Comparative Analysis of Inflorescence Architecture In

COMPARATIVE ANALYSIS OF INFLORESCENCE ARCHITECTURE IN

AQUILEGIA SPECIES

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfilment

Of the Requirements for the Degree

Master of Science

Biological Sciences

Michael R. Speck

2021

SIGNATURE PAGE

THESIS: COMPARATIVE ANALYSIS OF INFLORESCENCE ARCHITECTURE IN AQUILEGIA SPECIES

AUTHOR: Michael R. Speck

DATE SUBMITTED: Spring 2021

Department of Biological Sciences

Dr. Bharti Sharma Thesis Committee Chair Professor of Biological Science ______

Dr. Valerie Mellano Professor of Plant Science ______

Dr. Paul Beardsley Professor of Biological Science ______

ABSTRACT

The Aquilegia genus has undergone adaptive radiation over the last 1-5 million years. This has led to a wide array of diversity in flower size, plant height, and primary and secondary branching patterns of inflorescences. An inflorescence consists of flowers, branches, leaves, and bracts centered around the main stem. Some common inflorescence types in dicots and monocots include raceme, panicle, spike, thyrse, capitulum, and cymose. The Aquilegia genus is an example of a cymose inflorescence. Cymose patterns can be dichasial or monochasial and have primary, secondary, and tertiary branching.

While a lot of recent effort has gone into the study of genes involved in the floral development (ABC model) in the Aquilegia, only a few studies have looked in detail into inflorescence development and intraspecific morphological differences in lateral organs such as bracts, and leaves. This study was aimed to track Aquilegia formosa and Aquilegia coerulea to understand heteroblasty in lateral organs and observe inflorescence development. I noticed conserved morphological patterns in simple bracts, trifoliate bracts, and leaves with/without a petiole at three to four main nodes on the stem in both species. I also observed some differences between the species; this included height, number of buds, the requirement of cold to flower, the ramification of inflorescence, and the number of days plants remain in the reproductive state. In the future, I suggest comparative morphological analysis including more species from this genus.

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TABLE OF CONTENTS

SIGNATURE PAGE……………………………………………………………….……ii

ABSTRACT……………………………………………………………………………..iii

LIST OF TABLES………………………………………………………………...…….vi

LIST OF FIGURES………………………………………………………………...…..vii

CHAPTER 1: INTRODUCTION………………………………………………….……1

1.1 Inflorescence Types……………………...………………………………...... 1

1.2 (a) Review: Studies in Monocot (Grass) Model Systems………….………….7

1.2 (b) Review: Studies in Dicot Model Systems………………………….…….16

1.3 Aquilegia: A Dichasial, Basal Eudicot Model System…………..…………..23

CHAPTER 2: METHODS…………………………..…………………………………28

2.1 Growing Aquilegia Species……………………………………………….….29

2.2 Tracking Plants……………………………………………………………....30

2.3 Methods for Tracking 1-2-3 and 1-2-3-4 Patterns………………...…………32

2.4 Scanning Electron Microscopy Tissue Preparation for SEM…………….….42

CHAPTER 3: ANALYSIS………………………………………………………..……45

3.1 Analysis of A. formosa and A. coerulea……………………………..………45

3.2 Comparison of Primary Branches and Lateral Organs on Nodes in Stressed vs

Non-Stressed Plant in A. coerulea in the 1-2-3 and 1-2-3-4 Patterns………..…..79

3.3 Analysis of Phyllotaxy of Primary Branches in A. formosa and

A. coerulea…………………………………………...……………………..……91

3.4 Scanning Electron Microscopy of Apical Meristems of A. coerulea and A.

formosa………………………………………………………….……………….92

3.5 Analysis of Primary Branching in A. coerulea……………………..………..94

3.6 Tracking of A. formosa and A. coerulea………………………………...…...97

3.7 Differences between A formosa and A.coerulea……………….………….…98

CHAPTER 4: CONCLUSION AND FUTURE RESEARCH……………………...101

4.1 Conculsions from Research………………………………………………...101

4.2 Future Research……………………………………………………...…..…102

REFERENCES………………………………………………………………..……….106

LIST OF TABLES

Table 2.1. Steps involved in the dehydration of meristems for SEM……..…..…….…….43

Table 3.1. Comparison of leaf, bract, and petiole types in 1-2-3A and B pattern…….…46

Table 3.2. Comparison of leaf, bract, and petiole types in 1-2-3-4A and 1-2-3-4B….….53

Table 3.3. Comparison of most and least commonly found patterns….………….…...….60

Table 3.4. Comparison of leaf, bract, and petiole types in plants deviating from 1-2-3 and

1-2-3-4……………………………………………………………………………………68

Table 3.5. Comparison of frequencies of primary branching in A. coerulea…...…...……95

Table 3.6. Comparative analysis of A. coerulea and A. formosa…….…………….….....101

LIST OF FIGURES

Figure 1.1. Monochasial cyme inflorescence diagram….……..……..……………………2

Figure 1.2. Dichasial cyme inflorescence diagram……….……...... ……………………..3

Figure 1.3. Raceme inflorescence diagram………..……………..………...….…..………3

Figure 1.4. Thyrse inflorescence diagram……..……..……………..………..……………4

Figure 1.5. Panicle inflorescence diagram………...…………………..…..………………4

Figure 1.6. Spikelet inflorescence diagram……..…….………………...…..……………..5

Figure 1.7. Capitulum inflorescence diagram……..…………….………..…..…………...6

Figure 1.8. Meristem transitions in Zea mays……………..………………….…………..8

Figure 1.9. Meristem transitions in Brachypodium distachyon……..…….….………..…10

Figure 1.10. Meristem transitions in rice……..…...……………………..…..…………..12

Figure 1.11. Meristem transitions in wheat………………...…………..……………...…14

Figure 1.12. Meristem transitions in barley………..……..………………………...……15

Figure 1.13. Meristem transitions in Arabidopsis…….....…..…………………...………16

Figure 1.14. Meristem transitions in tomato…...…..….……...………………………….21

Figure 1.15. Stages of meristem transitions in Pisum sativum (Pea)……….……………23

Figure 1.16. Pictures of A. coerulea and A. formosa studied in this thesis …………….…24

Figure 1.17. Ramification of inflorescence in two Aquilegia species…………...... …...... …25

Figure 1.18 Meristem transitions in A. formosa and A. coerulea ……..…..…….………26

Figure 2.1. Primary (1°) and secondary (2°) branching in A. coerulea and A. formosa

………………………………………...………………………….………………………29

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Figure 2.2. Photographic tracking of Aquilegia species…….……..…………...……….31

Figure 2.3. A. coerulea ……...…………..…………..…..……...………………….……33

Figure 2.4. Observation 1 ………....……...... …………………………………………....34

Figure 2.5. Observation 2 ……………………..…………………………………..……..35

Figure 2.6. Observation 3………….………………...………………….……………….36

Figure 2.7. Observation 4 ……………….……………….…………………...………….37

Figure 2.8. Observation 5 ……………….………………………….…………...…….…39

Figure 2.9. Observation 6……….……………...…………………...……………...……40

Figure 2.10. Observation 7…………….……...…...……….……………………………41

Figure 2.11. Observation 8………….……………………………...……………………42

Figure 2.12. Equipment used in the analysis of the SAM……………..……..…….…..…44

Figure 3.1. Pictures of node 1 in 1-2-3A pattern in A. coerulea...... ,,,,,...... 47

Figure 3.2. Pictures of node 1 in 1-2-3B pattern in A. coerulea………..…..……………47

Figure 3.3. Pictures of node 1 in 1-2-3B pattern in A. formosa…………….…………….48

Figure 3.4. Pictures of trifoliate leaves and bracts at node 2 in 1-2-3A pattern in A. coerulea…...... 49

Figure 3.5. Pictures of trifoliate bracts leaves at node 2 in 1-2-3B pattern in A. coerulea…………………………………………….…………………………………….50

Figure 3.6. Pictures of trifoliate leaves and at node 2 in 1-2-3B pattern in A. formosa………….……………………………………………………………………….51

Figure 3.7. Pictures of trifoliate compound leaves at node 3 in 1-2-3A pattern in A. coerulea……………..……………………………………………………………………51

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Figure 3.8. Pictures of trifoliate sessile leaves at node 3 in the 1-2-3B pattern in A. coerulea…………………………………………………………………………………..52

Figure 3.9. Pictures of trifoliate sessile leaves at node 3 in the 1-2-3B pattern in A. formosa…………………………………………………………………………………..53

Figure 3.10. Pictures of node 1 in 1-2-3-4A pattern in A. coerulea…….………………..54

Figure 3.11. Pictures of node 1 in 1-2-3-4B pattern in A. formosa…………..………….54

Figure 3.12. Pictures of node 2 sessile trifoliate bracts in the 1-2-3-4A pattern in A. coerulea……………..……………………………………………………………………55

Figure 3.13. Pictures of node 2 variations in the 1-2-3-4B pattern in A. formosa………..56

Figure 3.14. Picture of trifoliate leaves at node 3 in 1-2-3-4A pattern in A. coerulea….…57

Figure 3.15. Pictures of trifoliate leaves at node 3 in 1-2-3-4B pattern in A. formosa.…58

Figure 3.16. Picture of node 4 in 1-2-3-4A pattern in A. coerulea……..…………………59

Figure 3.17. Pictures of node 4 in 1-2-3-4B pattern in A. formosa…..……..…..………..59

Figure 3.18. Pictures of the most common pattern found in A. coerulea (1-2-3A)…….…62

Figure 3.19. Pictures of the second most common pattern found in A. coerulea (1-2-

3B)………………………………………………………………………………………..63

Figure 3.20. Pictures of the third most common pattern found in A. coerulea (1-2-3-

4A)……………………………………………………………………………………….64

Figure 3.21. Picture of the outlier plant showing dramatic heteroblasty in A. coerulea…………………………………………………………………………,……….65

Figure 3.22. Pictures of the most common pattern found in A. formosa (1-2-3-4B).……66

Figure 3.23. Pictures of the second most common pattern found in A. formosa (same as outliers) (1-2-3B)…………………………..…………………………………………….67

Figure 3.24. Pictures A. formosa showing the 1-2-3B pattern………..………………….70

Figure 3.25. A. formosa showing dramatic heteroblasty…………….…....……………...71

Figure 3.26. Picture of A. formosa……...…………………………………………….….74

Figure 3.27. Nodes above node 1 (NA1) and below node 3 (NB3)

……………………………………………………………….…………………...………76

Figure 3.28. 1-2-3-4 extra node patterns…….……...... ………………………………….78

Figure 3.29. Pictures of A. coerulea wild type/normal, non-stressed plant and a stressed plant having powdery mildew infection………………………………………………….80

Figure 3.30. Pictures of non-stressed plants with branching/budding and stressed plants with no branching/budding in A. coerulea at node 2 in the 1-2-3 pattern…………………82

Figure 3.31. Pictures of stressed and non-stressed plants in A. coerulea at node 3 in the 1-

2-3 pattern………………………..………………………………………………………83

Figure 3.32. Pictures of a non-stressed plant and a stressed plant in Aquilegia coerulea showing the 1-2-3-4 pattern…………………..…………………………………………..85

Figure 3.33. Pictures of non-stressed and a stressed plant in A. coerulea at node 2 in the 1-

2-3-4 pattern………………………...……………………………………………………86

Figure 3.34. Pictures of a non-stressed plant and stressed plant in A. coerulea at node 3 in the 1-2-3-4 pattern…………..……………………………………………………………87

Figure 3.35. Pictures of a non-stressed plant and a fully stressed plant in A. coerulea at node 4 in the 1-2-3-4 pattern……………….…………………………………………..88

Figure 3.36. Comparison of a non-stressed branched plant to a stressed branched plant in

A. coerulea in the 1-2-3-4 pattern with some branches missing………………………….90

Figure 3.37. Pictures of the two opposite orientations in 1-2-3 pattern found in A. coerulea when looking at primary side shoots……………………………………………………..91

Figure 3.38. Pictures of the two opposite orientations in 1-2-3 pattern found in A. formosa when looking at secondary side shoots……………………………………..……………92

Figure 3.39. Scanning electron micrographs of shoot apical meristems (SAM) of A. formosa and A. coerulea…………………...……………………………………………..94

Figure 3.40. Pictures showing three and four primary branched plants in A. coerulea…………………………………………………………………………………..96

Figure 3.41. Tracking of A. formosa using a camera…….……………………..………..97

Figure 3.42. Tracking of A. coeurela using a camera…………………………………….98

Figure 4.1. Pictures of a non-stressed A. pubescence showing 1-2-3 pattern found in Canada…………………………………………………………………………….…….104

CHAPTER 1

INTRODUCTION

1.1 Inflorescence Types

An inflorescence consists of flowers, bracts, and branches, centered around the primary shoot (Kirchoff, 1986). Inflorescence architecture is important for plants because it determines the plant-pollinator interactions (Ohara & Higashi, 1994). Pollinators visit plants with specific inflorescence shapes and sizes for pollination (Ohara & Higashi,

1994). This interaction, if beneficial, ultimately ensures the reproductive success of plants (Schlinkert et al., 2016). Inflorescences are important for the agriculture industry in many ways. In crop plants, the number of branches and flowers can impact productivity. In the ornamental industry, showiness of inflorescences, flower number, and orientation are features that attract buyers (Elomaa et al., 2018).

