the exterior of the corolla tube, and includes a thickening of corolla tissue (arrow). C, Lateral oblique view of longitudinal section of M. lewisii showing curvature of adaxial filament, and abaxial filament about parallel with floral rnedian plane. Arrow indicates indentation in exterior of the corolla tube as seen in the interior of the corolla. A, C, Scales = 5.0 mm. B,

Scale = 1 .O mm.

302

Fig. 18 Longitudinal sections of buds of M. cardinalis and M. lewisii showing one stamen of each pair and corolla (DM). A, B, LM, cardinalis (A) and --M. lewisii (B) with pistil removed showing adaxial filaments curving toward their adjacent lateral walls. Note that in M cardinalis (A), the adaxial filaments are angled toward the rnedian plane before they curve toward their adjacent lateral walls- In M. lewisii (B), the adaxial filaments curve throughout their lengths toward the lateral walls. Arrows indicate indentations in exterior of the corolla tube as seen in the interior of the corolla. C, cardinalis with pistil removed showing elongated adaxial filaments roughly parallel with each other. D, Lateral view of M. lewisii showing elongated adaxial filaments still adjacent to the lateral walfs. Note divergence of thecae of the anthers and curvature of filament tips toward the median (arrowheads). Scales = 5.0 mm.

304

Fig. 19 Longitudinal dissections of M cardinalis and M. lewisii (DM). A, M cardinalis showing curvature of the abaxial filaments toward their adjacent lateral walls, and two trichorne - lined interior ridges (arrows) that occur on either side of the interior abaxial groove. B. M. lewisii showing curvature of the abaxial filaments toward their adjacent lateral walls (abaxial interior ridges are obscured by filaments). Note that in M. cardinalis, the abaxial filaments are adjacent to each other for a greater distance along the corolla throat than in M. lewisii (A, B).

C, M. cardinalis çhowing elongating abaxial filaments that curve toward their adjacent lateral walls, and two trichome-lined interior ridges (arrows) that occur on either side of the interior abaxial groove (arrows). D. M- lewisii showing elongating abaxial filaments that cuve toward their adjacent lateral walls. and two trichome-lined interior ridges (arrows) that occur on either side of the interior abaxial groove (arrows). Note that in cardinalis (C), the abaxial filaments are straight near their bases. whereas in M. lewisii, the entire length of the abaxial filaments curves prorninently toward their adjacent lateral wall. Scales = 5.0 mm.

306

Fig. 20 Dissected flowers of cardinalis and M. lewisii. A, 9,Adaxial view of & cardinalis (A) and M lewisii (B) with the abaxial wall of the corolla removed showing stamens and pistil along the median plane of the adaxial wall of the corolla throat. Scales = 10.0 mm.

308

Fig. 21 Dissected buds of cardinalis and M. lewisii (A, C, DI DM; 6,

SEM). A, Bases of abaxial filaments of cardinalis showing growth of filament tissue toward the median plane (arrows). B, Bases of abaxial filaments of lewisii at a sirnilar stage as A showing growth of filament tissue toward the median plane (arrows). CI Bases of elongated abaxial filaments of & cardinalis near anthesis showing growth of filament tissue as an elongated strip (arrows).

D, Bases of elongated abaxial filaments of M. lewisii near anthesis showing rounded growths of filament tissue that meet at the median plane (arrows).

Scales = 1.O mm.

310

Fig. 22 Transverse sections of corollas of cardinalis and M. lewisii midway along the length of the corolla throat (DM). A, View of cardinalis

(adaxial corolla length = ca. 4-5 mm.) showing general circular form, adaxial peak that corresponds with external ridge that occurs along the adaxial wall of the corolla throat (arrow), and interior trichome-lined shelves (arrowheads) on either side of the interior abaxial groove. B, View of M. lewisii (adaxial corolla length =

Ca. 4-5 mm.)showing pentagonal shape, adaxial peak (arrow), and interior trichorne-lined ridges (arrowhead) on either side of the interior abaxial groove.

