Transcript A generalist tymovirus of the tallgrass prairie

A generalist tymovirus of the
tallgrass prairie
•Michael Palmer1
•Andrew Doust1
•Rick Nelson2
•Min Byoung2
•Vaskar Thapa1
•Marilyn Roossinck2
•Ulrich Melcher1
1Oklahoma
State University
2Samuel Roberts Noble Foundation
Plant Virus Biodiversity and Ecology
PVBE
Methods
Preliminary Results
Confusions
Disclaimer: I am not a virologist
Plant Virus Biodiversity and
Ecology (PVBE)
An attempt to document the presence of viruses in
plants of the Nature Conservancy’s Tallgrass
Prairie Preserve, and to generate testable
hypotheses
PVBE People
Virologists,
ecologists,
geneticists,
bioinformaticians,
computer scientists
Known viruses species are mostly from cultivated plants
Plants of the Tallgrass Prairie Preserve
Abutilon theophrasti
Acalypha gracilens
Acalypha ostryifolia
Acalypha virginica
Acer negundo
Achillea millefolium
Aegilops cylindrica
Aesculus glabra
Agalinis aspera
Agalinis heterophylla
Agalinis purpurea
Agalinis tenuifolia
Agastache nepetoides
Ageratina altissima
Agrimonia parviflora
Agrimonia pubescens
Agrostis elliottiana
Agrostis gigantea
Agrostis hyemalis
Agrostis scabra
Agrostis stolonifera
Alisma subcordatum
Allium canadense
Allium stellatum
Allium vineale
Alopecurus
carolinianus
Amaranthus albus
Amaranthus hybridus
Amaranthus spinosus
Ambrosia
artemisiifolia
Ambrosia bidentata
Ambrosia psilostachya
Ambrosia trifida
Amelanchier arborea
Ammannia coccinea
Amorpha canescens
Amorpha fruticosa
Ampelopsis arborea
Ampelopsis cordata
Amphiachyris
dracunculoides
Amphicarpaea
bracteata
Anagallis minima
Andropogon gerardii
Andropogon ternarius
Andropogon
virginicus
Androsace
occidentalis
Anemone caroliniana
Antennaria neglecta
Antennaria
plantaginifolia
Apios americana
Apocynum
cannabinum
Aquilegia canadensis
Arabis canadensis
Arctium minus
Arenaria serpyllifolia
Argyrochosma
dealbata
Argythamnia
mercurialina
Arisaema dracontium
Arisaema triphyllum
Aristida longespica
Aristida oligantha
Aristida purpurascens
Aristida purpurea
Aristolochia
tomentosa
Arnoglossum
plantagineum
Artemisia
dracunculus??
Artemisia filifolia
Artemisia ludoviciana
Asclepias speciosa
Asclepias stenophylla
Asclepias sullivantii
Asclepias syriaca
Asclepias tuberosa
Asclepias verticillata
Asclepias viridiflora
Asclepias viridis
Asimina triloba
Asplenium
platyneuron
Asplenium
rhizophyllum
Asplenium
trichomanes
Aster anomalus
Aster drummondii
Aster ericoides
Aster falcatus
Aster lanceolatus
Aster oblongifolius
Aster oolentangiensis
Aster patens
Aster praealtus
Aster sericeus
Aster subulatus
Astragalus canadensis
Astragalus
crassicarpus
Astragalus
nuttallianus
Astranthium
integrifolium
Bacopa rotundifolia
Baptisia alba
Baptisia australis
Baptisia bracteata
Barbarea vulgaris
Bidens aristosa
Bidens bipinnata
Bidens frondosa
Boehmeria cylindrica
Bothriochloa
saccharoides
Botrychium
virginianum
Bouteloua
curtipendula
Bouteloua gracilis
Bouteloua hirsuta
Brickellia
eupatorioides
Bromus catharticus
Bromus japonicus
Bromus pubescens
Bromus tectorum
Buchloe dactyloides
Buchnera americana
Callirhoe alcaeoides
Callirhoe involucrata
Callitriche
heterophylla
Calylophus serrulatus
Calystegia sepium
Camassia scilloides
Cannabis sativa