One of the most interesting features of angiosperms is their wide array of inflorescence branching (Hake, 2008). Terminology defining inflorescence types has been used in confusing ways (Endress, 2010). One such example is in developmental genetics literature, where the term determinate is often used to describe a cymose branching pattern, and indeterminate is often used to describe a racemose branching pattern. In botanical literature, these terms are used in a slightly different context. The presence and absence of the terminal flower on the shoot apex determine if the inflorescence is determinate or indeterminate, respectively.

Inflorescences are classified into various types in literature, but amongst them, two major types differ strikingly, the cyme and the raceme (Wylder 1851; Celakovsky,

1893; Pilger, 1921; Troll, 1964, Endress, 2010). The other major type of inflorescence described in literature include thyrse, panicle, spikelet and capitulum. Below I will briefly review the structure of these major inflorescence types.

A cymose branching pattern can either be monochasial (Figure 1.1) (branch on one side of the plant) or dichasial (branch of both sides of the plants) (Figure 1.2), with the potential to have higher-order branching (Wydler 1851; Troll 1957, 1964, Endress,

2010). An example of dichasial cyme is Aquilegia coerulea (Sharma et al., 2019). As reviewed by Endress, racemes are determinate in which the primary shoot ends in a flower or indeterminate if it does not end in a flower (Endress, 2010) (Figure 1.3).

Racemose inflorescences have variable lateral branches but no third-order branches

(Endress, 2010). More flowers are added to the racemose inflorescence with the continuous growth of the primary shoot (Endress, 2010). Examples of plant species with racemes are Arabidopsis thaliana and Antirrhinum majus (Benlloch et al., 2007).

Figure 1.1 Monochasial cyme Inflorescence diagram. There is one lateral branch on right side coming of the main shoot in monochasium (Endress, 2010).

Figure 1.2 Dichasial cyme inflorescence diagram. There are two lateral branches coming of the main shoot in dichasium (Endress, 2010).

Figure 1.3 Raceme inflorescence diagram. The end of the primary shoot can either end in a flower or not (determinate vs indeterminate, (Endress, 2010)

A third type of inflorescence is referred to as a thyrse in which the inflorescence initiates as a racemose and has the higher-order branching of a cymose ( L. & A. Bravais

1837; Troll; 1964, as reviewed in Endress, 2010) (Figure 1.4). Another common branching pattern seen in grasses is the panicle, which can be highly branched (1.5).

Every order in panicle ends in a flower (Wydler, 1851; Celakovsky, 1893; Pilger, 1921;

Troll, 1964, Endress, 2010).

Figure 1.4 Thyrse inflorescence diagram. Inflorescence with features of both cymose and racemose (Endress, 2010)

Figure 1.5 Panicle inflorescence diagram. There is more branching at the base compared to the top in panicle (Endress, 2010)

In monocots such as wheat and barley, the inflorescence is called a spike, the flowering unit is called the spikelet (Figure 1.6), and the individual flower is called the floret. In spike inflorescences, the spikelet is attached directly to the inflorescence

(Koppolu & Schnurbusch, 2019).

Floret 3

Floret Palea

l emma

Rachilla

Floret 1

lower glume

Rachis

Figure 1.6 Spikelet inflorescence diagram (Kirby and Appleyard, 1987). Floret unit within the spikelet unit is shown.

Very interesting, unusual inflorescences are observed in the Asteraceae family. In this family, its arrangement has distinct geometric patterns on the inflorescence, which are at the same level and generally missing stalks. This inflorescence looks like a solitary flower and is called capitulum (Endress, 2010) (Figure 1.7). Examples of this inflorescence are found in Helianthus (Elomaa, 2018).

Figure 1.7 Capitulum inflorescence diagram. (Endress, 2010).

Besides morphological studies, the focus of scientific research in the last two decades have also been on understanding the genetic networks underlying diverse inflorescence architecture. Multiple studies on major model systems both from monocots and dicots are published that helps detangle the complex genetic pathways that control inflorescence development (selected reviewed in section 1.2).

1.2 Developmental and Molecular Changes Involved in Transitioning from a

Vegetative to Reproductive Phase

The generation of inflorescence architecture involves a complex interplay of meristem identity programs (Sharma 2019). The spatial and temporal expression of meristem identity genes determines the branching patterns of inflorescence. Complex genetic changes are involved in transitioning from Shoot Apical Meristem (SAM)-

Inflorescence Meristem (IM)- and Floral Meristem (FM). These transitions in meristems have been studied both in monocots (mainly grasses, examples include Zea mays,

Brachypodium distachyon, Oryza sativa, Triticum aestivum, Hordeum vulgare), and in dicots (examples include Arabidopsis thaliana , Solanum lycoprsicum, Pisum sativum and Aquilegia coerulea and Aquilegia Formosa).

Below I will briefly review the genetic studies on inflorescence development in monocots and dicots.

1.2 a) Review: Studies in Monocot (Grass) Model Systems

In grasses, meristems transition through discrete states to finally attain FM identity. Recent literature has unraveled the genetic basis of attaining different meristem identities. Zea mays is one of the best-studied grass model systems to understand inflorescence development and patterning. The transition from a vegetative to a reproductive state involves complex steps. First, the vegetative shoot apical meristem transitions to an inflorescence meristem, and then either becomes a branch meristem in the case of male inflorescence, tassel, which goes on to form a spikelet pair meristem

(SPM), or directly transitions into the SPM in a female inflorescence, the ear (Pnueli et al., 1998 as reviewed in Bartlett & Thompson, 2014) (Figure 1.8). The SPM, then transitions into a spikelet meristem (SM), which goes on to form the floral meristem

(Pnueli et al., 1998, Bartlett & Thompson, 2014, Whipple 2017).

Male Female BM

SPM

Figure 1.8 Meristem transitions in Zea mays ((Pnueli et al., 1998, reviewed in Bartlett & Thompson, 2014). Transitions in Zea mays can occur in a 5-6-step process.

Plants are in a continuous mode of development, and new organs are generated throughout a plant’s life cycle. The SAM of plants harbor stem cells that are required to maintain continuous organogenesis. These developmental changes are a result of complex gene expression patterns. The WUSCHAL (WUS) and CLAVATA (CLV) genes are very important to maintain the proliferation and differentiation of the stem cells

(Somssich et al.2016). The expression pattern of two WUS paralogues in maize

(ZmWUS1 and ZmWUS2), were studied using reporter lines (Nardmann & Werr, 2006, as reviewed in Bommert & Whipple, 2018). ZmWUS1 expression is detected in the SAM of seedlings in the shoot apex proceeding germination. ZmWUS2 is transcribed in leaf primordia (not the SAM) where it is expressed at the marginal tip (Nardmann & Werr,

2006, as reviewed in Bommert & Whipple, 2018). Functional studies on ZmWUS1 and

ZmWUS2 have not been reported. The FASICATED EAR2 gene of maize is an ortholog to

CLAVATA2 (CLV2), and the thick tassel dwarf1 (td1) is an ortholog of CLAVATA 1

(CLV1) (Taguchi-Shiobara et al.,2001; Bommert, 2005, as reviewed in Bommert &

Whipple, 2018). Both td1 and fea2 mutants had fascinated ear and thicker tassel

(Taguchi-Shiobara et al.,; Bommert, 2005 as reviewed in Bommert & Whipple, 2018).

Double mutants of td1/fea2 had more kernel rows, and leaf number was reduced

(Bommert et al., 2005, as reviewed in Bommert & Whipple, 2018). The Arabidopsis

CLV3 orthologs in Zea mays are ZmCLE7 and ZmCLE14 (Je et al., 2016; Somssich et al.,2016, as reviewed in Bommert & Whipple, 2018); they have not been functionally characterized.

The ERF transcription factor BRANCHED SILKLESS1(BD1) is involved in maize to specify SM identity (Colombo et al.,1998, Chuck et al., 2002). bd1 mutants in maize have highly branched ears (Colombo et al., 1998). In bd1-2 (allele of bd1), ears are more branched at the base, while mutant bd1-2 tassel is less branched compared to the wild type (Colombo et al., 1998). bd1-2 mutants do not form pistils and are female sterile

(Colombo et al., 1998). Three other genes RAMOSA1 (RA1), RAMOSA2 (RA2) and

RAMOSA3 (RA3) play an important role in SPM determinacy, and RA3 also in SM determinacy. RA1 encodes the C2H2 Zn-finger protein, RA2 encodes for LATERAL

ORGAN BOUNDARY, and RA3 encodes for trehalose 6 –phosphate phosphatase (TPP)

(Vollbrecht et. al. 2005, Tanaka et al. 2013). ra1, ra2, and ra3 mutants produce long branches in tassel and in ears; thus in wild-type plants, they function to limit the production of long branches by promoting determinacy of SPM.

In another grass model system, Brachypodium distachyon, the transition of SAM into FM does not involve the formation of SPM (Pnueli et al., 1998 as reviewed in

Bartlett & Thompson, 2014). Here, the vegetative meristem transitions into the inflorescence meristem. After this step, the branch meristem forms, which turns into the spikelet meristem, which then goes on to form the floral meristem (Pneuli et al., as reviewed in Bartlett & Thompson, 2014) (Figure 1.9).

Figure 1.9 Meristem transitions in Brachypodium distachyon (Pnueli et al., 1998 as reviewed in Bartlett & Thompson, 2014). Transitions in Brachypodium distachyon is a 5- step process.

In Brachypodium distachyon inflorescences, the SM is indeterminate resulting in spikelets producing multiple flowers (Bommert and Whipple 2018). Mutagenesis studies in Brachypodium showed defects in inflorescence development (Derbyshire and Byrne

2013). In the awi mutant, floret lemma did not develop awn and show defects in pistil development. The mutant nif1 failed to produce spikelets. The til1 mutant produced

fewer inflorescences and defective spikelets. A fourth mutant (mos1) in the same study was characterized more in-depth. mos1 mutants exhibited increased axillary meristem, which develops into branches and not spikelets. This phenotype was similar to the bd1 and fzp mutant phenotype from maize and rice, respectively.

In rice, the inflorescence meristem becomes the Primary Branch Meristem

(PBM). After this, the primary branch meristem can form a Secondary Branch Meristem

(SBM) or spikelet meristem, which goes on to form a floral meristem (Thompson &

Hake, 2009) (Figure 1.10). Genetic studies in rice have revealed that TILLERS

ABSENT1(TAB1), the WUS ortholog, initiates the development of axillary meristems

(Tanaka et al., 2015, as reviewed in Bommert & Whipple, 2018). FLORAL ORGAN

NUMBER 2 (FON2) encodes a CLE protein that is related to CLAVATA3. Functional studies revealed fon2 mutants had increased floral organ number. FON2 overexpression results in decreased flower number and floral organs. FON2 was implicated to have a role in meristem maintenance during the reproductive phase (Suzaki et al. 2006, Suzaki et al

2008).

PBM SBM

Figure 1.10 Meristem transitions in rice (Thompson & Hake, 2009). Transition to FM in rice occur in a 5-6 step process.

OSMAD34 encodes a rice MADS-BOX SEPALLATA-Like protein that regulates spikelet and inflorescence architecture (Gao et al., 2010, as reviewed in Bommert &

Whipple, 2018). Osmads34 mutants result in an alerted inflorescence morphology, displaying decreased secondary branches and spikelets, and increased primary branches.

ABERRANT PANICLE ORGANIZATION (APO1) encodes the F-box protein

UNUSUAL FLORAL ORGANS. Loss of function of apo1 results in reduced inflorescence branching and a reduced number of spikelets. Overexpression of APO has the opposite phenotype that produces a larger number of branches and spikelets (Ikeda et al., 2007,

Bommert & Whipple, 2018). This study implicated a functional diversification between

the APO1 and A. thaliana UFO. In rice, the transition of IM -SM and then to FM is suppressed by APO1, whereas in the core eudicot A. thaliana UFO promotes this (Ikeda et. al., 2007). The LFY homolog RFL in rice, controls flowering time and overall plant architecture. Loss of function of RFL results in delayed flowering, and its overexpression leads to early flowering (Rao et al., 2008).

The rice OsMADS14, OsMADS15, and OsMADS18 are three AP1/FRUITFULL- like transcription factors. Overexpression of all these paralogs promotes flowering (Lu et. al., 2012, and Yin et. al., 2019). All three paralogs also promote IM identity. Double mutants of osmads14 and osmads15 develop inflorescences with primary branch primordia but do not develop secondary branches or spikelets (Wu et al., 2017).

