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PONDWEEDS
MARIA KUZMINA UNIVERSITY OF GUELPH, CANADA
PONDWEEDS MARIA KUZMINA UNIVERSITY OF GUELPH, CANADA PONDWEEDS WITH - - PowerPoint PPT Presentation
PONDWEEDS MARIA KUZMINA UNIVERSITY OF GUELPH, CANADA PONDWEEDS WITH - - PowerPoint PPT Presentation
PONDWEEDS MARIA KUZMINA UNIVERSITY OF GUELPH, CANADA PONDWEEDS WITH US FORESTS IN THE WATER, UNDER AND ABOVE Linear-leaved Broad-leaved Diversifolius WHY MONITORING THEM AND WHAT MAKES IT DIFFICULT? Ecological studies : food and
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PONDWEEDS WITH US – FORESTS IN THE WATER, UNDER AND ABOVE
Linear-leaved Broad-leaved Diversifolius
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WHY MONITORING THEM AND WHAT MAKES IT DIFFICULT?
- Ecological studies: food and shelter for many
species of organisms
- Bioindication: species-specialists and
generalists towards the alkalinity and pollution
- Protection: 6 species at risk or special concern
in North America, among those 2 from Ontario (Hill’s and Ogden’s pondweeds)
- Aquatic habitats less accessible compared to
terrestrial ones
- Morphologic identification is limited by phenology
and microscopic characters
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WHAT ARE THEY AND HOW MANY?
ALISMATALES POTAMOGETONACEAE
Potamogeton (32-37 species) Stuckenia (4 species) Zannichellia (1 species)
30 species in Ontario
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BACKGROUND AND OBJECTIVES
Detection of the species of pondweeds (Potamogetonaceae) in water samples
- Invasive aquatic species
(Scriver et al., 2015) eDNA detection of plants
BROAD SPECIFIC
- Large-scale monitoring in soil
(Fahner et al., 2016) Requested and funded by the Species at Risk Research Fund
- f Ontario
(SARRFO, 2016- 2017,10K)
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PREPARING LOCAL REFERENCE LIBRARY
30 species of pondweeds reported from Ontario (VASCAN) 90 specimens: collection of DNA from CBG, part of “The Vascular Plants of Canada” project (dx.doi.org/10.5883/DS-POTAM) Potamogeton foliosus (Agriculture and Agrifood Herbarium, DAO) identified by Barre Hellquist*, the author of the treatment of pondweeds in the “Flora of North America”
* *
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g
- atpB-rbcL (Ito et al., 2014)
- ITS2 (China Plant BOL Group, 2011)
SELECTION OF THE MARKERS & DESIGNING THE PRIMERS
Datasets downloaded from GenBank (atpB-rbcL) & BOLD (ITS2) Datasets of atpB-rbcL & ITS2 generated for the local library
g
Designing primers specific for Potamogetonaceae Designing eDNA primers
eDNA marker (with mean length) Length (bp)
atpB-rbcL-117
101 ‒ 133
atpB-rbcL-184
161 ‒ 208
ITS2-157
133 ‒ 182
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SPECIES RESOLUTION WITH THE SELECTED eDNA MARKERS
Species- P. foliosus
- P. friesii
- P. subsibiricus
- P. vaseyi
- P. hillii
- P. pusillus
- P. strictifolius
- P. zosteriformis
- P. richardsonii
- P. perfoliatus
- P. alpinus
- P. illinoensis
- P. amplifolius
- P. pulcher
- P. epihydrus
- P. confervoides
- P. praelongus
- P. oakesianus
- P. natans
- P. gramineus
- P. crispus
- P. robbinsii
- P. spirillus
- P. bicupulatus
- S. filiformis
- S. vaginata
- S. pectinata
- Z. palustris
- P. foliosus
- P. friesii
- P. subsibiricus
- P. vaseyi
- P. hillii
- P. pusillus
- P. strictifolius
- P. zosteriformis
- P. richardsonii
- P. perfoliatus
- P. alpinus
- P. illinoensis
- P. amplifolius
- P. pulcher
- P. epihydrus
- P. confervoides
- P. praelongus
- P. oakesianus
- P. natans
- P. gramineus
- P. crispus
- P. robbinsii
- P. spirillus
- P. bicupulatus
- S. filiformis
- S. vaginata
- S. pectinata
- Z. palustris
- B. atpB-rbcL-184
- P. foliosus
- P. friesii
- P. obtusifolius
- P. subsibiricus
- P. vaseyi
- P. hillii
- P. pusillus
- P. strictifolius
- P. zosteriformis
- P. richardsonii
- P. perfoliatus
- P. alpinus
- P. nodosus
- P. illinoensis
- P. amplifolius
- P. pulcher
- P. epihydrus
- P. confervoides
- P. praelongus
- P. oakesianus
- P. natans
- P. gramineus
- P. crispus
- P. robbinsii
- P. spirillus
- P. bicupulatus
- S. filiformis
- S. vaginata
- S. pectinata
- Z. palustris
- P. foliosus
- P. friesii
- P. obtusifolius
- P. subsibiricus
- P. vaseyi
- P. hillii
- P. pusillus
- P. strictifolius
- P. zosteriformis
- P. richardsonii
- P. perfoliatus
- P. alpinus
- P. nodosus
- P. illinoensis
- P. amplifolius
- P. pulcher
- P. epihydrus
- P. confervoides
- P. praelongus
- P. oakesianus
- P. natans
- P. gramineus
- P. crispus
- P. robbinsii
- P. spirillus
- P. bicupulatus
- S. filiformis
- S. vaginata
- S. pectinata
- Z. palustris
- C. ITS2-157
Distances: number of substitutions per site (Tamura and Nei, 1993)
13 resolved species 17 resolved species
23 resolved species (77%)
atpB-rbcL-184 ITS2
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- Z. palustris
- S. filiformis
- S. pectinata
- P. crispus
- P. foliosus
- Z. palustris
- S. filiformis
- S. pectinata
- P. crispus
- P. foliosus
THE DETECTED SPECIES WERE UNAMBIGUOUSLY RESOLVED BY THE SELECTED eDNA MARKERS
atpB-rbcL-184 ITS2
ML Fast Tree (Price et al., 2010)
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SELECTION OF THE SITE FOR THE EXPERIMENT
Grand River at CHARITABLE RESEARCH RESERVE (rare), Ontario
- Easy to access: 30 km SW from Guelph, about 30 min drive
- Two species of pondweeds (Potamogeton crispus and Stuckenia
pectinata) were collected during the ecological survey (Telfer et al., 2015)
The goal of our experiment was to detect the species of pondweeds where they were AT LEAST previously documented!
