Relationships and Biology of Ambrosia Beetles and Fungi and the Development of Pre-Invasion Assessment of Potential Pests

RELATIONSHIPS AND BIOLOGY OF AMBROSIA BEETLES AND FUNGI AND THE DEVELOPMENT OF PRE-INVASION ASSESSMENT OF POTENTIAL PESTS

YOU LI

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2018

To my wife and supportive parents

ACKNOWLEDGMENTS

There are so many people who have made this effort possible, and I would not have enough room to name them all. First, I must thank my wife, Misa, who stood by me during this four years. I do not know where I would be without her support and wisdom.

My major advisor, Dr. Jiri Hulcr, has been a tremendous mentor and I feel fortunate he was kind enough to take me on as a student. His experience, and generosity throughout this process ensured the success of this. I would like to thank my committee members for their guidance and patience over the last few years. This includes Dr. Damian Adams, Dr. Huiping Yang, Dr. Jason A. Smith, and Dr. Matthew E.

Smith. Every member of my committee was very supportive and helped me grow during my Ph.D. work. Thank you for your time, expertise, and wisdom.

The samples of my research are most from China and USA. Many people help me in each chapter.

In Chapter 2, I am grateful to D. Rabern Simmons (University of Michigan) for teaching me how to write a manuscript. I would like to thank Changlin Zhao and Baokai

Cui (Beijing Forestry University, China) for sharing molecular data and Lukas Stelinski and Chris Gibbard provided several specimens.

In Chapter 3, I gratefully acknowledge Jianjun Guo (Guizhou University, China),

Ki-Jeong Hong and Moo-Sung Kim (Suncheon National University), Pham Hong Thai and Tran Thi Men (Vietnam National Museum of Nature) for their assistance in sample collecting and logistic support. Roger Beaver helped us identify Ambrosiophilus.

In Chapter 4, I would like to thank Jian Yao and Menglei Zhang (Institute of

Zoology, Chinese Academy of Sciences) for facilitating access to the collection records.

Additional thanks to Angie Macias and Matt Berger (West Virginia University) for help with DNA sequencing and molecular identification.

In Chapter 5, I would like to thank Adam Black and M. Patrick Griffith for their help and permission collecting beetles at the Montgomery Botanical Center (Miami, FL).

I thank the Pathology Laboratory for Translational Medicine at West Virginia University’s

School of Medicine for their assistance in histological preparations. Additional thanks to

Thomas H. Atkinson (University of Texas, USA) for training on platypodine identification.

In Chapter 6, I am grateful to Guangyu Liu, Jianjun Guo (Guizhou University) and

Paige Carlson (University of Florida) for aid in beetle collections.

In Chapter 7, I gratefully acknowledge Chengxu Wu and Zhen Zhang (Research

Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry,

China), Bo Wang (Xishuangbanna Tropical Botanical Garden, Chinese Academy of

Sciences), Zaihua Yang (Guizhou Academy of Forestry), Jianghua Sun (Institute of

Zoology, Chinese Academy of Sciences), Chi-yu Chen and Hou-Feng Li (National

Chung Hsing University), Liang-Jong Wang (Taiwan Forestry Research Institute) and

Ching-Shan Lin for their assistance in sample collecting and logistic support.

I am thankful for the continuous support from the members of Forest Entomology

Laboratory, School of Forest Resources and Conservation, University of Florida, my colleagues, Allan Gonzalez, Andrew J. Johnson, Caroline Storer, Craig Bateman,

Demian Gómez, James Skelton, Sedonia Steininger, Yin-Tse Huang, Zachary Nolen. I am grateful to each of them for their candid assistance at every stage of this project.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 9

LIST OF FIGURES ...... 10

LIST OF ABBREVIATIONS ...... 11

ABSTRACT ...... 12

CHAPTER

1 INTRODUCTION ...... 14

2 SYMBIOTIC FUNGI ASSOCIATED WITH AMBROSIODMUS IN USA ...... 17

Introduction to Chapter 2 ...... 17 Materials and Methods for Chapter 2 ...... 18 Isolation and Culturing of Fungi from Ambrosiodmus ...... 18 Identification of Fungi from Ambrosiodmus ...... 19 Microtome and Micro-CT ...... 20 Morphology of the Fungi from Isolation ...... 22 Results for Chapter 2 ...... 22 Isolation and Culturing of Fungi from Ambrosiodmus and Plants in the USA ... 22 Phylogenetic Analyses of Fungi from Ambrosiodmus in the USA ...... 23 Examination of Mycangia of Ambrosiodmus ...... 23 Morphology of Fungi from Ambrosiodmus ...... 23 Discussion for Chapter 2 ...... 24

3 SYMBIOTIC FUNGI ASSOCIATED WITH AMBROSIODMUS IN ASIA ...... 32

Introduction to Chapter 3 ...... 32 Materials and Methods to Chapter 3 ...... 34 Results to Chapter 3 ...... 35 Identification and Phylogenetic Analyses of Symbiotic Fungus Flavodon ambrosius in Asia ...... 35 Biology of Fungus Flavodon ambrosius in Asia ...... 36 Discussions to Chapter 3 ...... 36

4 DISTRIBUTION, HOST AND SYMBIOTIC FUNGI OF EUWALLACEA FORNICATUS IN CHINA ...... 42

Introduction to Chapter 4 ...... 42

Materials and Methods to Chapter 4 ...... 43 Results and Discussions to Chapter 4 ...... 43

5 SYMBIOTIC FUNGI ASSOCIATED WITH PLATYPODINAE IN THE SOUTHEASTERN USA ...... 49

Introduction to Chapter 5 ...... 49 Materials and Methods to Chapter 5 ...... 51 Platypodine Beetle Collection ...... 51 Isolation, Identification, Taxa Assignment, and Phylogenetic Analyses of Fungi Associated with Platypodine Beetle ...... 52 Community Analysis of Fungi from Platypodine Beetle ...... 54 Results to Chapter 5 ...... 55 Discussion to Chapter 5 ...... 56

6 DEVELOPMENT OF MYCANGIUM IN XYLOSANDRUS AMBROSIA BEETLES .. 64

Introduction to Chapter 6 ...... 64 Materials and Methods to Chapter 6 ...... 65 Experiment 1: Morphological Variation of Mycangia Test ...... 65 Experiment 2: Mycangial Development Test ...... 66 Hand Dissection of Xylosandrus ...... 67 Micro-CT Imaging ...... 67 Identification of Fungi from Mycangia ...... 68 Results to Chapter 6 ...... 68 Morphological Variation of Xylosandrus Mycangia ...... 68 Mycangial Development and Fungal Identification ...... 69 Discussions to Chapter 6 ...... 69

7 PATHOGENICITY EVALUATION OF CHINESE BARK AND AMBROSIA BEETLE-VECTORED FUNGI IN USA ...... 79

Introduction to Chapter 7 ...... 79 Materials and Methods to Chapter 7 ...... 81 Target Beetle Vector and Tested Object ...... 81 Collection Chinese Bark and Ambrosia Beetles ...... 81 Fungal Isolation and Identification ...... 82 Identification and Selection of Potential Pathogens Fungus ...... 83 Establishment of the Experimental Trees ...... 83 Inoculate Potential Pathogens ...... 84 Survey of the Pathogenicity ...... 85 Results to Chapter 7 ...... 85 Potential Pathogens Isolated from Chinese Bark and Ambrosia Beetles ...... 85 Pathogenicity to Local Pine Tree ...... 86 Pathogenicity to Local Oak Tree ...... 87 Symptoms in Local Pine Trees ...... 87 Symptoms in Local Oak Trees ...... 87

Discussion to Chapter 7 ...... 87

8 CONCLUSION ...... 101

LIST OF REFERENCES ...... 104

BIOGRAPHICAL SKETCH ...... 123

LIST OF TABLES

Table page

2-1 Species of Polyporales used for phylogenetic analyses ...... 27

2-2 Cultures of Flavodon cf. flavus and host information ...... 28

3-1 Primary symbionts isolated from Asian Ambrosiodmus and Ambrosiophilus beetle and the NCBI/GenBank accession numbers of their ITS and 28S rDNA sequences...... 40

4-1 Host trees of Euwallacea fornicatus specimens in the National Zoological Museum of China and new field collection from 2013 to 2015...... 47

5-1 Collection of platypodine beetle ...... 60

5-2 Cumulative frequency of fungal isolation from four platypodine beetles ...... 61

5-3 Colony forming units range and frequency of fungal isolation ...... 62

6-1 Collection information for Xylosandrus beetles in morphological variation of mycangia test...... 75

6-2 Xylosandrus beetles in morphological variation of mycangia test...... 75

6-3 Mycangia and fungal mass of callow and new mature beetles in Xylosandrus compactus and X. crassiusculus ...... 76

7-1 Collection of the potential plant pathogen ...... 92

7-2 Potential pathogens isolated from bark and ambrosia beetles from China ...... 93

7-3 Symptoms in loblolly pine trees ...... 94

7-4 Symptoms in slash pine trees ...... 95

7-5 Symptoms in live oak trees ...... 96

7-6 Symptoms in shumard oak trees ...... 96

7-7 Symptoms in local pine trees ...... 94

7-8 Symptoms in local oak trees ...... 98

LIST OF FIGURES

Figure page

2-1 Female Ambrosiodmus lecontei with eggs in plant host ...... 29

2-2 Wood decay as a result of colonization by the Ambrosiodmus-Flavodon symbiosis ...... 29

2-3 Best ML tree from GARLI analysis of combined nuclear ITS and 28S rDNA datasets ...... 30

2-4 Beetle mycangium and fungi ...... 31

3-1 The best Maximum likelihood tree of Flavodon inferred from ITS and 28S rDNA datasets with source beetle genera and GenBank accession numbers .... 40

3-2 Flavodon ambrosius and ambrosia beetles collected from China...... 41

4-1 The distribution of Euwallacea fornicatus in the south of China...... 48

4-2 The elytral declivity of Euwallacea fornicatus after being parasitized by an unknown natural enemy ...... 48

5-1 Best ML tree from RAxML analysis of 28S rDNA datasets ...... 63

6-1 Xylosandrus crassiusculus mycangia in various developmental stages ...... 76

6-2 Reconstruction of micro-CT scans of ambrosia beetle Xylosandrus amputatus in different developmental stages ...... 77

6-3 Mycangia with/without fungal mass and eversion of empty mycangia ...... 78

7-1 Size of stain in inoculated local pine ...... 99

7-2 Size of stain in inoculated local oak ...... 100

LIST OF ABBREVIATIONS

AFC Ambrosia Fusarium clade

BPP Bayesian posterior probabilities

BSA Bovine serum albumin

CFU Colony forming units

DMSO Dimethyl sulfoxide

Micro-CT Micro-computed tomography

PBS Phosphate buffered solution

PDA Potato dextrose agar

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

RELATIONSHIPS AND BIOLOGY OF AMBROSIA BEETLES AND FUNGI AND THE DEVELOPMENT OF PRE-INVASION ASSESSMENT OF POTENTIAL PESTS

You Li

May 2018

Chair: Jiri Hulcr Major: Forest Resources and Conservation

Ambrosia symbiosis is an obligate, farming-like mutualism between wood-boring beetles and fungi. It evolved at least 11 times and included many notorious invasive forest pests. Here, a series of surveys on the fungal symbionts of ambrosia beetles were presented.

First, I isolated the fungi associated with genus Ambrosiodmus in North America.

Histological sectioning and culture-independent sequencing of Ambrosiodmus mycangia revealed a single operational taxonomic unit identical to be within the Polyporales, closely related to Flavodon flavus. I identified this fungus as F. ambrosius and found it was also the primary mutualist of ambrosia beetles Ambrosiodmus and Ambrosiophilus in Asia. F. ambrosius is the only known ambrosial basidiomycete.

Secondly, two fungi associated with Euwallacea fornicatus in China have recorded: a putatively new Fusarium sp. belonging to Ambrosial Fusarium Clade and an anamorphic hypocrelean fungus, Sarocladium strictum. I also reported a local host plant list and distribution of E. fornicatus from records in the National Zoological Museum of

China and my samples.

For the first time, I surveyed the fungal community of four endemic platypodine beetles in the southeastern USA. Phylogenetic analyses of 28S rDNA sequences revealed that platypodines were associated with several genera within in

Ophiostomatales, especially Raffaelea. Platypodine beetle had a moderate association with Ceratocystiopsis spp. was first demonstrated.

Not only study on fungal symbiont, but also the process of mycangia associated with fungi. I choose most common ambrosia beetle in subtropics, Xylosandrus, as a model. With the hand dissection and micro-CT scan, I found three stages of the mycangium development. Mycangia are much more dynamic than previously thought, and their morphological changes correspond to the phases of the symbiosis. I proved fungus is not the trigger of mycangial development.

In the end, a pathogenicity test was conducted on fungi from exotic bark and ambrosia beetle. Forty fungi isolated from Chinese bark and ambrosia beetle were inoculated on native pine or oak tree. No fungus has proven to be a virulent pathogen of biosecurity concern, and most were not pathogenic. However, several strains of Asian

Leptographium spp., Ophiostoma spp., Fusarium oxysporum were mildly pathogenic to

Southeastern American pines.

CHAPTER 1 INTRODUCTION

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) are a polyphyletic collection of clades within the hyper-diverse bark beetles that have evolved the symbiotic evolutionary mode referred to as fungal farming. Over 3,000 ambrosia beetle species gain nutrition from growing fungi on abundant yet recalcitrant plant material and transport their fungal symbiont from one location, and thus, generation, to the next via a packet of fungal inoculum carried in a storage organ called a mycangium

(Francke-Grosmann, 1956; Hulcr and Stelinski, 2017). Mycangia are derived in different locations of the body (e.g. mandibular, elytral, mesothoracic) and, in most instances, are phylogenetically highly conserved (Hulcr and Cognato, 2010; Hulcr and Stelinski, 2017).

At least ten independent origins of the symbiosis within fungi, all known ambrosia fungi lie within lineages of the phylum Ascomycota and transported as budding pseudo- mycelium or conidia (Bateman et al., unpublished data; Dreaden et al., 2014; Kasson et al., 2013; Kolařík and Kirkendall, 2010; Massoumi Alamouti et al., 2009; Mayers et al.,

2015; Vanderpool et al., 2017). There is at least seven beetle species/clades in which fungus farming is assumed but not proved (Hulcr and Stelinski, 2017).

The majority of ambrosia beetles colonize freshly dead or dying trees and are harmless, but some damage lumber or attack live trees (Kendra et al., 2011; Mendel et al., 2012). In most cases, the damage is mainly due to the symbiotic fungi, either as a result of staining, structural weakness or pathogenicity.

Numerous non-native species were introduced to new regions by trade. Exotic wood-boring beetle damage in the US is estimated to cost more than $2.5 billion in local government expenditures and lost residential property values every year (Aukema et al.,

2011). At least one new alien forest insect pest is predicted to emerge as a significant problem every 5-6 years (Koch et al., 2010). Hence, ambrosia beetles are becoming increasingly economically and ecologically important. In several such cases, formerly harmless beetle species turned into invasive pests (Fraedrich et al., 2008; Hulcr and

Dunn, 2011; Ranger et al., 2010) and at least three cases, ambrosia beetle-fungus symbionts have triggered acute and ongoing epidemics among naive native host trees in the US (Fraedrich et al., 2008; Freeman et al., 2013; Ranger et al., 2010).

Even Platypodinae and fungus-feeding Scolytinae comprised ambrosia beetle since they have similar ecologies. In the same way of vectoring pathogenic fungi, some platypodine species are considered forest pests (Inácio et al., 2012a; Kile and Hall,

1988; Massoumi Alamouti et al., 2009). However, In phylogenetics, molecular data indicate a far relationship Platypodinae to Scolytinae (Gillett et al., 2014; Haran et al.,

2013).

Related to ambrosia beetles are bark beetles, which colonize and consume a more nutrient-rich phloem. Bark beetles, like ambrosia beetles, are also often associated with fungal symbionts, usually ascomycotan and rarely basidiomycotan fungi, and the intensity of association is more variable, ranging from facultative to obligate (Harrington, 2005; Hsiau and Harrington, 2003; Vega and Hofstetter, 2015).

Beetle-fungus consortia are responsible for spectacular outbreaks, driven either by climate, the pathogenicity of the fungus, or the mass-attacking nature of the beetles

(Freeman et al., 2013). Other insects also engage in symbioses functionally analogous to the ambrosia beetle-fungus symbiosis: attine ants (Schultz and Brady, 2008), termites (Aanen et al., 2002), ship timber beetles (Casari and Teixeira, 2011), and wood

wasps (Talbot, 1977). Though research focused on wood borer-fungus associations has recently increased, it has been almost entirely restricted to a minority of species that happen to be economically important. At the same time, thousands of beetle species remain unstudied, and each new exploration of the understudied taxa revealed interactions with unsuspected and undescribed fungal communities (Kasson et al.,

2013; Kolarik and Hulcr, 2009).

CHAPTER 2 SYMBIOTIC FUNGI ASSOCIATED WITH AMBROSIODMUS IN USA

Introduction to Chapter 2

Ambrosiodmus is a genus of 80 species within the largest group of ambrosia beetles, the Xyleborini (1,200 spp.; Coleoptera: Scolytinae) (Wood, 1982). The genus deserves closer scrutiny for several reasons. While its economic importance has been minimal, it is a very diverse and globally distributed genus. Many species are ecologically successful and abundant throughout their range. In the twentieth century, species have increasingly been established in non-native habitats via human-aided introductions. In the US alone, the originally Asian A. rubricollis Eichhoff has been present for nearly five decades (Bright, 1968). More recently, A. minor Stebbing, a species formerly known only from southern Asia (Wood and Bright, 1992), has spread across at least three southeastern states in the US (Halbert, 2011; Seltzer et al., 2013).

The habitat in which many Ambrosiodmus species live is different from that of most fungus-associated wood borers. While the majority of ambrosia fungi and ambrosia beetles depend on newly dead and relatively fresh tree tissues,

Ambrosiodmus species appear to be able to colonize wood throughout the process of its decay, including latter stages when xylem is co-colonized by competitive wood-rot fungi. The only report on mycangia and fungal symbionts in Ambrosiodmus examined A. rubricollis and determined the presence of preoral mycangia at the bases of the mandibles behind the labrum (Takagi, 1967). The mycangial content was described as

 Reprinted with permission from: Li, Y., Simmons, D. R., Bateman, C. C., Short, D. P., Kasson, M. T., Rabaglia, R. J., & Hulcr, J. (2015). New Fungus-Insect Symbiosis: Culturing, Molecular, and Histological Methods Determine Saprophytic Polyporales Mutualists of Ambrosiodmus Ambrosia Beetles. PloS one 10(9): e0137689.

globose conidia and short hyphal fragments, which yielded “white wooly mycelia only” when grown on agar.

To clarify the presence and identity of fungal symbionts of Ambrosiodmus, and to hypothesize upon the ecological habits and success of the genus, I characterized fungi associated with the ambrosia beetle genus and found entirely unexpected symbionts. I focused on three different Ambrosiodmus species: A. lecontei Hopkins (Figure 2-1), A. minor, and A. rubricollis, representing both the Eurasian and American fauna. I have used a comprehensive suite of complementary approaches including quantitative culturing for the main symbiont characterization, anatomical analysis of the beetle for the characterization of the mycangia and their content, and morphological cross- validation by microscopy of the symbiont identity in the mycangia and cultured fungi.