Capsella bursapastoris
Cardamine hirsuta
Carex amphibola
Carex annectens
Carex austrina
Carex bicknellii
Carex blanda
Carex brevior
Carex bushii
Carex caroliniana
Carex cephalophora
Carex davisii
Carex emoryi
Carex festucacea
Carex fissa
Carex flaccosperma
Carex frankii
Carex gracilescens
Carex granularis
Carex gravida
Carex lanuginosa
Carex leavenworthii
Carex microdonta
Carex muehlenbergii
Carex nigromarginata
Carya cordiformis
Carya illinoinensis
Carya texana
Ceanothus
americanus
Celastrus scandens
Celtis laevigata
Celtis occidentalis
Cenchrus carolinianus
Cenchrus longispinus
Cephalanthus
occidentalis
Cerastium fontanum
Cerastium
glomeratum
Ceratophyllum
demersum
Cercis canadensis
Chaerophyllum
tainturieri
Chaetopappa
asteroides
Chamaecrista
fasciculata
Chamaecrista
nictitans
Chamaesyce maculata
Chamaesyce
missurica
Chamaesyce nutans
Chamaesyce prostrata
Chamaesyce serpens
Chasmanthium
latifolium
Cheilanthes lanosa
Chenopodium album
Chenopodium
ambrosioides
Chenopodium simplex
Chloris verticillata
Chrysopsis pilosa
Cicuta maculata
Cinna arundinacea
Cirsium altissimum
Cirsium undulatum
Cirsium vulgare
Cissus incisa
Claytonia virginica
Clematis pitcheri
Clematis virginiana
Cocculus carolinus
Coelorachis
cylindrica
Commelina erecta
Commelina virginica
Conium maculatum
Convolvulus arvensis
Convolvulus equitans
Conyza canadensis
Conyza ramosissima
Coreopsis grandiflora
Coreopsis tinctoria
Cornus amomum
Cornus drummondii
Corydalis crystallina
Corydalis micrantha
Crataegus crus-galli
Crotalaria sagittalis
Croton capitatus
Croton glandulosus
Croton
lindheimerianus
Croton
monanthogynus
Croton texensis
Croton willdenowii
Cryptotaenia
canadensis
Cucurbita foetidissima
Cuscuta cuspidata
Cuscuta indecora
Cuscuta pentagona
Cycloloma
atriplicifolium
Cynanchum laeve
Cynodon dactylon
Cyperus acuminatus
Cyperus bipartitus
Cyperus echinatus
Cyperus erythrorhizos
Cyperus lupulinus
Cyperus
pseudovegetus
Cyperus setigerus
Cyperus squarrosus
Cyperus strigosus
Cystopteris protrusa
Cystopteris
tennesseensis
Dactylis glomerata
Dalea candida
Dalea multiflora
Dalea purpurea
Danthonia spicata
Dasistoma
macrophylla
Datura stramonium
Daucus carota
Daucus pusillus
Delphinium
carolinianum
Descurania pinnata
Descurania sophia
Desmanthus
illinoensis
Desmanthus
leptolobus
Desmodium canescens
Desmodium ciliare
Desmodium
cuspidatum
Desmodium
glutinosum
Desmodium illinoense
Desmodium
marilandicum
Desmodium obtusum
Desmodium
paniculatum
Desmodium
pauciflorum
Desmodium
sessilifolium
Dianthus armeria
Diarrhena obovata
Dicentra cucullaria
Dichanthelium
acuminatum
Dichanthelium boscii
Dichanthelium
clandestinum
Dichanthelium
linearifolium
Dichanthelium
malacophyllum
Dichanthelium
oligosanthes
Dichanthelium
scoparium
Dichanthelium
sphaerocarpon
Dicliptera brachiata
Digitaria cognata
Digitaria ischaemum
Digitaria sanguinalis
Diodia teres
Diospyros virginiana
Draba brachycarpa
Draba cuneifolia
Dracopis
amplexicaulis
Dryopteris marginalis
Echinacea pallida
Echinochloa crusgalli
Echinochloa muricata
Eclipta prostrata
Eleocharis
montevidensis
Eleocharis obtusa
Eleocharis palustris
Eleocharis parvula
Elephantopus
carolinianus
Eleusine indica
Ellisia nyctelea
Elymus canadensis
Elymus hystrix
Elymus villosus