ClView of cardinalis (adaxial corolla iength = ca. 10-11 mm.) showing pentagonal shape with curved lateral walls, and trichome distribution that now extends into the interior abaxial groove (arrow). D, View of M. lewisii (adaxial corolla length = Ca. 10-11 mm.) showing pentagonal shape with straight lateral walls, and trichome distribution that now extends into the interior abaxial groove

(arrow). Note the shorter trichornes of M. lewisii (D) versus those of cardinalis

(C). El View of M. cardinalis prior to anthesis showing spade shape, and much- elongated trichomes (arrow). Note the interior abaxial shelves that occur on either side of the interior abaxial groove have become pronounced and are more like ridges. F, View of M. lewisii prior to anthesis showing narrow spade shape of the corolla, and short trichomes (arrow) on the much pronounced interior abaxial ridges. Scaies = 1.O mm.

312

Fig. 23 Transverse sections of !&. cardinalis and M. lewisii midway along the length of the corolla throat (DM). A, View of !& cardinalis at anthesis showing oblong shape of the corolla with an acute adaxial peak (arrow), and long tnchornes along the interior abaxial ridges (arrowheads). Note the n'dges have become obscure as the relative transverse height of the corolla increased. B.

View of M. lewisii at anthesis showing spade shape of the corolla with an acute adaxial peak (arrow), and short trichomes along the distinct interior abaxial ridges

(arrowheads). Scales = 1.O mm.

314

Fig. 24 Transverse sections of M cardinalis and M lewisii at the distal

region of the corolla throat (DM). A, View of cardinalis (adaxial corolla length

= Ca. 5-6 mm.) showing general circular form of the corollz with adaxial peak

(arrow) that corresponds with the exterior adaxial ridge along the surface of the corolla throat. 6,View of M. lewisii (adaxial corolla length = Ca. 5-6 mm.) showing pentagonal shape of the corolla with an adaxial peak (arrow). and formation of interior abaxial ridges (arrowheads) that occur on either side of the interior abaxial groove. C, View of M cardinalis (adaxial corolla length = Ca. 15-

16 mm.) showing general circular shape of the corolla, and interior abaxial shelves (arrowheads) that occur on either side of the interior abaxial groove.

Note the long trichomes that occur along the two ridges. DlView of M. lewisii

(adaxial corolla length = ca. 15-16 mm.) showing pentagonal shape of the corolla, and distinct interior abaxial ridges (arrowheads). Note the short trichomes along the ridges, and their distribution, which extends into the interior abaxiai groove, and toward the lateral walls of the corolla. E, View of M. cardinalis at anthesis showing laterally narrow corolla with an acute adaxial peak

(arrow), and interior abaxial shelves (arrowheads). Note the densely arranged trichomes that are distributed along the shelves and in the interior abaxial groove. F, View of M. lewisii showing laterally broad corolla with a rounded adaxial peak, and tall interior abaxial ridges (arrowheads). Note the less densely arranged trichomes on the ridges and interior abaxial groove. Scales = 1.O mm.

316

Fig. 25 Line tracings of representative adaxial, lateral and abaxial corolla lobes from M. cardinalis and M. lewisii at anthesis. Average length (shown as vertical plane), width (shown as horizontal plane) and length : width ratio values are given in mm, as well as standard error. All values differ between the species.

Adaxial lobes are shown as the flower appears horizontally at anthesis. with the distal end of the flower to the right. Ad, Lat and Ab = adaxial, lateral and abaxial corolla lobe. -M. cardinalis -M. lewisii (16) (1 5)

Lat 31 8

Fig. 26 Regression lines showing growth rates of buds M; cardinalis and

--M. lewisii. The average growth rate of buds differs among the populations at the

P c 0.05 level (table 1). M. cardinalis

days from anthesis 320

Fig. 27 Allometric plots of log transformed abaxial corolla length (top of figure), and adaxial (middle of figure) and abaxial (bottom of figure) corolla lengths versus ovary length. Growth rate of the abaxial wall of the corolla is slower in M. cardinalis than in M. lewisii (top and bottom). Growth of the adaxial wall of the corolla is terminated sooner in M. iewisii than in fi cardinalis (middle). cardinalis lewisii

cardinalis @Sa lewisii

ovary length (mm) 322

Fig. 28 Allometric plots of adaxial (top of figure) and abaxial (bottom of figure) corolla tube lengths versus ovary length. Growth of both the adaxial and abaxial walls of the corolla tube terminates later in M. lewisii than in !&. cardinalis. cardinalis oega