Materials and Methods for Chapter 2

Isolation and Culturing of Fungi from Ambrosiodmus

To isolate the dominant fungal symbionts in Ambrosiodmus, three beetle species were sampled: A. lecontei (native to the South-Eastern US), A. rubricollis (native to

Japan, but widely established in the US) and A. minor (native to SE Asia and recently established in the SE US). Female specimens of all three species were collected by light-trapping (August 2014 through April 2015) and from moribund-to-rotten wood of

Myrica cerifera (A. lecontei), Liquidambar styraciflua (A. rubricollis), and Platanus occidentalis (A. minor), in Gainesville, Florida, USA. Males have not been included in this analyses as all Xyleborini ambrosia beetle males are flightless, rare, and lack mycangia necessary for transporting symbiotic fungi (Kirkendall et al., 2015). Live beetles and wood sections were transported to the laboratory in clean containers with a tissue moistened with sterile water. After identification in the laboratory, whole beetles

were surface-washed by vortexing for 1 min in 1 mL of sterile distilled water with one small drop of Tween 80. Beetle heads were aseptically removed, crushed, and placed in a 500 µL solution of sterile phosphate buffer saline and vortexed for 30 s. Resulting solutions were diluted to 1:100 and 1:1000 concentrations and 100 µL of each dilution were used to inoculate BD Difco™ PDA medium, which was strengthened with additional agar (5 g/L). CFUs within mycangia were estimated for each morphotype by multiplying the number of colonies on a plate by the inverse of the inoculum dilution. To determine symbionts in the ambrosial gardens, fungi were collected from

Ambrosiodmus tunnels in Liquidambar styraciflua and Platanus occidentalis using sterile scalpels. Dominant morphotypes were quantified and purified by the methods described above.

Identification of Fungi from Ambrosiodmus

Fungal DNA was isolated with Extract-N-Amp Plant PCR kits (Sigma-Aldrich Co.

LLC.), with the modification of using 3% BSA in the place of a dilution solution.

Sequences of the nuclear internal transcribed spacers ITS1-5.8S-ITS2 (ITS) and nuclear 28S ribosomal DNA (rDNA) regions were amplified with the primer combinations ITS1/ITS4 (White et al., 1990) and LROR/LR5 (Vilgalys and Hester,

1990), respectively, and ExTaq polymerase kits (Clontech-TaKaRa). Amplified products were visualized with SYBR® Green I Stain (Lonza Group Ltd.) on a 1% agarose gel in

1X TAE buffer. Products were purified with ExoSAP-IT (Affymetrix, Inc.) and submitted to the University of Florida Interdisciplinary Center for Biotechnology Research for

Sanger sequencing. Chromatograms were assembled and inspected in Geneious

(Geneious version 7.1.8).

Ribosomal DNA sequences from three representative fungal isolates were aligned to ITS and nuclear 28S rDNA sequences from additional Polyporales and

Russulales taxa (Table 2-2) (Tomšovský et al., 2010; Zhao and Cui, 2014) in Geneious using the MAFFT alignment tool (Katoh et al., 2002). Data matrix deposited in

TreeBASE. The Akaike information criterion (AIC) in jmodeltest 0.1.1 (Guindon and

Gascuel, 2003; Posada, 2008) was used to select a nucleotide substitution model for the dataset. Maximum likelihood (ML) phylogenetic analyses were conducted using the suggested model parameters in GARLI 2.01 (Zwickl, 2006) and additional AIC recommended settings to determine the best tree topology. Bootstrap support values were calculated in GARLI from 100 search replicates, which were summarized with

(Sukumaran and Holder, 2010). A Bayesian phylogenetic analysis was conducted using the same AIC recommended model parameters in MrBayes 3.1.2 (Ronquist and

Huelsenbeck, 2003), in which two runs of four chains each were executed simultaneously from 5 000 000 generations, with sampling every 500 generations. The resulting 7501 trees retained after a burn-in of 2500 trees in SumTrees were used to compute BPP. The new fungal species will be taxonomically described elsewhere.

Microtome and Micro-CT

Given the phylogenetic position of Ambrosiodmus within the Xyleborini with preoral mycangia (Hulcr and Cognato, 2010) and a previous examination of the genus

(Takagi, 1967), the beetle heads were targeted for examination.

Eleven female of Ambrosiodmus lecontei from a cryopreserved beetle depository in the Hulcr lab were examined to determine the fungi in the mycangia. Individuals examined were collected 29 November 2010 from redbay trees (Persea borbonia) near

Lake Alfred, Polk County, Florida and preserved in cryotubes containing a 95% ethanol

solution, which was stored at -80°C . Heads were aseptically removed from the pronotum before sectioning. Once removed, beetle heads were fixed in 10% neutral buffered formalin for 24 h and then soaked in phenol for 6 d to soften the cuticle

(Fraedrich et al., 2008). Heads were treated with an automated tissue processor to allow desiccation of tissues and infiltration of paraffin and subsequently embedded in paraffin blocks. Heads were orientated with the most anterior end facing downward to permit transverse sectioning and simultaneous bilateral visualization of the mycangia. A

Microm HM 325 rotary microtome (Walldorf, Germany) was used to cut 10-μm transverse sections. Selected slides confirmed by immediate viewing were then dried at

60°C for three days, double-stained with Harris-hematoxylin and eosin-phloxine, and examined and photographed using a Nikon Eclipse E600 compound microscope (Nikon

Instruments, Melville, New York) equipped with a Nikon Digital Sight DS-Ri1 high- resolution microscope camera and Nikon NIS-Elements BR 3.2 imaging software.

Two female of Ambrosiodmus minor from a cryopreserved beetle depository in the Hulcr lab were examined to determine the mycangia. Individuals examined were collected April 2015 from sweetgum trees (Liquidambar styraciflua) near Lake Alice,

Gainesville, Florida and preserved in cryotubes containing a 95% ethanol solution, which was stored at -80°C. Prior to scanning, specimens were dehydrated with in graded series of ethanol baths (from 95% to 100%) and dried at the critical point

(Hitachi hcp-2, Hitachi Inc., Tokyo, Japan). All beetles were scanned with a MicroXCT-

400 (Xradia Inc., California, USA; beam strength: 60 kV, absorption contrast) at the

Institute of Zoology, Chinese Academy of Sciences, China.

Morphology of the Fungi from Isolation

To document the morphology of the fungal symbionts of Ambrosiodmus, I used six isolates that were recovered from heads of all three Ambrosiodmus species, as well as two isolates recovered from the galleries of A. minor and A. rubricollis. Isolates were cultured on PDA medium and incubated at 20 °C for several weeks. Hyphae were aseptically removed from the edges of the representative fungal colonies and examined microscopically with a Nikon Eclipse 55i equipped with a ProgRes® SpeedXT core 3 camera (Jenoptik Optical Systems).

Results for Chapter 2

Isolation and Culturing of Fungi from Ambrosiodmus and Plants in the USA

Individuals of each of the three Ambrosiodmus yielded a single fungal morphotype in at least two head extract isolation events and estimated CFUs of the same dominant morphotype from different isolation events ranged from 10 to 4000

(Table 2-1). Representative subcultures were cryoarchived in PDA slant vials, immersed in 15% glycerol, and stored at -80 °C, and deposited in the Hulcr collection at the

University of Florida and the Forestry & Agricultural Biotechnology Institute (FABI) fungal culture collection (CMW) in Pretoria, Gauteng Province, South Africa.

Active Ambrosiodmus galleries were routinely found in decayed wood. The wood adjacent to the Ambrosiodmus gallery was typically soft and spongy, and the area was surrounded by dark lines (Figure 2-2). These lines are a common result of antagonistic interactions among genetically distinct decay fungi in co-colonized dead wood. Regular ambrosia fungi do not form such zones of defense.

Phylogenetic Analyses of Fungi from Ambrosiodmus in the USA

The best ML phylogeny (Figure 2-3) placed the representative fungal isolates from A. lecontei and A. minor heads in a monophyletic group within the phlebioid clade of the Polyporales (Binder et al., 2013) with 98% BPP but only 48% ML bootstrap support. These isolates are sister to GenBank accession JN710543 (Miettinen et al.,

2012), identified as Flavodon flavus (Klotzsch) Ryvarden (Ryvarden, 1973), a placement supported by 100% BPP and bootstrap value. TreeBASE accession number

S17679 (access: http://purl.org/phylo/treebase/phylows/study/TB2:S17679?x-access- code=78dea9e953e4b2e5ca3c028608a5dbae&format=html). GenBank accessions

KR119072–KR119080 and KR871005–KR871009.

Examination of Mycangia of Ambrosiodmus

Serial transverse sections from the heads of 11 female A. lecontei and 2 female

A. minor confirmed the presence of one pair of preoral mycangia at the base of the mandibles examined (Figure 2-4, A-B). The mycangial content was a tightly packed mass of hyphae of varying diameters (Figure 2-4, C). No conidia or budding pseudo- mycelium were observed.

Morphology of Fungi from Ambrosiodmus

On PDA medium, isolates 6853_white_myce, 6855_white_myce, and

6860_sub_white_myce produced dimitic hyphae, clearly of two diameters, but also with some variation in size. Smaller, generative hyphae (Figure 2-4, D) were present at colony margins but were rapidly obscured by larger, skeletal hyphae (Figure 2-4, E). No reproductive structures and no clamp connections were observed.

Discussion for Chapter 2

All analyses indicated that Ambrosiodmus species carry in their mycangia, and culture in their galleries, previously unknown ambrosial Basidiomycotan symbionts closely related to Flavodon flavus (Basidiomycota: Polyporales). The condition of the host trees in which the galleries of Ambrosiodmus were collected, and the structure of the wood surrounding the beetle gallery, indicate that the fungal symbiont is a true wood-degrading saprophyte (Figure 2-2). There are very few known cases in which a fungus converts degraded lignocellulose into a complete diet for its animal symbiont.

Consequently, the metabolic aspects of the Ambrosiodmus-Flavodon symbiosis deserve further scrutiny.

The majority of the ambrosial beetle-fungus symbioses investigated thus far involve Ascomycotan fungi, typically from the orders Ophiostomatales or Microascales.

These relatives of plant pathogens are excellent at extracting simple nutrients from freshly dead tree tissue, but their cellulolytic capacity is limited (Licht and Biedermann,

2012). Consequently, most ambrosia insect groups are early colonizers of dying or freshly dead trees and are unable to utilize older wood colonized by wood-rot

Basidiomycotan fungi, whose enzymatic machinery for lignocellulose degradation is superior. A few basidiomycetous mutualists of wood boring insects exist. Amylostereum

(Russulales), associated with Siricidae wood wasps, is a true saprotroph, but it does not supply the entire nutrition to its host (Harrington, 2005). Several phloem-feeding bark beetles and attine ants require basidiomycete mutualists for their development, but these fungi are not competitive wood-decay saprophytes (Harrington, 2005; Mueller et al., 2001). A recent report of a basidiomycete fungus (Antrodia) from an unrelated ambrosia beetle Anisandrus dispar (Hsiau and Harrington, 2003) may not refer to a

nutritional symbiont, which is likely Ambrosiella hartigii (Batra, 1967; Happ et al., 1976).

The only other case in which a cellulolytic saprotroph appears to provide a complete nutrition is the Termitomyces mutualist of fungus-farming termites (Batra, 1967).

Nuclear 28S rDNA sequences of fungal isolates derived from A. minor differed from those of fungi cultured from A. lecontei and A. rubricollis by a single transitional pyrimidine mutation. Additionally, the ITS rDNA sequences from A. minor and A. lecontei heads are 94% similar, whereas the A. minor sequences themselves are identical. Ambrosiodmus minor was introduced to the US recently, compared to the long-established A. rubricollis and the native A. lecontei. Taken together, these comparisons suggest the propagation of lineage-specific fungal strains corresponding to the established US species and the recent immigrant species.

All the isolates are approximately equally related to sequences reportedly derived from Flavodon flavus (Figure 2-3). Given the 100% supported monophyly and the matching hyphal morphology, these isolates are referred as Flavodon cf. flavus until further analyses support their differentiation. Flavodon flavus is a free-living wood saprophyte described from northern Europe, a location far outside of the distribution of any Ambrosiodmus (Miettinen et al., 2012). This indicates that the fungal genetic markers have not yet diverged from a free-living relative, or that the association is asymmetrical, in which beetles depend on the fungus but the fungus may still be capable of aposymbiotic lifestyle. Most symbioses between insects and Basidiomycotan fungi are similarly asymmetrical; this includes not only the Siricidae wood wasps and the bark beetles mentioned above, but also some fungus-growing termites and some fungus growing attine ants (Mueller et al., 2005).

A novel form of symbiont dispersal mode in ambrosia fungi was recorded: non- budding hyphal growth (Figure 2-4, B). The A. lecontei mycangia appeared to be packed with hyphae of varying diameters (Figure 2-4, C), corroborating the presence of a dimitic Basidiomycotan fungus. The fungus Flavodon flavus was described as dimitic and lacking clamp connections (Ryvarden, 1973), and this description conformed with the hyphae I observed in mycangia (Figure 2-4, C) and from A. minor (Figure 2-4, D-E) that I molecularly characterized as Flavodon cf. flavus (Figure 2-3).

The Ambrosiodmus lecontei mycangia are almost certainly homologous with the usual preoral mycangia in some Xyleborini ambrosia beetles; thus it appears that the unusual fungus transmission mode (hyphae) is a feature of the new fungal symbiont, not of the beetle carriers. Note that the term “preoral” used here for xyleborine mycangia that were traditionally called “mandibular”; the term “mandibular” is preoccupied by the true mandibular mycangia in Dendroctonus (Six, 2003a).

By an association with a saprophytic Basidiomycotan, the beetle genus

Ambrosiodmus has gained access to a ubiquitous resource previously unavailable to any other bark or ambrosia beetle. Given the number of ambrosia beetle and fungus species worldwide and the increasing research focuses on them, human should only expect more such interactions and evolutionary innovations.

Table 2-1. Species of Polyporales used for phylogenetic analyses, with information of Ambrosiodmus symbionts Flavodon cf. flavus. GenBank accession no. Species Clade ITS 28S Ingroup: Albatrellus syringae (Parmasto) Pouzar Residual Polyporoid JN710607 JN710607 Antrodia albida (Fr.) Donk Antrodia DQ491414 AY515348 Antrodiella americana Ryvarden & Gilb. Residual Polyporoid JN710509 JN710509 A. semisupina (Berk. & M.A. Curtis) Ryvarden Residual Polyporoid EU232182 EU232266 Ceraceomyces serpens (Tode) Ginns Phlebioid AF090882 AF090882 Ceriporia viridans (Berk. & Broome) Donk Phlebioid KC182777 – Ceriporiopsis alboaurantia C.L. Zhao Phlebioid KF845947 KF845954 C. aneirina (Sommerf.) Domański #1 Phlebioid FJ496683 FJ496704 C. aneirina #2 Phlebioid KF845945 KF845952 C. balaenae Niemelä #1 Residual Polyporoid FJ496669 FJ496717 C. balaenae #2 Residual Polyporoid FJ496668 FJ496718 C. consobrina (Bres.) Ryvarden Residual Polyporoid FJ496667 FJ496716 C. gilvescens (Bres.) Domański #1 Phlebioid FJ496685 FJ496719 C. gilvescens #2 Phlebioid FJ496684 FJ496720 C. guidella Bernicchia & Ryvarden Phlebioid FJ496687 FJ496722 C. pseudogilvescens (Pilát) Niemelä & Kinnunen #1 Phlebioid FJ496673 FJ496702 C. pseudogilvescens #2 Phlebioid FJ496679 FJ496703 C. pseudogilvescens #3 Phlebioid FJ496680 FJ496700 C. pseudoplacenta Vlasák & Ryvarden #1 Phlebioid JN592499 JN592506 C. pseudoplacenta #2 Phlebioid JN592497 JN592504 C. pseudoplacenta #3 Phlebioid JN592498 JN592505 C. resinascens (Romell) Dom. Phlebioid EU340896 EU368501 C. semisupina C.L. Zhao, B.K. Cui & Y.C. Dai #1 Phlebioid KF845949 KF845956 C. semisupina #2 Phlebioid KF845950 KF845957 C. semisupina #3 Phlebioid KF845951 KF845958 Cinereomyces lindbladii (Berk.) Jülich Gelatoporia FN907906 FN907906 Climacocystis borealis (Fr.) Kotl. & Pouzar Residual Polyporoid JQ031126 JQ031126 Coriolopsis caperata (Berk.) Murrill Core Polyporoid AB158316 AB158316 Dacryobolus karstenii (Bres.) Oberw. Ex Parmasto Antrodia EU118624 EU118624 Earliella scabrosa (Pers.) Gilb. & Ryvarden Core Polyporoid JN165009 JN164793 Flavodon flavus (Klotzsch) Ryvarden Phlebioid JN710543 JN710543 Flavodon cf. flavus Phlebioid KR119072 KR119075 Flavodon cf. flavus Phlebioid KR119073 KR119076 Flavodon cf. flavus Phlebioid KR119074 KR119077 Ganoderma lingzhi Sheng H. Wu, Y. Cao&Y.C. Dai Core Polyporoid JQ781858 – Gelatoporia subvermispora (Pilát) Niemelä #1 Gelatoporia FJ496694 FJ496706 G. subvermispora #2 Gelatoporia FN907911 FN907911 Gloeoporus pannocinctus (Romell) J. Erikss. Phlebioid EU546099 FJ496708 G. dichrous (Fr.) Bres. Phlebioid EU118627 EU118627 Grammothelopsis subtropica B.K. Cui & C.L. Zhao Core Polyporoid JQ845096 JQ845099 Hornodermoporus martius (Berk.) Teixeira Core Polyporoid FJ411092 FJ393859 Hypochnicium lyndoniae (D.A. Reid) Hjortstam Residual Polyporoid JX124704 JX124704 Junghuhnia nitida (Pers.) Ryvarden Phlebioid EU118638 EU118638 Mycoacia fuscoatra (Fr.) Donk Phlebioid JN649352 JN649352 M. nothofagi (G. Cunn.) Ryvarden Phlebioid GU480000 GU480000 Obba rivulosa (Berk. & M.A. Curtis) Miettinen & Rajchenb. Gelatoporia FJ496693 FJ496710 O. valdiviana (Rajchenb.) Miettinen & Rajchenb. Gelatoporia HQ659235 HQ659235 Oligoporus lacteus (Fr.) Gilb. & Ryvarden Antrodia KC595939 KC595939 Perenniporia medulla-panis (Jacq.) Donk Core Polyporoid FJ411088 FJ393876 Perenniporiella neofulva (Lloyd) Decock & Core Polyporoid FJ411080 FJ393852 RyvardenPhanerochaete chrysosporium Burds. Phlebioid HQ188436 GQ470643 Phlebia livida (Pers.) Bres. Phlebioid AF141624 AF141624 P. radiata Fr. Phlebioid HQ604797 HQ604797 P. subserialis (Bourdot & Galzin) Donk Phlebioid AF141631 AF141631 P. unica (H.S. Jacks. & Dearden) Ginns Phlebioid EU118657 EU118657

Table 2-1. Continued GenBank accession no. Species Clade ITS 28S Piloporia sajanensis (Parmasto) Niemelä Tyromyces HQ659239 HQ659239 Podoscypha venustula (Speg.) D.A. Reid Residual Polyporoid JN649367 JN649367 Polyporus tuberaster (Jacq. ex Pers.) Fr. Core Polyporoid AF516598 AJ488116 Postia alni Niemelä & Vampola Antrodia KC595932 KC595932 P. floriformis (Quél.) Jülich Antrodia KC595937 KC595937 P. guttulata (Peck ex. Sacc.) Jülich Antrodia EU118650 EU118650 P. sericeomollis (Romell) Jülich Antrodia KF112878 KF112878 Pouzaroporia subrufa (Ellis & Dearn.) Vampola #1 Residual Polyporoid FJ496661 FJ496723 P. subrufa #2 Residual Polyporoid FJ496662 FJ496724 Sebipora aquosa Miett. Gelatoporia HQ659240 HQ659240 Skeletocutis amorpha (Fr.) Kotl. & Pouzar Tyromyces FN907913 FN907913 S. jelicii Tortič & A. David Tyromyces FJ496690 FJ496727 Steccherinum fimbriatum (Pers.) J. Erikss. Residual Polyporoid EU118668 EU118668 S. ochraceum (Pers.) Gray Residual Polyporoid JQ31130 JQ31130 Trametes pubescens (Schumach.) Pilát Core Polyporoid AY684173 AY855906 Truncospora ochroleuca (Berk.) Pilát Core Polyporoid FJ411098 FJ393865 Outgroup: Stereum hirsutum (Willd.) Pers. AB733150 AB733325

Table 2-2. Cultures of Flavodon cf. flavus and host information. Host or associated Estimated Subculture Original culture Isolation source beetle CFU 6853 6853_white_myce Ambrosiodmus minor Head extract 1500 6855 6855_white_myce A. minor Head extract 1500 6860 6860_sub_white_myce A. lecontei Head extract 4000 7324 7324_white_myce A. lecontei Head extract - Gallery in Platanus 7346 7346_white_myce A. minor - occidentalis 7353 7326_white_myce A. rubricollis Head extract 30 7354 7341_white_myce A. rubricollis Head extract 100 Gallery in Liquidambar 7373 7325_white_myce A. rubricollis - styraciflua

Figure 2-1. Female Ambrosiodmus lecontei with eggs in plant host. Note white fungal mycelial garden (arrows) in gallery. Photo courtesy of author.

