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Beyond rDNA: a multigene phylogenomic approach to resolution of the Mortierellaceae

Authors:

Abstract

The Mortierellales comprise a polyphyletic lineage of early diverging, globally distributed, soil fungi. Several Mortierella species are known to interact with roots and promote plant growth, even though they do not make traditional mycorrhizal structures. Mortierella also harbor bacterial endosymbionts and have unique oleaginous biology that makes these fungi industrially important. Inter- and intra-species diversity in the Mortierellales has been most thoroughly sampled in continental Europe and North America and characterized using the ITS and LSU rDNA regions. In order to better understand the biogeography and global diversity of the Mortierellales, we are making efforts to isolate and identify these fungi from geographically distinct and undersampled environments including North American caves, Australia, Fiji, and Africa by baiting from soils using chitin as a substrate. Preliminary results indicate the presence of novel Mortierella species from our sampled regions, but also that some species, such as M. elongata, are globally distributed. Most phylogenetic analyses of Mortierellales are performed using rDNA. However, strong sequence divergence limits the possible resolution of the overall structure of this polyphyletic order. To address this limitation we are pursuing two non-rDNA approaches to improve phylogenetic resolution: multi-locus sequence typing and low-coverage genome sequencing. We will compare these phylogenetic approaches in their ability to resolve the phylogeny and to distinguish species based on genealogical concordance. Finally, we plan to use this phylogeny as a framework within which to place previously unidentified ITS sequences from GenBank having affinity to Mortierella. Contributed Talk C10.6
Beyond rDNA:
a multigene phylogenomic approach
to resolution of the Mortierellaceae
Natalie Vande Pol & Gregory Bonito
August 9, 2016
84th Mycological Society of America Meeting
MUCOROMYCOTA
Spatafora et al., 2016 (in
press)
Background
Polyphyletic, early diverging filamentous soil
fungi
Hosts endobacteria
Globally ubiquitous
Plant associated
Broad plant host range
No mycorrhizal structures
Plant growth promoting (PGP)
Protects plants against heat stress
Diversity and Phylogeny of
Mortierellaceae
6 genera
Aquamortierella, Dissophora, Gamsiella,
Lobosporangium, Modicella, Mortierella
Polyphyletic lineage
LSU region cannot resolve phylogeny
ITS region does not align across genera
7 clades
High proportion of “unidentifiable” OTUs
Diversity
Nagy et al., 2011
Unassigned MOTUs have been described but not
sequenced
Duplicate species descriptions
92 MOTUs
Inferred 120-170 authentic Mortierellaceae species
Phylogeny
Wagner et al., 2013
LSU and full ITS region
Seven clades
Methods
Baiting Soil for Mortierella
Africa
Fiji
Australia
New Zealand
Sequenced ITS region
Strains from Collaborators
Sequence data
Genbank
Collaborators & an NGS project by Tedersoo et al., 2014
Sequence Analysis
MAFFT Aligned
>3500 sequences
Partially Trimmed LSU regions
Full-length Dereplication
>2600 unique sequences
BLASTed to Clade
100 unassigned to clade
Sequence Analysis by Clade
MAFFT alignments in Mesquite
Trimmed to ITS2
Discarded incomplete sequences
Clustered at 97%
Total of 448 OTUs across all clades
MUSCLE aligned and built phylogenetic trees in MEGA7
Partial deletion treatment of gaps
Maximum Likelihood