Elymus virginicus
Elytrigia repens
Equisetum hyemale
Equisetum laevigatum
Eragrostis capillaris
Eragrostis cilianensis
Eragrostis
curtipedicellata
Eragrostis hirsuta
Eragrostis intermedia
Eragrostis pectinacea
Eragrostis pilosa
Eragrostis spectabilis
Eragrostis trichodes
Erechtites
hieraciifolia
Erigeron annuus
Erigeron
philadelphicus
Erigeron strigosus
Erigeron tenuis
Eriochloa contracta
Eryngium
leavenworthii
Eryngium yuccifolium
Erythronium
mesochoreum
Escobaria
missouriensis
Eupatorium
altissimum
Eupatorium
coelestinum
Eupatorium
perfoliatum
Eupatorium serotinum
Euphorbia corollata
Euphorbia
cyathophora
Euphorbia dentata
Euphorbia
heterophylla
Euphorbia marginata
Euphorbia spathulata
Euthamia
gymnospermoides
Evax prolifera
Evolvulus nuttallianus
Evonymus
atropurpureus
Festuca arundinacea
Festuca pratensis
Festuca
subverticillata
Fimbristylis puberula
Fragaria virginiana
Fraxinus americana
Fraxinus
pennsylvanica
Fraxinus
quadrangulata
Froelichia floridana
Froelichia gracilis
Fuirena simplex
Galactia regularis
Galium aparine
Galium circaezans
Galium pilosum
Galium virgatum
Gamochaeta purpurea
Gaura coccinea
Gaura lindheimeri
Gaura longiflora
Gaura parviflora
Geranium
carolinianum
Geranium pusillum
Geum canadense
Glandularia
bipinnatifida
Glandularia
canadensis
Gleditsia triacanthos
Glycyrhiza lepidota
Gnaphalium
obtusifolium
Gossypianthus
lepidota
Gratiola neglecta
Grindelia lanceolata
Grindelia squarrosa
Gymnocladus dioicus
Gymnopogon
ambiguus
Hackelia virginiana
Hedeoma hispida
Hedyotis nigricans
Helenium amarum
Helenium autumnale
Helianthus annuus
Helianthus hirsutus
Helianthus
maximiliani
Helianthus mollis
Helianthus
pauciflorus
Helianthus salicifolius
Heliopsis
helianthoides
Heliotropium tenellum
Hibiscus trionum
Hieracium longipilum
Hordeum pusillum
Houstonia pusilla
Hymenopappus
scabiosaeus
Hypericum
drummondii
Hypericum
hypericoides
Hypericum
perforatum
Hypericum punctatum
Hypoxis hirsuta
Ipomoea pandurata
Iresine rhizomatosa
Iris variegata?
Iva annua
Juglans nigra
Juncus acuminatus
Juncus brachycarpus
Juncus diffusissimus
Juncus dudleyi
Juncus interior
Juncus marginatus
Juncus tenuis
Juncus torreyi
Juncus validus
Juniperus virginiana
Justicia americana
Koeleria macrantha
Krigia cespitosa
Kummerowia
stipulacea
Kummerowia striata
Lactuca floridana
Lactuca ludoviciana
Lactuca serriola
Lamium amplexicaule
Laportea canadensis
Lechea mucronata
Lechea tenuifolia
Leersia oryzoides
Leersia virginica
Lepidium densiflorum
Lepidium virginicum
Leptochloa
fascicularis
Lespedeza bicolor
Lespedeza capitata
Lespedeza cuneata
Lespedeza intermedia
Lespedeza
procumbens
Lespedeza repens
Lespedeza stuevei
Lespedeza violacea
Lespedeza virginica
Leucanthemum
vulgare
Leucospora multifida
Liatris aspera
Liatris punctata
Liatris pycnostachya
Lindernia dubia
Linum medium
Linum rigidum
Linum sulcatum
Lithospermum incisum
Lobelia cardinalis
Lolium perenne
Lomatium
foeniculaceum
Lotus unifoliolatus
Ludwigia alternifolia
Ludwigia palustris
Ludwigia peploides
Luzula bulbosa
Lycopus americanus
Lythrum alatum
Maclura pomifera
Malva rotundifolia
Malvastrum hispidum
Marrubium vulgare
Marsilea vestita
Matelea gonocarpos
Medicago lupulina
Medicago minima
Medicago sativa