Wheat is an important crop plant. There are diploid, tetraploid, and hexaploid wheat varieties (Shitsukawa et. al., 2009). The transition to flowering in wheat involves steps that include the vegetative meristem transitioning into the inflorescence meristem which then transitions to a spikelet meristem (Koppolu & Schnurbusch, 2019). The spikelet meristem makes 10-12 floral meristems with a distichous pattern placed on the rachilla (Koppolu & Schnurbusch, 2019). The inflorescence meristem ends with a terminal spikelet meristem (Koppolu & Schnurbusch, 2019) (Figure 1.11). Comparative developmental studies have revealed that during the early stages of IM formation, the 2n,

4n, and 6n are very similar but a clear distinction in development is noted at the floret differentiation stage (Shitsukawa et al., 2009). The variant of tetraploid wheat called bh wheat displays ectopic long lateral branches bearing florets. The WHEAT FRIZZY

PANICLE (WFZP) gene (Dobrovolskaya et al., 2015, Gauley & Boden, 2019) controls spike branching in wheat. TEOSINTE BRANCHED1 (TB1) is a class II TCP transcription

factor. TB1 controls inflorescence branching in wheat in a dosage-dependent manner

(Dixon et al., 2018). The AP2-like gene Q in wheat is involved in inflorescence development (Gauley & Boden, 2019). Ectopic expression of Q leads to a more spiked compact inflorescence and stunted height in wheat. (Simons et al., 2006 as reviewed in

Gauley & Boden, 2019).

FM FM

FM FM FM FM SM

Figure 1.11 Meristem transitions in wheat (Koppolu & Schnurbusch, 2019). Transitions in wheat occur in a 5-step process.

In barley, the vegetative meristem transitions into IM which then develops into the triple spikelet meristem (TSM). The TSM transitions into two lateral spikelet meristems and the central spikelet meristem. In the six-rowed type, the central and lateral spikelets both are fertile, while in the two-rowed type only the central spikelet is fertile.

(Koppolu & Schnurbusch, 2019) (Figure 1.12). Five different loci have been reported that

can convert two-rowed barley into the six-row type mutants with lateral florets that have greater size and fertility; these genes are vulgare row-type spike 1(Vrs1), 2, 3, 4, and intermedium spike-c (Gustafsson & Lundqvist, 1980; FUKUYAMA et al. 1975, Koppolu

& Schnurbusch, 2019, Komatsuda et al., 2007, Gauley & Boden, 2019).

TSM

LSM CSM LSM

FM FM FM Figure 1.12 Meristem transitions in barley (Koppolu & Schnurbusch, 2019). Transitions to FM in barley occur in a 5-step process.

As highlighted above, studies in grass model systems show some species-specific patterns of meristem transition. For example, SPM development is seen in maize while not in rice or wheat. Overall the diversity in inflorescence branching within the grass family is astounding and the genetic studies of mutants has helped us to understand how branching is regulated at a molecular level.

1.2 (b) Review: Studies in Dicot Model Systems

Arabidopsis is a good model system for studying both floral and inflorescence development. The process of transitioning to flowering proceeds in the following pathway in Arabidopsis. First, the vegetative meristem (VM) transitions to an inflorescence meristem (IM), and then it transitions to a floral meristem (FM) which forms floral organs (Figure 1.13) (Pnueli et al., 1998, Bartlett & Thompson, 2014;

Balanza et al., 2019). This is a simple explanation of the three-step process. The genetic changes that determine the transition into each of these stages are very complex (Pnueli et al., 1998, Bartlett & Thompson, 2014). Arabidopsis plants display a racemose inflorescence. Multiple genes, including WUS, CLV, FT, TFL1, LFY, UFO, and PIN1, regulate the development and overall architecture of the inflorescence branching in

Arabidopsis.

Figure 1.13 Meristem transitions in Arabidopsis (Pnueli et al., 1998, Bartlett & Thompson, 2014; Balanza et al., 2019). Transitions in Arabidopsis occur in a 3-step process.

In Arabidopsis, the stem cell differentiation is maintained by the CLV-WUS signaling pathway, which is an autoregulatory negative-feedback loop (Brand, 2000;

Somssich et. al., 2016, reviewed by Bommert & Whipple, 2018). WUS is signaled from the organizing center to the meristem apex where it promotes the fate of stem cells.

Differentiated stem cells express CLV, which represses WUS (Brand et. al., 2000;

Somssich et. al. 2016, reviewed by Bommert & Whipple, 2018). Repression of WUS by

CLV3 results in a loss of stem cell activity. In clv mutants, WUS expression expands and there is an accumulation of stem cells (Brand et.al., 2000; Somssich, et. al., 2016 as reviewed Bommert & Whipple, 2018). The CLV3 peptide is recognized by receptors-like proteins that repress WUS. (Somssich, et al., 2016 as reviewed Bommert & Whipple,

2018) BARELY ANY MERISTEM (BAM), BAM1, BAM2, and BAM3 are CLV1 related receptor- like kinases (DeYoung et al., 2006; Somssich, et al., 2016 as reviewed

Bommert & Whipple, 2018). bam mutants showed reduced shoot meristem size.

(DeYoung et al., 2006; Somssich et. al., 2016 as reviewed Bommert & Whipple, 2018).

In Arabidopsis, the homeobox gene STIMPY (WOX9/STIP) positively regulates the expression of WUSCHEL (WUS) (Wu et. al., 2005). WOX9 and WUS interact to promote vegetative SAM growth (Wu et. al. 2005). The SBP-box gene paralogous

SPL9/15 in Arabidopsis controls the juvenile to adult phase transition. These genes are also suggested to have a role in establishment of IM and FM identity. (Schwarz et. al.

2008, Bommert & Whipple, 2018). The SBP-box genes are targets of miR156 and miR157 (Gandikota et al., 2007 as reviewed in Poethig, 2009). SPL3 contains a miRNA responsive element (MRE) which is complementary to miRNA157 and miR156

(Gandikota et al., 2007 as reviewed in Poethig, 2009). Genetic studies have revealed

“functional redundancy” in SPL family transcription factors (Poethig, 2009). Constitutive expression of SPL3, or its closely related paralogs SPL4 and SPL5, causes the early flowering phenotype (Gandikota et al., 2007, Wu et., 2009).

The phosphatidylethanolamine-binding proteins (PEBPs) are reported to play an important role in both flowering time and inflorescence development. There are three major clades of this family FLOWERING LOCUS T or FT-like, TERMINAL FLOWER or

TFL-like, and MOTHER OF FT or MFT-like. FT induces flowering in Arabidopsis, and this function of FT has been reported in many other angiosperms (Adeyemo et al., 2017).

FT protein moves from leaves to the SAM through the phloem, FT binds with FD (basic leucine-zipper transcription factor), and this complex activates meristem identity genes

(Andres et al., 2015). Woody perennial poplar has two duplicate copies of FT, FT1, and

FT2, with diverged functions (Hsu et al., 2011). FT1 is involved in reproductive onset in response to winter temperatures, and FT2 is involved in bud inhibition and vegetative growth (Hsu et al., 2011). In Arabidopsis, TFL promotes indeterminacy of the inflorescence and delays flowering, while tfl1 shows a terminal flower and bolts early

(Bradley, 1997, as reviewed by Park, Eshed, & Lippman, 2014). Defects such as making fewer leaves and shoots are also observed in tfl mutants (Conti & Bradley, 2007).

LEAFY (LFY), APETALA (AP1), and CAULIFLOWER (CAL) are floral meristem identity genes. In lfy mutants, phenotypes display an increased number of secondary inflorescences and the development of abnormal flowers (Weigel et al.,1992, Benlloch et al., 2015). APETELA (AP1) is activated by LFY, which on activation positively feeds back on LFY. (Wagner, 1999, Benlloch et al., 2015). In ap1-1 mutants, bract-like structures form in the first-whorl, and these mutants lack petals (Irish & Sussex, 1990;

Weigel et al, 1992; Wagner, 1999). CAULIFLOWER (CAL), which encodes a MADS- box transcription factor, is closely related to AP1 expression. (William et al., 2004, as

reviewed in Benlloch et al., 2015). LFY directly regulates CAL and AP1 (Wagner et. al.

1999, William et al., 2004, Benlloch et al., 2015).

Often the arrangement of vegetative organs on the primary stem is different than the arrangement of flowers on the inflorescences (Endress and Doyle 2007). The study by

Okada et al. 1991 reported that “auxin polar transport system(s)” are necessary for inflorescence and bud formation. The pin 1 mutants display abnormal phyllotaxis and inflorescence development. (Okada et al. 1991, Bartlett & Thompson, 2014). Polar localization of PIN determines phyllotaxis by redirecting “auxin fluxes” and creating

“auxin gradients”, which are needed to form organ primordia (Benkova et al., 2003,

Bartlett & Thompson, 2014).

In Arabidopsis, MONOPTEROS (MP) are involved in primordium initiation

(Weijers & Wagner, 2016, as reviewed in Zhu & Wagner, 2020). MP regulates the transcription factor FILAMENTOUS FLOWERS (FIL). Mutant fil plants show abnormality in number, shape, and arrangement of flowers and floral organs (Sawa et al.

1999). Auxin promotes the accumulation of FIL to initiate flower primordia (Wu et al.,

2015, as reviewed in Zhu & Wagner, 2020). Mutants in MP lead to naked inflorescences lacking flowers (Yamaguchi et al. 2013, Chung et al., 2019, reviewed in Zhu & Wagner,

2020). ARFs ETTIN (ETT), ARF4 directly, and MP indirectly promote reproductive shoots by the silencing of SHOOTMERISTMLESS (STM) (Chung et al., 2019 as reviewed in Zhu & Wagner, 2020).

Auxin and Cytokinin play a crucial role in the maintenance of the stem cells.

ARABIDOPSIS RESPONSE REGULARTOR7 (ARR7) and ARABIDOPSIS RESPONSE

REGULARTOR15 (ARR15) are repressed by auxin in SAM: however, their expression is

induced by cytokinin (Zhao et al., 2010, Bartlett & Thompson, 2014). In arf5 (mp) mutants, ARR7 and ARR15 are increased exclusively in the apices (Zhao et al., 2010, as reviewed in Bartlett & Thompson, 2014). In grasses, this system has not yet been discovered (Bartlett & Thompson, 2014).

Inflorescences in the Solanaceae family are very complex. The family is important economically and from an agricultural perspective because it includes many crop plants. Genetic research on many Solanaceae species has been reported amongst them tomato has emerged to be a useful model system. Tomato grows in a sympodial pattern. The sympodial shoot units or meristems (SYM) are formed from the vegetative meristem, which makes three leaves and then terminates with an inflorescence (Lippman et al., 2008; Pnueli et al., 1998 as reviewed Bartlett & Thompson, 2014). The compound inflorescence grows in a repetitive pattern. The “sequential one-nodal inflorescence sympodial units (ISUs)” is terminated by one flower (Schmitz & Theres,1999; Lippman et al., 2008, Thouet et al., 2008). During inflorescence development, the SIM (sympodial inflorescence meristem) produces a new SIM laterally (sympodial inflorescence meristem) before forming the FM (Lippman et al., 2008). This is a repeating pattern with

SIMs creates a zigzag pattern in the inflorescence (Lippman et al., 2008) (Figure 1.14

Pnueli et al., 1998 as reviewed Bartlett & Thompson, 2014).

Figure 1.14 Meristem transitions in tomato (Pnueli et al., 1998 as review Bartlett & Thompson, 2014). Transitions to FM in Tomato occur in a 4-step process.

The tomato FALSIFLORA (FA) and ANANTHA (AN) genes are orthologs of LFY and UFO respectively (Risseeuw et al., 2013; Molinero-Rosales et al.,1999; Lippman et al., 2008). FA and AN are involved in the floral specification (MacAlister et al., 2012).

Comparing the tomato fa mutant phenotype to the wild type phenotype, fa does not form flowers, but instead forms only secondary inflorescence shoots (Molinero-Rosales et al.,

1999). In an mutant in tomato, the inflorescence meristem does not stop proliferating, leading to abnormal organ types as compared to flowers in wild type (Allen & Sussex,

1996). In tomato, the mutant tmf affects the meristem, causing early flowering and changes an inflorescence with multiple flowers into a solitary flower (MacAlister et al.,

2012, reviewed in Park, Eshed, & Lippman, 2014). SELF PRUNING (SP) controls the transition of the vegetative to reproductive phase in tomato (Molinero-Rosales et al.,

2004). The SP is a homolog of CENTRORADIALIS and TERMINAL FLOWER 1 (Pneeli et al., 1998). The homolog of the Lateral suppressor gene in tomato is required for axillary meristem growth (Schumacher et al., 1999). ls mutant plants are only able to form axillary meristems in the axils of the two leaf primordia “preceding the

inflorescence” (Schumacher et al., 1999). LATERAL SUPPRESSOR is also required for axillary meristem development in other plants including Arabidopsis, which are under control by this similar mechanism. These processes highlight the complexity of tomato development (Greb et al. 2003 as reviewed in Zhu & Wagner, 2020).

Legumes have a compound indeterminate inflorescence. In pea, the vegetative meristem transitions into a primary inflorescence meristem (I1) which then produces the secondary inflorescence meristem (I2). The secondary inflorescence meristem then generates the floral meristems (Sussmilch et al., 2015) (Figure 1.15 Pnueli et al., 1998 as reviewed in Bartlett & Thompson, 2014; Berbel et al., 2012). VEGETATIVE1 (VEG1)), is a AGL79 LIKE MADS-box gene; it is required to establish the I2 meristem identity.

Homozygous veg1 mutants do not form flower buds (Reid & Murfet, 1984, Berbel et. al.,

2012). This mutant form lateral branches producing shoots, instead of an inflorescence and flowers. (Reid & Murfet, 1984). DET (TFL homolog) and PIM (AP1 homolog) are two genes that repress VEG1 in the I1 and floral meristem respectively (Berbel et. al.

