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Diversity of Foliar Endophytes in Wild and Cultured Metrosideros Polymorpa Inferred from Environmental PCR and ITS Sequence Data

Maleo Intro

Above me, the forest was alive. Tree fern fronds brushed against each other, honeycreepers piped melodies as they flitted between towering branches, and the trees stood anchored strongly in the soil, their waxy leaves stretching 200 feet higher into the canopy. As my gaze shifted to the fallen leaves surrounding my black rubber boots, I spotted a tiny white mushroom, almost angelic in its presence. The mushroom was no more than an inch tall, with a fragile opaque stem topped by a gilled white cap. It was growing out of an ohi'a leaf that was dark and slimy after many days on the damp and decaying forest floor. As I inspected the other fallen leaves, I noticed that only those of ohi'a trees hosted these tiny fungi. I was astounded that the mushroom was species-specific in its choice of host, and I began to wonder how this mushroom had originally become associated with ohi'a trees. It dawned on me that not only is the forest a system, but each individual tree is an intricately connected hub of ecological activity. Each leaf, even, could be host to many species of fungi.

Figure 1: Ohi'a tree

Figure 1: Ohi'a tree


Fungal foliar endophytes are fungi that live within healthy leaves. The relationship between these fungi and their hosts is symbiotic in some cases: the fungi benefit their hosts via increased drought tolerance, protection against herbivores, and resistance to pathogens (Higgins 2007). In other cases, the relationship is antagonistic. Fungal symbioses with plants are key determinants of biomass, nutrient cycling, and ecosystem productivity (Arnold 2007). Despite many years of research and hundreds of journal articles that mention endophytic fungi, the diversity, phylogenetic position, and relationship of foliar endophytes with their plant hosts, are still largely unknown.

The fossil record indicates that plants have been associated with endophytic fungi for more than 400 million years, and that these fungi were likely associated with the first plants to colonize land. Thus, the fungal community within plants has played a significant role in the evolution of life on land. This coevolution involves the reduction of enzymatic capabilities in the fungi, increasing their dependence on the host plant to provide nutrients for growth, and an increase in their production of particular secondary metabolites beneficial in the process of symbiosis (Rodriguez 2009). Very little work has been done on studying the endophytic communities associated with foliage in Hawai'i, and even less on those of native Hawaiian plants. Given the apparent significance of fungal endophytes to the evolution and overall survival of plant species across the globe, I was very excited to embark on a critical study of the fungal foliar endophytic communities associated with the native Hawaiian plants in my backyard.

Work by Hoffman et al. in 2009 points to a need to compare endophytic communities within different sites and plant communities, which would indicate the role of surrounding species on endophytic community composition. Rodriguez et al. questioned in 2009 the evolutionary dynamics of habitat-adapted symbiosis. It is apparent that a study involving the endophytic community of a native Hawaiian species would not only be groundbreaking, but also a crucial element in understanding the role of the foliar endophytic community in the overall health of the plant host within different habitats, natural or not.

The first step in such an understudied field is the most fundamental. With functional application toward conservation in mind, the species chosen for study should be common and widespread in the habitat. Study of the foliar fungal endophytic community of a threatened rare species might seem more critical, but a baseline study utilizing a more dominant species is certainly more functional. Ohi'a lehua (Metrosideros polymorpha) is a common native Hawaiian plant. The word polymorpha means many forms, because ohi'a trees are common in tropical jungles near the ocean shore as well as along the volcanic rim on our island mountains. The towering tree whose slimy leaves I had noticed in the rain forest near my home is an ideal model for native plant communities across the island chain. As native species become more threatened, garden and greenhouse propagation, followed by out planting into natural environments, will become increasingly common. Because we know so little about endophytic fungi, the appropriate place to start would be to investigate whether the environment or the host plays the principal role in determining the species of fungal foliar endophytic community structures. The endophytic community within an ohi'a that has grown from seed in a greenhouse may be different from that of an ohi'a plant that has grown in its natural environment, surrounded by other native species that may have contributed crucial elements to its fungal foliar endophytic community.