Melica nitens
Melilotus officinalis
Melothria pendula
Menispermum
canadense
Mentzelia
oligosperma
Mimosa quadrivalvis
Mimulus alatus
Minuartia patula
Mirabilis albida
Mirabilis nyctaginea
Mollugo verticillata
Monarda citriodora
Monarda fistulosa
Monolepis nuttalliana
Morus alba
Morus rubra
Muhlenbergia bushii
Muhlenbergia
capillaris
Muhlenbergia
schreberi
Muhlenbergia
sobolifera
Myosotis verna
Myriophyllum
pinnatum
Najas guadalupensis
Nasella leucotricha
Nelumbo lutea
Nemastylis
geminiflora
Nepeta cataria
Nothoscordum bivalve
Nuttallanthus
canadensis
Oenothera biennis
Oenothera laciniata
Oenothera linifolia
Oenothera speciosa
Oenothera triloba
Oenothera villosa
Onosmodium molle
Ophioglossum
engelmannii
Ophioglossum
vulgatum
Opuntia macrorhiza
Osmorhiza longistylis
Ostrya virginiana
Oxalis dillenii
Oxalis stricta
Oxalis violacea
Panicum anceps
Panicum capillare
Panicum
dichotomiflorum
Panicum flexile
Panicum obtusum
Panicum rigidulum
Panicum virgatum
Parietaria
pensylvanica
Paronychia fastigiata
Parthenium
hysterophorus
Parthenium
integrifolium
Parthenocissus
quinquefolia
Pascopyrum smithii
Paspalum floridanum
Paspalum laeve
Paspalum pubiflorum
Paspalum setaceum
Passiflora incarnata
Pedicularis
canadensis
Pediomelum
argophyllum
Pediomelum
esculentum
Pediomelum
linearifolium
Pellaea atropurpurea
Peltandra virginica
Penstemon cobaea
Penstemon tubiflorus
Penthorum sedoides
Perideridia americana
Phalaris canariensis
Phalaris caroliniana
Phleum pratense
Phryma leptostachya
Phyla cuneifolia
Phyla lanceolata
Physalis angulata
Physalis longifolia
Physalis pubescens
Physalis pumila
Physalis virginiana
Phytolacca americana
Pilea pumila
Plantago aristata
Plantago lanceolata
Plantago major
Plantago patagonica
Plantago pusilla
Plantago rugelii
Plantago virginica
Platanus occidentalis
Pleopeltis
polypodioides
Poa annua
Poa arida
Poa compressa
Poa pratensis
Poa sylvestris
Polanisia dodecandra
Polygala incarnata
Polygala verticillata
Polygonatum biflorum
Polygonum
arenastrum
Polygonum aviculare
Polygonum
convolvulus
Polygonum
hydropiper
Polygonum
hydropiperoides
Polygonum
lapathifolium
Polygonum
pensylvanicum
Polygonum persicaria
Polygonum punctatum
Polygonum
ramosissimum
Polygonum scandens
Polygonum tenue
Polygonum
virginianum
Polytaenia nuttallii
Populus deltoides
Portulaca oleracea
Portulaca pilosa
Potamogeton foliosus
Potamogeton nodosus
Potentilla arguta
Potentilla simplex
Prenanthes aspera
Prionopsis ciliata
Proboscidea
louisianica
Prunella vulgaris
Prunus americana
Prunus angustifolia
Prunus mexicana
Prunus serotina
Psoralidium
tenuiflorum
Ptelea trifoliata
Ptilimnium nuttallii
Pycnanthemum
tenuifolium
Pyrrhopappus
carolinianus
Pyrrhopappus
grandiflorus
Pyrus communis
Quercus macrocarpa
Quercus marilandica
Quercus
muehlenbergii
Quercus prinoides
Quercus shumardii
Quercus stellata
Quercus velutina
Ranunculus abortivus
Ranunculus hispidus
Ranunculus sceleratus
Ratibida columnifera
Rhus aromatica
Rhus copallinum
Rhus glabra
Rhynchospora
glomerata
Rhynchospora harveyi
Ribes aureum
Rorippa sessiliflora
Rosa carolina
Rosa multiflora
Rubus allegheniensis
Rubus flagellaris
Rubus occidentalis
Rubus ostryifolius
Rudbeckia hirta
Ruellia humilis
Ruellia strepens
Rumex altissimus