2012, Benlloch et al., 2015). VEG1 and DET repress the expression of each other to control the formation of the apical primary inflorescence (I1) and the meristems of the secondary inflorescence (I2) (Benlloch et al., 2015). DET have been functionally characterized as an I1 identity gene. In det mutants the I1 meristem converts into I2 meristem laterally, and terminates into a stub. PIM a MADS-box gene, is important for specifying the establishment of floral meristem identity. Loss of function of pim leads misexpression of VEG1 promoting formation of I2 meristems (Berbel et. al. 2001,

Benlloch et al., 2015). VEG2, a FD homolog, is essential for flowering (Sussmilch et al.,

2015, Murfet and Reid, 1993; Reid et al., 1996; Weller et al., 2009, reviewed by Benlloch

et al., 2015). In mutant line veg2-1, flowering does not occur, this phenotype is similar to veg1 mutants (Weller et al., 2009, reviewed in Benlloch et al., 2015). In a weaker line veg2-2 there is a delay of flowering and the I2 inflorescence resembles the I1 (Murfet and

Reid, 1993; Benlloch et al., 2015). In addition, a study reported mutations in alleles of the gene NEPTUNE (NEP) nep-1 and nep-2, which show increases in flowering on I2 meristem (Singer et al., 1999, as reviewed in Benlloch et al., 2015). NEP has a role in limiting the number of flowers produced by I2.

a) b)

Figure 1.15 Stages of meristem transitions in Pisum sativum (pea) (Pnueli et al., 1998, reviewed by Bartlett & Thompson, 2014; Berbel et al., 2012). Transitions to FM in Pisum sativum occur in a 4-step process.

1.3 Aquilegia: A Dichasial, Basal Eudicot Model System

The Aquilegia genus originated approximately around 6 Mya and has undergone adaptive radiation (Bastida et al. 2010), leading to 70 species (Kramer & Hodges, 2010).

A phylogeny by Fior et al. shows the relationship amongst Aquilegia species that inhabit

North America, Asia, and Europe (Fior et al., 2013). The phylogeny was based on sampling 84 individuals from 62 species of Aquilegia. Next generation 454 sequencing was applied to sequence chloroplast DNA, 21 intergenic spacers and three introns were

used in this study along with a portion of matK coding sequence.

Owing to differences in morphological traits such as petal color and spur length within this genus, pollinators of these species can be different (Figure 1.16) (Hodges &

Kramer, 2007). Pollinators have played an integral role in Aquilegia speciation (Ballerini

& Kramer, 2011). Within the Aquilegia genus the inflorescence pattern is dichasial cymose (Sharma et al., 2019), however, it is important to note that the higher order branching and overall ramification of cymes is variable among species (Figure 1.17 A,

B).

A B

Figure 1.16: Pictures of two Aquilegia species studied in this thesis. (A) A. formosa and (B) A. coerulea.

A B

Figure 1.17. Ramification of inflorescence in two Aquilegia species. This figure above (A, B) represent the cymose arrangement of this genus. (A) A. formosa with multiple primary and secondary branches and (B) Aquilegia coerulea with fewer primary branches.

In our study, we have done comparative morphological analysis on two Aquilegia species, A. coerulea and A. formosa. The habitat of A. formosa extends all the way from

California to Alaska (Groh, 2018). The habitat of A. coerulea ranges from Colorado, to

Arizona, and also Utah (Thairu & Brunet, 2015).

In A. formosa and A. coerulea, the meristem transitions from the VM to the IM and then reaches the FM (Figure 1.18) (Ballerini & Kramer, 2011, Sharma et al., 2019).

Figure 1.18 Meristem transitions in A. formosa and A. coerulea (Ballerini & Kramer, 2011, Sharma et al 2019).

Cold treatment or vernalization is critical to flowering in Aquilegia species

(Ballerini & Kramer, 2011). One gene that is expressed during flowering time is AqFT.

Expression of AqFT is increased in the vegetative state and peaks in the fourth week in vernalization and then decreases. Similarly, the Aquilegia FPI (Floral Pathway

Integrators) homologs AqLFY, AqAGL24.1, AqAGL24.2, and AqFL1 increase after vernalization. The AqSOC in comparison slightly decreases after vernalization. The

AqLFY gene specifically, increases over “two-fold” when vernalized. This increase in

AqLFY is greater than any other of the FPI genes.

Similar to A. formosa, A. coerulea transitions from the VM to the IM and then reaches the FM (Sharma et al., 2019 a) (Figure 1.23). Developmental and transcriptomic studies were conducted in A. coerulea to study genetic players involved in flowering. The developmental data showed presence of vegetative SAM at week 0 (when the plant is 6-8 leave stage and ready to be put in vernalization). Inflorescence meristems was observed after 3 weeks of vernalization. The floral meristem was distinctly visible week 4 and week 6 (at week 4 plants were removed from vernalization, week 6 refers to 2-weeks post-vernalization). Transcriptome analysis of apical meristems at time points weeks 0, 4,

6 and of young inflorescences was performed. Differential expression of the three FT homologs AqFT, AqFT2, and AqFT3 during floral transition was reported. Expression of

AqUFO1, AqUFO2 AqUFO2A, and AqLFY was observed at week 6.

Further functional analysis of AqLFY and AqUFO1/AqUFO2 (Sharma et al., 2019 b) confirmed their role in promoting the transition of IM to FM. In aqlfy silenced plants, extra sepals and bract/sepal chimeras were noticed however, petal identity was not disrupted. In aqufo1, aqufo2, and aqufo1/2 silenced plants, extra sepals, loss of petal identity, reduction in stamen number was observed. Functional studies to understand the role of AqFT homologs will perhaps shed more light into flowering time genetics in

Aquilegia.

While the above studies have provided a wealth of knowledge, it is imperative to mention that our knowledge of the genetic pathways underlying diverse inflorescence types is still limited. More comparative studies both at morphological and genetic level, especially in non-traditional model systems, will help understand the evolution of diverse inflorescence types. Within the Ranunculaceae family distinct inflorescence branching patterns are observed for example Nigella and Delphinium make racemes while Aquilegia displays a cymose branching pattern. Interestingly, the degree of complexity of cymose branching within the species of Aquilegia varies.

CHAPTER 2

METHODS

In the last two decades, Aquilegia has emerged as a model system for developmental genetics, ecological, and genomics studies (Sharma et al., 2019). Within the genus, there are species such as A. coerulea that produce approximately four primary inflorescence branches and about three secondary inflorescence branches (Figure 2.1A).

The genus also has species like A. formosa, where the ramification of the inflorescence is more complex, and it produces about 7-8 primary branches and around 18 secondary branches on average (Figure 2.1 B). In this study, I used A. coerulea and A. formosa as model systems to study the heteroblasty seen in lateral organs of the inflorescence. I measured the following aspects in this study: a) arrangement and count of leaf and bracts on nodes and their morphology, b) inflorescence growth over time, and c) observation of

SAM and bud primordia through SEM. Methods used in this study are described below.

A B

1° 2° 2°

1°

1° 2°

1°

Figure 2.1 Primary (1°) and secondary (2°) branching in A. coerulea (A) and A. formosa (B) Picture of Aquilegia coerulea (A) and A. formosa (B) displaying different primary and secondary branching patterns.

2.1 Growing Aquilegia Species

Seeds for A. coerulea ‘Origami Red and White’ were obtained from Swallowtail

Seeds (Santa Rosa, CA, USA). Aquielgia. coerulea seeds were put on a moistened soil tray with PRO-MIX BX Mycorrhizae soil mix and allowed to germinate. The seeds took about two weeks to germinate. When the seedlings measured 3 inches in length, they were then transplanted into 4-inch pots with PRO-MIX soil. After being transplanted, plants were watered daily in summer months and every 2 days during winter. The plants were grown at the Cal Poly Pomona campus's greenhouses. Daily removal of dead leaves was required to deter Downy mildew infection. After 8-9 weeks, plants attained an eight- leaf stage, and were then moved to a growth chamber for 4 weeks to give a cold treatment

or vernalization to induce flowering. After four weeks in vernalization, plants were moved back to the greenhouses and flowering took place 3-4 weeks after cold treatment.

Aquielgia. formosa were plants purchased (over 20) from local nurseries (Theodore

Payne Foundation, Sun Valley, CA, USA) and the California Botanic Garden

(Claremont, CA, USA) and allowed to bud and flower. Watering varied depending on the time of the year. In summer months watering was needed daily, and in fall and winter watering was required every 2-3 days.

2.2 Tracking Plants

I used approaches that included making drawings of patterns, taking measurements, and photography to track plant growth. Measurements and readings were taken every 2-3 days to document the observed: 1-2-3 A, 1 -2-3 B, 1-2-3-4A, and 1-2-3-

4B patterns which describe bract and leaf patterns on the inflorescence (Figure 2.2 A-D).

A B

3/7/18 3/28/18

C D

6/6/2018 6/29/2018

Figure 2.2 Photographic tracking of Aquilegia species. The pictures show the inflorescence development in both species over a period of three weeks. Pictures of A. coerulea (A) and (B) and A. formosa (C) and (D) were taken using a digital camera multiple times a week.

2.3 Methods for Tracking 1-2-3 and 1-2-3-4 Patterns

Aquilegia vegetative leaves are compound trifoliate which can have long petioles and variable length petiolules that tend to become more elongated as the plant ages.

Leaflets are deeply lobed. In the inflorescence, the bracts exhibit heteroblasty. The bracts can be simple or trifoliate, weakly lobed or ovate, obtuse, lanceolate, and sessile or free.

In this study, the purpose of my observations was to record the pattern of heteroblasty in inflorescence of both Aquilegia species.

As I began. my observations, I noticed the following patterns distinctly, which I named as 1-2-3A and B and 1-2-3-4A and B. I would start the observation of the inflorescence from the base. I looked for the first fully fused bract on the main stem

(bracts can be simple, difoliate or trifoliate). I then noted the morphology of lateral organs on the nodes below until I saw a compound trifoliate leaf which was distinct from other nodes above it. I notate the first fully fused bract as node 1. The nodes under node 1 were noted as 2, 3, or 4 depending on if it is the 1-2-3 or 1-2-3-4 pattern. I observed

th heteroblasty was gradual in the case of the 1-2-3-4 pattern where the 4 node displays a trifoliate compound leaf. In the case of the 1-2-3 pattern, a trifoliate compound leaf is observed at node 3. Hence, lateral organs on inflorescence including leaves and bracts on top few nodes are less complex as compared to the leaves on the bottom nodes.

There was one or two nodes in between the trifoliate compound leaf (or trifoliate sessile leaf) and the bract. In A. coerulea node 3 was a compound trifoliate leaf (Figure

2.3 C, D). Node 2 was the intermediate node displaying a weakly lobed trifoliate leaf

(Figure 2.3 B, D). Node 1 was a sessile trifoliate bract (Figure 2.3 A). The nodes above

node 1 has a bract (as seen in Figure 2.3D). There were also nodes above node 1 in A. formosa. In most cases in A. formosa there were one to multiple nodes below the noted 3 or 4 position. In these nodes the lateral organs were trifoliate compound leaves. I notated the morphologies of leaves or bracts seen at nodes 1, 2, and 3 (Data discussed in Chapter

3).

A B C

2 1 3

Figure 2.3 A. coerulea: A) Showing trifoliate leaf with petiole at node 3, B) an intermediate node at 2 and C) trifoliate bract at node 1.

To denote the 1-2-3 and 1-2-3-4 patterns I made 8 observations in sequential order. My first major observation was that A. coerulea had 3 buds near terminal flower

(Figure 2.4). Three primary branches are coming off the main shoot that terminates into a flower.

Bud 1

Bud 2 Bud 3

Figure 2.4 Observation 1. A. coerulea were found to have only three primary branches occurring.

The second observation was in A. coerulea. Several biological replicates (data in chapter 3) lateral organs seen at nodes 1, 2 and 3 had similar morphology. Sessile bracts were seen at node 1, trifoliate bracts and leaves were observed at node 2, and compound leaves were observed at node 3 (Figure 2.5).

A B

2 2 3

Figure 2.5 Observation 2. A. coerulea were found to have three primary branches and contain similar arrangements of sessile bracts (node 1), trifoliate bracts and leaves (node 2), and leaves (node 3) at three nodes along the primary axis (A, B).

The third observation noticed was in A. formosa, biological replicates (i.e. samples seen) had similarities in sessile bracts (node 1) trifoliate bracts and leaves (node

2), and leaves (node 3) (Figure 2.6 A, B)

A B

1 1

2 2

Figure 2.6 Observation 3. Plants were found in A. formosa (A, B), that contained similarities in bract and leaf features as seen in coerulea in the 1-2-3 node pattern.

The fourth observation was noted in biological replicates which showed a deviation from the 1-2-3 pattern. I observed the transition from complex leaf to a simple bract was more gradual, instead of one node there were two nodes in between a compound leaf and bract. This was noted as the 1-2-3-4 pattern (Figure 2.7 A, B). From this observation, it was determined heteroblasty can be gradual in some plants.

A B

1 3

2 3 4

Figure 2.7 Observation 4. Plants were found that show two nodes between the 1 and 4 nodes. Trifoliate bracts and leaves at node 2 and 3 was observed at these two intermediate nodes. A compound tri foliate leaf was observed at node 4. A. coerulea (A) and formosa (B).