PURPOSE

Figure 2: Ohi'a lehua blossoms

Figure 2: Ohi'a lehua blossoms


The main objective of this study is to determine the composition of the foliar fungal endophytic community associated with Ohi'a lehua (Metrosideros polymorpha), a common native Hawaiian tree species (Figure 1 and 2). The endophytic communities of M. polymorpha from different elevations (8,100 feet and 350 feet) and from wild and cultivated trees were investigated using live cultures, environmental PCR, cloning, and molecular sequencing. The variable of elevation was introduced in addition to the variable of environment in order to compare two phenotypes of the same species and to broaden the application and implications of this project.

The results of this investigation will further our understanding of the similarities between a plant's endophytic community and its surrounding environment. This relationship has implications that could be a critical element of native plant conservation. If individual ohi'a trees growing at different elevations are shown to harbor similar fungal foliar endophytic communities, then it can be inferred that host selection for particular fungal endophytes exists. This inference could be imperative to outplanting efforts. If the communities of fungi are different, then it can be inferred that the surrounding environment largely influences the composition of the fungal foliar endophytic community in a particular tree.

METHODS

Figure 3: collecting leaves

Figure 3: collecting leaves


Figure 4

Figure 4


Figure 5

Figure 5


Figure 6

Figure 6


Leaf Collection
 

Approximately 20 mature leaves per tree were collected. Leaves with galls or other markings were avoided (Figure 3). The samples were kept refrigerated in sealed bags until sterilization. Four trees (two replicates from each elevation) were sampled from the Common Garden in Volcano (elevation 3,600 feet). Leaves of two trees at each elevation (350 feet and 8,100 feet) from the same aged lava flow were collected as well, for a total of eight samples. Figure 4 shows an ohi'a plant that originated from a population at 350 feet elevation growing in the Common Garden. Figure 5 shows an ohi'a plant growing about 50 meters away that originated from a population at 8,100 feet elevation. The leaves in Figure 4 are waxy and large, whereas those of Figure 5 are smaller and pubescent (furry).

Surface Sterilization

Leaves were individually rinsed with running tap water to remove loose surface particles, then submerged in a series of solutions to surface-sterilize the leaves so that the only DNA being analyzed is that inside the leaf. The submersion sequence is as follows: 30 seconds in 95% ethanol, two minutes in 10% chlorine bleach, and two minutes in 70% ethanol (Figure 6).

Drying

In preparation for DNA extractions, four leaf disks per tree were placed into drying tubes to ensure a sterile environment.

DNA Extractions

Tissue samples (5-10 mg of dried leaf material) were lysed in Lysing Matrix A Tubes using the FastPrep instrument for 20 seconds at a speed setting of 4.0. Total genomic DNA, including that of fungal endophytes, was isolated using the DNeasy Plant Mini Kit (Qiagen Inc.), following the manufacturer's protocol.

PCR

The fungal-specific primers ITS1F and ITS4 were used to amplify the internal transcribed spacer (ITS) region of the fungal nuclear genome. PCR protocol consisted of an initial denature for three minutes at 96?C, followed by 35 cycles of 94?C for 30 seconds, 55? to 58?C for 60 seconds, and 72?C for 60 seconds, followed by a final extension at 72?C for 10 minutes.

Gel

PCR quality and yield were analyzed via gel electrophoresis using a 1% agarose gel.

Cloning

Cloning reactions were performed using a TOPO-TA Cloning Kit for Sequencing (Invitrogen Inc.), following the manufacturer's protocol. For each cloning reaction, 6 to 10 colonies were isolated and screened for the appropriate fungal DNA insert, using restriction digest analysis and PCR.

