Putting endophytes and epiphytes into evolutionary context: a study of the vine Smilax rotundifolia Christopher B. 1 Zambell, James F. 2 White, & Robert T. 3 O’Connor 1. Graduate Program in Ecology & Evolution, and Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901; 2. Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901; 3. Undergraduate Program in Biotechnology, Rutgers School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ 08901 Introduction: 1. Pestalotiopsis Past research in our lab has shown the woody vine Smilax rotundifolia (Smilacaceae, Liliales) to house a rich associated community of surface and endophytic fungi. However, like many ecological surveys, this work is at risk of leaving only a very vague record of multiple "morphospecies" of common plant associated genera. If on the other hand strong taxonomic identities at the species level can be established then this study has greater value beyond the immediate hypotheses that the study was designed to test. It can serve as a solid record for future reference and contribute towards a growing understanding of fungal niches, hostsymbiont evolutionary history, and structure of plant-fungal ecological networks. Figure 5. Portion of tree from Bayesian analysis of a partitioned alignment of genes ITS (K80+I+G model) and Tef1 (JC model) using our own isolates (indicated in green) and the alignment/dataset of Gomes et al. (2013) downloaded from treeBASE, study 13943. Posterior probabilities are shown at nodes. Recent publication of multi-gene phylogenies for some notoriously difficult taxonomic groups is now making such reliable identification possible. We used published phylogenies, and a multi-gene, multiisolate approach to establish the phylogenetic placement of morphotypes of Phyllosticta, Colletotrichum, Phomopsis and Pestalotiopsis that were previously found to be strongly associated with healthy Smilax leaves and woody stems. Neighbor Joining Results: ITS – Species “1” clusters with 99% bootstrap support. Tef1 – Species “1” clusters with 100% bootstrap support. Figure 3. Bayesian tree based on analysis of a partitioned alignment of genes ITS (HKY+G model) and Tef1 (GTR+I+G model) including our own isolates (indicated in green) and sequences downloaded from Genbank according to the accession numbers given in the dataset of Maharachchikumbura et al. (2012). Red branches are attached to species for which only the ITS gene was available. Asterisks indicate ex-type or ex-epitype cultures. Posterior probabilities are shown at the nodes. Figure 1. Sampling locations Figure 2. Smilax rotundifolia vines Objectives: • Determine if morphotypes represent individual species by criteria of genealogical concordance (Taylor et al. 2000) - defined in this study as the formation of monophyletic clades in neighbor joining, distancebased phylogenies for each individual gene. • Determine if the morphotypes represent previously described species. Neighbor Joining Results: ITS – Species “1D” clusters with 82% bootstrap support, sp. “4” clusters with 87% support, while sp. “1L” and sp. “2” are not monophyletic. Tef1 – Species “1D” clusters with 99% support, “1L” clusters with 89% support, sp. “4” clusters with 100% support, and sp. “2” with 100% support. Discussion: A third gene locus is needed since ITS does a poor job of resolving spp. “2” and “1L”. It is notable that the four morphospecies are well spread out in the tree rather than close relatives. 4. Colletotrichum acutatum complex Figure 6. Portion of tree from Bayesian analysis of a partitioned alignment of genes ITS (K80+I model) and GAPDH (JC model) including our own isolates (indicated in green) and the alignment/ dataset of Damm et al. (2013), downloaded from treeBASE (study 12762), and reduced in size to speed computing times. The ex-type strain of C. fioriniae was accidentally removed from the dataset. Posterior probabilities are shown at the nodes. Methods: View publication stats Discussion: Our Phomopsis morphotype receives strong support as a species from both genes. Its closest relative is a pathogen of grape vines, perhaps hinting at a host shift among tangled vines in the distant past. The rest of the clade contains species from trees of diverse plant families. 2. Phyllosticta • Observe any patterns in terms of host association or endophytism in the context of the larger phylogenies. • Isolates: In August of 2011 we sampled epiphytic and endophytic fungi from a total of 54 S. rotundifolia plants across three main locations in New Jersey (Fig. 1): (1) deciduous forest and small woodlots on Rutgers Campus, (2) coastal scrub and maritime forest of Sandy Hook Gateway National Recreation Area, and (3) conifer dominated sites in the Pine Barrens of Brendan T. Byrne State Forest. • Sequences: ITS sequences were acquired for all isolates. Sequences of one other selected gene were acquired for each genus based on literature review of what might be useful. Lab work was conducted at Rutgers - procedures/primers are omitted here for brevity. • Alignment: Reference alignments were taken from treeBASE (treebase.org) or sequences were directly downloaded from Genbank based on accession numbers given in the literature. After adding our own sequences alignment was done using MAFFT (G-INS-I algorithm) followed by minor manual adjustments, or in some cases manual addition of our sequences to the previous treeBASE derived alignment. • Neighbor Joining: Neighbor joining trees were generated for each individual gene using Mega 5.2.1 with 10,000 bootstrap replications under the conditions, “Method: p-distance”, “Substitutions to Include: d: Transitions + Transversions”, and “Gaps/Missing Data Treatment: Pairwise Deletion.” • Bayesian phylogeny: Each gene alignment was then tested for optimum model in jModelTest v2.1.4 using 3 possible nucleotide substitution schemes, and the final model chosen according to AICc. MrBayes v3.2.1 was used to apply separate models to each gene, and then run per defaults for 3 million to 6 million generations until standard deviation of split frequencies was <0.01, and 25% of trees were discarded as burn-in before the final tree was constructed. Trees were edited in FigTree v1.4.0 and Serif DrawPlus. 3. Diaporthe (Phomopsis) Neighbor Joining Results: C. acutatum type isolates were interspersed amongst the C. fioriniae clade with bootstrap support of 95% (ITS) and 100% (GAPDH) for the clade as a whole. Note that a third Pine Barrens isolate was included in the ITS dataset. Discussion: Our isolates are members of the species C. fioriniae, described by Damm et al. (2012) as a multi-host fruit rot pathogen, an endophyte, and in one instance found as an entomopathogen of scale insects. Acknowledgements: Figure 4. Bayesian tree based on analysis of a partitioned alignment of genes ITS (SYM+I+G model) and Tef1 (HKY+G model) including our own sequences (indicated in green) combined with Genbank sequences that were downloaded according to accession numbers given in Glienke et al. (2011), Su and Cai (2012) and Zhang et al. (2013a, 2013b). Also included are the top 10 Blast search ITS results (shown in red) after Blast searching one of each of our Phyllosticta morphotypes. Asterisks indicate ex-type, ex-epitype, or ex-neotype cultures. Neighbor Joining Results: ITS – Species “1” clusters with 99% bootstrap support and sp. “3” clusters with 93% support. Tef1 - Species “1” clusters with 100% support and sp. “3” clusters with 99% support. Discussion: These both seem to be solidly supported species. In the Bayesian tree, species “1” is sister to a clade of Phyllosticta with coniferous evergreen hosts. Species “3” seems to be closely related to a number of Phyllosticta spp. that occupy other evergreen plants, with the exception of the Fraxinus species. S. rotundifolia’s stem is evergreen and some of the leaves survive through late into the winter. We are very grateful to the New Jersey Mycological Association for a student research grant that made this study possible. 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