"O%, Co%, lewisii 0 Q O O hi O 3& O

O 2 4 6 8 10 12 ovary length (mm)

ovary length (mm) 324

Fig. 29 Allometric plots of adaxial (top of figure) and abaxial (bottom of figure) stamen lengths versus ovary length. Growth of both the adaxial and abaxial stamens teminates sooner in M. lewisii than in M. cardinalis. M. cardinalis 9. d*l 8 a 0 M. lewisii am l

CI .-Cu x O nCU 51 m O 2 4 6 8 10 12 ovary length (mm)

O 2 4 6 8 10 12 ovary length (mm) 326

Fig. 30 Allometric plot of style length versus ovary length. Growth of the style terminates sooner in M. lewisii than in M. cardinalis. M. cardinalis je

0 M. lewisii ,ah#

O 2 4 6 8 10 12 ovary length (mm) 328

Fig. 31 Allornetric plot of abaxial corolla length (measured as tube plus throat) versus adaxial corolla length. Growth trajectories of the abaxial and adaxial walls of the corolla are similar in both species until the length of the corolla = ca. 1O mm. M. cardinalis

0 M. lewisii I

adaxial corolla length (mm) Table 1 Sfopes t SE (b) and Regression Coefficients (P) of Regression of Adaxial Calyx Length vs. Time (as Days from Anthesis)

M. cardinalis M. lewisii F P df Table 2

Floral Organ Lengths (mean I SE, (n)) in mm at Anthesis.

Population Calyx Adaxial Abaxial Ovary Style Abaxial Abaxial Adaxial Adaxial corolla corolla stamen corolla stamen corolla tube tube -M. cardinalis 31.7 10.3 41.2 10.4 28.2 I0.3 9.9 t 0.1 42.3 i 0.6 34.2 & 0,4 7.8 i 0.2 31.0 + 0.4 7.5 10.2

Note. All means differ between the species at the P c 0.05 level. See fig. 2 for dimensions measured. 332

Table 3 Slopes + SE (b) and Regression Coefficients (6)of Regression of Log Transformed Adaxial and Abaxial Corolla Lengths vs. Ovary Length (mm)

-M. cardinalis --M. lewisii F P df

log b 0.1510.05 adaxial ? 0.95 corolla Iength vs. ovary

log b 0.12 k0.05 abaxial ? 0.94 corolla length VS. ovary Note. See fig. 2 for dimensions measured- 333

Discussion

In the previous chapters, the floral ontogenies of three populations of cardinalis, and the two races (represented by two populations each) of M. lewisii were described, and the observed differences were interpreted as evolutionary changes. Finally, the ontogenies of the two sympatric populations from Yosemite

National Park, CA, of each species were compared and interpreted.

Within each species, differences in ontogeny were slight as would be expected given the low degree of morphological variation. However, developmental differences were observed that were not reflected in the mature flowers.

for M. lewisii and M. cardinalis, early floral ontogeny is defined as the period from the initiation of the floral apex, to the time of trichome initiation on the surface of microsporangia of the anthers. Timing of trichome initiation works well as a marker as it is easily identifiable, it occurs at a similar stage in both species, and it roughly corresponds with meiosis in the microsporangia, and initiation of ovules on the placentas. Also, by this point, al1 floral organslwhorls have initiated.

Mid - ontogeny is defined as the period following initiation of trichornes on the anthers, to the formation of the corolla tube. By this point, al1 major floral structures have formed. Late ontogeny is the period foliowing formation of the corolla tube to anthesis. Using these definitions, no change in growth of a floral structure in late ontogeny can disrupt the initiation or formation of another floral structure. 334

Infraspecific variation

Characteristics of calyx development were the only ones to Vary in all three developmental trajectories. Timing of calyx initiation, although consistent within a population, fluctuated among the populations and races. The same is true for the rate of calyx growth along its length, and the timing of termination of growth. It was suggested that calyx characteristics are more susceptible to minor evolutionary alterations than other floral structures because even changes that occur in early development (e.g., initiation) in the calyx are unlikely to be disruptive to subsequent development.