Figure 2-2. Wood decay as a result of colonization by the Ambrosiodmus-Flavodon symbiosis. Photo courtesy of author.

Figure 2-3. Best ML tree from GARLI analysis of combined nuclear ITS and 28S rDNA datasets. Flavodon cf. flavus isolates, with host beetle in parentheses, within phlebioid clade of Polyporales. Values at nodes indicate >70% ML bootstrap support, and thickened branches indicate >95% Bayesian posterior probability support values. Photo courtesy of author.

Figure 2-4. Beetle mycangium and fungi. A. Micro-CT scan transverse cross sections of adult female Ambrosiodmus minor heads showing position of mycangia (yellow arrow) within mouthparts; B. Compacted fungal hyphae constituting A. lecontei mycangial inoculum; C. Loose hyphae from A. lecontei mycangial packet, with both small (black arrows) and larger (red arrow) hyphae, likely representing dimitic generative and skeletal hyphae, respectively. D-E. Flavodon cf. flavus with dimitic generative (D) and skeletal (E) hyphae on the media. Key: m, mycangium. Photo A courtesy of author. Photo B and C courtesy of Dylan P. G. Short and Matt T. Kasson. Photo D and E courtesy of D. Rabern Simmons.

CHAPTER 3 SYMBIOTIC FUNGI ASSOCIATED WITH AMBROSIODMUS IN ASIA

Introduction to Chapter 3

Flavodon ambrosius is the primary mutualistic fungus of beetles in the genera

Ambrosiodmus and Ambrosiophilus in North America (Scolytinae: Xyleborini) (Kasson et al., 2016; Li et al., 2015b; Simmons et al., 2016b). These two beetle genera form a monophyletic clade (Storer et al., 2015), suggesting a single origin of Flavodon farming.

Four beetle species have been recorded in association with F. ambrosius:

Ambrosiodmus lecontei, Ambrosiodmus minor, Ambrosiodmus rubricollis (Li et al.,

2015b), and Ambrosiophilus atratus (Kasson et al., 2016). All of these species, except

Ambrosiodmus lecontei, were recently introduced to North America from Asia (Atkinson et al., 1990; Bright, 1968; Wood and Bright, 1992) and are now established and common. Phylogenetically, Ambrosiodmus is sister to Ambrosiophilus, an unusual genus of ambrosia beetles, some of which have lost their capacity to culture their own fungal gardens and depend on mycoclepty, or fungus theft (Hulcr and Cognato, 2010).

Such unique ecological features and phylogenetic position suggest the possibility of association with fungi different than those typically associated with other ambrosia beetles, yet virtually nothing is known about the Ambrosiodmus symbionts in Asia.

Many more species of Ambrosiodmus and Ambrosiophilus occur in Asia, Africa, and South America, but their fungal symbionts have either not been studied (Beaver and Liu, 2010; Knížek, 2011; Wood, 1982) or have been poorly identified (Takagi, 1967;

 Reprinted with permission from: Li, Y., Bateman, C. C., Skelton, J., Jusino, M. A., Zachary, J. N., Simmons, D. R., & Hulcr, J. (2017). Wood decay fungus Flavodon ambrosius (Basidiomycota: Polyporales) is widely farmed by two genera of ambrosia beetles. Fungal Biology 121(11), 984-989.

Yamashita, 1966). Aside from F. ambrosius, the only described species of Flavodon are the saprophytic and free-living polypore species F. flavus (Klotzsch) Ryvarden

(Ryvarden, 1973), found in southern Asia, tropical Africa to South Africa, Australia, and

Jamaica (Corner, 1987; Maas, 1967; Miettinen et al., 2012; Ryvarden and Johansen,

1980), and “F. cervinogilvus” (Corner, 1987), an invalid species first recorded from the eastern coast of the Island of Hawaii (Simmons et al., 2016b). It is therefore unclear whether Asian Ambrosiodmus and Ambrosiophilus 1) also farm F. ambrosius, 2) farm a different symbiotic species of Flavodon, or 3) farm a non-wood-decaying Ascomycete fungus species, as would be far more typical among ambrosia beetles.

The goal of this work was to test whether Asian Ambrosiophilus and

Ambrosiodmus ambrosia beetle are associated with F. ambrosius in their native regions. The possibility of F. ambrosius playing the same symbiotic role around the world may indicate a scenario of unusual dominance of a single ambrosia fungus over many beetle species separated by continental-scale barriers and about 20 Ma (Jordal and Cognato, 2012). Such strict associations between the

Ambrosiodmus/Ambrosiophilus clade and their fungi would be in contrast to the related lineages of ambrosia beetles, namely Xyleborus and Euwallacea, most of which carry diverse and often loose symbiotic fungi in both native and non-native regions

(Harrington et al., 2010; Hulcr and Cognato, 2010; Kostovcik et al., 2015; O’Donnell et al., 2015; Ploetz et al., 2017). The results expand the understanding of the symbiotic relationship between basidiomycetous wood-decaying ambrosia fungi and their beetle vectors.

Materials and Methods to Chapter 3

Two Ambrosiodmus and two Ambrosiophilus species from Asia were sampled in four locations throughout China, South Korea, and Vietnam (October 2015 through May

2016; Table 3-1). All beetles were excised directly from active galleries in wood. In addition to gallery excision, Ambrosiophilus atratus was also caught in ethanol-baited traps in South Korea. To ensure finding the actual primary symbiont of each beetle species, i focused on fungi isolated strictly from the oral mycangia (excised by cutting off the frontal part of the head) and from active galleries of Ambrosiodmus and

Ambrosiophilus beetles. Cultures obtained from beetle hosts were isolated by dilution- plating of mycangial contents and gallery wood chips on PDA medium, as described by

Li et al. (2015b). CFUs of fungal isolations were recorded. Pure isolates were imported into and studied in, a quarantine facility in Gainesville, FL, USA, under the USDA/APHIS permit No. P526P-16-02872. Beetle and fungus voucher specimens are preserved and stored at the Forest Entomology Laboratory, School of Forest Resources and

Conservation, University of Florida, Gainesville, FL, USA.

DNA was extracted from pure subcultures. Sequences of the nuclear internal transcribed spacers ITS1-5.8S-ITS2 (ITS) and nuclear 28S ribosomal DNA (rDNA) regions were amplified. The primer pairs for PCR amplification were ITS1/ITS4 (White et al., 1990) and LROR/LR5 (Vilgalys and Hester, 1990). PCR amplification of ITS and

28S followed protocols of Li et al. (2015b). Sequencing of the PCR products in forward and reverse directions and editing and assembling of the nucleotide sequences were accomplished following Simmons et al. (2016b).

To place Asian Flavodon isolates within the known phylogenetic context, I selected 28S rDNA sequences from Simmons et al. (2016b) and ITS rDNA sequences

from Li et al. (2015b), Kasson et al. (2016) and Miettinen et al. (2012), which included F. ambrosius and related taxa. I aligned these sequences with those from my cultured

Ambrosiodmus and Ambrosiophilus-associated fungi using default settings in ClustalX

2.0 (www.clustal.org). The alignments are deposited in TreeBASE (access URL: http://purl.org/phylo/treebase/phylows/study/TB2:S20605?x-access- code=a5b8cb957aff2c80c1985d844175f6ee&format=html). Sequence evolution model for the Maximum likelihood (ML) analyses was selected using Mega 7 (Hall, 2013) setting the initial tree for ML calculations to NJ/BioNJ. Tamura 3+G was selected for the

ITS dataset and Kimura 2+G model was selected for the 28S dataset. ML analyses were also conducted with Mega 7, setting the number of fast bootstrap replicates to

1000.

Results to Chapter 3

Identification and Phylogenetic Analyses of Symbiotic Fungus Flavodon ambrosius in Asia

The ML phylogenetic analyses of both the ITS and 28S markers recovered nearly identical topology for the Flavodon clade (Figure 3-1). The topology corroborated the placement of F. ambrosius in the genus Flavodon together with F. flavus in the phlebioid clade in the Polyporales (Li et al., 2015b). All Asian Flavodon sequences were placed within the homogeneous Flavodon ambrosius clade, which consisted of sequences from the F. ambrosius holotype and other isolates from Florida and West Virginia, from both native and non-native North American beetle species. The F. ambrosius sequences did not show any divergence or lineages specific to different beetle vectors or locations.

DNA sequences: GenBank accessions LC216225-LC216231(ITS) and LC215903-

LC215909(28S).

The monophyly of all F. ambrosius isolates studied here strongly supports the hypothesis that beetles in the Ambrosiodmus/Ambrosiophilus clade share the same symbiotic fungus in Asia and North America.

Biology of Fungus Flavodon ambrosius in Asia

The incidence and habits of F. ambrosius in Asia were consistent with those observed in North America. All branches containing F. ambrosius and their beetle vectors were notably decayed (Figure 3-2, A). In many cases, both Ambrosiodmus and

Ambrosiophilus tunnel entrances were closely clustered, rather than being randomly distributed on the tree. For example, the F. ambrosius samples from China were collected from multiple galleries of Ambrosiodmus rubricollis and Ambrosiophilus subnepotulus found immediately adjacent to one another on the same branch. Galleries of both beetle species were covered by white filamentous fungal growth (Figure 3-2, D).

Discussions to Chapter 3

In total, five ambrosia beetle species in Ambrosiophilus and Ambrosiodmus are now confirmed to employ F. ambrosius as their primary symbiotic fungus (Li et al.,

2015b) (Kasson et al., 2016). The symbiosis between F. ambrosius and all its vectors has been maintained in both deep evolutionary time during the natural spread of ancestral Ambrosiodmus around the world, as well as during recent human-assisted spread and introductions.

The monophyly of F. ambrosius in two markers commonly used in fungal systematics indicates that all the isolates are members of the same phylogenetic species. The ITS region is typically variable enough to detect sub-species divergence in fungi of Basidiomycota (Lindner and Banik, 2011; Schoch et al., 2012), but it did not recover any divergence within F. ambrosius. If multiple lineages exist within F.

ambrosius, they are either unrecoverable using the two markers selected here, or additional Flavodon diversity occurs in geographic regions not sampled for this work, such as in South America.

All Flavodon strains isolated in this work were morphologically uniform. F. ambrosius isolated from Ambrosiophilus atratus in West Virginia displayed two types of colony morphology, but both were genetically identical (Kasson et al., 2016). An ambrosia fungus that was likely Flavodon was previously recorded from Ambrosiodmus rubricollis from Japan (Takagi, 1967; Yamashita, 1966) and described as “white wooly mycelia” without further identification. Isolates of this fungus were not available to us, but the brief description fits the morphology of F. ambrosius.

Most ambrosia beetles maintain their symbiont associations through time and through introductions into non-native regions. Mayers et al. (2015) suggest that in some groups, each beetle species is associated with a unique fungus species, and each fungus is typically only found associated with one beetle species. In other beetle clades, co-phylogenetic analysis has suggested that symbiotic partners may frequently be swapped over evolutionary time (O’Donnell et al., 2015). The association between

Ambrosiodmus/Ambrosiophilus and F. ambrosius appears to represent an entirely different scenario. My results suggest an association of a single fungus and many beetle species, a dominance of a single fungus that has been maintained throughout evolutionary history as the beetles have radiated into many species and spread around the world. Why has F. ambrosius not also radiated into diverse lineages of fungal symbionts that are each specialized for its own vector beetle, as has been observed in other closely related ambrosia beetle clades? Could the unique ecological traits of this

sole known white-rotting basidiomycete ambrosia fungus explain these unique relationships? These questions offer avenues for future inquiry and remain at the forefront of symbiosis and co-evolutionary research.

My observations support the hypothesis that some Ambrosiophilus species are mycocleptic, colonizing the vicinity of galleries established by other ambrosia beetles carrying the same fungus (Figure 3-2, B-C) (Hulcr and Cognato, 2010). The one species of Ambrosiophilus that has been well-studied (A. atratus) has oral mycangia with the capacity to transmit and preserve fungal inoculum, and frequently creates its own galleries (Kasson et al., 2016). However, the Ambrosiophilus species which are more often reported as mycocleptic have not been studied for their mycangia, and are typically found associated with ambrosia beetles from the genus Beaverium (Hulcr and

Cognato, 2010). This is the first report of multiple mycocleptic associations between

Ambrosiophilus and Ambrosiodmus.

The evolutionary transition of Flavodon from a free-living species to the ambrosial phenotype seen in F. ambrosius deserves future study. As in the USA, the

Asian F. ambrosius is also known only from ambrosia beetles, and no fruiting body has been reported (Kasson et al., 2016; Li et al., 2015b; Simmons et al., 2016b). The closely related free-living species F. flavus is common in tropical regions of Asia, and frequently produces sexual fruiting bodies (Dai, 2012). My data do not allow to ascertain whether

F. ambrosius participates in sexual reproduction, or whether the fungus is horizontally acquired by the beetles from free-living populations, but neither has been reported.

Many unexplored species of Ambrosiodmus occur throughout Africa and South

America, but they were not available during this project. In addition, the genus

Beaverium, widespread in Asia and Oceania, is also likely associated with Flavodon, as suggested by the frequent co-colonization by Ambrosiophilus. If all these beetles indeed carry F. ambrosius as their symbiont, it would make this fungus one of the most widespread fungal symbionts of animals.

Table 3-1. Primary symbionts isolated from Asian Ambrosiodmus and Ambrosiophilus beetle and the NCBI/GenBank accession numbers of their ITS and 28S rDNA sequences. Voucher Taxon Locality Beetle vector CFU Isolate part no. Flavodon Huaxi, Guizhou, Ambrosiodmus oral LL51 16000 ambrosius China rubricollis mycangia Huaxi, Guizhou, Ambrosiodmus F. ambrosius LL53 N/A gallery China rubricollis Nanming, Ambrosiodmus oral F. ambrosius LL70 500 Guizhou, China rubricollis mycangia Nanming, Ambrosiophilus oral F. ambrosius LL71 1500 Guizhou, China subnepotulus mycangia Tam Đảo, Ambrosiodmus oral F. ambrosius V12236 1500 Vietnam minor mycangia Gwangyang, Ambrosiophilus oral F. ambrosius V12544 1100 South Korea atratus mycangia Gwangyang, Ambrosiophilus oral F. ambrosius V12546 1000 South Korea atratus mycangia

Figure 3-1. The best Maximum likelihood tree of Flavodon inferred from ITS and 28S rDNA datasets with source beetle genera and GenBank accession numbers. Values above branches represent ML bootstrap percentages >50 % for that node from a summary of 1000 replicates. The beetle vectors are indicated in squares and circles. Photo courtesy of author.

Figure 3-2. Flavodon ambrosius and ambrosia beetles collected from China. A: White- rot wood with galleries of ambrosia beetles. B: Gallery of Ambrosiophilus subnepotulus with F. ambrosius. C: Boring holes of Ambrosiophilus sp. (middle) and Ambrosiodmus rubricollis (right and left) on the bark. D: Galleries of Ambrosiophilus subnepotulus (right) and Ambrosiodmus rubricollis (left) on the same branch; Red arrows: F. ambrosius; Bars: 1 mm. Photo courtesy of author.

CHAPTER 4 DISTRIBUTION, HOST AND SYMBIOTIC FUNGI OF EUWALLACEA FORNICATUS IN CHINA

Introduction to Chapter 4

Euwallacea is a genus of mostly Asian ambrosia beetles (Storer et al., 2015).

The genus includes over 50 recognized species and is increasingly important due to several globally invasive pest species (Eskalen et al., 2013; Li et al., 2014; Mendel et al., 2012). Currently, the most damaging are several populations within the species complex called Euwallacea fornicatus, associated with fungal mutualists in the

Ambrosia Fusarium Clade (AFC) and the fungal genus Raffaelea (Freeman et al., 2013;

Kasson et al., 2013). This beetle-fungus complex is able to injure or kill trees by the mass attack, each of which inoculates the mildly pathogenic symbiont (Smith and Hulcr,

2015). E. fornicatus has a vast distribution throughout Asia and Oceania, and has recently been introduced and established in Mesoamerica and several locations in the

US (Kirkendall and Ødegaard, 2007; Rabaglia et al., 2006). This species (or complex of species) has a broader host range than previously thought (Browne, 1961;

Danthanarayana, 1968), and has much broader distribution (CABI, 2015; James, 2007).

Little is known regarding the distribution of this increasingly important pest in

China (Li et al., 2015a; Li et al., 2014). Even the Catalog of Scolytidae and Platypodidae

(Wood & Bright 1992), an essential reference for scolytine biogeography, contains few records of this species from China. Browne (1961) and Danthanarayana (1968) had

 Reprinted with permission from: Li, Y., Gu, X., Kasson, M. T., Bateman, C. C., Guo, J., Huang, Y., Li, Q., Rabaglia, R. J., & Hulcr, J. (2016). Distribution, host records, and symbiotic fungi of Euwallacea fornicatus (Coleoptera: Curculionidae: Scolytinae) in China. Florida Entomologist 99(4), 801-804.

comprehensively recorded its host range in Sri Lanka, India, and Southeast Asia, but neither included any records from China. Consequently, any research on the biogeographic, ecological and climate-related aspects of this beetle is currently likely to suffer a significant gap in the baseline data.