Restriction Digest Analysis

Restriction digest analysis was performed using a TOPO-TA Cloning Kit for Sequencing (Invitrogen Inc.), following the manufacturer's protocol and using the endonuclease enzyme EcoR1.

PCR (to Analyze Transformants)

PCR was performed to analyze transformants using a TOPO-TA Cloning Kit for Sequencing (Invitrogen Inc.), following the manufacturer's protocol and using the primers ITS1F and ITS4.

PCR Purification

PCR was purified for sequencing using a QIAquick PCR Purification Kit (Qiagen Inc.) according to the manufacturer's protocol.

Sequencing

Cleaned PCR products were submitted to Elim Biopharmaceuticals Inc. for sequencing.

Data Analysis

ITS data were recovered from 39 clones. Sequence data were screened and manually edited using Sequencher 4.0. BLAST was used to obtain closest match to existing sequences in the NCBI GenBank Database. MacClade was used to manually align 37 clones (clones 1F and 4B were removed as outliers), and GARLI was used to generate a maximum likelihood phylogenetic hypothesis. PAUP* was used to calculate pairwise genetic distances between the cloned ITS sequences.

DATA ANALYSIS AND DISCUSSION

This study was conducted to document the diversity of the foliar fungal endophytic community associated with ohi'a lehua (Metrosideros polymorpha) to assess whether the environment or the host determines the endophytic community structure of different elevational phenotypes. Sequencing of the ITS region was utilized to identify the fungal endophytes associated with wild and cultivated trees. In the Common Garden, the trees had been cultivated in an environment foreign to that which they had adapted to, surrounded by species such as kahili ginger (Hedychium gardnerianum), hapu'u pulu (Cibotium splendens), and banana poka (Passiflora tripartite). At the low elevation site, the dominant species present were uluhe (Dicranopteris linearis) and strawberry guava (Psidium cattleianum). At the high elevation site, the species composition was almost entirely native, consisting of a'ali'i (Dodenaea viscosa)and pukiawe (Styphelia tameiameiae).

Restriction digest analysis was performed using the endonuclease enzyme EcoR1 to remove the fungal DNA insert from the E. coli plasmid. Unfortunately, an EcoR1 site was found to exist within the middle of the DNA insert, so the EcoR1 enzyme not only digested the EcoR1 sites on each end of the insert, but also cleaved the insert in half. This method was found to be useful to take a baseline assessment of the DNA present, as sequences can be compared by fragment length along the ladder.

Fig. 7

Figure 7: Phylogenetic relationships among 36 sequences of endophytes obtained via environmental PCR of leaves from low elevation (blue/green) and high elevation (orange/red) trees. This phylogram indicates that the endophytes within M. polymorpha leaves of the same elevational phenotype harbor genetically similar communities, largely independent of growing environment. The outlier (clone 4E) may indicate that the environment likely plays a limited role in contributing some (but not a majority of) endophytes.


Figure 8

Figure 8.


Figure 9: Mean Pairwise Genetic Distances Among Endophytic Communities of Wild and Cultivated Elevational Phenotypes of M. polymorpha

Figure 9: Mean Pairwise Genetic Distances Among Endophytic Communities of Wild and Cultivated Elevational Phenotypes of M. polymorpha


As shown in Figure 7, the ITS sequences recovered from M. polymorpha leaves are divided into two distinct clades, indicating that high and low elevation phenotypes harbor distinct endophytic communities. Each of these clades in turn is composed entirely of endophytes from both wild and cultivated trees of a single elevational phenotype. This suggests that the host, not the growing environment, determines endophytic community composition. Figure 8 confirms that the ITS sequences recovered from environmental PCR are nearly identical within elevational phenotypes, and not within growing environments. Figure 9 illustrates that mean pairwise genetic distances between sequenced endophytes of low elevation phenotypes are greater than those of high elevation phenotypes. This suggests that low elevation phenotypes of M. polymorpha harbor greater genetic diversity than do those at high elevation. Additionally, the mean pairwise genetic distances of endophytes within cultivated trees of both elevational phenotypes are greater than those in a wild environment.