Features of early development of the inner floral organs were consistent within and between the species. The timings of initiation of corolla, stamen and pistil primordia were indistinguishable among al1 populations studied. Differences in shape of primordia (e-g.,petal and stamen primordia) were observed among some of the populations and races. Differences in growth trajectories of the inner floral organs were of rate and timing of termination.

Within ail populations, individual growth trajectories were largely invariant, suggesting they are governed by a conserved developmental program.

However, additional variations in morphologies were generated by combinations of trajectories working in opposition or in concert. Between M. lewisii and M. cardinalis, the early floral ontogenies are virtually indistinguishable. During mid and late ontogenies of the species, dramatic differences were observed with respect to corolla size and shape, stamen and pistil length, nectary characteristics, and trichorne characteristics on both the corolla and anthers. 335

Heterochronic and allornetric analyses provided valuable insight into the developmental changes that must have occurred during the evolution of (what has been hypothesized here to be) M. lewisii from a & cardinalis-like anceçtor.

All of the allometric changes observed were those of timing of termination of growth. As the heterochronic evolutionary processes responsible were a combination of progenesis (e-g., early termination of growth of the stamens and style in M. lewisii relative to & cardinalis) and hypemorphosis (e-g., delayed termination of growth of the corolla tube in M. lewisii relative to M cardinalis), the floral fom in M. lewisii cannot be easily described as either peramorphic or paedomorphic. Instead, the derived floral form of M. lewisii is best described as a mosaic of paedomorphic organs (e-g. stamens), peramorphic organs (e-g. corolla tube), and features resulting from developmental novelties (e-g., highly differentiated hinge-like nectary, dorso-ventrally narrow corolla throat).

Tucker (1984) hypothesized that morphological features that are diagnostic of low taxonomic levels (e-g., species) would be determined late in ontogeny. That is, changes in development that yield interspecific variation occur late in development. She proposed as a reason for this, that early changes in development are more stable than late changes. Changes in early ontogeny are more likely to disrupt subsequent developrnent and possibly be detrimental to the organism. Therefore, once a change in ontogeny is established, a subsequent reversion may render the organism inviable, thus rendering the original change stable. From this, it was expected that rnorphological variation among the populations of M. cardinalis, and the rnorphological variation between the races 336 of M. lewisii (putative species) would be determined during late stages of ontogeny.

Among the populations of M cardinalis, developmental differences were observed at early, mid, and late stages. In the context of heterochrony and allometry, the predominant changes below the species level were those of rate of growth, and timing of temination of growth. As changes in rate of growth occur early in ontogeny. this contradicts Tucker's hypothesis. The changes in ontogeny that generate infraspecific variation in this Mimulus complex may be of suficiently low magnitude that the inclusion or exclusion of these changes from the developmental program would not be detrimental to subsequent development. If this is true, then Tucker's hypothesis cannot be universally valid below the level of species.

Between the races (putative species) of M. lewisii, patterns of developmental variation were similar to those observed among the populations of

-M. cardinalis. Developmental differences were observed at early, mid and late stages of ontogeny. Perhaps the most diagnostic floral features of the Rocky

Mountain (magenta) race of M. lewisii are the colour of the corolla (magenta versus pale pink) and shape of the corolla aperture (wider and more rounded in the Rocky Mountain race versus the Sierra Nevada race). The differences in corolla shape occur in mid to late ontogeny. Although change in corolla colour is unlikely to be detrimental to further developrnent regardless of when it occurs, if corolla shape is diagnostic of the race (putative species) then Tucker's hypothesis is supported. 337

The early stages of ontogeny of M cardinalis and M. lewisii are virtually indistinguishable. During the middle stages, minor variation occurs (e-g., trichorne distribution on anthers, shape of corolla lobes). During late stages, the heterochronic/allometric changes occur that generate many of the diagnostic features of each species, and the differences in corolla shape become prominent. The M. cardinalis - M. lewisii group show the patterns of developmental variation predicted by Tucker's hypothesis.