Tamura-Nei model
Gamma distributed rate variation
500 bootstrap iterations
Clade 4- /dissophora
Clade4 OTU97
Clade4 OTUs
Clade4 OTUs
Clade4 OTUs
Clade4 OTUs
Clade4 OTU26
Clade4 OTUs
Clade4 OTU42
Clade4 OTU61
Clade4 OTU57
Clade4 OTU23
Clade4 OTU94
Clade4 OTU22
Clade4 Mortierella globulifera strain CBS746.68
Clade4 Mortierella globulifera CBS858.70
Clade4 OTU1
Clade4 OTUs
Clade4 Mortierella echinula strain CBS282.71
Clade4 OTU86
Clade4 OTU47
Clade4 OTU118
Clade4 OTU105
Clade4 OTUs
Clade4 OTU3
Clade4 OTU27
Clade4 OTU11
Clade4 OTU8
Clade4 OTUs
Clade4 Gamsiella multidivaricata CBS227.78
Clade4 OTU7
Clade4 OTU84
Clade4 OTUs
Clade4 OTU64
Clade4 OTU37
Clade4 OTU43
Clade4 OTUs
Clade4 OTU13
Clade4 OTU10
Clade4 Mortierella horticola CBS254.76
Clade4 OTU96
Clade4 Mortierella stylospora CBS211.32
Clade4 OTU9
Clade4 Dissophora decumbens st rain CBS592.88
Clade4 OTU4
Clade4 Dissophora ornata CBS347.77
Clade4 OTU12
Clade4 OTU85
Clade4 Mortierella angusta CBS293.61
Clade4 Mortierella simplex strain CBS751.68
Clade4 OTU5
93
24
40
59
89
100
100
35
99
40
74
99
96
19
88
88
50
50
96
76
13
55
46
88
76
38
33
8
35
30
68
84
17
54
62
96
48
4
36
17
2
11
0
0
67
238
12
25
11
21
8
0.20
Wagner et al., 2013
Clade2 OTU4
Clade2 Mortierella epigama strain CBS489.70
Clade2 OTU3
Clade2 OTU7
Clade2 OTU19
Clade2 OTU18
Clade2 OTU14
Clade2 OTU17
Clade2 OTU13
Clade2 OTU12
Clade2 Mortierella minutissima var. dubia CBS307.52
Clade2 Mortierella horticola CBS305.52
Clade2 OTU2
Clade2 Mortierella horticola strain CBS869.68
Clade2 OTU8
Clade2 OTU20
Clade2 Mortierella epicladia strain CBS246.75
Clade2 OTU6
Clade2 Mortierella minutissima strain CBS226.35
Clade2 Mortierella clonocystis s train CBS176.74
Clade2 OTU10
Clade2 OTU21
Clade2 OTU9
Clade2 Mortierella clonocystis s train CBS357.76
Clade2 Mortierella epicladia strain CBS355.76
Clade2 OTU5
Clade2 Mortierella verticillata strain CBS315.52
Clade2 OTU11
Clade2 OTU16
Clade2 Mortierella humilis strain CBS745.68
Clade2 OTU1
Clade2 Mortierella humilis CBS222.35
Clade2 Mortierella vertic illata strain CBS220.58
Clade2 Mortierella vertic illata strain CBS374.95
99
88
64
0
37
72
28
17
40
28
66
48
11
13
0
21
24
36
19
0.050
Clade 2- /humilis
Wagner et al., 2013
Multi-Locus Sequence Typing
14 loci
Amplicons 750-2000 bp
384 isolates
Non-ribosomal Markers
Mortierella Phylogeny
Approach #2: Low-coverage genome
sequencing
92 isolates, overlapping with MLST
Simpler preparation
Fewer a priori assumptions
More expensive per sample
Conclusions & Future Work
More diversity than previously recognized
Need additional geographical sampling &
isolates
Ribosomal markers are inadequate for
phylogenetic structure, but useful for species
identification
Stay tuned for MLST data & low-coverage
genomes
Questions/Feedback?
PI: Gregory Bonito
Alessandro Desiro
Nico Benucci
Nils Benning
Collaborators:
Kerry O’Donnell, USDA
Jason Stajich, UC Riverside
Rytas Vilgalys, Duke University
Andy Miller,
Andrea Porras-Alfaro
Martin Chilvers, Michigan State Univ.
Leho Tedersoo,
1. ITS1f-LR5
2. RPB1 largest subunit
3. EF-1alpha
4. CTP synthase
5. chitin synthase
6. Xanthine dehydrogenase
7. Calcium-translocating P-type ATPase
8. Class V Myosin motor head
9. Acyl-CoA oxidase
10.Hypothetical protein, Amino acid transporters
11. Hypothetical protein, DNA replication licensing factor, MCM5 component
12.onanonoxo-7-onima-8-eninoihtemlysoneda (biotin biosynthesis)
13.delta-12 fatty acid desaturase
14.Glycosyltransferase Family 21 protein
14 Loci
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