The fifth observation was noted at node 3. The trifoliate compound leaves can have a petiole 1-2-3A (Figure 2.8 A-D) and can be without an elongated petiole in 1-2-

3B. (Figure 2.8 E-H). Here the A and B added to 1-2-3 denotes presence or absence of petiole. Every petiole leaf contained petiolules.

A B

1 2

3 1

C D

1 2

3 2

E F

1 1

3 2

G H

2 1

3 2

Figure 2.8 Observation 5. Plants were found that contained a 3-node displaying a trifoliate compound leaf with a petiole (A-D). This is called 1-2-3A. Trifoliate leaves were also found that had no petiole and is named as 1-2-3B (E-H). Two biological replicates for each pattern is shown above.

The sixth observation was noticed in the 1-2-3-4 pattern in A. coerulea. At node

4 the compound leaf always had a petiole and the leaflets had petiolules. I classified this as 1-2-3-4A (Figure 2.9 A, B).

A B

Figure 2.9 Observation 6. The pattern 1-2-3-4A, is an elongated petiole attached to the leaflets which fuses with the stem at node 4. Two biological replicates showing these patterns are shown above (A, B).

The seventh observation was made in A. formosa. At node 3 in the 1-2-3 pattern there was only one variation observed. The trifoliate leaf is sessile, and no elongated petiole were seen. This was called 1-2-3B. The 1-2-3B pattern in A. formosa is similar to the 1-2-3B in A. coerulea, there is no petiole attached to trifoliate leaf and the leaf is sessile (Figure 2.10 A, B).

A B

3 3

Figure 2.10 Observation 7. Similar to 1-2-3B in A. coerulea there is no elongated petiole attached to the trifoliate leaf which are fused to the stem at the 3 nodes in A. formosa.

The eighth observation was made in A. formosa. At node 4 in the 1-2-3-4 pattern there was only one variation observed, the trifoliate leaf is sessile, and no elongated petiole is observed (Figure 2.11 A, B)

A B

1 2 3 4

Figure 2.11 Observation 8. Similar to 1-2-3B in A. coerulea and formosa, compond leaf at node 4 node is sessile.

2.4 Scanning Electron Microscopy Tissue Preparation for SEM

SEM on A. formosa (pictures by Uriah sanders and preparations by Michael

Speck) and A. coerulea (pictures by Timothy Batz) were used to for a comparative morphology analysis of shoot apical meristem. I used the method published by Sharma et. al. 2019.

The dissected SAM was fixed in formaldehyde-acetic acid alchohol (FAA).

Meristems were then put through a series of ethanol treatments with increasing concentrations (Table 2.1). The critical point drying technique was used to delicately desiccate the samples prior to their introduction into the SEM vacuum chamber (Figure

2.12 A). Sputter coating of desiccated meristems was conducted using the 108 Auto

Sputter Coater (Ted Pella, INC., Redding, CA) with a gold target (Figure 2.12 B). The

gold coated meristems were then loaded into the SEM vacuum chamber (Hitachi

SU3500, Hitachi High Technologies America, Inc.) and observed filament settings at 10-

15 kV (Figure 2.12 C, D). SEM of A. coerulea and A. formosa were taken.

SEM procedures in A. coerulea and A. formosa Table 2.1 Steps involved in the dehydration of meristems for SEM. Proceeding FAA fixation, meristems in both A. formosa and A. coerulea were dehydrated (Table: 1.1)

Step EtOH Time (hrs)

1 70% 2

2 80% 2

3 90% 2

4 95% 2

5 100% 2

6 100% 2

A B

C D

Figure 2.12 Equipment used in the analysis of the SAM. Critical point drying was performed on the meristems before coating and after dehydration (A). A sputter coater was used before SEM where the SAM was plated with gold (picture taken by Tim Batz) (B). Using the computer interface, pictures were taken of A formosa and A. coerulea SAM using SEM (C, D) (pictures taken by Tim Batz)

CHAPTER 3

ANALYSIS

3.1 Analysis of A. formosa and A. coerulea

In this study, my overall sample size for A. coerulea was 86 and A. formosa was

16 plants. In this sample size, I observed the 1-2-3 and 1-2-3-4 patterns. A smaller sample of 43 plants which included plants from the 86 sample in A. coerulea, and all 16 A. formosa were documented using photography and drawings, for further analysis. The morphological aspects analyzed after bolting included positioning, numbering, and morphology of bracts (simple sessile, trifoliate), and leaves (weakly lobed, trifoliate compound deeply lobed, leaves with or without petiole and petiolule). I noticed the following patterns of leaf and bract types arranged on the inflorescence and describe them as 1-2-3A, 1-2-3B, and 1-2-3-4A patterns in A. coerulea and 1-2-3B, and 1-2-3-4B in A. formosa respectively (Table 3.1and 3.2).

Analysis of 1-2-3A and 1-2-3B in A. coerulea and A. formosa Table 3.1 Comparison of leaf, bract, and petiole types in 1-2-3A and B pattern. Based on position (node1-3), number and heteroblasty observed in lateral organs including bract, leaf, and petiole, in the 1-2-3A and 1-2-3-B patterns in A. coerulea and A. formosa were noted. The 1-2-3A pattern was not observed in A. formosa, however the 1- 2-3 B patterns was observed.

A. coerulea A. coerulea A. formosa (1-2-3A) (1-2-3B) (1-2-3B) Trifoliate sessile 12 6 5 bract (node 1) Difoliate sessile 4 6 0 bract (node 1) Simple sessile 4 4 0 bract (node 1)

Trifoliate leaf 11 10 5 (node 2) Trifoliate bract 9 6 0 (node 2) Petiole (node 3) 20 0 0

No petiole (node 0 16 5 3) Total sample (1- 20 16 5 2-3)

In A. coerulea at node 1, bracts were sessile lobed or non-lobed and can be trifoliate, simple, or difoliate, in the 1-2-3A pattern (Figure 3.1 A-C). I observed the 1-2-

3A pattern in 20 biological replicates n=43. Trifoliate sessile bracts occurred in 12 biological replicates, simple sessile bracts occurred in 4 biological replicates, and difoliate sessile bracts occurred in 4 biological replicates in 1-2-3A from the 43 plants observed.

A B C

1 1 1

Figure 3.1 Pictures of node 1 in 1-2-3A pattern in A. coerulea. Pictures show the sessile bract types at node 1 in 1-2-3A in A. coerulea in three different biological replicates. These three biological replicates were plants with the most observed pattern 1- 2-3. Bracts can be trifoliate (A), simple (B), or difoliate (C).

In A. coerulea, bracts in the 1-2-3B pattern at node 1 were sessile lobed or non- lobed and can be trifoliate, simple, or difoliate (Figure 3.2 A-C). Trifoliate sessile bracts occurred in 6 biological replicates, difoliate sessile bracts occurred in 6 biological replicates, and simple sessile bracts occurred in 4 biological replicates in the1-2-3B pattern. The 1-2-3B pattern was observed in 16 biological replicates.

A B C

1 1

Figure 3.2 Pictures of node 1 in 1-2-3B pattern in A. coerulea. Pictures of the sessile bract types at node 1 in 1-2-3B pattern in A. coerulea was observed in three different biological replicates as shown above. Bracts can be trifoliate (A), simple (B), or difoliate (C).

In A. formosa, in the 1-2-3B pattern, bracts at node 1 were lobed and were always trifoliate and sessile (Figure 3.3 A-C). The total sample size for A. formosa was 16 biological replicates with 5 showing the 1-2-3B pattern.

A B C

1 1 1 \

Figure 3.3 Pictures of node 1 in 1-2-3B pattern in A. formosa. Pictures of sessile bracts in the axil of a budding branch at node 1 in the 1-2-3B pattern (A-C) in three biological replicates. Bracts are lobed trifoliate sessile.

Trifoliate bracts and leaves in the 1-2-3A pattern at node 2 had variations. Node 2 is an intermediate node above node 3 and below node 1. There was more variation in lateral organs at node 2 (trifoliate bracts and leaves) in A. coerulea, as compared to A. formosa.

In A. coerulea bracts in the 1-2-3A pattern at node 2 were either a) partially sessile showing 3 petiolule (one central and two lateral) but no petiole (Figure 3.4 A), or b) completely sessile trifoliate lobed without any petiolule (Figure 3.4 B, C), or c) partially sessile with a central petiolule and sessile lateral leaflet (D).

In A. coerulea, another variation observed was the presence of trifoliate leaves instead of bracts on node 2, the leaves displayed variation in degree of lobing observed in different biological replicates (Figure 3.4 E, F, G). In some biological replicates, a) the trifoliate lobed leaves have an elongated central petiolule and sessile lateral leaflets

(Figure 3.4 E, F), b) while in some biological replicates trifoliate lobed leaves were

partially sessile that had 3 petiolule but no petiole (Figure 3.4 G). I observed the 1-2-3A pattern in 20 biological replicates, 7 of which are shown below. Trifoliate bracts occurred in 9 biological replicates, while trifoliate leaves occurred in 11 biological replicates.

A B C 2 2

D E F

2 2

Figure 3.4 Pictures of trifoliate leaves and bracts at node 2 in 1-2-3A pattern in A. coerulea. Pictures of trifoliate bracts at node 2 (A, B, C, D), which can be partially sessile has petiolule but no petiole (A), sessile (B, C), partially sessile with elongated central petiolule and sessile lateral leaflets (D). Trifoliate leaves (E, F, G) are also shown. What differentiates position node 1 from 2 is an increase in complexity.

Both trifoliate bracts or leaves were seen in the 1-2-3B pattern at node 2

(intermediate node), in A. coerulea. The trifoliate bracts were non-lobed (Figure 3.5 A-C) while leaves were lobed (Figure 3.5 D). Bracts have entire margin and were ovate.

Trifoliate leaves have an elongated central petiolule and sessile lateral leaflets. The 1-2-

3B pattern was observed in 16 biological replicates from a sample of 43 plants. Trifoliate bracts were seen in 6 biological replicates, while trifoliate leaves were seen in 10 biological replicates.

A B C

2 2

Figure 3.5 Pictures of trifoliate bracts and leaves at node 2 in 1-2-3B pattern in A. coerulea. Bracts (A-C) and trifoliate leaves (D) were seen at node 2 in the 1-2-3B pattern which are non-lobed and lobed respectively. 5 biological replicates are shown above. What differentiates position node 1 from 2 is an increase in complexity in lateral organs.

Trifoliate leaves were seen 5 times, showing the 1-2-3B pattern at node 2

(intermediate node), from a sample of 16 A. formosa. Trifoliate lobed leaves have a slightly elongated central petiolule and lateral leaflets that were sessile (Figure 3.6 A, B).

This was seen in the greenhouse (A) and wild (B). Trifoliate leaves were also partially sessile (has 3 petiolule but no petiole) (Figure 3.6 C).

A B C

2 2 2

Figure 3.6 Pictures of trifoliate leaves at node 2 in 1-2-3B pattern in A. formosa. Pictures were taken of trifoliate leaves at node 2. Trifoliate leaves have an elongated central petiolule and sessile lateral leaflets (A, B). Slight elongation of petiolule in central and lateral leaflets were also seen (C).

In A. coerulea, at node 3 in the 1-2-3A pattern trifoliate compound leaves with an elongate petiole was seen (Figure 3.7 A-C). Each of the leaflets are distinctly lobed into three segments and petiolules are connected to the petiole making this node non- sessile. The 1-2-3A pattern in A. coerulea, was seen in 20 biological replicates from a total sample of 43 plants.

A B C

3 3 3

Figure 3.7 Pictures of non-sessile trifoliate compound leaves at node 3 in 1-2-3A pattern in A. coerulea. Pictures were taken of trifoliate compound leaves at node 3 (A- C). 3 different biological replicates are shown above. Dramatic heteroblasty is observed in lateral organs attached at the node 3.

In A. coerulea, at node 3 in the 1-2-3B pattern a trifoliate leaf (distinctly lobed into three segments) without an elongated petiole was seen making it partially sessile

(Figure 3.8 A-C). The central and lateral leaflets had petiolules. My sample size for node

3 in 1-2-3B in A. coerulea was 43 biological replicates, in which 16 were 1-2-3B.

B A C

3 3 3

Figure 3.8 Pictures of trifoliate partially sessile leaves at node 3 in the 1-2-3B pattern in A. coerulea. Pictures of trifoliate leaves at node 3 without an elongated petiole (sessile, 1-2-3B) (A-C). Dramatic heteroblasty is observed in lateral organs attached at the node 3.

Trifoliate leaves (distinctly lobed into three segments) were seen in the 1-2-3B pattern at node 3 in A. formosa (Figure 3.9 A-C). Leaves did not have elongated petiole connected to petiolules, which was also seen in 1-2-3B trifoliate compound leaves in A. coerulea. This makes leaves at node 3 partially sessile. Trifoliate leaves without a petiole were seen in all 5 biological replicates of 1-2-3B from a total sample of 16 plants.

A B C

3 3 3

Figure 3.9 Pictures of trifoliate partially sessile leaves at node 3 in the 1-2-3B pattern in A. formosa. The leaves lack an elongated petiole and are mostly sessile (1-2- 3B) (A-C). Three biological replicates are shown above. Dramatic heteroblasty as compared to node 1, and 2, was observed in lateral organs attached at the node 3.