Rumex crispus
Rumex hastatulus
Sabatia campestris
Sagina decumbens
Sagittaria latifolia
Salix caroliniana
Salix nigra
Salsola kali
Salvia azurea
Sambucus canadensis
Samolus valerandi
Sanicula canadensis
Sanicula odorata
Sapindus saponaria
Schedonnardus
paniculatus
Schizachyrium
scoparium
Scirpus americanus
Scirpus atrovirens
Scirpus koilolepis
Scirpus pendulus
Scleria pauciflora
Sclerochloa dura
Scrophularia
marilandica
Scutellaria lateriflora
Scutellaria ovata
Scutellaria parvula
Sedum nuttallianum
Selaginella rupestris
Senecio plattensis
Senna marilandica
Setaria glauca
Setaria parviflora
Setaria viridis
Sibara virginica
Sida spinosa
Sideroxylon
lanuginosum
Silene antirrhina
Silene stellata
Silphium integrifolium
Silphium laciniatum
Sisymbrium officinale
Sisyrinchium
campestre
Smilax bona-nox
Smilax herbacea
Smilax tamnoides
Solanum americanum
Solanum carolinense
Solanum dimidiatum
Solanum
elaegnifolium
Solanum ptychanthum
Solanum rostratum
Solanum sarrachoides
Solidago canadensis
Solidago gigantea
Solidago
missouriensis
Solidago nemoralis
Solidago radula
Solidago rigida
Solidago ulmifolia
Sonchus asper
Sorghastrum nutans
Sorghum halepense
Spartina pectinata
Spermolepis
divaricata
Spermolepis inermis
Sphenopholis obtusata
Spiranthes cernua
Spiranthes lacera
Spiranthes vernalis
Sporobolus
compositus
Sporobolus
cryptandrus
Sporobolus
heterolepis
Sporobolus
pyramidatus
Sporobolus
vaginiflorus
Stachys tenuifolia
Staphylea trifolia
Stellaria media
Stipa comata
Stipa curtiseta
Strophostyles helvula
Strophostyles
leiosperma
Stylosanthes biflora
Symphoricarpos
orbiculatus
Talinum calycinum
Talinum parviflorum
Taraxacum
laevigatum
Taraxacum officinale
Tephrosia virginiana
Teucrium canadense
Thalictrum
dasycarpum
Thelesperma filifolium
Thlaspi arvense
Torilis arvensis
Toxicodendron
radicans
Tradescantia
occidentalis
Tradescantia ohiensis
Tradescantia tharpii
Tragia betonicifolia
Tragopogon dubius
Tribulus terrestris
Tridens flavus
Tridens strictus
Trifolium arvense
Trifolium campestre
Trifolium pratense
Trifolium repens
Trifolium vesiculosum
Triodanis leptocarpa
Triodanis perfoliata
Tripsacum dactyloides
Typha domingensis
Typha latifolia
Ulmus americana
Ulmus rubra
Utricularia gibba
Valerianella radiata
Verbascum thapsus
Verbena bonariensis
Verbena bracteata
Verbena canescens
Verbena hastata
Verbena simplex
Verbena stricta
Verbena urticifolia
Verbesina alternifolia
Verbesina encelioides
Verbesina virginica
Vernonia arkansana
Vernonia baldwinii
Veronica arvensis
Veronica peregrina
Viburnum rufidulum
Vicia sativa
Viola bicolor
Viola pedata
Viola sororia
Vitis riparia
Vulpia octoflora
Woodsia obtusa
Xanthisma texanum
Acidity, sand
Tallgrass
Prairie
crosstimbers
Sandstone
Sandstone outcrops
savannas
Shortgrass prairie /
limestone outcrops
Forest cover
Limestone or
bottomland
mesic forest
PVBE schema
Sample plants
Extract nucleic
acids and
sequence
Mine data and
interpret
Current status
Propose and
test ecological
hypotheses
Plants are sampled without regard for symptoms
PVBE schema
Broad survey
“target plant species”
Ambrosia psilostachya
Asclepias viridis
Panicum virgatum
Sorghastrum nutans
Vernonia baldwinii
Ruellia humilis
Extract nucleic
acids and
sequence