Materials and Methods to Chapter 4

Here I present previously unpublished host records of Euwallacea fornicatus deposited in the National Zoological Museum of China (NZMC), Institute of Zoology,

Chinese Academy of Sciences, Beijing, and from extensive field investigation in China from 2013 to 2015. Chinese host tree names were associated with scientific names according to Iconographia Cormophytorum Sinicorum Tomus website

(http://pe.ibcas.ac.cn/tujian/tjsearch.aspx). The collection at the National Zoological

Museum of China in Beijing contains 193 specimens of E. fornicatus collected from

1960 to 1999. Dr. Huifen Yin and Dr. Fusheng Huang identified the specimens

Results and Discussions to Chapter 4

The collection data show that this beetle in mainly distributed in the humid and subtropical Southern China, but it also occurs in distinctly temperate and dry habitats

(Figure 4-1). Seven tree species are recorded for the first time as host plants of E. fornicatus (Table 4-1). Three of them belong to plant families from which the beetle has not been recorded before: Actinidiaceae, Oleaceae and Pinaceae. I observed a complete family (eggs, larvae, pupae and adults) on a weakened Pinus massoniana in

October 2015. This is the first record of E. fornicatus from a conifer. Although a single record from a particular host plant is not necessarily indicative of a stable host association, E. fornicatus is known to be broader host tree specificity, and it suggests that the fungal mutualist is viable in conifers.

My data suggest that in its native habitat E. fornicatus is capable of colonizing still-living tissues of angiosperm hosts. This may help explain the beetle’s unique semiochemical ecology (Kendra et al., 2011). However, most of the data do not suggest that the beetle is an aggressive colonizer of live and healthy trees, since nearly all individuals in my collection were collected from weak, diseased, or dead host plants. I was not able to corroborate the supposed aggressive attacks on Litchi chinensis in the south of China reported previously (Wang and Yuan, 2003) even after I visit to the sites from which the event was recorded. I only found this beetle mass attacking relatively healthy Acer buergerianum and Platanus orientalis in the urban area of Kunming city.

The NZMC collection labels do not contain information on whether the trees were killed by the beetle.

In Guiyang, Guizhou (26.3857°N, 106.6731°E) on a log of black locust Robinia pseudoacacia, I found more than ten dead E. fornicatus bearing distinct signs of having been parasitized by an unknown natural enemy. The parasitoid consumed the abdomen of E. fornicatus and bored an exit hole through the elytral declivity (Figure 4-2).

Unfortunately, the parasitoid was not collected. This symptom is known from other

Scolytinae beetles parasitized by Hymenoptera (Nierhaus-Wunderwald, 1993), and this observation suggests that a search for natural enemies as a part of biocontrol efforts may be fruitful.

Additionally, two fungi isolated from E. fornicatus and its gallery in Guizhou were identified. The first fungus was morphologically similar to a recently described

Paracremonium pembeum, a known mycangial commensal of the polyphagous shot hole borer (E. fornicatus species complex) in California and Vietnam (Lynch et al.,

2016). About 15000 CFUs of it were isolated from the oral mycangia of one individual.

Cultures were slimy to moderately floccose and pale pink to salmonaceous in color. The conidia were generated in simple verticillate phialides. The fungus was identified by amplifying the ribosomal DNA (rDNA) internal transcribed spacer (ITS) and querying the

NCBI GeneBank database. Three representative ITS rDNA sequences (Hulcr12051 and

LL84) were 100% identical to the Hypocrealean fungus Sarocladium strictum and an uncultured Acremonium (Genbank accessions KM249080 and HG936339, respectively). Sarocladium has been previously reported to be associated with bark and ambrosia beetles throughout the Northern temperate region (Hutchison, 1999;

Jankowiak and Kolařík, 2010; Jankowiak et al., 2007), as well as from mites

Steneotarsonemus spinki (Acari: Tarsonemidae) in Taiwan (Hsieh et al., 1980).

Acremonium sp. had been isolated from E. fornicatus (Freeman et al., 2016).

The second fungus was consistent with the known nutritional mutualist of

Euwallacea, a representative of the AFC (Kasson et al., 2013). It produced abundant aerial mycelia and clavate macroconidia forming in sporodochia; 8000 CFUs were isolated from the oral mycangia of one individual. Portions of the translation elongation factor 1-α (EF1-α) and the second largest subunit of RNA polymerase 2 (RPB2) were used to confirm placement among known AFC members (Kasson et al., 2013). Initial

GenBank BLAST searches revealed isolate 12049A, 12049B, and LL74 RPB2 sequences were 99-100% identical to Fusarium euwallaceae strains NRRL 62626 and

FD31 ACVI (KU171702 and JX892009, respectively). A BLAST search of EF1 sequences revealed that strains Hulcr12049 and LL74 were 99% similarity to Fusarium sp. AF-12, AF-5, and AF-4 and Fusarium ambrosium (KM406629, KC691542,

KC691537, and KC691528). Conclusively, my sequencing results indicate that the

Fusarium sp. associated with E. fornicatus in China is a member of the monophyletic

AFC. However, sequencing of additional loci is needed to confirm whether or not these strains represent a novel species.

Table 4-1. Host trees of Euwallacea fornicatus specimens in the National Zoological Museum of China (NZMC) and new field collection from 2013 to 2015. Number of Province Location Family of host Host Source specimens Beijing Beijing Malvaceae Theobroma cacao NZMC 3 (greenhouse) Chongqing Taojiaxiang Rutaceae Citrus sp. NZMC 2 Fujian Nanjing Sapindaceae Litchi chinensis NZMC 20 Fujian Zhangpu Euphorbiaceae Ricinus communis NZMC 43 Fujian Zhaoan Sapindaceae Litchi chinensis NZMC 9 Guangdong Jiangmen Euphorbiaceae Ricinus communis NZMC 10 Guangdong Zhanjiang Euphorbiaceae Hevea brasiliensis NZMC 10 Guangdong Zhongshan Sapindaceae Litchi chinensis NZMC 32 Guizhou Guiyang Fabaceae Robinia pseudoacacia field 8 Guizhou Guiyang Oleaceaea Ligustrum compactumb field 16 Guizhou Guiyang Pinaceaea Pinus massonianab field 11 Hainan Ledong Euphorbiaceae Hevea brasiliensis NZMC 1 Hainan Wuzhishan Fabaceae Acacia sp. NZMC 1 Sichuang Chengdu Fabaceae Robinia pseudoacacia NZMC 11 Sichuang Emei Mountain Fabaceae Robinia pseudoacacia NZMC 4 Tibet Motuo Actinidiaceaea Saurauia tristylab NZMC 1 Tibet Motuo Euphorbiaceae Mallotus barbatusb NZMC 2 Tibet Motuo Fagaceae Castanopsis fargesiib NZMC 1 Yunnan Kunming Fabaceae Dalbergia odorifera NZMC 2 Yunnan Kunming Platanaceae Platanus orientalisb field 5 Yunnan Kunming Sapindaceae Acer buergerianum field 3 Yunnan Xishuangbanna Betulaceae Betula alnoides NZMC 1 Yunnan Xishuangbanna Euphorbiaceae Hevea brasiliensis field 5 Yunnan Xishuangbanna Euphorbiaceae Ricinus communis NZMC 7 Yunnan Xishuangbanna Fabaceae Acacia mearnsii NZMC 25 Yunnan Xishuangbanna Fabaceae Cassia siamea NZMC 3 Yunnan Xishuangbanna Fabaceae Erythrina variegata NZMC 1 Yunnan Xishuangbanna Fagaceae Castanea sp.b NZMC 1 Yunnan Xishuangbanna Theaceae Camellia sinensis NZMC 3 a New record of host plant family; b New record of host plant species.

Figure 4-1. The distribution of Euwallacea fornicatus in the south of China. Photo courtesy of author.

Figure 4-2. The elytral declivity of Euwallacea fornicatus after being parasitized by an unknown natural enemy. Photo courtesy of author.

CHAPTER 5 SYMBIOTIC FUNGI ASSOCIATED WITH PLATYPODINAE IN THE SOUTHEASTERN USA

Introduction to Chapter 5

There are more than 1400 species in the weevil subfamily Platypodinae (Wood,

1993). More than 90% platypodine species are distributed in the tropical region. And less than 10% reach wet temperate areas (Jordal, 2015; Wood, 1993). All but two species in the subfamily Platypodinae maintained nutritional symbioses with fungal cultivars (Jordal, 2014). Platypodinae was estimated to originated in the mid-Cretaceous

(119–88 Ma), much earlier than Scolytinae and the other farming insects (Jordal, 2015).

Usually, platypodine species attracted dying or very severely injured trees rather than trees in health (Hubbard, 1896; Jordal, 2015).

Platypodines are dominated by ambrosia fungi species of the genus Raffaelea and other genera in order Ophiostomatales (Beaver, 1989; Bellahirech et al., 2014;

Dreaden et al., 2014; Kubono and Ito, 2002; Latifa, 2011; Yun et al., 2015). Platypodine species and their symbiotic fungi are increasingly important as the international trade of lumber continuous to grow and more exotic species were introduced by wooden production (Brockerhoff et al., 2006). For instance, Platypus quercivorus which vectors the plant pathogen Raffaelea quercivora causes significant damage on oak trees in

Japan (Ito et al., 2003; Kubono and Ito, 2002). Similarly, Platypus cylindrus kills the

European oak by vectoring the oak pathogen Raffaelea montetyi (Inácio et al., 2012a;

Latifa, 2011). Megaplatypus mutatus, which is associated with Raffaelea spp., attacks poplars in Argentina and Italy (Alfaro et al., 2007; Ceriani-Nakamurakare et al., 2016).

While the platypodines and their fungi have received some attention in Asia,

Europe, and Oceania (Faulds, 1977; Inácio et al., 2012a; Kubono and Ito, 2002; Tarno

et al., 2016), the fungal symbionts of the American fauna is poorly known (Batra, 1961;

Ceriani-Nakamurakare et al., 2016; Farris and Funk, 1965). Compared to the high diversity of platypodine species in Asia, Africa, and Neotropics, North America has few species, including only seven native species of platypodine beetles (Wood, 1993). Four of these, Euplatypus compositus, E. parallelus, Myoplatypus flavicornis, and

Oxoplatypus quadridentatus, are only recorded from southeastern USA (Atkinson,

2004).

There have been no previous accounts of the fungi that are associated with the four native platypodine species in the southeastern USA. For the two unusual species

Myoplatypus flavicornis and Oxoplatypus quadridentatus, the biology has never been comprehensively studied, let alone their relationships with symbiotic fungi. Based on previous collection information, Myoplatypus flavicornis usually infests weakened pine trees (Pinus) with the other bark-feeding scolytine beetle and Oxoplatypus quadridentatus preferred oak trees (Fagaceae) (Atkinson, 2000; Atkinson and Peck,

1994). Euplatypus compositus and E. parallelus are common in some areas, and light attracts them. In the only two reports of fungal isolations from southeastern platypodines, Batra et al. (1961) and Verrall (1943) isolated the yeast fungi

Ambrosiozyma monospora from Euplatypus compositus in Mississippi. E. parallelus indigenous to the southeastern USA, but it has recently become a global invasive species, and now found throughout Africa, Asia, Central America, and parts of Oceania

(Beaver, 2013). This beetle species has been reported to attack live rubber trees Hevea brasiliensis in Brazil (Pereira da Silva et al., 2014) and China (Li et al. in press), as well as Indian Rosewood Dalbergia sissoo in Bangladesh (Boa and Kirkendall, 2004).

Several reports from Asia indicated it as a suspected vector of fungal pathogens of

Burmese Rosewood Pterocarpus indicus (Boa and Kirkendall, 2004; Bumrungsri et al.,

2008; Sanderson et al., 1996; Tarno et al., 2016).

Studying the fungal symbionts of platypodine species is difficult for three reasons: 1) Platypodine species are preferring to infest deep within the lower trunk of large trees (Atkinson and Peck, 1994), making the collection of these beetles is laborious; 2) The presence and locations of mycangia of most platypodine species are uncertain (Hulcr and Stelinski, 2017; Nakashima, 1975; Wood, 1993); 3) A robust phylogenetic placement of Raffaelea and related genera within the Ophiostomatales has not been fully resolved, leading to taxonomic uncertainty of recovered fungi. In contrast, the study of scolytine beetles and their relationship with fungi are more popular as their fungal isolation were clear (Hulcr and Stelinski, 2017; Vanderpool et al., 2017).

In this study, I identified the fungal symbionts from four endemic platypodine species using fungal culturing and community analyses. To strengthen the identification of Ophiostomatalean fungi, I generated a phylogenetic tree to accurately place the isolates among previously sequenced specimens available via Genbank NCBI BLAST

(National Center for Biotechnology Information; Basic Local Alignment Search Tool) or rough phylogenetic analysis before (Bumrungsri et al., 2008; Inácio et al., 2012b; Tarno et al., 2016).

Materials and Methods to Chapter 5

Platypodine Beetle Collection

All known species of platypodine beetle, Euplatypus compositus, E. parallelus,

Myoplatypus flavicornis and Oxoplatypus quadridentatus, in the southeastern USA, were collected from 2015 to 2017 (Table 5-1). In total 49 individuals. To decrease the

effect of opportunistic and transient commensal fungi, every beetle species was acquired from at least two locations or more than two times from the same area except for Euplatypus parallelus. Specimens that came from the same field sites were taken from different plant individual at least 100m apart. When possible, both the male and female were taken from every gallery as one sample. The beetle specimens were stored alive at room temperature with water-moistened sterile paper towels for one day after collection.

Isolation, Identification, Taxa Assignment, and Phylogenetic Analyses of Fungi Associated with Platypodine Beetle

To eliminate opportunistic fungi attached to the exoskeleton of beetles, live beetles were washed by vortexing for 10 seconds in a sterile solution of 1mL water and one drop of Tween 80. A second wash was performed using a solution of only sterile water. Due to a high diversity and limited knowledge of mycangia in Platypodinae, I focused on the isolation from the head, pronotum, surface wash, and gallery. Beetles were held with forceps under a dissecting microscope while the head, thorax, and abdomen were separated using a sterile scalpel as previously described (Fraedrich et al., 2008; Kasson et al., 2013). The head and pronotum were transferred into 2mL microcentrifuge tubes containing 1mL of PBS and crushed using sterilized micropestels.

The tube containing a macerated body segment and second wash was then serially diluted and plated at concentrations of 1/10, 1/100, and 1/1000 on PDA media as described by Li et al. (2015). The PDA media was amended with 0.05 g/L cycloheximide to limit the growth of non-Ophiostomatalean fungi (Harrington, 1981). CFUs were estimated for each morphotype by multiplying the number of colonies on a plate by the inverse of the inoculum dilution. If the beetle was collected from wood, the surface layer

of the gallery was scraped and streaked on the PDA media using a sterile scalpel. Fungi were allowed to grow at 25°C for 5-7 days and examined at intervals.

Extraction of genomic DNA from fungal cultures was performed by scraping approximately 5-10μg of mycelium from pure cultures and adding it to 20μL extraction solution from the Extract-N-Amplify Plant PCR kit (Sigma-Aldrich). Samples were then incubated at 96°C for 30 minutes. Following incubation, 20μL of 3% BSA solution was added, vortexed, and spun down. The upper 20uL of the supernatant was used as the

PCR template.

PCR amplification for Sanger sequencing was performed on portions of the 28S large subunit ribosomal DNA (rDNA) loci using the primer pair LR0R/LR5 (Vilgalys and

Hester, 1990). Final PCR volumes of 25µL consisted of 1 µL of template DNA, 1 µL of

LSU forward and reverse primer, 1 µl of DMSO, 12.5 µL Premix TaqTM (Ex TaqTM

Version2.0; Takara Bio Inc.), and 9.5 µL sterile water. Amplified products were cleaned using the ExoSAP-IT (Affymetrix Inc.) kit according to manufacturer’s instructions.

Sanger sequencing was performed by Interdisciplinary Center for Biotechnology

Research (ICBR) at University of Florida (Gainesville, FL) or GENEWIZ (South

Plainfield, NJ)

Sequence chromatograms were inspected for quality and assembled in

Geneious 9.1.5. Ophiostomatalean fungi were binned into operational taxonomic units

(OTUs) at 100% similarity. Identification of sequences was first made to the lowest possible taxonomic rank via Genbank NCBI. I further classified Ophiostomatalean fungi using phylogenetic analysis because these fungi are known to be widely crucial to

platypodine beetle biology and because this group is not well represented or curated in

Genbank.

Additional LSU sequences were included from representative taxa within the

Raffaelea after a study by Simmons et al. (2016a). Bayesian inference analyses were performed using MrBayes 3.2.6 (Ronquist and Huelsenbeck, 2003), and Maximum likelihood (ML) analyses were performed using RAxML2.0 (Stamatakis, 2014) on the

University of Florida supercomputer (HiperGator2.0). The tree was edited in FigTree

1.4.3 (http://tree.bio.ed.ac.uk/software/figtree) and Adobe Photoshop CS4. Isolate names were created based on monophyly and closest relative. For isolates that were lost to contamination, identification was inferred by sequences of representatives.

Representative sequences for all taxa were uploaded to GenBank (accession number

LC363534-LC363555).

Community Analysis of Fungi from Platypodine Beetle

Fungal CFU and frequency data were processed by using Microsoft Office Excel

365 ProPlus. When specimens were collected from wood, I accounted all the fungal isolations of each male and female pair together as one sample in CFUs and frequency statistics. In the mycobiota, and quantitative analysis, Saccharomycetales yeast fungi (Candida spp., Pichia spp., or Ambrosiozyma spp.) and Hypocreales fungi

(Fusarium spp.) were lumped in each category without further identification to genus or species level. Because yeast and Fusarium fungi are not primary mutualist, they merely always associated with bark and ambrosia beetle (except Euwallacea) (Bateman et al.,

2016; Ceriani-Nakamurakare et al., 2016; Hulcr and Stelinski, 2017; Kasson et al.,

2013; Kostovcik et al., 2015; Musvuugwa et al., 2015; Yun et al., 2015). The other fungal species were not included if they were only found from a single sample.

Results to Chapter 5

In total, 66 Ophiostomatalean fungal isolates were recovered. After assignment at 100% similarity in LSU sequences, I recovered 44 fungal isolates representing 12 fungal OTUs in Raffaelea, two fungal isolates representing two fungal OTUs in Esteya, one fungal isolates representing one fungal OTUs in Leptographium, eight fungal isolates representing two fungal OTUs in Ophiostoma, and 11 fungal isolates representing four fungal OTUs in Ceratocystiopsis (Figure 5-1; Table 5-2). After phylogenetic analysis, 19 Ophiostomatalean fungi were identified (Table 5-2). All

Raffaelea fungi belong to Raffaelea s. str. clade except sole isolation Raffaelea sp.8 in

Raffaelea sulphurea complex. Every platypodine species was associated with at least one Raffaelea fungal species. No fungus in the Raffaelea lauricola complex was found during my isolation.