Analysis of 1-2-3-4A and 1-2-3-4B in A. coerulea and A. formosa Table 3.2 Comparison of leaf, bract, and petiole types in 1-2-3-4A and 1-2-3-4B. Of the total observed plants, some plants exhibited the 1-2-3-4A and 1-2-3-4B patterns in A. coerulea and A. formosa respectively. 1-2-3-4A has a petiole at node 4, while 1-2-3-4B does not have a petiole at node 4.

A. coerulea (1-2-3-4A) A. formosa (1-2-3-4B) Trifoliate sessile bract 0 6 (node 1) Difoliate sessile bract 1 0 (node 1) Mono bract (node 1) 5 0 Trifoliate leaf (node 2) 0 2 Trifoliate bract (node 2) 6 0 Trifoliate leaf fully sessile 0 4 (node 2) Trifoliate leaf (node 3) 3 4 Trifoliate bract (node 3) 3 0 Trifoliate leaf fully sessile 0 2 (node 3)

Petiole (node 4) 6 0 No petiole (node 4) 0 6

Total sample (1-2-3-4) 6 6

At node 1 bracts in the 1-2-3-4A pattern were sessile and simple (Figure 3.10 A) in A. coerulea. A sessile difoliate bract was observed in one biological replicate (Figure 3.10

B). I observed the 1-2-3-4 A pattern in 6 biological replicates in A. coerulea from a total sample of 43 plants.

A B

1 1

Figure 3.10 Pictures of node 1 in 1-2-3-4A pattern in A. coerulea. Bracts in the 1-2-3- 4A pattern are shown in two pictures above in different biological replicates.

In A. formosa, the 1-2-3-4B pattern, bracts at node 1 were lobed and were always trifoliate sessile. This was observed in 6 biological replicates from a total sample of 16 plants (Figure 3.11 A-C).

A B C

1 1 1

Figure 3.11 Pictures of node 1 in 1-2-3-4B pattern in A. formosa. Bracts in the 1-2-3-4 pattern were always sessile trifoliate lobed at node 1 (A-C).

In A. coerulea, at node 2, in the 1-2-3-4A pattern, a trifoliate bract was always observed (Figure 3.12 A-C). This was observed in 6 biological replicates from a total sample of 43 plants

A B C

2 2 2

Figure 3.12 Pictures of node 2, fully sessile and partially sessile trifoliate bracts in the 1-2-3-4A pattern in A. coerulea. Sessile trifoliate bracts at node 2 (A, B) and partially sessile (C) trifoliate bracts occurred in all biological replicates. What differentiates position 1 from 2 is an increase in complexity.

In A. formosa, two variations at node 2 were seen. A deeply lobed trifoliate leaf

(Figure 3.13 A) and a fully fused weakly lobed leaf to stem (Figure 3.13 B). The central leaflet of the trifoliate leaf had a slightly elongated central petiolule (partially sessile), with lateral leaflets that were sessile. A total of 6 biological replicates displayed 1-2-3-4B pattern from a sample of 16 plants. A trifoliate leaf occurred in 2 biologicial replicates and a fully fused leaf occurred in 4 biological replicates

A B

2 2

Figure 3.13 Pictures of node 2 variations in the 1-2-3-4B pattern in A. formosa. Trifoliate leaves (partially sessile) (A) and a fully sessile leaf node 2 (B) were seen at node 2 in A. formosa. The difference between a trifoliate leaf and a fully fused leaf is a trifoliate leaf has central petiolule while fully fused leaf does not.

In A. coerulea, at node 3 (an intermediate node) in the 1-2-3-4A pattern, there were 2 variations observed, a trifoliate bract (Figure 3.14 A, B, C) and leaf (Figure

3.14D). Trifoliate bracts are ovate (partially sessile). Trifoliate leaves have an elongated central petiolule and sessile lateral leaflets (partially sessile). There was variation in being lobed and non-lobed displayed by trifoliate bracts. My sample size for node 3 in 1-2-3-

4A in A. coerulea was 6 biological replicates from a total sample of 43 plants. Trifoliate bracts and trifoliate leaves both occurred in three biological replicates in A. coerulea at 1-

2-3-4A.

A B C

3 3 3

Figure 3.14 Picture of trifoliate leaves at node 3 in 1-2-3-4A pattern in A. coerulea. Trifoliate bracts (A, B, C) and leaves at node 3 (D). Weak lobing in the bracts B and C and the leaf in picture D shows increased level of complexity as compared to the ovate bracts.

In A. formosa, there were three variations seen at node 3 (an intermediate node) in a sample of 6 biological replicates of the 1-2-3-4B pattern, from a total sample of 16 plants (Figure 3.15 A-D). Trifoliate leaves have an elongated central petiolule and sessile lateral leaflets (partially sessile) (Figure 3.15 A, B), completely sessile leaves are attached to the stem (Figure 3.15 C), and partially sessile leaves showing three petiolule but no petiole which are not lobed into three segments (Figure 3.15 D). Trifoliate leaves were seen in 4 biological replicates, while the fully fused leaf was seen in 2 biological replicates in A. formosa 1-2-3-4B.

A B C

3 3 3

Figure 3.15 Pictures of trifoliate leaves at node 3 in 1-2-3-4B pattern in A. formosa. Pictures show lobed, trifoliate leaves at node 3 (A- D). What differentiates position 3 from 2 is an increase in complexity or observed heteroblasty.

In A. coerulea, at node 4 in the 1-2-3-4A pattern, there was only 1 variation, a trifoliate compound leaf (non-sessile). Trifoliate compound leaves (Figure 3.16 A, B) have leaflets distinctly lobed into three segments and leaflets have petiolules attached to a petiole which connects to the main stem. The sample size for node 4 in 1-2-3-4A in A. coerulea was 6 biological replicates from a total sample of 43 plants.1-2-3-4B was not observed in A. coerulea.

A B

4 4

Figure 3.16 Picture of node 4 in 1-2-3-4A pattern in A. coerulea. These trifoliate compound leaves with a petiole attached to the main stem were observed in the 1-2-3-4A pattern (A, B).

At node 4 in A. formosa in the 1-2-3-4B pattern, a distinctly lobed compound trifoliate leaf with no petiole was always seen (partially sessile) (Figure 3.17 A, B). This is similar to node 3 in the 1-2-3B pattern observed in A. formosa and in A. coerulea. The sample size for node 4 in 1-2-3-4B in A. formosa was 6 biological replicates from a total sample of 16 plants. Central and lateral leaflets have petiolules but the leaf is sessile.

A B

4 4

Figure 3.17 Pictures of node 4 in 1-2-3-4B pattern in A. formosa. Pictures are from two biological replicates (A, B) showing node 4. These sessile trifoliate leaves were found without a petiole, however, the central and lateral leaflets had petiolules (1-2-3- 4B).

In summary, I analyzed dramatic heteroblasty in the inflorescence, and I have described my observation using the 1-2-3A, or B and 1-2-3-4A, or B patterns in A. coerulea and A. formosa. Some patterns were more common than others (Table 3). The

1-2-3A, or B and 1-2-3-A pattern were documented from 43 biological replicates of A. coerulea plants. These 43 plants in which I characterized morphology of lateral organs came from the original sample of 86 plants and pictures from later samples collected. The

1-2-3B and 1-2-3-4B was documented in 16 A. formosa, observations were made on plants that were photo documented (Table 3.3).

Analysis of 1-2-3-4A, 1-2-3-4B, 1-2-3A, and 1-2-3B in A. coerulea and A. formosa Table 3.3 Comparison of most and least commonly found patterns. The 1-2-3 and 1- 2-3-4 patterns were seen to occur at different incidence. Variation from these two general patterns were also seen.

A. coerulea A. formosa

1-2-3 Pattern 70 (n= 86 ) 81.3% 5 (n=16) 31.2%

1-2-3-4 Pattern 16 (n= 86) 18.6% 6 (n=16) 37.5%

1-2-3A Pattern 20 (n= 43) 46.5% 0

1-2-3B Pattern 16 (n=43) 37.2% 5 (n= 16) 31.2%

1-2-3-4A Pattern 6 (n= 43) 13.9% 0

1-2-3-4B Pattern 0 6 (n=16) 37.5%

Variation from 1-2-3 and 1 (n=43) 2.3% 5 (n=16) 31.2

1-2-3-4

Greenhouse n=86 n=13

Wild/Bio Trek 0 n=3

Total Sample (Original n=86 n=16

Survived)

*Orignal sample (lost to n=200

mildew and heat)

The 1-2-3 pattern was seen 70 times while 1-2-3-4 was observed 16 times in a sample size of 86 in A. coerulea. From the 86 plants, some were photo documented to make up my sample size of 43 plants in A. coerulea in which I notated morphological variations of leaf and bract patterns. Plants separate from the original 86 plant sample, from a second batch were also photo documented and included in the 43-plant sample.

Based on observations on morphology of lateral organs the plants were classified as 1-2-

3A, 1-2-3B, and 1-2-3-4A patterns. The 1-2-3A pattern was the most common pattern found (Figure 3.18 A-D).

A B C

3 1

Figure 3.18 Pictures of the most common pattern found in A. coerulea (1-2-3A). Pictures were taken of A. coerulea (A-D) showing nodes 1, 2 and 3. Node 3 was a trifoliate compound leaf with a petiole in the axil of a budding branch.

The 1-2-3B pattern was the second most common pattern, documented 16 times in A. coerulea in a sample size of 43 plants grown in the greenhouse (Figure 3.19 A-D).

The 1-2-3 pattern was seen 70 times in a sample size of 86 plants.

A B C

1 2 3

Figure 3.19 Pictures of the second most common pattern found in A. coerulea (1-2- 3B). Pictures were taken from biological replicates in A. coerulea (A-D) at three different nodes. Node 3 was a trifoliate leaf without a petiole in the axil of a budding branch.

The 1-2-3-4A pattern was the third most common pattern in A. coerulea. In a sample size of 43 plants grown in the greenhouse this pattern occurred 6 times (Figure

3.20 A-D). The 1-2-3-4A pattern was seen 16 times in in a sample size of 86 A. coerulea plants.

A B C

1 3 2

1 2

Figure 3.20 Pictures of the third most common pattern found in A. coerulea (1-2-3- 4A). Pictures were taken from an A. coerulea biological replicate (A-E) at four different nodes. Node 4 was a trifoliate compound leaf with a petiole.

The 1-2-3-4B pattern was the most common pattern documented, occurring 6 times in A. formosa in a sample size of 16 plants grown in the greenhouse and BioTrek

(Figure 3.21 A-E)

A B C

1 2 3 D

4 ‘

Figure 3.21 Pictures of the most common pattern found in A. formosa (1-2-3-4B). Pictures were taken from a biological replicate in A. formosa (A-E). Node 4 was a fused trifoliate leaf without a petiole.

The 1-2-3B pattern was observed 5 times in A. formosa in a sample size of 16 plants grown in the greenhouse (Figure 3.22 A-D).

A B C

2 1 3

Figure 3.22 Pictures of the second most common pattern found in A. formosa (1-2- 3B). Pictures from A. formosa biological replicate (A-D) at three different nodes. Node 3 was a sessile trifoliate leaf without a petiole.

The 1-2-3B pattern in A. formosa was also seen growing in the BioTrek (Figure

3.23 A, B). This is the second most common pattern seen in A. formosa .

A B

2 1

2 3

Figure 3.23 Pictures A. formosa showing the 1-2-3B pattern. Pictures showing lateral organs at nodes 1,2, and 3 of A. formosa growing in Bio Trek. Pictures (A, B) were taken from the same plant

Analysis of variations in patterning in A. coerulea and A. formosa Table 3.4 Comparison of leaf, bract, and petiole types in plants deviating from 1-2-3 and 1-2-3-4. Of the total observed plants, some plants deviated from the 1-2-3-4 A, B and 1-2-3 A, B patterns in both A. coerulea and A. formosa. This was determined by using observations described in the methods section (chapter 2).

A.coerulea A.formosa A. formosa A. formosa

Variation 1 Variation 1 Variation 2 Variation 3

Trifoliate sessile bract 1 2

(node 1)

Mono bract (node 1) 1

Trifoliate leaf (node 1) 2

Trifoliate leaf fully 2

sessile (node 2)

No petiole trifoliate 1 1 2

sessile leaf (node 2)

Trifoliate leaf (node 3) 1

Trifoliate leaf fully 1

sessile (node 3)

Trifoliate leaf (node 4) 2

No petiole trifoliate 2

leaf (node 5)

Total Sample 1 (n=43) 1 (n=16) 2 (n=16) 2 (n=16)

Variations from the 1-2-3 and 1-2-3-4 pattern in both A. coerulea and A. formosa.

In variation 1 in A. coerulea, a simple sessile bract and a trifoliate leaf without a petiole was seen starting at the base of the plant and going up (Figure 3.24 A, B). In variation 1 in A. formosa (Figure 3.24 C), a sessile trifoliate bract and trifoliate leaf without a petiole was seen as you go up from the base of the plant. Rather than transitioning into a weakly lobed trifoliate bract or leaf, direct transitioning into a sessile bract was observed, hence heteroblasty is very dramatic in this case. (Figure 3.24 A, B, C).