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THE WORKFLOW OF THE EXPERIMENT
SLIDE 13
- All three markers performed highly specifically and
consistently across the locations
- ITS2-157 detected more species compared to atpB-rbcL
Abundance of reads after filtering Percent of reads hit the local reference library Number of pondweed species detected
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WHY REFERENCE LIBRARY MATTERS?
Incompleteness and conflicting taxonomy affect the results
Top Hit Identification Location Library eDNA Marker and Raw Data Treatment atpB-rbcL-117 atpB-rbcL-184 ITS2-157
Dereplicated Clustered Dereplicated Clustered Dereplicated Clustered
Potamogeton crispus
2
DS- POTAM
270 275
Gen Bank
275
Potamogeton foliosus
1
DS- POTAM
1,363 1,367 2
DS- POTAM
454 453 3
DS- POTAM
14,062 13,954 1,267 1,274
Potamogeton gayi
3
Gen Bank
13,938
Potamogeton pusillus
1
Gen Bank
1,367 2
Gen Bank
453 3
Gen Bank
1,274
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Top Hit Identification Location Library eDNA Marker and Raw Data Treatment atpB-rbcL-117 atpB-rbcL-184 ITS2-157
Dereplicated Clustered Dereplicated Clustered Dereplicated Clustered
Stuckenia filiformis
1
DS-POTAM
778
2
DS-POTAM
6,412
3
DS-POTAM
4,031 Stuckenia pectinata
1
DS-POTAM
47,979 49,141 191,594 180,965 9,263 9,267
Gen Bank
49,141 180,967 9,267
2
DS-POTAM
295,614 307,529 226,484 212,250 30,684 30,775
Gen Bank
307,779 212,272 30,775
3
DS-POTAM
329,810 335,372 344,475 324,822 34,610 34,639
Gen Bank
335,372 324,824 34,639
WHY RAW DATA TREATMENT MATTERS?
Analysis of dereplicated (=identical) sequences revealed a species,
- therwise hidden in the post-clustering analysis
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NEGATIVES AND NON-TARGET DNA
Top Hit Identification Location Library eDNA Marker and Raw Data Treatment atpB-rbcL-117 atpB-rbcL-184 ITS2-157
Dereplicated Clustered Dereplicated Clustered Dereplicated Clustered
Stuckenia pectinata
PCR NEGATIVE DS- POTAM
12 12
Gen Bank
12
Panicum capillare
2
Gen Bank
34
Charophyta
1
Gen Bank
25
Cyanobacteria
1
Gen Bank
60 2
Gen Bank
13
Proteobacteria
DNA NEGATIVE Gen Bank
77 1
Gen Bank
229 2
Gen Bank
153 3
Gen Bank
40
SLIDE 17
WE FOUND TWO SPECIES PREVIOUSLY DOCUMENTED …AND THREE MORE!
Species documented at the experimental site (Telfer et al., 2015):
- Potamogeton crispus
- Stuckenia pectinata
rare checklist (Jenna Quinn, rare, personal communication):
- Potamogeton amplifolius*
- Potamogeton crispus
- Potamogeton pusillus**
- Stuckenia pectinata
New to both previous reports:
- Potamogeton foliosus
- Stuckenia filiformis
- Zannichellia palustris
* Potentially was not at the experimental site
** Potential misidentification of P. foliosus?...MK
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CONCLUSIONS
- eDNA effectively detects different species of pondweeds in water
samples, revealing their potential under-estimated diversity in the aquatic communities
- The developed protocol can be used for ecological surveys,
biomonitoring, and locating rare and protected species
- Using both chloroplast and nuclear markers improves species
resolution among the selected species of pondweeds and increases reliability of the results
- The results of eDNA detection strongly depend on the completeness
and accuracy of the reference libraries.
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Species at Risk Research Fund for Ontario (SARRFO) Ministry of Natural Resources
Alex Borisenko Nataly Ivanova Vasco Elbrecht Stephanie Pedersen Elizabeth Sears Jenna Quinn