Hypothesis of speciation

Original hypotheses suggested that M. cardinalis and M. lewisii share a recent cornmon ancestor (presumably very similar to one of the species) or that one is derived from the other (Hiesey et al. 1971). It was long held that cardinalis was derived from an M- lewisii - like ancestor because the majority of species within Mimulus are bee - pollinated. Ongoing work at the University of

Washington, Seattle, on the phylogeny of this group hypothesizes that M. lewisii forms a rnonophyletic clade embedded within a group of hummingbird - pollinated species and is more closely related to cardinalis than it is to any other species (P. Beardsley pers. comm.). From this, it is hypothesized here that --M. lewisii is the derived species that has evolved frorn a humrningbird - pollinated M. cardinalis - iike ancestor.

Recent studies have elucidated the genetic architecture of floral traits within these two species that are hypothesized to confer adaptation to their individual specific pollinators (Bradshaw et al. 1995; Bradshaw et al. 1998).

Further to this, the adaptive values of some of these traits were quantified 338

(Schemske and Bradshaw 1999). However, these studies were done with an 9 priori assumption that M. lewisii is ancestral to & cardinalis. One issue that confounded this hypothesis was that ten of the essential floral traits within fi cardinalis are govemed by alleles that are recessive to those of M. lewisii and thus more dificult to establish within a population (Bradshaw et al. 1998). It was proposed that this difficulty could be overcome by even a low rate of selfing.

That M. lewisii is the derived species is a plausible hypothesis that would not require an explanation of the establishment of dominantly inherited mutations into a population of an M. cardinalis - Iike ancestor. With the genetic, phylogentic and floral adaptation data provided, data from this study cm be used to hypothesize the major evolutionary events that were associated with speciation.

If the common ancestor had a floral form simiiar to M. cardinalis as it exists today, elongation of the abaxial wall of the corolla throat would bring the abaxial base of the lateral corolla lobes fonvard and distal to their adaxial base.

The resulting oblique base would cause the lateral lobes to flex abaxialty (as seen in M. lewisii). QTLs that explain variation in corolla length (on any wall) were not identified, thus genetic linkage of this trait to others cannot be confirmed. However, if this change occurred, changes in degree of lobe refiexation may have been concomitant. If the corolla lobes were still reflexed, the position of the abaxial lobe would interfere with the position of the lateral lobes, which would likely interfere with each other. QTLs that explain variation in degree of lobe refiexation were found on five linkage groups; four of which were common to the lateral and adaxial petals. This suggests that changes in reflexation of the lateral lobes were accompanied by changes in reflexation of the adaxial lobe. Although not included in the QTL analyses, the reflexation of the abaxial lobe is likely genetically linked to the reflexation of the other lobes.

Therefore, al1 lobes would change from being reflexed to recurved. Seven of the eight QTLs that explain variation in corolla reflexation were found to be recessive for the & cardinalis allele. Hypotheses of self-fertilization would not need to be invoked to explain the establishment of the M. lewisii alleie into a population. As well, elongation of the lateral corolla walls would necessarily accompany elongation of the abaxial wall, and would likely result in a wider corolla as seen in

--M. lewisii. The eiongated corolla along the abaxial surface, with the changes in lobe reflexation to become recurved would provide a landing platform for bees as seen in the modern M. lewisii. As lobe reflexation decreased the projected area of the corolla in polar view (i.e., the area seen by an approaching pollinator), the new form would have a much-increased projected area, which has been deemed to be important in attracting bees. QTLs that explain variation in corolla projected area mapped together on shared linkage groups with QTLs that explain variation in corolla aperture height, and lateral petal width. This indicates that much of the change in projected area was concurrent with change in lobe reflexation and corolla shape change. Schemske and Bradshaw (1 999) point out that multiple morphological characteristics likely contribute to the projected area of the corolla.

Following the change in corolla shape more suited to bee - pollination, changes in pistil length from longer to shorter would affect efficiency of pollen transfer from a visiting bee to the stigma. This study of ontogeny determined that 340 variation in pistil length was almost entirely explained by variation in style length.