Mine data and
interpret
Propose and
test ecological
hypotheses
Selected target species
Ambrosia psilostachya, western ragweed
Sorghastrum nutans, Indiangrass
Asclepias viridis, antelope-horn milkweed
Ruellia humilis, wild/prairie petunia
Panicum virgatum, switchgrass
Vernonia baldwini, western ironweed
Vegetation
monitoring
plots
10m
PVBE schema
Broad survey
Extract
nucleic
Virus-like
acids
and
particle
sequence
isolation
Mine data and
interpret
“target plant species”
Ambrosia psilostachya
Asclepias viridis
Panicum virgatum
Sorghastrum nutans
Vernonia baldwinii
Ruellia humilis
dsRNA isolation with
454 pyrosequencing
Propose and
test ecological
hypotheses
PVBE schema
Broad survey
Extract nucleic
Virus-like
acids and
particle
sequence
isolation
Mine data and
interpret
Propose and
test ecological
hypotheses
“target plant species”
Ambrosia psilostachya
Asclepias viridis
Panicum virgatum
Sorghastrum nutans
Vernonia baldwinii
Ruellia humilis
dsRNA isolation with
454 pyrosequencing
Intense study of
Asclepias viridis
by RT-PCR
Laboratory method for double-stranded RNA purification
Mix vigorously
to form
emulsion
Young leaves (5 g)
Centrifuge
Grind in
liquid
Transfer into 50 ml tube
nitrogen
containing 10 ml
extraction buffer and 10
ml Ph:Ch
Transfer top phase into
new tube
Final aqueous
phase
Repeat Ph:Ch extraction
Decant and
resuspend in 0,1
mM EDTA / 0.3
M NaOAC
Centrifuge
to pellet
dsRNA
Wash in 6 time
with application
buffer
Precipitate with
NaOAc and
EtOH overnight
at -20ْ C
Transfer eluate to
a 15 ml tube
1
Transfer to a
microcentrifuge tube and
fill with cold ethanol to
reprecipitate
Add absolute
proof ethanol
(16,5% of
aqueous
volume)
Add elution
buffer
Resuspend in 50 mkl 0.1 mM
EDTA
2 3 4 Lad 1 kb
bp
12,2
2
1,6
Check the dsRNA by
electrophoresis on a
1.5% agarose gel in
0.5X TBE II
Pass through
enocolumn
containing
CF11powder
cellulose binding
dsRNA (if ethanol
concentration is
16,5%)
Vernonia baldwinii (line 1) and
Flavoparmelia sp. (line 4) have no dsRNA.
Total NA (for bar
coding and making
hybridization target)
Buffers
Application buffer:
Extraction buffer:
0.1 M NaCl
0.1 M NaCl
50 mM Tris, pH8
50 mV Tris, pH 8
0.5 mV EDTA, pH8
1 mV EDNA, pH 8
16.5% Ethanol
1% SDS
Elution buffer:
0.1 M NaCl
50 mV EDTA, pH 8
1
506,5
396
344
298
Ambrosia psilostachya (line 2) and Parmelia
sp1. (line 3) show bands for dsRNA
Protocol for ds RNA isolation adopted from M. Roossinck, 2005
454 sequencing
Sophisticated, high-throughput system –
but easier said than done
Preliminary (2006) Results
592 specimens analyzed
296 plant species in 307 genera and 91 families
308 specimens are positive for dsRNA (i.e. probable viruses)
2007-2008 Results
not yet tabulated on project database
high rate of novelties detected by main methods
many ‘fungal’ viruses
BLAST results:
One virus is
predominantly found in
Asclepias viridis that
appears that is distinct
from, but has high
similarity to,
Kennedya yellow
mosaic tymovirus
http://virology.wisc.edu
From ICTVdB
Tymovirus
Icosahedral virion
ssRNA
Genome 6000-7000nt
Infects dicots
Symptoms none to severe;
appear in leaves
Transmitted by beetles
Virus-like particle
Asclepias viridis
Other species
Negative,
74
Positive, 9
Negative,
7