A B

2 2

Figure 3.24 A. formosa and A. coerulea showing dramatic heteroblasty. Lower arrow is pointing to a trifoliate leaf (A, B, C) and the upper arrow points to a simple sessile bract (A, B) or trifoliate sessile bract (C). A. coerulea (A, B) and A. formosa (C)

In variation 2 in A. formosa, a trifoliate fused leaf was seen at nodes which I would normally call node 2 and 1. I did not observe a tri bract at what I have called

position 1(Figure 3.25 A-C).

A B

1 2 2

Figure 3.25 Picture of A. formosa. The above A. formosa had a trifoliate leaf with petiolules both in central and lateral leaflets, below the trifoliate leaf with only a central petiolule.

The third type of variation occurred twice (Figure 3.26 E-I, P and J-N, Q), it had a fully sessile tri-bract at node 1(Figure 3.26 E, J). Like the 1-2-3B and 1-2-3-4B (Figure

3.26 A-D, O) patterns, this variant had an increase in complexity in leaf morphology

noted from top to down in the primary shoot. Nodes 2, 3, and 4 (Figure 3.26 F-H, K-M) were intermediate nodes. Node 2 was a fully fused leaf (Figure 3.26 F, K). Node 3 was a somewhat partially sessile trifoliate leaf (Figure 3.26 G) in one case and a fully fused trifoliate leaf in the other (Figure 3.26 L). Node 4 was a trifoliate leaf (Figure 3.26 H, M) in both cases. The compound fused trifoliate leaf (Figure 3.26 I, N) at node 5 had petiolules both in central and lateral leaflets. This is a plant in which the heteroblasty was even more gradual than compared to the 1-2-3-4 pattern. This what I will call 1-2-3-4-5 pattern.

E J

1 1

A F K

1 2 2

G B L

2 3

C H M

3 4

D I N

4 5 5

O P 1 1

2 2 3

4 3

4 5

Q 1

Figure 3.26 Picture of A. formosa. The above A. formosa pictures show the 1-2-3-4 pattern (A-D, O) and the outlier pattern (E-I, P and J-N, Q) which I call 1-2-3-4-5B.

It is imperative to note that there were nodes above node 1 (notated as node above node 1 (NB1)) or nodes below the number 3 node, especially in A. formosa (notated as node below node 3 (NB3)). As mentioned above in methods, my observations included first starting at the base of the inflorescence and going up. I then looked for the first fully

fused bract on the main stem, (bracts can be simple, difoliate or trifoliate). I then notated the morphology of lateral organs on the nodes below this bract until I saw a compound trifoliate leaf which was distinct from other nodes above it. I notated the first fully fused bract as node 1, the node below as node 2, 3, or 4 depending on if it is the 1-2-3 or 1-2-3-

4 pattern. This rule applies to both A. formosa and A. coerulea.

In the 1-2-3A pattern in A. coerulea there was one biological replicate that had an additional node (NA1) above node 1, but no additional nodes were seen below the 3 node

(NB3) (Figure 3.27A). In the 1-2-3B pattern in A. coerulea I observed one biological replicate that had an additional node below the 3 node (NB3) but no additional nodes above the 1 node (NA1) (Figures 3.27 B).

Similarly, in A. formosa I noticed a biological replicate where there were nodes below node 3 (NB3), but no node above the number 1 node (NA1) in the 1-2-3B pattern

(Figure 3.27 C). In A. formosa there was another example where I observed a node below node 3 (NB3), and a node above the number 1 node (NA1) in the 1-2-3B pattern (Figures

3.27 D). While notating readings I observed the 1-2-3 nodes are apparent first, but it is not until a couple weeks later I observed NB3 and NB1.

A B

NA 1 1

2 3 NB 3

C D

2 2

3 NB NB

Figure 3.27 Nodes above node 1 (NA1) and below node 3 (NB3) are seen the in 1-2-3 pattern in A. coerulea (A, B) and A. formosa (C, D).

I observed no nodes above 1 (NA1) or below 4 (NB4) in A. coerulea (Figure 3.28

A) in the 1-2-3-4A pattern. This lack of nodes above the 1 node (sessile simple bracts)

and below the 4 node (trifoliate compound leaves) was seen in all biological replicates in

A. coerulea observed. In the 1-2-3-4B pattern, in A. formosa I noticed two biological replicates in which a node below the 4 node (NB4), which had trifoliate leaflet without a petiole was observed (Figure 3.28 B, C). No nodes above the sessile trifoliate bracts at node 1 were observed

A B

1 2

1 2 NB 3

1 2

4 NB

Figure 3.28 1-2-3-4 extra node patterns. In all A.coerulea found with the 1-2-3-4A pattern, there were only four nodes total (A). There were never more than four nodes. In A. formosa in the 1-2-3-4A pattern (B, C) there are nodes below node 4 (NB4) but not above node 1 (NA4).

3.2 Comparison of Primary Branches and Lateral Organs on Nodes in Stressed vs

Non-Stressed Plant in A. coerulea in the 1-2-3 and 1-2-3-4 Patterns.

I also observed the lateral organs, bracts and leaves in stressed plants that suffered from powdery mildew infection (year-around) and that were heat stressed (during summer) in A. coerulea. In stressed plants, there was both no branching or fewer primary branches. The flower buds were also missing in many cases and only the terminal flower was seen. In some plants only 1-3 flowers were noted. Sessile bracts, trifoliate bracts and leaves, trifoliate compound leaves, and leaflets with a petiole at nodes were found to have the same position as compared to an A. coerulea that were wild type. Hence, I can conclude that in plants, the morphology and placement of the sessile bract (node 1), non- sessile bract or leaf (node 2), and leaf (node 3) was similar despite being stressed or not in the 1-2-3 pattern. A few exceptions with monofoliate leaves at node 2 and 3 were noted in a few stressed plants.

An example of a 1-2-3 non-stressed plant (Figure 3.29 A-C, G) was compared to a stressed plant (Figure 3.29 D-F, H). The stressed plant had no branches and displayed only a terminal flower.

A B C

1 2 3

D E F 3

1 2

H G

1 1

3 2

Branched/Budding Branched/Budding Not Stressed at Stressed at Node/S Node/S (BNS) (SNB)

Figure 3.29 Pictures of A. coerulea wild type/normal, non-stressed plant and a stressed plant having powdery mildew infection. A non-stressed plant with buds and primary branches coming off the 1-2-3A nodes were seen (A-C, G). Pictures of A. coerulea with no buds and primary branches at 1-2-3A nodes (D-F) with a terminal flower was observed (H). Bracts and leaves morphology was similar in stressed and non- stressed plant

Five variations were noted in trifoliate bracts and leaves at node 2 in wild type non-stressed plants (Figure 3.4). These variations were also seen in stressed plants. The variations seen at node 2 in the 1-2-3 pattern in stressed plants include sessile and non- sessile trifoliate bracts (Figure 3.30 A-G). A-C shows fully sessile trifoliate bracts at nodes that were stressed (A, B. SNB) and non-stressed (C. BNS). Figure D shows a stressed partially sessile lobed trifoliate bract (SNB), while figure E shows a partially- sessile lobed trifoliate bract that’s non-stressed (BNS). Figure F shows a partially sessile stressed bract a node 2 (SNB). Figure G shows a partially sessile non-stressed trifoliate bract at node 2 (BNS).

SNB BNS

A B C

2 2 2

D E

2 2

F G

2 2

Figure 3.30 Pictures of non-stressed plants with branching/budding and stressed plants with no branching/budding in A. coerulea at node 2 in the 1-2-3 pattern. Pictures of stressed plants with no branches/buds and non-stressed plants with branches/buds were taken of A. coerulea at node 2 (A-G).

Three examples of non-sessile stressed trifoliate compound leaves were seen, with no branches/buds coming off the 3 node in the 1-2-3A pattern (Figure 3.31 A-C). Wild type non-sessile trifoliate compound leaves at node three displayed a similar phenotype

(Figure 3.31 D, E).

A B C

3 3 SNB D E

BNS

Figure 3.31 Pictures of stressed and non-stressed plants in A. coerulea at node 3 in the 1-2-3 pattern. Stressed A. coerulea with no buds and branches at 3 node (1-2-3A) in the axil of a trifoliate compound leaf (A-C). Pictures of non-stressed plant with buds and branches coming off the 3 nodes (D, E).

1-2-3-4A non-stressed (all branches and buds present) and stressed (no branches or buds) plants were compared in A. coerulea. In the two plants compared (Figure 3.32

A-J), both stressed and non-stressed plants had a number 1 node that was a simple sessile bract (Figure 3.32 A, E). The number 2 node was a trifoliate bract in both the non- stressed plant (Figure 3.32 B), and stressed plant (Figure 3.32 F). The number 3 node was a trifoliate lobed leaf in the non-stressed plant (Figure 3.32 C) and trifoliate bract in the stressed plant (Figure 3.32 G). The number 4 node was a non-sessile trifoliate compound leaf (Figure 3.32 D, H), which has similar morphology in both non-stressed and stressed plants.

A B C

1 2 3 BNS

4 BNS

E F G

1 2 3 SNB H

4 SNB

I J

1 2 1

4 2

3 4

BNS SNB

Figure 3.32 Pictures of a non-stressed plant and a stressed plant in A. coerulea showing the 1-2-3-4 pattern. Non-stressed plant of A. coerulea with buds and branches coming off the 1-2-3-4A nodes (A-D, I). Pictures of a stressed plant with no buds and branches coming off the 1-2-3-4A nodes was also noticed (E-H, J). Morphology of simple sessile bracts, trifoliate non-lobed leaf and bracts, and trifoliate compound leaves was observed.

Morphological similarities and differences were noticed in trifoliate bracts in stressed and non-stressed plants at node 2. Plants with no buds or branches (Figure 3.33

A-C) and plants with buds and branches (Figure 3.33 D, E) were compared in the 1-2-3-

4A pattern.

A B C

2 2 2

SNB

D E

2 2

BNS

Figure 3.33 Pictures of non-stressed and a stressed plant in A. coerulea at node 2 in the 1-2-3-4 pattern. Stressed plant pictures were taken of A. coerulea with no buds and branches coming off the number 2 node (A-C). Non-stressed pictures were taken of plants with buds and flowers coming off the number 2 node (D, E). A-E were trifoliate bracts.

Morphological variation was noticed in trifoliate bracts and trifoliate leaves at node 3 in the 1-2-3-4A pattern. These similarities and differences were found in both stressed plants (Figure 3.34 A-C) and non-stressed plants (Figure 3.34 D, E).

A B C

3 3 SNB D E

3 3 BNS

Figure 3.34 Pictures of a non-stressed plant and stressed plant in A. coerulea at node 3 in the 1-2-3-4 pattern. Stressed plant pictures were taken of A. coerulea with no buds and branches coming off the number 3 node (A-C). Non-stressed plant pictures were taken of A. coerulea with buds and branches coming off the number 3 node (D, E). A-C were trifoliate bracts, while D and E were trifoliate lobed leaves

Morphological similarities were noticed in the three unfused leaves with a petiole at node 4 in the 1-2-3-4A pattern. These similarities were seen in both stressed plants

(Figure 3.35 A, B) and non-stressed plants (Figure 3.35 C, D).

A B

4 SNB 4 C D

4 4

BNS

Figure 3.35 Pictures of a non-stressed plant and a fully stressed plant in A. coerulea at node 4 in the 1-2-3-4 pattern. Stressed plant of A. coerulea with no buds and branches coming off the number 4 node (A, B). Picture of a non-stressed plant with buds and branches coming off the 4 node was also seen (C, D).

Examples of the 1-2-3-4A pattern was noticed where there were some branches/budding at nodes due to stress such as mildew. Comparison of stressed (Figure

3.36 E-L, N, O) vs non-stressed (having all its branches) (Figure 3.36 A-D, M) was looked at. These stressed plants had 1 (Figure 3.36 E) – 3 (Figure 3.36 I, J, K) primary branches present at nodes. Even when there were branches/budding missing, nodes in the

1-2-3-4A pattern (simple sessile bracts, trifoliate bracts and leaves, and non-sessile trifoliate compound leaves) remained roughly the same with similarities in morphology as compared to a non-stressed plant.

A B C

2 3 BNS

4 BNS

E F G

2 3 1 SNB

4 SNB I J K

2 3 1 SNB

SNB

M N

1 2 3 1 2 4 3 4

Figure 3.36 comparison of a non-stressed branched plant to a stressed branched plant in A. coerulea in the 1-2-3-4 pattern with some branches missing. A. coerulea with 1-3 branching/budding nodes in two different A. coerulea (E-L, N, O) was seen. Pictures of a non-stressed plant showing buds coming off all the nodes was also seen (A- D, M). Simple sessile bracts, trifoliate bracts and leaves, and trifoliate compound leaves remained at the same positions along the main stem in all these plants

3.3 Analysis of Phyllotaxy of Primary Branches in A. formosa and A. coerulea

Another aspect that I have observed in this study was the inflorescence branch phyllotaxy in the 1-2-3A and B patterns with respect to bracts position. When lined up with the first fused sessile trifoliate bract (node 1) facing forward, there were two orientations of alignment of primary branching when comparing the 1-2-3A and B patterns in A. coerulea (Figure 3.37 A, B).