Euplatypus compositus and Oxoplatypus quadridentatus were associated with the most amount of Raffaelea fungi, each with four species. For Euplatypus compositus, two of fungi, Raffaelea cf. campbellii1 and Raffaelea sp.6, from Florida were also present with beetles from Georgia. Both were also obtained from Florida every collection since 2014 The fungi of Oxoplatypus quadridentatus are less specific: all

Raffaelea spp. were isolated in low quantities and CFU. Only Raffaelea cyclorhipidia appeared on collections from three locations (Washington, D.C., Florida, and Virginia) but in low frequency. E. parallelus was only associated with one Raffaelea which were isolated from all individuals (Table 5-2; Table 5-3). Myoplatypus flavicornis was associated with three Raffaelea fungi. Raffaelea sp.5 were appeared with a relatively high frequency (Table 5-3) and obtained every year since 2014 in Florida.

Esteya and Leptographium are sporadic in Oxoplatypus quadridentatus and

Myoplatypus flavicornis. Ophiostoma fungi also associated with Oxoplatypus quadridentatus and Myoplatypus flavicornis, but appeared at only one location or one year during isolation. Two unknown species of Ceratocystiopsis were found in all platypodine species except Myoplatypus flavicornis. Ceratocystiopsis sp.1 appeared at three beetle vectors. Ceratocystiopsis sp.2 was only associated with Oxoplatypus quadridentatus and appeared in both Washington, D.C. and Virginia.

Fusarium was recovered from two platypodine species during the isolations but was less common than Raffaelea. Saccharomycetales yeasts fungi (Candida spp.,

Pichia spp., Ambrosiozyma spp.) were quite abundant on the beetle.

Discussion to Chapter 5

Farris and Funk (1965) had found Treptoplatypus wilsoni associated with a fungus resembling Tuberculariella sp.. Afterwards, one species of Tuberculariella was transferred to Raffaelea by Harrington et al. (2010). This is likely the oldest record of platypodine-Ophiostomatalean in North America.

I found some evidence of consistent and wide-spread species-level associations between platypodines and Ophiostomatalean fungi. This study support some earlier research (Bellahirech et al., 2014; Dreaden et al., 2014; Kinuura, 2002; Payton, 1989), especially some Raffaelea fungi consistently appeared from single platypodine species on different locations and time, such as two species of Raffaelea (sp.6 and cf. cambellii1) recovered from Euplatypus compositus in Florida and Georgia, and

Raffaelea sp.5 continuously present in isolation from Myoplatypus flavicornis for three years. Those repetitious emergences suggest that Raffaelea cf. campbellii1 and

Raffaelea sp.6 are the primary fungi of Euplatypus compositus and Raffaelea sp.5 is the

primary fungus of Myoplatypus flavicornis. Besides, Raffaelea cf. campbellii1, Raffaelea sp.6, and Raffaelea sp.5 were isolated with high frequency and abundant CFU.

Euplatypus parallelus is getting more attention lately because of its rapid spread and because of reports of it becoming a vector of wilt pathogens in Asia (Beaver, 2013;

Bumrungsri et al., 2008; Gümüş and Ergün, 2015). This is surprising as neither

Raffaelea nor any other Ophiostomatalean fungi were found during previous isolations of Euplatypus parallelus (Bumrungsri et al., 2008; Tarno et al., 2016). The same happens to Euplatypus segnis from Mexico (Alvidrez-Villarreal et al., 2012). In the present study, I found that Raffaelea sp.7 associated with Euplatypus parallelus in

Florida. It is still unclear whether Raffaelea sp.7 fungi is primary of Euplatypus parallelus since a few samples were included from one USA location. However, Raffaelea sp.7 also appeared at Euplatypus parallelus from Hainan, China (Li et al., unpublished data).

Due to the Geographical distance between China and USA, this relationship between

Raffaelea sp.7 and Euplatypus parallelus appears to be strong. The previous no-

Raffaelea result is probably caused by missing antibiotics during isolation, because

Fusarium and yeast are quite abundant on the plates when isolation of Euplatypus parallelus from Asia (Li et al., unpublished data). The rapid growth of Fusarium fungi would easily surpass and cover the other fungi in the media.

One Raffaelea species, R. cyclorhipidia, was consistently present with

Oxoplatypus quadridentatus at different locations. Compare with R. cf. campbellii 2, R. cyclorhipidia is more likely mutualist of Oxoplatypus quadridentatus, though this fungi was rare in our isolation with low CFU and frequence. Oxoplatypus quadridentatus has specificity to oak as well as the three most well-known platypodine pests Platypus

quercivorus, Platypus cylindrus and Platypus koryoensis whose symbiotic fungi belong to the Raffaelea sulphurea complex (Kim et al., 2009; Kubono and Ito, 2002; Latifa,

2011; Simmons et al., 2016a). These fungal identifications indicate the primary fungi of

Oxoplatypus quadridentatus is not in that complex even if their host range is similar.

The phenomenon of multiple Raffaelea associated with single species were previously recorded in the Platypodinae, such as Megaplatypus mutatus, Platypus quercivorus, Platypus cylindrus and Platypus koryoensis, Platypus hamatus, and

Dinoplatypus flectus (Ceriani-Nakamurakare et al., 2016; Endoh et al., 2011; Inácio et al., 2012a; Park, pers. comm.)(Skelton et al., in press). Even though Raffaelea s.l. fungi are also widespread across several unrelated Scolytine beetle tribes including Corthylini and Xyleborini, usually one Raffaelea was dominant (Bateman et al., 2015; Biedermann et al., 2013; Harrington et al., 2011; Ploetz et al., 2017; Simmons et al., 2016a). One possibility is suggested by hypotheses related to their mycangial structure (Nakashima,

1975; Nobuchi, 1993). They had multiple potential mycangia including exoskeletal pores and internal sac (Nakashima, 1975, 1979, 1982; Nobuchi, 1993). In this study, only

Euplatypus compositus and Oxoplatypus quadridentatus have exoskeletal pores on the pronotum, while Euplatypus parallelus and Myoplatypus flavicornis lack of exoskeletal pores. It is not clear whether they have internal mycangia, and, therefore, the crushing of the head and the pronotum were applied during isolations.

I demonstrated that platypodine beetles have a moderate association with

Ceratocystiopsis. Ceratocystiopsis is a group of morphological and molecular distinct species that are strongly associated with bark beetles and divided from genus

Ophiostoma (Seifert et al., 2013; Zipfel et al., 2006). This genus appeared in the

isolation of three endemic platypodine beetles. There are limited reports showing the association of Ceratocystiopsis with platypodine beetles. Inácio et al. (2012b) and

Bellahirech et al. (2014) has been recovered a fungus Ophiostoma sp.X (accession number JF909532) from Platypus cylindrus that appeared to be a Ceratocystiopsis sp.2

(Figure 5-2). The specificity and ecological significance of the association between

Ceratocystiopsis and platypodine beetles await further investigation.

My study found the yeast Saccharomycetales species account for significant proportion of fungal community in platypodine beetles which corresponds with other recent studies of platypodine beetles (Tarno et al., 2016; Yun et al., 2015) (Skelton, in press). Preliminary results based on dual-culture of Raffaelea and yeast indicate a compatible relationship exists between yeast species and Raffaelea (Yun et al., 2015).

This compatible connection would allow both to grow together as co-inhabitants in the galleries of bark beetles (Davis, 2015). A future bioassay test will involve at least platypodine-fungus fidelity, total diversity, and the promiscuous relationship between platypodine beetle, yeast, and Raffaelea.

Table 5-1. Collection of platypodine beetle Number of Species Location Source Date specimens Euplatypus Gainesville, FL, USA light trap 2015-III-15 4 compositus Gainesville, FL, USA Oak 2015-VIII-27 4 Gainesville, FL, USA light trap 2017-III-15 2 Cleveland, GA, USA light trap 2017-VII-18 1 Euplatypus Miami, FL, USA light trap 2015-VIII-14 5 parallelus Myoplatypus Gainesville, FL, USA Pine 2015-VII-9 5 flavicornis Gainesville, FL, USA Pine 2016-V-4 3 Gainesville, FL, USA Pine 2017-III-5 5 Oxoplatypus Gainesville, FL, USA Oak 2015-VII-28 5 quadridentatus Front Royal, VA, USA Oak 2016-XI-24 9 Washington, D.C., USA Oak 2016-IX-26 5

Table 5-2. Cumulative frequency of fungal isolation from four platypodine beetles Euplatypus Euplatypus Oxoplatypus Myoplatypus compositus parallelus quadridentatus flavicornis GA, FL, FL, D.C., FL, VA, FL, USA USA USA USA USA USA USA (1) (10) (5) (5) (5) (9) (13) Ophiostomatales Raffaelea Raffaelea cf. campbellii1 1 4c Raffaelea cf. campbellii2 5 Raffaelea cyclorhipidia 2 1 2 Raffaelea fusca 2 Raffaelea sp.1 3 Raffaelea sp.2 3b Raffaelea sp.3 1 Raffaelea sp.4 1 Raffaelea sp.5 8c Raffaelea sp.6 1 4c Raffaelea sp.7 5 Raffaelea sp.8 1 Esteya Esteya vermicola 1 Esteya sp. 1 Leptographium Leptographium sp. 1a Ophiostoma Ophiostoma cf. quercus 3a Ophiostoma sp. 5a Ceratocystiopsis Ceratocystiopsis sp.1 5a Ceratocystiopsis sp.2 3a 2 1 Hypocreales Fusarium spp. 2a 7a Saccharomycetales Candida spp./Pichia 1 6b 5 3 5 5 8b spp./Ambrosiozyma spp. a indicate this fungus appeared only from the beetle’s surface and gallery. b and c indicate this fungus appeared in various collections from the same location (b two collections; c three collections.

Table 5-3. Colony forming units range and frequency (%) of fungal isolation* Euplatypus Euplatypus Oxoplatypus Myoplatypus

compositus parallelus quadridentatus flavicornis Ophiostomatales Raffaelea cf. campbellii1 45(20-4000) Raffaelea sp.6 45(80-3000) Raffaelea sp.2 27(100) Ceratocystiopsis sp.2 27(100) 40(100-1000) 5(20) Raffaelea sp.3 9(100) Raffaelea sp.7 100(200-3000) Ceratocystiopsis sp.1 26(90-1500) Raffaelea cf. campbellii2 26(19-190) Raffaelea cyclorhipidia 26(20-380) Ophiostoma cf. quercus 16(100) Raffaelea sp.4 5(80) Raffaelea sp.8 5 Esteya vermicola 5(10-40) Leptographium sp. 5 Raffaelea sp.5 62(130) Ophiostoma sp. 38 Raffaelea sp.1 23(10-30) Raffaelea fusca 15(30-100) Esteya sp. 8(200) Hypocreales Fusarium spp. 40 54 Saccharomycetales Candida spp./Pichia 64(40-7000) 100(100-3000) 53(95-4700) 62(40-1400) spp./Ambrosiozyma spp. * Note: numbers in the bracket indicate CFU.

Figure 5-1. Best ML tree from RAxML analysis of 28S. Values at nodes represent ML bootstrap percentages ≥70% from a summary of 500 replicates, and branches in bold represent BPP ≥95%. Photo courtesy of author.

CHAPTER 6 DEVELOPMENT OF MYCANGIUM IN XYLOSANDRUS AMBROSIA BEETLES

Introduction to Chapter 6

Xylosandrus is an ambrosia beetle genus of about 40 species within a large group of ambrosia beetles, the Xyleborini (Dole et al., 2010; Wood and Bright, 1992).

Within Xyleborini, Xylosandrus and four other genera (Anisandrus, Cnestus,

Eccoptopterus, and Hadrodemius) together represent a large monophyletic clade characterized by the mesothoracic mycangium (Cognato et al., 2011a; Dole et al., 2010) and by highly specific association with Ambrosiella ambrosia fungi (Ascomycota:

Microascales) (Harrington et al., 2014; Lin et al., 2017; Mayers et al., 2015).

Xylosandrus is a globally distributed genus of which many species are invasive, ecologically dominant and economically important, and increasingly popular as a model genus for studies of the ambrosia symbiosis (Bateman et al., 2015; Harrington et al.,

2014; Kostovcik et al., 2015; Mayers et al., 2015; Ranger et al., 2015). Reasons for the interest include the economic impact and high abundance of some species, but also the fact that the mycangium is large and can be easily sampled for fungi. The mycangium plays a crucial role in the protection, transmission, and proliferation of the symbiotic fungi and thus in the host beetle ecology (Batra, 1985; Francke-Grosmann, 1966;

Kajimura and Hijii, 1992). In contrast with much research of the dynamics of symbiotic fungi in mycangia and gallery, morphological variation of mycangia and relationship between fungi and morphogenesis of mycangia was less systematic documented or observed (Bateman et al., 2016; Biedermann et al., 2013; Francke-Grosmann, 1956;

Kajimura and Hijii, 1992; Kinuura et al., 1991; Yang et al., 2008). In some symbiotic associations between animals and microorganisms, the symbiont-induced organ

development was found, such as Squid-Vibrio and Butterfly-Wolbachia (Narita et al.,

2007; Nyholm and McFall-Ngai, 2004). During my studies, it has become apparent that the mycangium is a dynamic organ with dramatic and variable morphological changes.

To aid in the understanding of the organ that is critical for one of the most widespread symbioses, I studied the development of the mycangium in five widely distributed

Xylosandrus species and tested whether symbiotic fungi trigger the mycangial growth.

Materials and Methods to Chapter 6

Experiment 1: Morphological Variation of Mycangia Test

My sampling included five species of ambrosia beetle species with a large, mesonotal mycangium, a large internal organ with an opening between the pronotum and the mesonotum (Mayers et al., 2015) (Francke-Grosmann, 1967), Xylosandrus amputatus (Blandford), X. compactus (Eichhoff), X. crassiusculus (Motschulsky), X. discolor (Blandford) and X. germanus (Blandford). These five Xylosandrus species are all native to Asia, and four of them were introduced to the continental United States

(Anderson, 1974; Cognato et al., 2011b; Weber and MacPherson, 1982) and elsewhere. Several species have become important nursery and lumber pests (Ploetz et al., 2013; Ranger et al., 2015), and have come to dominate the ambrosia beetle community in the Eastern US forests. Each species was represented by 21 to 61 individuals, either from China or USA (Table 6-1). Beetles used for hand dissection or micro-CT scan were preserved in 95% to 100% ethanol and stored at −80°C.

All available beetles were sorted into three ontogenetic stages (Table 6-1): 1)

Callow, teneral adults inside the parental gallery, typically with a light-colored exoskeleton. This category was available only for X. compactus and X. crassiusculus; 2)

Dispersing adults: mature adults are leaving the parental gallery and searching for the

new substrate to establish their own new galleries; 3) The foundresses: adult females are boring into a new branch and creating a gallery which contained eggs or larvae.

Males were not sampled since in the haplo-diploid inbred Xyleborini, males do not fly and probably do not participate in fungus transmission.

Experiment 2: Mycangial Development Test

Live X. compactus and X. crassiusculus in the mycangial development test were separately breed from twigs of beauty berry (Callicarpa americana) and xylem of sweetgum (Liquidambar styraciflua) in Gainesville, FL, USA at September 2017.

The fungus–free adults, were acquired following an improved disinfestation method of Batra (1985) and Francke-Grosmann (1967). The larva in the late prepupal stage was taken from its natural galleries in the wood to a petri dish. As soon as larvae become pupae by peeling the skin, they were transported to a new petri dish. When pupae became young callow adults with a light-colored exoskeleton and exuviated skin still sticks to the abdomen, they were carried to a new sterile petri dishes again. All transportations are suggested to get maximum sterile of pupae and adults in a biosafety cabinet. All plates contained prepupa and pupa were aseptic and kept moist by clean wet tissue.

At least five live young callow adults of each species were used to check mycangial development by hand dissection. Mycangial development condition of mature adults, with a dark-colored exoskeleton, were also inspected by dissection after mature in clean tissue or autoclaved artificial galleries. Both mature conditions were represented by at least five individuals. All mycangia checked were also isolated on the

Potato Dextrose Agar (PDA) media followed protocols of Li et al. (2017) to test whether fungi exist. Artificial galleries were made of twigs of beautyberry and xylem of sweetgum

which bored by an electric drill. Galleries were autoclaved for one hour before inoculated with callow adults.

Hand Dissection of Xylosandrus

Inspection by dissecting mostly followed protocols of Bateman et al. (2016). For the assessment of presence or absence of fungal mass in mycangia and mycangial development, beetles were dissected manually under a dissection microscope using

30X magnification. First, abdomen and elytra were affixed by minute pins 000 insect pins to a paraffin platform. Then, the head and pronotum were held by fine forceps. The scutellum was grasped and pulled slowly using fine forceps until the membranous tissue or the mycangium between the scutellum and pronotum was exposed. In case of live beetle in mycangial development test, eversion of mycangia was also inspected by pushing the head with pin or forceps.

Micro-CT Imaging

Xylosandrus amputatus and X. crassiusculus were stored in ethanol (95-100%) for dehydration. Before scanning, they were dried at critical point (Hitachi hcp-2, Hitachi

Inc., Tokyo, Japan). X. amputatus were scanned with MicroXCT-400 (Xradia Inc.,

California, USA; beam strength: 60 kV, absorption contrast). X. crassiusculus were scanned with a Phoenix v|tome|x M (GE's Measurement & Control business, Boston,

USA) at the University of Florida’s Nanoscale Research Facility, using a 180kv x-ray tube with a diamond-tungsten target, at 90 kV, 400 mA, and with a one second detector time, averaging of 3 images per rotation and a resulting voxel resolution of 2.01µm. Raw x-ray data were processed using GE’s proprietary datos|x software v 2.3 to produce a series of tomogram images. These Micro-CT image stacks were then viewed, sectioned, measured and analyzed using Amira 5.4.1 (Visage Imaging, San Diego,

USA) and VG StudioMax 2.2 (Volume Graphics, Heidelberg, Germany). Final figures were prepared with Photoshop and Illustrator (CS5, Adobe).

Identification of Fungi from Mycangia

All fungi acquired from isolation were identified following the protocols of Li et al.

(2017). Sequences of the nuclear internal transcribed spacers ITS1-5.8S-ITS2 (ITS) was amplified with primer ITS1F/ITS4 (White et al., 1990). I do not further classify fungi to species, so sequences were only identified by using Basic Local Alignment Search

Tool (BLAST).

Results to Chapter 6

Morphological Variation of Xylosandrus Mycangia

During dissection and micro-CT scan, three different morphology of mesothoracic mycangia with varying amount of fungi in all five species of Xylosandrus were found: 1) deflated mycangium without fungal mass [Figure 6-1, A-D; Figure 6-2, B-D; Figure 6-3,

A]; 2) not full-inflated mycangia with small fungal mass [Figure 6-3, B] or an asymmetrical mycangium on just one side [Figure 6-1, C]; 3) inflated mycangia full of fungal mass [Figure 6-2, A-C; Figure 6-3, C].