CONCLUSION

The findings of this experiment from both live cultures and environmental PCR and ITS sequence data support the conclusion that the host, not the environment, determines the foliar fungal endophytic community of M. polymorpha. Furthermore, high and low elevation phenotypes harbor distinct endophytic communities. Host, not locality (wild versus cultivated), determines endophytic community composition. Low elevation phenotypes of M. polymorpha harbor a greater genetic diversity of fungal endophytes than do those of high elevation. Samples of M. polymorpha from a cultivated environment harbor greater genetic diversity of fungal endophytes than do those of a wild environment.

These findings indicate that there is likely a previously undocumented level of selection by plant hosts of fungal endophytes in Hawaiian botanical communities. This study is the first significant one of its kind. The foliar fungal endophytic community of M. polymorpha has never been studied using environmental PCR. These findings are very intriguing because they are novel and because they present a conclusion which has implications to play a large role in conservation efforts of native Hawaiian plants. These findings may suggest a previously undocumented evolutionary relationship between foliar fungal endophytes and their hosts. Further studies should study the surrounding species in each sampled environment to determine the role of surrounding community on contributing to the endophytic community of M. polymorpha.

This study is the first one of its kind. The foliar fungal endophytic community of M. polymorpha has never been researched so extensively. My findings have implications for the conservation of native Hawaiian plants and possibly of plants worldwide. Further studies would indicate whether or not the introduced endophytes are harmful or not, as well as the individual role of each endophyte within the plant's endophytic community. A study comparing the fungi discovered within ohi'a trees to those in a sampling of surrounding plants would be helpful to determine the true effect the environment has on foliar fungal endophytes. I hope that with this research as a baseline, the role of foliar fungal endophytes in native Hawaiian plants can be assessed and one day utilized in conservation efforts so that the breathtaking forestscape of my backyard is a sight for my children's children to behold.

ACKNOWLEDGEMENTS

I would like to thank Dr. Brian Perry for his constant guidance and mentorship. The funding for this project was provided by his lab fund at the University of Hawai'i at Hilo. My mother, Betsy Kodis, and my advisor, Jamie Nekoba, both also provided invaluable support so that I could complete this research.

BIBLIOGRAPHY

Arnold, Elizabeth, et al. "Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR" Mycologia 99.2 (2007): 185-206.

Arnold, Elizabeth, and Francois Lutzoni. "Diversity and Host Range of Foliar Fungal Endophytes: Are Tropical Leaves Biodiversity Hotspots?" Ecology 88.3 (2007): 541-549.

Brown, Alfred E. Benson's Microbiological Applications. 10th ed. Boston: McGraw Hill, 2007, pp. 95-96.

Hata, K., R. Atari, and K. Sone. "Isolation of endophytic fungi from leaves of Pasania edulis and their within-leaf distributions." Mycoscience 43.5 (2002): 369-73. Retrieved from the World Wide Web on 5 Oct 2009. http://www.springerlink.com/content/h2utgd8tknvpmgv1

Hoffman, Michelle, et al. "Molecular Analysis Reveals a Distinctive Fungal Community Associated with Foliage of Montane Oaks in Southeastern Arizona." Journal of the Arizona-Nevada Academy of Science 401.1 (2008): 99-100.

Higgins, Lindsay, et al. "Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three major plant lineages" Science Direct 42 (2007): 543-555.

Rodriguez, R.J., et al. "Fungal endophytes: diversity and functional roles." New Phytologist 182 (2009): 313-330.

White, D.A. "Mycology and Taxonomy." 20 Nov 2009. Retrieved from the World Wide Web on 10 Dec 2009. http://www.ontarioprofessionals.com/weird3.htm

Zimmerman, Naupaka. Telephone interview. 3 Nov 2009.

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