A B

3 2

Left (3), Right (2), Center (1) Right (3), Left (2), Center (1)

Figure 3.37 Pictures of the two opposite orientations in 1-2-3 pattern found in A. coerulea when looking at primary side shoots. Opposite orientations in A. coerulea in primary branching was seen in the 1-2-3 A and B patterns. Figure A was left (node 3), right (node 2), center (node 1) and figure B was Right (node 3), Left (node 2), Center (node 1).

Similarly, in A. formosa, I noted both orientations in the 1-2-3A and B patterns (Figure

3.38 A, B).

A B

1 2

3 2

Left (3), Right (2), Center (1) Right (3), Left (2), Center (1)

Figure 3.38 Pictures of the two opposite orientations in 1-2-3 pattern found in A. formosa when looking at secondary side shoots. Two opposite orientations in A. formosa were seen in primary branching of the 1-2-3B pattern nodes. The first fully fused sessile trifoliate bract was faced forward. Figure A was left (node 3), right (node 2), center (node 1) and figure B was right (node 3), left (node 2), center (node 1).

3.4 Scanning Electron Microscopy of Apical Meristems of A. coerulea and A. formosa.

The number of flowers and branches observed vary strikingly in A. formosa and

A. coerulea. Aquilegia species require vernalization a cold treatment required for transitioning to flowering. Vernalization period for A. formosa is 8 weeks while for A. coerulea it is 3 weeks. While in both species the inflorescence is a dichasial cyme, the ramification seen is dense in A. formosa due to the presence of multiple secondary

branches. In A. coerulea transition to flowering happens in the last 3-4 weeks of vernalization, inflorescence meristems (IM’s) are visible by week 3 of vernalization and

FM’s by week 4 (Sharma et. al 2019). In A. formosa IM’s can be noticed around 6 weeks and FM’s around 8 weeks of vernalization. Both species take 3-4 week to bolt after they have been vernalized. Around 3-4 weeks post vernalization the apical meristems from both species were dissected to compare the arrangement of FM’s and lateral organs. From the scanning electron micrographs, it is clear that FM’s for most buds are already formed in both species. Since A. coerulea produces fewer primary, secondary branches and flowers as compared to A. formosa (Figure 3.39 A, B) the apical meristems in A. coerulea (Figure 3.39 C, D) are less dense as compared to A. formosa.

Because of limited samples I was unable to do a comparative analysis of number of flowers and their arrangement on the SAM of both species but that is a great future direction as a next step.

A B

FB FB VS FB FB

C D

VS FB FB FB

Figure 3.39 Scanning electron micrographs of shoot apical meristems (SAM) of A. formosa and A. coerulea. In A. formosa compactly packed SAM has many floral buds (FB) and vegetative structures (bracts and leaf (VS)) (A, B). The SAM of A. coerulea is was less compact as compared to A. formosa (C, D). Pictures A and B were taken by Uriah Sanders (preparations done by Michael Speck), C, D by Timothy Batz.

3.5 Analysis of Primary Branching in A. coerulea

I noticed the following ratios in primary branching on the inflorescence in A. coerulea. I describe this below (Table 3.5).

A. coerulea branching features. Table 3.5 Comparison of frequencies of primary branching in A. coerulea. Primary branches of 25 A. coerulea plants were counted to get an idea of the primary branch frequencies along the primary shoot.

Number of Primary Branches Frequency

3 Primary Branches 7 Times

4 Primary Branches 14 Times

5 Primary Branches 3 Times

6 Primary Branches 1 Times

The 1-2-3A and 1-2-3B pattern occurred in both 4 (Figure 3.40 A) and 3 (Figure

3.40 B) primary branched plants in A. coerulea. The 1-2-3-4A pattern was found in stressed and non-stressed plants with 1-4 (Figure 3.40 C, D) primary branches present in

A. coerulea.

A B

2 2

C D

2 3 1 4

2 3 4

Figure 3.40 Pictures showing three and four primary branched plants in A. coerulea. Figures A and B show the 1-2-3A and 1-2-3B pattern in 4 and 3 primary branched plants respectively. Figures C and D show the 1-2-3-4A pattern with 4 to 1 primary branches respectively.

3.6 Tracking A. formosa and A. coerulea

Patterning of leaves or bracts on nodes 1, 2, 3 and 4 was observed from emergence of first bud to dehiscence in A. formosa and A. coerulea. This tracking was done over a 7-week period in A. formosa (Figures 3.41 A-G) Photography and diagrams were methods used to document these two species.

Week 1 Week 2 Week 3 A B C

D E F

Week 4 Week 5 Week 6 G

Week 7

Figure 3.41 Tracking of A. formosa using a camera. Pictures of A. formosa were taken weekly over a 7-week span (Figures A-G).

Patterning of leaves or bracts on nodes 1, 2, 3 was observed from emergence of first bud to dehiscence in A. coerulea over a 6-week span. In A. coerulea (Figure 3.42 A-

F). Observations were noted every 2-3 day. Photography and diagrams were methods used to document these two species.

Week 1 Week 2 Week 3 A B C

D E F

Week 4 Week 5 Week 6 Figure 3.42 Tracking of A. coeurela using a camera. Pictures of A. coeurela (A-F) were taken weekly over a 6-week span.

3.7 Difference between A. formosa and A. coerulea

Based on the tracking shown above in 3.4, I noted morphological features of the inflorescences that included height of plant, flower size, number of primary and secondary branches, number of buds, and noted the time from budding to dehiscence. A. formosa was sampled from plants growing in BioTrek and greenhouse. All A. coerulea plants were grown in the greenhouse (Table 3.6).

The average height of A. formosa grown in gallon pots in the greenhouse was

46.95 cm (n=20) and the height of the biological replicates from BioTrek was 71.5 cm

(n=8). The average height of the A. coerulea plants was 14.7 cm (n=10). The average flower size was 3.86 cm (n= 20) in A. formosa and the average flower size was 6.25 cm

(n=11) in A. coerulea. The average number of primary branches was 7.1 in the greenhouse (n=20) and the average number of primary branches was 7.8 in the BioTrek

(n= 8) in A. formosa. The average of primary branches was 3.92 (n=26) in the greenhouse in A. coerulea. The average number of secondary branches was 18.14 (n= 7) in the greenhouse and BioTrek combined in A. formosa. The average number of secondary branches was 3.3 (n=10) in the greenhouse in A. coerulea. In A. formosa the average number of buds was 71.5 in the BioTrek (n=8) and 38.5 in the greenhouse (n=19). In A. coerulea the average number of buds was 8.4 (n=10). To measure budding to dehiscence, these two species were tracked every 2-3 days and documented by photography. It took

6.37 weeks in the greenhouse (n= 4) for A. formosa to completely dehisce and 7.25 weeks in the BioTrek (n= 4) for A. formosa to completely dehisce. It took 4.85 weeks (n=7) for

A. coerulea to completely dehisce in the greenhouse.

Analysis of differences between A. coerulea and A. formosa Table 3.6 Comparative analysis of A. coerulea and A. formosa. Morphological characters including plant height, flower size, number of primary and secondary branches, number of buds, and the time period from budding to dehiscence was looked at.

Greehouse (GH) BioTrek A. formosa (GH and/or A. coerulea (only in GH)

(BT) BT)

Height (GH and BT) GH 46.95 cm (STDEV GH 14.17 cm (STDEV

5.26) 1.5)

BT 71.5 cm (STDEV

11.7)

Flower Size (GH) GH 3.86 cm (STDEV GH 6.25 cm (STDEV .8)

1.03)

Primary Branches GH GH 7.1 (STDEV 1.44) GH 3.92 (STDEV .759)

and BT BT 7.8 (STDEV 1.24)

Secondary Branches GH 18.14 in GH and BT GH 3.3 (STDEV 2.75)

and BT Combined (STDEV 2.26)

Number of Buds GH and GH 38.5 (STDEV 17.61) GH 8.4 (STDEV 3.025)

BT BT 71.5 (STDEV 11.71)

Budding to Dehiscence GH 6.37 Weeks (STDEV GH 4.85 Weeks (STDEV

GH and BT 1.1) 2.6)

BT 7.25 Weeks (STDEV

1.25)

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CHAPTER 4

CONCLUSION AND FUTURE RESEARCH

4.1 Conclusions from Research

The comparative analysis of lateral organs on inflorescences of A. coerulea and A. formosa demonstrates:

1) In both Aquilegia species heteroblasty in the inflorescence is noted. In A. coerulea, increasingly simple leaves were observed in the upper nodes found in the inflorescence stem. At node 1, bracts were observed. At node 2, either weakly lobed bracts or weakly lobed leaves were observed. At node 3 a weakly lobed partially sessile leaf or non-sessile compound trifoliate leaf is observed. In A. formosa, at node 1 a bract is observed, but nodes 2, 3, and 4 are mostly compound leaves with increasing complexity, respectively.

The compound leaves at node 2 were fused with the node, and petiolule is only observed in the central leaflet. In some biological replicates, lateral organs at node 3 displays a similar morphological pattern as observed in node 2 (1-2-3-4 pattern), in others petiolules were observed in the central and the lateral leaflets.

2) A. coerulea (1-2-3A and 1-2-3B) and A. formosa (1-2-3B) have variation with respect to the orientation of primary branching in the 1-2-3 pattern. In these two species orientations, branching can be left (3), right (2), center (1) or right (3), left (2), center (1).

To find these patterns, the first tri-bract at node 1 was faced forward and then the branching orientations were notated. However, this needs to be analyzed further since there were nodes above node 1. This data serves as a basis for future phyllotaxis studies.

101

3) SAM of A. formosa are more compactly arranged with flowers and lateral organs as compared to A. coerulea. Ramification of A. formosa infloresence is complex as compared to A. coerulea and this can be observed in early development as shown by the

SEM of the apical meristems. While this was a preliminary study, a detailed in-depth analysis will be required in the future to comprehensively analyze the differences in inflorescence pattering at early stages of development.

4) In stressed plants (showing no branches or flowers), mostly the morphology of lateral organs on nodes in the 1-2-3A, 1-2-3-B, 1-2-3-4A patterns was similar as compared to a non-stressed plant in A. coerulea with a few exceptions.

4.2 Future Research

Morphological and developmental studies in grasses have detailed inflorescence development (Reinheimer et al., 2009 and Kellogg et al., 2013) work. Studies on inflorescence development in some angiosperms models have been reported: such as in tomato (Molinero-Rosales et al., 1999, Lippman et al., 2008). However, in angiosperms there is a great variability in inflorescence branching pattern. Aquilegia coerulea can be a good model system for understanding the development of dichasial cyme.

Due to adaptive radiation of the Aquilegia genus, some morphological features have changed between species in this genus (such as length of petal spurs). From observations in this study, it was determined there is conservation in other morphological features (such as placement and morphology in bracts and leaves). In many plant species

LFY, TFL, AP1 are the major factors that control reproductive meristem behavior

102

(Benlloch et al., 2007). A similar process could be occurring in A. coerulea, where genes are activated and downregulated in the shoot apical meristem to determine how many primary branches come off the main shoot at the nodes. Comparative transcriptomics of developing meristems in different species could be done to identify candidate genes that may underlie the differences seen in branch numbers. Transcriptomic analysis of A. coerulea and A. formosa can also be done to identify the genes that leads to heteroblasty, displaying different lateral organs such as, sessile bracts, trifoliate bracts and trifoliate compound leaves. One morphological characteristic that can be studied further is how early in development the establishment of phyllotaxy in the inflorescence occur. In my preliminary analysis, I observed the buds are already formed even before the inflorescence bolted. Compactness on the inflorescence was less in A. coerulea as compared to A. formosa.

Future studies in the Aquilegia genus can look at ramification of inflorescence in other species. From the pictures of A. pubescence (Figure 4.1 A-D) and A. yangii (Li et al.,2018) it seems some inflorescence branching patterns might be conserved. From the A. pubescence pictures it can be deduced that a lobed and fully sessile trifoliate bract is present at node 1, (Figure 4.1 B). At node 2 a partially sessile trifoliate bract (Figure 4.1

C) and at node 3 a trifoliate leaf consisting of leaflets (in three segments) without a petiole can be observed (Figure 4.1 D). As noticed with the other species looked at in this study, there was an increase in complexity going from the sessile trifoliate bract, to the partially sessile trifoliate bract, and to the trifoliate leaf without a petiole in A. pubescence.

103

1 2

B C

Figure 4.1. Pictures of a non-stressed A. pubescence showing 1-2-3 pattern found in Canada. Pictures A-D are of A. pubescence, with similar morphology to the 1-2-3B. Picture of a fully sessile trifoliate bract was found at node 1 (B). Picture of a trifoliate bract was found at node 2 (C). Picture of trifoliate leaf without a petiole was found at node 3 (D). Pictures taken by Carole Weinstein.

104

A. yangii (Li et al.,2018) an Asian columbine species had a simple sessile bract at node 1, most similar to some variants at the 1-2-3A and 1-2-3B pattern in A. coerulea.

The number 2 node was a trifoliate lobed leaf with an elongated central petiolule and sessile lateral leaflets. This is similar to A. coerulea and A. formosa 1-2-3A, and 1-2-3B patterns. A trifoliate compound leaf with a petiole was found at the number 3 node, which is most similar to A. coerulea 1-2-3A. As noticed with the three other Aquilegia species, heteroblasty was noted in A. yangii.

105

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