The mesothoracic mycangium in all five species of Xylosandrus undergoes a uniform sequence of developmental stages (Table 6-2): The young callow adults do not have fungal mass in mycangia. Mycangia were undetectable by micro-CT scan, but there is a simple membrane connecting the pronotum and mesonotum could be only observed by dissection. (Figure 6-1, A; Figure 6-3, A). In almost all mature dispersers, the pro-mesonotal membrane is deeply invaginated into a paired, semi-spiral pouch. It is typically filled with the fungal inoculum (Figure 6-3, C), but not always. All dissected dispersing of X. compactus, X. discolor and X. germanus possessed fully developed

mycangia (Figure 6-2, A-C). In one X. amputatus and all X. crassiusculus dispersers, the mycangia are variable in size and symmetry. Some dispersers have an only small piece of fungal mass in mycangia (Figure 6-3, B), or just one of the mycangial pouches is formed. (Figure 6-1, B-C). After a foundress establishes her new gallery, the fungal inoculum appears to get discharged, and the invaginated mycangium reverts to a simple flat membrane. All dissected X. amputatus and X. discolor foundresses lost their inflated mycangia (Figure 6-2, B-D). In X. compactus, X. crassiusculus and X. germanus, some foundresses retained very small or asymmetrical mycangia.

Mycangial Development and Fungal Identification

All live adults of X. compactus and X. crassiusculus could evert their mycangia, and none contains fungal mass. Seven isolations of Xylosandrus mycangia presented same morphological fungal colony; the rest are sterile on the culture. No fungal mass visible during dissection. The young callow adults own mycangial structure which is only available to observe when eversion. The mature adults breed from both tissue, and artificial gallery are also owning empty mycangia. All fungus were morphologically similar to Penicillium fungi. About 1-20 CFUs of it were isolated from mycangia of each.

ITS rDNA sequences of all culture were 99% identical to Penicillium verruculosum

(GenBank accessions HM469420).

Discussions to Chapter 6

Most published reports on the mycangial morphology of Xylosandrus focused on the disperser stage: X. crassiusculus (Harrington et al., 2014; Kinuura, 1995; Mayers et al., 2015), X. discolor (Schedl, 1962) and X. germanus (Francke-Grosmann, 1967;

Mayers et al., 2015; Takagi and Kaneko, 1965). Therefore, most descriptions are of a full, well-formed mycangium, while its developmental plasticity has been

underestimated. It had been observed the mycangia contained asymmetric fungal mass and varied after winter (Francke-Grosmann, 1956, 1958, 1966). Casually, few morphological variation of mycangia were also seen on the other Xyleboines beetle, such as Xyleborus affinis and Cnestus mutilatus (Kajimura and Hijii, 1992; Schneider,

1987). Even though scholars agree ambrosia beetle got symbiotic fungi from their parental gallery and colonized in the new gallery for a long time (Batra, 1985; Francke-

Grosmann, 1966; Nakashima, 1982; Schedl, 1962; Six, 2003a), my result reinforce it with evidence of mycangial morphology.

The majority of the samples conformed to the inflation-deflation scenario well.

However there was also a significant number of non-conforming cases. The variability of the mycangial structure of Xylosandrus crassiusculus seen in the micro-CT scans

(Figure 6-1, B-C) was also corroborated by numerous hand dissections, which showed that X. crassiusculus often has mycangia that are smaller than their full possible extent, or asymmetrical. However, these results may also have been caused by three additional factors. First, the specimen drying process may shrink or collapse the membranous mycangia if they are not filled with a fungal mass. Second, both mycangia and fungal inoculum collapsed while drying. Third, it is possible that the morphological changes are gradual, not abrupt, and that I sampled across different degrees of the change. When dissecting a dead beetle, it is difficult to distinguish a flat mesothoracic membrane from an invaginated but completely empty membranous mycangium (Figure 6-3, C). Due to tissue lose elasticity right after dead, mycangial eversion was not visible on specimens from alcohol immersion. Therefore, all mycangia of live individuals were evaginated to confirm the development.

Xylosandrus is one of five genera in the large xyleborine clade characterized by the synapomorphic mesothoracic mycangium (Cognato et al., 2011a; Dole et al., 2010).

Morphological variation of mycangia in other species in this clade has not been assessed as systematically as for the five species included here, but I have observed signs of the same mycangial development in the following species: Xylosandrus morigerus and Diuncus haberkorni. In D. haberkorni, this observation seems to invalidate the earlier suggestion that the entire genus Diuncus has lost its mycangia as a result of its fungus-stealing habit (Hulcr and Cognato, 2010). While the fungus- stealing habits are undisputed, the mycangium has not been lost. Instead, it is present only in the dispersal stage, while Hulcr and Cognato (2010) only examined foundresses extracted from galleries. Likewise, the foundresses of X. crassiusculus and X. germanus were from early-stage galleries, and many still had small size mycangia that may eventually complete the deflation. The mechanism of the fungus inoculation into the new gallery is still unknown. Whether it is a sudden discharge or a gradual release process could be visualized in the future by a time-series of micro-CT scans.

I documented the main morphological changes of the mycangium, but not attempt to explore the changes in the abundance of the fungi or the fungus community composition at a different stage of adult. The mycangium likely has more functions than just serving as a mechanical receptacle, and probably maintains an environment in which the primary mutualist proliferates at the expense of non-mutualistic fungi

(Kinuura, 1995; Kostovcik et al., 2015; Six, 2003). Documenting the complete dynamics of the organ will be important in the understanding of not only of the basic ecology of the

insect but also of the transmission of plant pathogens and the phenology of the attacks on economically important plants. (Batra, 1963, 1967; Beaver, 1989; Six, 2003a).

After notice of mycangium swollen by fungal mass, the second test determined whether the invagination is triggered by the presence of the fungus. In the young callow beetle, the mycangium may form slowly, and slowly get filled and stretched by the fungal inoculum as previously suggested in genera Dendroctonus and Xylosandrus

(Barras, 1973; Kinuura, 1995; Mayers et al., 2015). However, all live young callow adults were all available to evaginate their mycangia in my test (Figure 6-3, D-G) indicated that mycangia had been developed when eclosion.

All five Xylosandrus beetle are tightly associated with ambrosia fungi Ambrosiella which is absent in isolation from mycangia of callow adults in this study. The sterile treatments to acquire young callow and mature adults, avoided the involvement of symbiotic fungi from gallery and skin as much as possible. Even though few Penicillium fungi appeared on the isolation from X. crassiusculus (Table 3), I do not suggest mycangia is undergoing symbiont-induced developmental changes due to Penicillium is common opportunistic fungi in fungal culture and not presented in X. compactus test.

The vast majority of isolation are sterile and new young callow adults are born with the structure of empty mycangium are adequate evidence to exclude the interference factor of fungus. However, it is still uncertainty of fungus may have some effect on the ultimate size or function of mycangia after beetle mature, because I noted the eversion of mycangia of callow adults are still smaller than mycangia of adults in dispersal stage

(Figure 6-2, A; Figure 6-3, G).

The micro-structure of the mycangium of this clade of beetles was also documented by Stone et al. (2007) in Cnestus mutilatus. The work demonstrated the complexity of the organ, such as the presence of secretion glands and its membranous basis. Ontogenetic changes have not been assessed, but the mycangium is almost certainly homologous since Cnestus and Xylosandrus are very closely related (Dole et al., 2010) and both tightly associated with fungi Ambrosiella (Lin et al., 2017; Mayers et al., 2015). The dynamics of fungi in mycangia of C. mutilatus had also been studied by

Kajimura and Hijii (1992). Even though morphological variation and development of mycangia have not been directly described, fungi Ambrosiella were observed and isolated from mycangia of callow adults emerged from aseptic conditions and natural galleries (Kajimura and Hijii, 1992). Whereas no Ambrosiella was isolated from callow adults in this test. A similar phenomenon has also been observed by Yang et al. (2008) from callow adults of Xylosandrus germanus. It is potential contributed by more restricted sterile sterilization.

Other clades of ambrosia beetles and fungus-feeding bark beetles

(Curculionidae: Scolytinae) have evolved different types of mycangia (Bateman et al.,

2017; Francke-Grosmann, 1956, 1967; Hulcr and Stelinski, 2017; Schedl, 1962). In some cases, the mycangia are rigid chitinous structures and not flexible in which developmental plasticity is unlikely (such as Dendroctonus spp., Platypus spp. and

Pityoborus spp. )(Furniss et al., 1987; Nakashima, 1975; Six, 2003b; Yuceer et al.,

2011). However, in many other clades, mycangia are membranous and their ontogeny is currently being examined (Hulcr and Stelinski, 2017). In contrast with Platypodines

(Curculionidae: Platypodinae), which is close related to scolytids in both ecological

niche and phylogeny, the development of the mycangial structure of Xylosandrus was brought forward. Mycangia of Crossotarsus niponicus were not detectable until adults matured (Nakashima, 1979).

There is no evidence for symbiotic fungal triggers of these developmental events of mycangia in Xylosandrus. This phenomenon of symbiont-induced tissue modification in squid has not been observed (Nyholm and McFall-Ngai, 2004). Wherefore, one of the major questions remains: what triggers the changes in the mycangial morphology? The sampling employed in this work did not allow us to distinguish whether it is a deterministic ontogenesis or an endosymbiotic trigger, it appears that all factors are probably at play and their importance will need to be identified with an experimental approach.

Table 6-1. Collection information for Xylosandrus beetles in morphological variation of mycangia test. Beetle Locality Capture type Number of beetles Xylosandrus Guiyang, Guizhou, China disperser 4 amputatus Fuzhou, Fujian, China disperser 12 Gainesville, FL, USA disperser 2 Guiyang, Guizhou, China foundress 5 X. compactus Gainesville, FL, USA callow, disperser and 51 foundress X. crassiusculus Guiyang, Guizhou, China disperser 13 Fuzhou, Fujian, China disperser 10 Branford, FL, USA callow and foundress 4 Gainesville, FL, USA callow and foundress 13 Hosford, FL, USA disperser 6 Miami, FL, USA disperser 15 X. discolor Guiyang, Guizhou, China disperser 3 Fuzhou, Fujian, China disperser 8 Fuzhou, Fujian, China foundress 10 X. germanus East lake, NC, USA disperser 15 State College, PA, USA foundress 10 Total 181

Table 6-2. Xylosandrus beetles in morphological variation of mycangia test. Proportions of beetle with different mycangial shapesa Beetle Callow Disperser Foundress N S I N S I N S I Xylosandrus amputatus N/A N/A N/A 1 0 17 5 0 0 X. compactus 0 0 13 0 0 32 2 4 0 X. crassiusculus 0 0 7 0 39 5 4 6 0 X. discolor N/A N/A N/A 0 0 11 10 0 0 X. germanus N/A N/A N/A 0 0 15 3 7 0 a Letters in brackets mean different shapes- N: not inflated mycangia; I: inflated mycangia full of fungal mass; S: not full-inflated mycangia with small fungal mass or an asymmetrical mycangium on just one side.

Table 6-3. Mycangia and fungal mass of callow and new mature beetles in Xylosandrus compactus and X. crassiusculus Beetle Mature Fungal mass Mycangium Fungi isolate Adult stage a species condition in mycangium eversion from mycangia Xylosandrus Young callow (5) N/A No Yes No compactus Mature (5) Clean tissue No Yes No Mature (5) Artificial gallery No Yes No X. Young callow (8) N/A No Yes Yes (2) b crassiusculus Mature (6) Clean tissue No Yes Yes (2) b Mature (8) Artificial gallery No Yes Yes (3) b a The number in brackets were represented individuals in each test. b 2 or 3 isolation of mycangial isolation from X. crassiusculus appear fungi Penicillium sp..

Figure 6-1. Xylosandrus crassiusculus mycangia in various developmental stages. A, B, C, D: Single micro-CT sections through the mesonotal region. The small beetle lateral profiles are digitally reconstructed cross-resections for illustration only, not actual photographs. Photo courtesy of author.

Figure 6-2. Reconstruction of micro-CT scans of ambrosia beetle Xylosandrus amputatus in different developmental stages (dissected pronotum with head scanned by MicroXCT-400, stacked images generated by Amira). A, B: Internal view of the mesothorax, longitudinal profile. C, D: Ventral view of the thorax and head; Scale bars = 0.5 mm. Photo courtesy of author.

Figure 6-3. Mycangia with/without fungal mass and eversion of empty mycangia. A, B, C, F, G. Xylosandrus crassiusculus. D, E. Xylosandrus compactus. A. empty mycangium. B. mycangium with small fungal mass. C. mycangia filled with fungal mass. D, F. eversion of empty mycangia, lateral. E, G. eversion of empty mycangia, dorsal. Red arrows, empty mycangia. White arrows, scutellum. Black arrows, fungal mass. Blue arrows, everted mycangia. Photo courtesy of author.

CHAPTER 7 PATHOGENICITY EVALUATION OF CHINESE BARK AND AMBROSIA BEETLE- VECTORED FUNGI IN USA

Introduction to Chapter 7

Range expansions and invasions of bark and ambrosia beetle (Coleoptera:

Curculionidae: Scolytinae and Platypodinae) have been facilitated by increased human global movement of infested wood materials (Aukema et al., 2010; Brockerhoff et al.,

2006). Although only a few non-native species could successfully colonize in new host and environment, several severe damages in landscape and economic by those special cases already been found worldwide (Batra, 1985; Beaver, 2013; Cognato et al., 2005;

Harrington et al., 2011). These destructive beetle often associated with fungal symbionts that attack species phylogenetically similar to their historical hosts (Aukema et al., 2011; Bertheau et al., 2010; Hulcr et al., 2017; Hulcr and Lou, 2013; Li et al.,

2016). The bark and ambrosia beetle with associated fungi usually cause minor damage in their original range. However, due to absence of resistance to the pest in the novel host, the damages can be especially unexpected.

A tight symbiosis occurs between bark and ambrosia beetle and their fungal community. Symbiotic fungi not only function as the nutritional resource but also assist beetle breakdown the defense systems of the host plant (DiGuistini et al., 2011;

Hammerbacher et al., 2013; Hulcr and Stelinski, 2017). Moreover, some mutualistic fungi are plant pathogen which could cause more severe damage than beetle even kill the plant in the special condition. Well-known examples include the introduction of

Dutch elm disease (Ophiostoma ulmi and Ophiostoma novo-ulmi) epidemic by bark beetle Scolytus multistriatus and Hylurgopinus rufipes (Brasier, 1991; Brasier, 2001), and Laurel wilt (Raffaelea lauricola) along with its invasive exotic vector beetle, the red

bay ambrosia beetle Xyleborus glabratus (Fraedrich et al., 2008). Since Xyleborus glabratus arrive the USA at 2002, led to over 90% tree mortality of redbay trees in about

100 counties of the Southeast coastal plains (Spiegel and Leege, 2013). Moreover, one species of beetle vector always carry more than one species fungal symbionts

(Jankowiak et al., 2017; Ploetz et al., 2017). It sharply increases the risk of invasive beetle. These damaging beetle-fungus complexes are so pervasive that, in some cases, the future of several tree species lies in jeopardy.

Over two dozen bark and ambrosia beetle species have already become established in various forested regions of the US (Haack, 2001). As new exotic beetles species continue to enter the US, an important question is whether their associated fungi are pathogenic to native tree species (Haack, 2001). All of the most detrimental ambrosia beetles and fungi are from non-native regions (Hulcr and Dunn, 2011). It is difficult for regulatory agencies to make an evidence-based decision to distinguish which species can be ignored if introduced, which require close monitoring and further research, and which species need eradication if introduced. To address this question, the Hulcr lab developed a safe and feasible approach to assessing the threat of fungi associated with exotic ambrosia beetles before their establishment in the US. I used the approach to test the potential plant pathogens with bark and ambrosia beetle from

China.

In this study, ambrosia beetles were collected from China mainland and Taiwan island where their symbiotic fungi were isolated. China mainland and Taiwan island are located in Southeast Asia where the weather and latitude are similar to the southeastern

USA. Two most known severe invasive ambrosia beetle and their fungal partner were

introduced from Asia (Hulcr and Lou, 2013; Stouthamer et al., 2017). Scolytine beetle owns a high diversity in Southeast Asia, and most of their fungal symbionts are poorly understood. In most cases, the identity of the fungal symbionts is not known (Six,

2003a), let alone symbiont diversity in ambrosia beetles and pathogenicity in the host plant. The fungi isolated from Chinese bark and ambrosia beetle will be utilized to pathogenicity test.

Materials and Methods to Chapter 7

Target Beetle Vector and Tested Object

For the present study, I selected fungal species that likely associated with bark and ambrosia beetle which usually infest on pine and oak. Because two species of pine are [loblolly pine (Pinus taeda) and slash pine (Pinus elliottii)] and oak [live oak

(Quercus virginiana) and shumard oak (Quercus shumardii)] which are widely distributed the southeastern USA (Barnett and Sheffield, 2005; Edwards, 1990; Harms,

1990; Huggett et al., 2013; McKeand et al., 2003; Wear and Greis, 2012). In the southeastern region of the United States, loblolly and slash pines have been planted on more than 10 million and 4.2 million ha, respectively (Barnett and Sheffield, 2005;

Huggett et al., 2013; Wear and Greis, 2012). The area of two oak tree species is unknown, but they are common in both rural and urban areas (Edwards, 1990; Harms,

1990). These species are not previously exported, frequently to the origin of symbiotic beetle. Non-native beetle also prefers the similar plant in its host range when it was first introduced to the new area.

Collection of Chinese Bark and Ambrosia Beetles

During five long-term collecting trips in China mainland (CHN) from 2013 to 2017 and two in Taiwan island (TPE) at 2015 (Table 7-1), I collected bark beetles and their

galleries from wood. When access bark and ambrosia beetle from wood, both beetle and gallery were utilized in isolation to confirm the primary fungi and secondary fungi.

When beetle acquired from traps, only the species whose host range are primary on pine or oak were selected.

Fungal Isolation and Identification

Isolate method of symbiotic fungi refers to Li et al. (2015) and Bateman et al.

(2015). I isolated fungi by three processes depends on what species of bark and ambrosia beetle. 1) For bark beetle, only surface wash and woodchips from the gallery were isolated. 2) For ambrosia beetle species that were clearly known to have a mycangium, the mycangial prat was isolated with serial diluted to 1/10, 1/100, and

1/1000 dilutions. 3) For beetle species that were unknown to have a mycangium, the fungi from the beetle surface and head were isolated with serial diluted. All isolation were plated on standard PDA media.

Detection of fungi by the molecular method is always after inoculating the subculture fungi on the new plate for 1 or 2 weeks. The subculture should be without any contamination. 10 µL of mycelium was added to 20 µL of Sigma-Aldrich Extraction

Solution in 0.25 mL PCR tubes, which were then incubated in a thermocycler at 96°C for 20 min. After incubation, 20 µL of 3% BSA was added to each tube, shake thoroughly and then centrifuged at 2000 rpms for 30s. The upper half of this solution was used as genomic DNA template for PCR. Basic PCR reactions used in sequencing consisted of a final volume of 25 µL: 1 µL of template DNA, 1 µL of LSU forward and reverse primer (LROR and LR5) (Vilgalys and Hester, 1990), 1 µl of DMSO, 12.5 µL

Premix TaqTM (Ex TaqTM Version2.0; Takara Bio Inc.), and 9.5 µL sterile water. PCR products were purified using Exosap-IT, following the manufacturer’s protocols. PCR

reactions were performed was as follows: initial denaturation at 95°C for 4 min, followed by 34 cycles at 94°C for 30s, 55°C for 40s, and 72°C for 120s, and a final extension of

72°C for 5 min. For some fungus that not match LROR and LR5, I also attempted to amplify with ITS sequence region with primer (ITS1F and ITS4) (White et al., 1990).