While change in the length of the style rnay have been crucial, ali QTLs that explained variation in pistil length shared linkage groups with QTLs that explained variation in stamen length and were mapped closely together. This is supported by the similar nature of the developmental change of stamen and style length (Le., early temination of growth in M, lewisii). Therefore, changes in much of the length of both stamens and style were likely concurrent. It is pointed out here that Bradshaw et al. (1998) did not actually study stamen length, merely the distance of the abaxial anthers from the base of the caiyx. However, this study of development suggests that rnost of the variation in the distance between the base of the calyx and the abaxial anthers is a function of stamen (filament) length. Other factors (e.g., length of the corolla tube. filament curvature) contribute only minor variation.

QTLs that explain variation in pistil and stamen lengths share Iinkage groups with QTLs that explain variation in colour and shape characteristics of the corolla. Much of the change in corolla characteristics may have been concurrent with much of the change in stamen and pistil lengths. Virtually, al1 of these QTLs are recessive or additive for the M cardinalis allele.

It is proposed here that the first changes to occur during speciation from an cardinalis - like ancestor to an M. Iewisii - like descendent were an extension of growth of the abaxial corolla wall (which caused or was associated with a series of corolla shape changes), and an abbreviated period of growth of the style and stamens. Quantitatively. much of the change was likely concurrent based on the shared linkage groups of many of the QTLs involved, and based on the tight coupling of early developmental processes governing growth of the corolla, stamens and pistil.

McKinney (1988) proposed that in cases of rapid evolution, the first developmental changes to occur would be of high magnitude, and that subsequent "fine - tuningn of these and associated traits would follow (Le., many subsequent changes of smaller magnitude). The evolution of M. lewisii seems to fit this pattern. The first developmental changes Le-, growth of the abaxial corolla surface, stamens and style, likely involved a few, if not a single change in timing of termination. Following this were smaller developmental changes in many traits such as those seen in corolla and trichorne characteristics, calyx characteristics, stamen architecture, etc.

Schemske and Bradshaw (1999) found that colour of the corolla, and nectar volume characteristics had a significant effect on pollinator visitation. In short, the red colour of a corolla decreased (but did not prevent) bee visitation.

Greater nectar volume increased hummingbird visitation dramatically, but did not affect bee visitation. If M. lewisii is ancestral to cardinalis as was previously proposed (Bradshaw et al. 1995; Bradshaw et al. 1998; Schemske and

Bradshaw 1999), then one of the initial changes would have to have been an increase in nectar volume. If & cardinalis is ancestral, the change in nectar volume would not have been as important, therefore, could have occurred in later stages of evolution. The red colour of the corolla would iikely have greatly decreased bee visitation but not prevented it altogether. The aforementioned 342

shape changes may have increased bee visitation and effective pollination so

that a persistent population of this variant arose in which change from a red to a

pink corolla could occur. This characteristic is under simple genetic control and

governed by one gene recessive for the & cardinalis allele. Rapid establishment

of this phenotype into a population is plausible.

Conciusions

Natural selection can only affect evolutionary change once phenotypic

variation is avaiiable. Therefore, in order to understand how evolution is working

in a given group, it is necessary to understand the mechanisms through which

diversity of fom is generated from the genetic to ontogenetic levels.

Comparative studies of floral development can elucidate particular mechanisrns

of evolutionary change (as opposed to trends associated with changes in plant

form over IO'S or 100's of millions of years). However, these studies must be

done in the context of an explicit hypothesis of phylogeny, Le., the study

organisms must be closely related genealogically (members of a monophyletic

W0uP)- References

Alberch P (1982) Developmental Constrainsts in Evolutionary Processes

JT Bonner ed., Springer-Verlag, Berlin, pp 313-332.

Alberch PI SJ Gould, GF Oster, DB Wake 1979 Size and shape in ontogeny and phylogeny. Paleobiology 5: 296-3 17.

Beardsley PI R Olmstead (1999) Abstract XVI International Botanical

Congress, St Louis, USA

Bradshaw HD, KG Otto, BE Frewen, JK McKay, DW Schemske 1998

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