Sanger sequencing was performed by the Interdisciplinary Center for Biotechnology

Research (ICBR) at University of Florida (Gainesville, FL) or GENEWIZ (South

Plainfield, NJ).

Identification and Selection of Potential Pathogens Fungus

Sequence chromatograms were inspected for quality and assembled in

Geneious (Geneious version 9.1.5). Identification of sequences was made to the lowest possible taxonomic rank via Genbank NCBI BLAST (National Center for Biotechnology

Information; Basic Local Alignment Search Tool). Base on the result with the highest identities, I selected plant pathogen or primary symbiotic fungi for inoculation, such as

Fusarium, Geosmithia, and Ophiostomatoid. Sometimes the selected fungus is both primary symbiotic fungus and potential plant pathogen. In the meantime, the record of host plant range of bark and ambrosia beetles are considered.

Establishment of the Experimental Trees

Pine trees were provided by Weyerhaeuser and Rayonier. Oak trees were provided by Half Moon Growers. All trees were potted and grown in a quarantined greenhouse facility at the Division of Plant Industry (DPI), Department of Agriculture and

Consumer Services in Gainesville, Florida, USA under the USDA/APHIS permit

No.P526P-16-02872. The greenhouse achieved in a biosafety level 2 (P2) facility. All seedling were living in pots. After export to the greenhouse in DPI, they were individually inspected and kept for two weeks to ensure the absence of any native plant

pathogens or insect damage and an additional pesticide treatment was used. The initial height of the pine tree was 1.5–2.0 m with a trunk diameter of 2.0–2.5 cm at 15.0 cm above soil level. The initial height of the pine tree was 1.0–1.5 m with a trunk diameter of 1.5–2.0 cm at 15.0 cm above soil level. The seedlings were maintained under natural light conditions, watered every two days, and kept under a night-day temperature regime averaging at 27°C . No additional treatments (e.g., fertilization or pesticide) were applied. All the seedlings were individually identified with writing a unique number by waterproof marker pen.

Inoculate Potential Pathogens

A single hole was drilled at a downward angle (approximate 45 degrees) into the xylem of each seedling using a 1.98 mm (5/64 inch) drill bit. Holes were made within the basal 15–20 cm of the stem and were up to 4 cm deep. Spore suspensions were pipetted into the xylem in 50 µL aliquots (simulate a bark or ambrosia beetle boring with fungal spores). Number of spores are inferred from the CFU counts of isolationas from each beetle species. If fungus was isolated from the gallery, number of spores would refer to close related beetle or fungal isolation. Wound sites were wrapped in parafilm immediately following inoculation. Spore suspensions were brought back to culture on the PDA media again to check if been contaminated. Native weak pathogen Diplodia quercivora and Ophiostoma ips are chosen as the positive control for oak and pine separately. Clean water was set as a negative control in this experiment.

Due to the restriction of space in the greenhouse and progress of fungal collection, Inoculation was divided into five rounds. The first round was from October to

December 2013. The second round was from May to August 2015. The third round was

from August to October 2015. The fourth round was from August to October 2016.The fifth round was from August to October 2017

Survey of the Pathogenicity

Seedlings were monitored. All signs and symptoms (including resin bleeding, canker, and mortality) in pathogenic development were recorded every five days. Trees were destructively sampled ten weeks after inoculation to quantify fungal infection and host response. Bark was removed from the boring hole of inoculation by carpet knife.

Resin blooding and canker lesions were recorded. Next, lesion (width and length) on the phloem triggered by inoculation were measured by caliper. Then, trees were cut longitudinally, through the point of inoculation, to uncover sapwood area. The discolored xylem (stain width and stain length) were measured by caliper and transparent soft ruler. Finally, small tissue samples surrounding the point of inoculation were surface- sterilized and placed on PDA plates at 25° C incubator again for re-isolation of fungi to confirm the lesion was caused by the inoculated spore suspensions.

Differences in pathogenicity (lesion vertical, lesion horizontal, xylem vertical, and xylem depth) will be tested. Means and standard deviation of each lesion were calculated by R (version 3.1.2). Charts and diagrams were made by Microsoft Office

Excel 365 ProPlus.

Results to Chapter 7

Potential Pathogens Isolated from Chinese Bark and Ambrosia Beetles

In total, 40 species pathogens fungi were isolated from Chinese bark and ambrosia beetles (Table 7-2). 29 fungi were assigned to native pine, and 11 fungi were assigned to native oak based on their collecting data and host range of their beetle vector. 21 fungi were from mainland China and 19 fungi from Taiwan Island. 23 fungi

were isolated from bark-feeding scolytine beetle. 14 fungi were isolated from fungus- feeding scolytine beetle. Three fungi were isolated from the platypodines beetle. Fungi

Sporothrix sp. LL99, Ophiostoma pulvinisporum LL152, and Grosmannia huntii LL206 were isolated from multiple bark beetle species repeatedly, whereas the other fungi were from single beetle species.

Pathogenicity to Local Pine Tree

Assess 29 fungal pathogenicities (and virulence) on pine (Figure 7-1, Tables 7-3,

7-4). 15 fungi Graphilbum sp. CB2013, Ophiostoma microcarpum 10254, Sporothrix nigrograna LL195, Sporothrix sp. LL99, Ceratocystiopsis minuta 10195, Leptographium pini 11414, Leptographium s. l. sp. 7085, Leptographium koreanum 10237, Grosmannia huntii LL206, Ophiostoma ips CB2013, Leptographium sp. LL112, Ophiostoma pulvinisporum LL152, Leptographium sp.10223, Ophiostoma ips LL257, Leptographium s. l. sp. 7083, Diplodia seriata LL151, and Ophiostoma clavatum LL120 were found to cause larger lesions on the phloem than positive controls in loblolly pine. Two fungi

CHN Diplodia seriata LL151 and Ophiostoma ips LL257 were found to cause larger lesions on the phloem than positive controls in slash pine. Only two fungi

Leptographium s. l. sp. 7083 and Leptographium koreanum 10237 were found to cause more xylem discoloration than positive controls in loblolly pine. Three fungi Ophiostoma ips CB2013, Leptographium s. l. sp. 7083 and Ophiostoma ips LL257 were found to cause more xylem discoloration than positive controls in slash pine. Despite larger lesions and more sapwood discoloration after fungal inoculation, no reductions in needle water potentials were observed between treatments. No tree died during the experiments.

Pathogenicity to Local Oak Tree

Assess 11 fungal pathogenicities (and virulence) on oak (Figure 7-2, Table 7-5,

Table 7-6). Only one fungus Raffaelea quercivora 7069 was found to cause larger lesions on the phloem than positive controls both in live oak and shumard oak. No fungi was found to cause more xylem discoloration than positive controls in both oak tree species. No wilt in leaf or twig was observed between treatments. No tree died during the experiments.

Symptoms in Local Pine Trees

All fungal symptom was checked during and after inoculation (Table 7-7). All cause canker or bleeding or both symptoms from the area surrounding the point of inoculation except Sporothrix sp. LL99. The bleeding always breaks the parafilm.

Sixteen fungi cause bleeding in loblolly pine and 24 fungi cause bleeding in slash pine.

Twenty-two fungi cause canker in loblolly pine and 18 fungi cause canker in slash pine.

Symptoms in Local Oak Trees

All fungal symptom was checked during and after inoculation (Table 7-8). All cause canker or bleeding or both symptoms from the area surrounding the point of inoculation except Pseudozyma aphidis 7335. The bleeding did not break the parafilm but visible on the boring hole when inspected. Four fungi cause bleeding in live oak and seven fungi cause bleeding in shumard oak. Three fungi cause canker in live oak and five fungi cause canker in shumard oak.

Discussion to Chapter 7

There are many reported inoculation studies with bark and ambrosia beetle fungi on the plant, but they were usually restricted to few fungi or beetle vectors (Kusumoto et al., 2015; Matusick and Eckhardt, 2010; Musvuugwa et al., 2016). Lesion length or

lesion area following fungal inoculation on phloem and xylem is most often used to assess the pathogenicity to plant (Kajii et al., 2013; Kusumoto et al., 2014; Matusick and

Eckhardt, 2010; Nevill et al., 1995). My test included 40 potential fungal pathogens from bark and ambrosia beetle in China. No pathogenicity tests have been previously performed in the majority of my fungi. Most of bark and ambrosia beetles sampled in isolation have not yet shown any economic or ecological impact in China except the

Dendroctonus valens, Tomicus spp., and Dendroctonus armandi. Dendroctonus valens was introduced from New World to China at the 1990s (Miao et al., 2001). The outbreak infested over 0.5 million hectares of forests of Chinese red pine Pinus tabuliformis and other pines and killed more than six million trees (Li et al., 2001). Leptographium procerum CMW25626 is the primary fungi associated with Dendroctonus valens in

China (Lu et al., 2010). However, Leptographium procerum had not caused bigger lesions than the other fungi in this test. Leptographium sp. LL112 (previously described as Leptographium qinlingensis) was isolated from Dendroctonus armandi which is a serious pest on Chinese white pine Pinus aramdii in Central China (Pu and Chen,

2008). Leptographium sp. LL112 make larger lesion on loblolly pine. The pine shoot beetles of the genus Tomicus are among the more damaging insects in Eurasian pine forests (Kirkendall et al., 2008). Three fungi Leptographium koreanum 10237,

Sporothrix sp. LL99, and Geosmithia sp. CB2013 were isolated from Tomicus spp..

Only Leptographium koreanum 10237 cause longer lesion in loblolly pine. Even though the other beetle vectors had not caused significant damage in their historical distribution and host plant, the high risk will be in the new host by limited resistance and insect rapidly adapt to a new climate (Brasier, 2001). The lesion of Fusarium oxysporum

10271, and Leptographium s. l. sp. 7083 on loblolly pine are approach or exceed positive control, but their beetle vector Xyleborus pincola and Pityophthorus sp. are secondary pest in Asia. Similar circumstances in Ophiostoma ips CB2013, Ophiostoma pulvinisporum LL152, Ophiostoma ips LL257, and Leptographium s. l. sp. 7083 with their beetle Ips chinensis, Cryphalus sp., Euwallacea interjectus, Cocootrypes sp. and

Orthtomicus sp. Although these fungi were not lethal pathogens or carried by non- aggressive beetle, they were able to grow on the new host tree and make considerable lesion as a positive control.

Native oak tree species are more tolerable to the fungal inoculation, the potential pathogen failed to cause lesions larger than positive control. Even though the oak wilt disease pathogen Raffaelea quercivora, they had not reached half of the positive control in the lesion. Raffaelea quercivora has been considered to be a high virulence to several Fagaceae species with unique sapwood discoloration in Asia (Kusumoto et al.,

2014; Kusumoto et al., 2015).

In the present studies, bleeding and canker followed by inoculation were observed. Bleeding is more frequently in pine than oak. Previous work has shown that resistance to fungal infection is associated with the oleoresin production by pine (Owen et al., 1987). Movement of water through wood could be significantly altered by fungal invasion (Croisé et al., 2001; Joseph et al., 1998). Cankers commonly result in the death of branches or entire stems (Chen et al., 2013). However, no symptom of water conduction and dead branches were observed.

Few of those isolated fungi were only pathogenic on a few plant species. Why do some fungal species cause serious damages, while others form the close or same

group are completely harmless? Beetles serve as vectors should play an important role.

Several fungi were considered to be a weak pathogen to plant. But when inoculated via a beetle mass attack, the weak pathogens may exhaust the plant defense mechanism.

For example, Thousand Cankers Disease is caused by Geosmithia morbida and white pine root decline is caused by Leptographium procerum. Generally, Both Geosmithia morbida and Leptographium procerum have been considered to be a weak pathogen

(Ginzel and Juzwik, 2014; Matusick et al., 2012; Wingfield, 1986). With the help from beetle vector, those fungi massacre the walnut (Juglans spp.) in the eastern USA and

Chinese red pine (Pinus tabuliformis) in China separately (Kolarik et al., 2011; Lu et al.,

2010). Therefore, even there is a low probability (about 10%) that exotic species become invasive (Jeschke and Strayer, 2005), never underestimate the weak pathogens by their small lesions in this study. When conduct risk assessment, the selectivity of the host by beetle vectors should be considered, as well as the resistance of different species of native plant. However, overinterpretation of my results is not necessary. My analysis did not include the natural biology of beetles or fungi, which may be dominant factor. Another limitation of this analysis is that it relies on several assumptions, including that same disease resistance by different pine and oak varieties and no impacts from climate. The species composition of the pine forest of the has changed over the years. Before the ranges for both loblolly pine and slash pine have been expanded through the extensive planting of seedlings from tree improvement programs in the southeastern USA, longleaf pine (Pinus palustris) are predominant

(McKeand et al., 2003).

The direct threat of those beetle and fungi from China will be minor, due to log export is not available in China. China is the world's largest timber importer and second largest consumer (Yang and Zhai, 2012). Wood furniture and plywood are making up the majority of woodware export in China (Zhu, 2016). Northern China now faces a shortage of mature and usable forests. Consequently, in order to start a new phase wherein China protects natural forests, China had banned all commercial logging in natural forests in key forest zones in 2015 and will completely stop commercial logging in forests in 2017 (State Forestry Administration, 2015). However, most of fungi and beetle vector in this research are not endemic in China. They are widely distributing in

Southeast Asia. This pathogenicity evaluation can be used by policymakers to assess whether to invest in proactive measures that reduce the rate of arrival of Asian bark and ambrosia beetle (Rabaglia et al., 2008) and develop management of control in advance, like trade and phytosanitary policy, early detection and rapid response (EDRR).

Table 7-1. Collection of the potential plant pathogen Date Location Target host plant and plantation Jun 2013 Kunming, Yunnan, China Pine (Pinus yunnanensis, P. kesiya) Xishuangbanna, Yunnan, Pine (P. yunnanensis, P. kesiya) Jun-Aug 2014 China and oak Mar 2015 Nantou, Taiwan (China) Pine (P. taiwanensis) and oak Guiyang and Weining, Sep-Oct 2015 Pine (P. yunnanensis, P. armandii) China Oct 2015 Nantou, Taiwan (China) Pine (P. taiwanensis) and oak Oct 2015 Ninshan, Shaanxi, China Pine (P. armandii) Oct-Nov 2015 Fuzhou, Fujian, China Pine (P. massoniana) Nov 2016 Quanzhou, Fujian, China Pine (P. massoniana) May 2017 Putian, Fujian, China Pine (P. massoniana)

Table 7-2. Potential pathogens isolated from bark and ambrosia beetles from China Native No. Isolated fungus Beetle vector Location CFU host 1 Graphilbum sp. CB2013 Ips chinensis Yunnan Pine 2 Ophiostoma ips CB2013 Ips chinensis Yunnan Pine 3 Geosmithia sp. CB2013 Tomicus minor Yunnan Pine 4 Ascomycete sp. 7694 Cyrtogenius sp. Yunnan Pine 10000 5 Ceratocystiopsis sp. 7744 Polygraphus sp. Yunnan Pine 12000 6 Geosmithia pallida 7686 Webbia pabo Yunnan Oak 22000 7 Leptographium procerum Dendroctonus valens Yunnan Pine 7000 CMW25626 8 Ophiostoma sp. 7690 Cyrtogenius sp. Yunnan Pine 10000 9 Ophiostoma sp. 7712 Webbia pabo Yunnan Pine 11500 10 Ophiostoma sp. 7736 Cyclorhipidion aff. fukiense Yunnan Oak 10000 11 Ophiostoma clavatum LL120 Dendroctonus armandi Shaanxi Pine 4000 12 Leptographium sp. LL112 Dendroctonus armandi Shaanxi Pine 5000 13 Ophiostoma cf. abietinum LL98 Hylurgops longipillus Guizhou Pine 5000 14 Sporothrix sp. LL99 Hylurgops longipillus, Guizhou Pine 5000 Polygraphus sp.,Tomicus minor 15 Raffaelea rapaneae LL134 Xyleborus pinicola Guizhou, Fujian Pine 7000 16 Diplodia seriata LL151 Cryphalus sp. Fujian Pine 4000 17 Ophiostoma pulvinisporum LL152 Cryphalus sp., Fujian Pine 4000 Euwallacea interjectus, Cyrtogenius luteus, and Cocootrypes sp. 18 Grosmannia huntii LL206 The same as above Fujian Pine 4000 19 Sporothrix nigrograna LL195 Euwallacea interjectus Fujian Pine 4000 20 Raffaelea subfusca LL138 Euwallacea validus Fujian Pine 1500 21 Ophiostoma ips LL257 Orthotomicus sp. Fujian Pine 5000 22 Geosmithia sp. 7333 Hypothenemus birmanus Taiwan Oak 900 23 Raffaelea quercivora 7047 Cyclorhipidion ohnoi Taiwan Oak 10000 24 Raffaelea quercivora 7069 Crossotarsus emancipatus Taiwan Oak 6000 25 Leptographium s.l. sp.7083 Polygraphus sp. Taiwan Pine 200 26 Leptographium s.l. sp. 7085 Xyleborus pinicola Taiwan Pine 500 27 Ophiostoma sp. 7080 Crossotarsus emancipatus Taiwan Oak 5000 28 Raffaelea crossotarsa 7081 Crossotarsus emancipatus Taiwan Oak 990 29 Pichia sp. 7018 Cryphalus fulvus Taiwan Pine 2600 30 Pseudozyma aphidis 7335 Urocorthylus fanii Taiwan Oak 5000 31 Raffaelea cyclorhipidia 7049 Cyclorhipidion ohnoi Taiwan Oak 12000 32 Sporothrix sp. 7335 Urocorthylus fanii Taiwan Oak 200 33 Ceratocystiopsis minuta 10195 Cryphalus sp. Taiwan Pine 4000 34 Ophiostoma microcarpum 10254 Cryphalus sp. Taiwan Pine 5000 35 Mariannaea elegans10179 Hylastes sp. Taiwan Pine 5000 36 Fusarium oxysporum 10271 Pityophthorus sp. Taiwan Pine 5000 37 Leptographium koreanum 10237 Tomicus sp. Taiwan Pine 1000 38 Ophiostoma ips 10187 Xyleborus pinicola Taiwan Pine 5000 39 Leptographium sp. 10223 Xyleborus pinicola Taiwan Pine 5000 40 Leptographium pini 11414 Xyleborus pinicola Taiwan Pine 5000

Table 7-3. Symptoms in loblolly pine trees* Phloem Phloem Xylem Xylem Fungal species vertical horizontal vertical horizontal CHN Ascomycete sp. 7694 0.50 0.23 0.40 0.20 0.60 0.19 0.32 0.11 CHN Ceratocystiopsis sp. 7744 0.82 0.13 0.68 0.19 1.12 0.59 0.32 0.29 CHN Diplodia seriata LL151 2.57 0.17 1.13 0.22 2.85 0.19 0.71 0.14 CHN Geosmithia sp. CB2013 0.75 0.18 0.58 0.14 1.28 0.22 0.68 0.48 CHN Graphilbum sp. CB2013 1.09 0.29 0.69 0.33 1.85 0.53 1.21 0.69 CHN Grosmannia huntii LL206 1.57 0.57 0.82 0.11 2.86 0.42 0.48 0.02 CHN Leptographium procerum 0.93 0.06 0.40 0.10 1.63 0.59 0.40 0.00 CMW25626 CHN Leptographium qinlingensis 2.26 1.73 0.49 0.15 4.64 0.84 0.43 0.03 LL112 CHN Ophiostoma sp.7690 0.70 0.07 0.40 0.10 2.10 1.92 0.84 1.21 CHN Ophiostoma cf. abietinum LL98 0.99 0.10 0.67 0.14 1.56 0.49 0.44 0.08 CHN Ophiostoma clavatum LL120 3.48 0.36 0.95 0.50 4.00 1.34 0.49 0.05 CHN Ophiostoma ips CB2013 1.78 0.63 0.80 0.26 3.73 1.52 0.56 0.71 CHN Ophiostoma pulvinisporum 2.26 0.82 0.60 0.26 2.35 0.81 0.56 0.05 LL152 CHN Raffaelea fusca LL138 1.01 0.32 0.63 0.15 1.12 0.49 0.44 0.06 CHN Raffaelea rapaneae LL134 0.97 0.24 0.60 0.13 1.47 0.51 0.38 0.02 CHN Sporothrix nigrograna LL195 1.32 0.37 0.57 0.06 1.76 0.25 0.48 0.05 CHN Sporothrix sp. LL99 1.44 0.42 0.60 0.18 1.65 0.24 0.42 0.03 CHN Ophiostoma ips LL257 2.17 0.11 0.87 0.07 2.84 0.48 0.66 0.17 TPE Ceratocystiopsis minuta 10195 1.44 0.19 0.70 0.22 2.35 0.42 0.37 0.07 TPE Fusarium oxysporum 10271 1.04 0.38 0.47 0.06 5.28 1.24 0.41 0.07 TPE Leptographium koreanum 10237 1.79 1.22 0.54 0.13 5.59 0.71 0.49 0.15 TPE Leptographium s. l. sp. 7083 2.34 1.60 0.81 0.35 5.91 5.79 0.39 0.02 TPE Leptographium s. l. sp. 7085 1.50 0.68 0.69 0.43 3.08 1.95 0.46 0.06 TPE Leptographium sp.10223 2.44 1.38 0.60 0.08 3.70 1.86 0.38 0.02 TPE Mariannaea elegans 10179 1.09 0.18 0.47 0.10 2.46 0.96 0.34 0.07 TPE Ophiostoma ips 10187 1.11 0.36 0.49 0.02 2.70 1.98 0.35 0.05 TPE Ophiostoma microcarpum 10254 1.33 0.65 0.47 0.05 3.85 1.26 0.39 0.03 TPE Pichia sp. 7018 0.92 0.33 0.79 0.43 1.40 0.60 0.37 0.01 TPE Leptographium pin 11414 1.59 0.22 0.60 0.15 2.59 1.22 0.53 0.10 Negative control (water) 0.60 0.35 0.40 0.23 0.88 0.37 0.48 0.45 Positive control (Ophiostoma ips) 1.03 0.68 0.73 0.40 5.36 3.41 0.60 0.37 *Note: Means followed by standard deviation after .

Table 7-4. Symptoms in slash pine trees* Phloem Phloem Xylem Xylem Fungal species vertical horizontal vertical horizontal CHN Ascomycete sp. 7694 0.66 0.09 0.56 0.11 0.74 0.11 0.28 0.15 CHN Ceratocystiopsis sp. 7744 0.52 0.22 0.44 0.11 1.44 1.17 0.24 0.09 CHN Diplodia seriata LL151 1.97 0.38 1.07 0.38 2.33 0.45 0.66 0.34 CHN Geosmithia sp. CB2013 0.79 0.25 0.59 0.33 1.34 0.45 0.69 0.69 CHN Graphilbum sp. CB2013 0.88 0.09 0.71 0.10 2.24 0.78 0.65 0.46 CHN Grosmannia huntii LL206 1.02 0.24 1.09 0.61 1.60 0.13 0.54 0.02 CHN Leptographium procerum 1.10 0.20 0.43 0.06 2.03 0.21 0.37 0.06 CMW25626 CHN Leptographium qinlingensis 1.48 0.52 0.54 0.07 1.76 0.51 0.40 0.03 LL112 CHN Ophiostoma sp.7690 0.64 0.22 0.52 0.19 0.98 0.41 0.30 0.02 CHN Ophiostoma cf. abietinum 1.17 0.80 0.59 0.06 0.93 0.06 0.32 0.03 LL98 CHN Ophiostoma clavatum LL120 1.54 0.75 0.69 0.12 2.96 1.48 0.43 0.14 CHN Ophiostoma ips CB2013 1.55 0.45 0.95 0.31 4.08 1.42 1.35 0.86 CHN Ophiostoma pulvinisporum 2.01 0.98 0.75 0.25 3.76 1.82 0.49 0.08 LL152 CHN Raffaelea fusca LL138 0.73 0.04 0.66 0.14 1.22 0.25 0.33 0.05 CHN Raffaelea rapaneae LL134 0.64 0.01 0.56 0.02 1.18 0.56 0.31 0.04 CHN Sporothrix nigrograna LL195 0.74 0.25 0.68 0.06 1.27 0.33 0.39 0.09 CHN Sporothrix sp. LL99 0.52 0.13 0.45 0.09 1.07 0.20 0.36 0.04 CHN Ophiostoma ips LL257 2.33 0.48 0.84 0.06 4.12 0.22 0.66 0.03 TPE Ceratocystiopsis minuta 0.77 0.05 0.47 0.08 1.19 0.58 0.28 0.04 10195 TPE Fusarium oxysporum 10271 0.73 0.07 0.37 0.03 1.18 0.58 0.34 0.04 TPE Leptographium koreanum 1.55 0.24 0.58 0.02 1.56 0.44 0.40 0.03 10237 TPE Leptographium s. l. sp. 7083 1.66 1.68 0.68 0.13 4.72 3.33 0.52 0.22 TPE Leptographium s. l. sp. 7085 1.02 0.54 0.58 0.11 2.82 0.93 0.34 0.09 TPE Leptographium sp.10223 0.87 0.24 0.56 0.11 1.98 0.43 0.41 0.04 TPE Mariannaea elegans 10179 0.79 0.06 0.55 0.11 1.25 0.49 0.32 0.01 TPE Ophiostoma ips 10187 1.13 0.85 0.46 0.09 1.08 0.16 0.34 0.01 TPE Ophiostoma microcarpum 0.79 0.22 0.63 0.05 1.23 0.42 0.30 0.04 10254 TPE Pichia sp. 7018 1.00 0.46 0.63 0.06 1.13 0.35 0.33 0.06 TPE Leptographium pin 11414 1.07 0.36 0.54 0.07 2.15 0.42 0.40 0.05 Negative control (water) 0.55 0.24 0.47 0.17 0.72 0.23 0.43 0.36 Positive control (Ophiostoma ips) 1.99 2.28 1.06 0.82 3.34 2.43 1.08 1.44 *Note: Means followed by standard deviation after .

Table 7-5. Symptoms in live oak trees* Phloem Phloem Xylem Xylem Fungal species vertical horizontal vertical horizontal CHN Geosmithia pallida 7686 0.62 0.15 0.48 0.04 1.54  0.59 0.30 0.12 CHN Ophiostoma sp. 7712 0.66 0.15 0.54 0.11 1.82  0.75 0.28 0.08 CHN Ophiostoma sp. 7736 0.74 0.38 0.48 0.16 2.80  1.44 0.34 0.05 TPE Geosmithia sp. 7333 0.73 0.06 0.60 0.10 0.73  0.25 0.53 0.15 TPE Grosmannia serpens 0.60 0.20 0.43 0.06 1.10  0.17 0.40 0.10 7047TPE Ophiostoma sp. 7080 0.70 0.10 0.50 0.00 2.77  1.62 0.47 0.06 TPE Pseudozyma aphidis 0.70 0.00 0.50 0.00 1.33  0.70 0.40 0.10 7335TPE Raffaelea crossotarsa 0.70 0.00 0.50 0.10 1.03  0.84 0.37 0.06 7081TPE Raffaelea 1.33 0.71 0.83 0.31 1.10  0.56 0.50 0.00 quercivora7069TPE Raffaelea sp . 7049 0.70 0.10 0.47 0.06 1.20  0.10 0.33 0.06 TPE Sporothrix sp. 7335 0.63 0.21 0.40 0.10 0.67  0.15 0.40 0.10 Negative control (water) 0.63 0.19 0.48 0.12 0.96  0.36 0.33 0.05 Positive control 1.18 0.48 0.52 0.13 6.52  6.00 0.51 0.26 (Diplodia quercivora) *Note: Means followed by standard deviation after .

Table 7-6. Symptoms in shumard oak trees* Phloem Phloem Xylem Xylem Fungal species vertical horizontal vertical horizontal CHN Geosmithia pallida 7686 0.64 0.13 0.56 0.05 2.52 2.43 0.36 0.18 CHN Ophiostoma sp. 7712 0.64 0.42 0.46 0.15 1.50 0.87 0.20 0.12 CHN Ophiostoma sp. 7736 0.96 0.29 0.62 0.15 1.88 0.89 0.30 0.07 TPE Geosmithia sp. 7333 0.70 0.20 0.50 0.00 1.37 0.76 0.37 0.15 TPE Grosmannia serpens 0.83 0.38 0.50 0.17 1.50 0.46 0.33 0.06 7047TPE Ophiostoma sp. 7080 0.80 0.17 0.50 0.10 1.50 0.66 0.43 0.06 TPE Pseudozyma aphidis 0.53 0.25 0.40 0.10 0.87 0.57 0.63 0.58 7335TPE Raffaelea crossotarsa 0.60 0.10 0.37 0.12 1.07 0.51 0.33 0.06 7081TPE Raffaelea quercivora 1.20 0.87 0.53 0.06 1.50 0.87 0.27 0.15 7069TPE Raffaelea sp. 7049 0.63 0.12 0.40 0.00 1.63 0.90 0.40 0.00 TPE Sporothrix sp. 7335 0.53 0.12 0.47 0.15 1.07 0.45 0.40 0.10 Negative control (water) 0.67 0.23 0.40 0.14 1.00 0.52 0.37 0.06 Positive control 1.18 0.59 0.61 0.22 8.88 8.91 0.51 0.12 (Diplodia quercivora) * Note: Means followed by standard deviation after .

Table 7-7. Symptoms in local pine trees Native host pine Isolated fungus Beetle vector Loblolly pine Slash pine Bleeding Canker Bleeding Canker Ascomycete sp. 7694 Cyrtogenius sp. * * * Ceratocystiopsis minuta 10195 Cryphalus sp. * * * * Ceratocystiopsis sp. 7744 Polygraphus sp. * * * Diplodia seriata LL151 Cryphalus sp. * * * * Fusarium oxysporum 10271 Pityophthorus sp. * * * * Geosmithia sp. CB2013 Tomicus minor * * * Graphilbum sp. CB2013 Ips chinensis * * Grosmannia huntii LL206 Coccotrypes sp., Cyrtogenius luteus and * * * gallery Leptographium koreanum Tomicus sp. 10237 * * * Leptographium pini 11414 Xyleborus pinicola * * * Leptographium procerum Dendroctonus valens CMW25626 * * * Leptographium s. l. sp. 7083 Polygraphus taiwanensis * * * Leptographium s. l. sp. 7085 Xyleborus pinicola * * * * Leptographium sp. LL112 Dendroctonus armandi * * * Leptographium sp. 10223 Xyleborus pinicola * Mariannaea elegans 10179 Hylastes sp. * * * Ophiostoma cf. abietinum LL98 Hylurgops longipillus * * Ophiostoma clavatum LL120 Dendroctonus armandi * * * Ophiostoma ips CB2013 Ips chinensis * * * * Ophiostoma ips 10187 Xyleborus pinicola * * * Ophiostoma microcarpum Cryphalus sp. 10254 * * * * Ophiostoma pulvinisporum Cryphalus sp., Euwallacea LL152 interjectus and Cocootrypes * sp. gallery Ophiostoma sp. 7690 Cyrtogenius sp. * * * * Ophiostoma ips LL257 Orthotomicus sp. * * * Pichia sp. 7018 Cryphalus fulvus * * Raffaelea rapaneae LL134 Xyleborus pinicola * * Raffaelea subfusca LL138 Euwallacea validus * Sporothrix nigrograna LL195 Euwallacea interjectus * * Sporothrix sp. LL99 Hylurgops longipillus, Polygraphus sp., Tomicus minor

Table 7-8. Symptoms in local oak trees Native host pine Isolated fungus Beetle vector Live oak Shumard oak Bleeding Canker Bleeding Canker Ophiostoma sp. 7712 Webbia pabo * * Geosmithia pallida 7686 Webbia pabo * Ophiostoma sp. 7736 Cyclorhipidion aff. fukiense * * Geosmithia sp. 7333 Hypothenemus birmanus * Raffaelea quercivora Cyclorhipidion ohnoi * * * 7047 Raffaelea quercivora Crossotarsus emancipatus * * * * 7069 Ophiostoma sp. 7080 Crossotarsus emancipatus * Pseudozyma aphidis Urocorthylus fanii 7335 Raffaelea crossotarsa Crossotarsus emancipatus * * 7081 Raffaelea cyclorhipidia Cyclorhipidion ohnoi * 7049 Sporothrix sp. 7335 Urocorthylus fanii * *

Figure 7-1. Size of stain in inoculated local pine. Photo courtesy of author.

Figure 7-2. Size of stain in inoculated local oak. Photo courtesy of author.

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CHAPTER 8 CONCLUSION

Several thousand beetle-fungus symbioses remain unstudied and promise unknown and unexpected mycological diversity and enzymatic innovations. This investigation has revealed three symbioses:

Ambrosiodmus-Flavodon symbiosis in North America and Asia. The

Ambrosiodmus-Flavodon symbiosis is unique in several aspects. It is the first reported association between an ambrosia beetle and a basidiomycotan fungus; and the mycosymbiont is a white-rot saprophyte rather than an early colonizer: a previously undocumented wood borer niche. Few fungi are capable of turning rotten wood into complete animal nutrition. The North America and Asia samples indicate that this single species of the white-rot basidiomycete ambrosia fungus Flavodon ambrosius is the primary associate of all Ambrosiodmus and Ambrosiophilus ambrosia beetles in the northern hemisphere, making it the most widespread known ambrosia fungus species, both geographically and in terms of the number of beetle species. The Flavodon-beetle symbiosis appears to employ an unusually strict mechanism for maintaining fidelity, compared to the symbioses of the related Xyleborini beetles, which mostly vector more dynamic fungal communities.

Euwallacea-Fusarium symbiosis in China. Since Euwallacea fornicatus invasive to the USA in the early 2000s, its symbiotic fungus Fusarium were isolated and recorded from many countries. Nonetheless, China as a country of Euwallacea fornicatus origin, this study is the first report of this symbiosis. This investigation has not only specified the distribution of E. fornicatus in China based on records in the National

Zoological Museum of China and my samples from two-year collections, but also update

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the host plant list including the first family-level host records in the Actinidiaceae,

Oleaceae, and Pinacae.

Platypodines-Raffaelea symbiosis in the southeastern USA. I report that fungal community of four endemic platypodine beetles Euplatypus compositus,

Euplatypus parallelus, Myoplatypus flavicornis and Oxoplatypus quadridentatus in the southeastern USA. Fungi from isolation were analyzed by quantitative culture and DNA barcode identification. Phylogenetic analyses of 28S rDNA sequences and fungal community analyses revealed that platypodines were associated with several genera within in Ophiostomatales, Especially Raffaelea. I also first demonstrated that platypodine beetle had a moderate association with Ceratocystiopsis.

The approaches described above serve as a model for rapid and robust exploration and characterization of this frontier of symbiology. Given the number of ambrosia beetle and fungus species worldwide and the increasing research focuses on them, more discoveries of such interactions and evolutionary innovations should be expected.

To further understand how mycangia work with symbiosis, I collected, bred and dissected the most common ambrosia beetle Xylosandrus in subtropics. I dissected by hand or scanned with micro-CT the mycangia in various developmental stages in five

Xylosandrus ambrosia beetle species that possess a large, mesonotal mycangium to determine the ontogeny of the mycangium. All five species display three stages of the mycangium development: 1) Young callow adults have an empty mycangium without fungal mass. 2) In fully mature adults during dispersal, the pro-mesonotal membrane invaginates, and most individuals develop mycangium of variable size and symmetry,

102

mostly filled with the symbiont. 3) After successful establishment of their new galleries, most females discharge the bulk of the fungal inoculum and deflate the mycangium.

Mycangia are much more dynamic than previously thought, and their morphological changes correspond to the phases of the symbiosis. I proved fungus is not the trigger of mycangial development. Studies of the fungal symbionts or plant pathogen transmission in ambrosia beetles need to consider which developmental stage to sample from.

Illustrations of the different stages, including microphotography of dissections and micro-CT scans were provided.

Finally, from an applied research perspective, i test whether fungi associated with exotic beetles are pathogenic to native tree species. Due to the cryptic nature of the fungi and their beetle vector and new non-native beetle-fungus symbiosis continue to enter the USA by trade, preventing introductions is therefore extremely difficult.

Regulatory agencies need evidence-based references or appropriately experienced scientists to perform risk analyses to distinguish which species can be ignored if introduced, which require close monitoring and further research, and which species need eradication if introduced. To address this question, a safe and feasible approach was developed to assessing the threat of fungi associated with exotic ambrosia beetles before their establishment in the US. For the 40 potential pathogens isolated from

Chinese bark and ambrosia beetles. Most were found to be not pathogenic, but some had mildly pathogenic to native pine, such as fungi Leptographium spp. from Xyleborus pinicola, Tomicus sp. and Dendroctonus armandi, fungi Ophiostoma spp. from

Orthtomicus sp. and Ips chinensis, fungus Fusarium oxysporum from Pityophthorus sp..

However, no fungus has proven to be a virulent pathogen of biosecurity concern.

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BIOGRAPHICAL SKETCH

You Li was born and raised in Fuzhou, Fujian province, China. He has always loved to observe insects. He then attended the Fujian Agriculture and Forestry

University an in the fall of 2007 and graduated in the spring of 2011 with a Bachelor of

Plant Protection. He immediately started working on her MS program in the summer of

2011 at the Guizhou University Joint training in Institute of Zoology, Chinese Academy of Sciences in Beijing and graduated with his MS degree in summer of 2014. He enrolled at the University of Florida immediately and earned his Ph.D. in forest resources and conservation under the supervision of Dr. Jiri Hulcr in spring 2018.

During his Ph.D. program, You Li attended many professional conferences including

Mycological Society of America Conference and International Congress of Entomology.

He also attended some workshops in Asia to teach people how to identify bark and ambrosia beetles.

Before his doctoral program at the University of Florida, he proposed to and married fiancée, Misa Lin. Their first child was born in March 2015.

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