Positive, 5
dsRNA (454)
Negative,
612
Positive,
10
Negative,
7
Positive, 8
Tymovirus in Asclepias viridis
VLP
Negative
No data
dsRNA
Negative No data Positive Grand Total
3
3
1
7
4
Positive
Grand Total
7
5
9
7
3
10
10
9
26
Species
Ambrosia psilostachya
Amorpha fruticosa
Asclepias viridis
Asplenium rhizophyllum
Cephalanthus occidentalis
Croton monanthogynus
Cyperus acuminatus
Desmanthus illinoensis
Lythrum alatum
Panicum virgatum
Ruellia humilis
Solanum rostratum
Vernonia baldwinii
# of positives
1
1
36
1
1
1
1
1
1
2
2
1
1
Asclepias viridis intensely sampled from 3
plots, (and sequenced from selections of two)
Plot 208 (Swale)
2005, 2006, 2007
20
(-, -,+)
4
7
4
5
4
3
4
4
10
4
0
3
7
3
8
4
Distance (m)
15
4
6
4
2
4
1
5
4
8
1
3
9
6
9
3
1
81
1
0
-15
1
2
7
-10
-5
2
5
3
2
5
0
4
9
2
7
2
6
3
3
3
6
3
4
1
8
5
2
1
5
1
6
1
4
1
30
2
3 0
8
3
1
2
0
9
-5
2
0
1
7
5
2
4
2
2
2
1
1
9
2
3
10
15
4
7
4
6
4
5
Plot 307 (Sand creek)
(+,+,+)
-15
4
3
4
3
1
93
8
Distance (m)
Distance (m)
4
8
4
7
4
4
4
5
4
206
4
2
4
1
7
1
0
-25
4
-15
3
3 10
8
Distance (m)
5
-35
1
1
4
6
9
7
-25
3
1
-15
5
1
5
40
9
1
4
2
0
1
3
0
2
2 8
3
9
0 3
3
23
3
4
3
6
1
7
5
2
5
buffer
-5
2
7
3
1
3
5
10m
-15
2
1
1
9
1
6
1
5
-5
2
6
2
6
2
5
1
4
1
5
0
5
2
7 2
8
2
3
9
1
3
4
2
1
12 5
61
2
2 0
7
0
1
9
15
-5
-10
-15
Distance (m)
buffer
1
0
1
3
0
3
32
3
3
7
2
25
m
4
9
15
5
30
-5
40
m
4
0
3
9
4
0
2
Probability of equal
infection in each transect
<0.01
4 35
2
5
8
3
60m 50m
4
3
Plot 343 (Stadia)
(+,-,+)
1
1
1
2
1
6
70
m
40
3
7
9
25
1
2
45
4
4
3
-10
5
8
4
8
20
2
2
1
8
15
2
4
2
3
3
5
3
6
30
m
Red- Tymovirus infection
Black – no Tymovirus
2
3
2
4
25
Presence of tymovirus in Asclepias viridis over time and space
Plot
208
2005
Negative
2006
Negative
2007
343 remote
343 (+,-)
Positive (36%,18/50)
307 (+,+)
307
Positive
Positive
Positive
(66%, 33/50)
343
Positive
Negative
Positive
(82%, 41/51)
208 (-, -)
208 remote
RT-PCR results for Asclepias viridis
positive
negative
Source of samples
2007 plots
2008 plots
2008 TGPP
2008 not TGPP
0%
20%
40%
60%
% positive
80%
100%
Tymovirus presence in A. viridis tubers in plot 208 (RT-PCR)
Plot 208 (Swale)
(+/+)
(Plant/ tuber)
(-/-)
20
47
45
48
43
44
15
46
42
41
10
40
Distance (m)
38
39
37
4
5
1
18 20
5
2
6
9
3
11
10
8
16
50
12
7
49
15
17
14
13
21
22 24
19
23
0
-10
-5
(-/-)
25
26
32
27
28
31 0
30
29
5
-5
33
35
36
34
buffer
-15
-10
-15
Distance (m)
buffer
10
(+/+)
15
20
Most viruses have relatively low proportion of reads in VLPs
Exception: a new tymovirus, but only in Asclepias viridis.
Virus Genus
Plant species
# plant
specimens
# Reads/
specimen
Read
%
Ascovirus
Artemisia ludoviciana
1
3
2.0
Badnavirus
Ambrosia psilostachya
2
10
1.3
Sorghastrum nutans
1
3
1.0
Chrysovirus
Asplenium rhizophyllum
1
27
11.9
Comovirus
Asclepias viridis
1
4
0.7
Flexivirus
Ambrosia psilostachya
2
97
25.4
Metapneumovirus
Desmanthus illinoensis
1
3
1.5
Coccolithovirus
Festuca subverticillata
1
2
0.8
Sorghastrum nutans
1
2
0.8
Ranavirus
Panicum virgatum
1
2
0.7
lambda-like phage
Ambrosia psilostachya
1
2
0.4
Tymovirus
Amorpha fruticosa
1
2
0.7
Asclepias viridis
10
315
85.7
Asplenium rhizophyllum
1
33
14.6
Cephalanthus occidentalis
1
4
1.1
Desmanthus illinoensis
1
7
3.5
Interpretation:
high titers
Tymovirus reads from Virus-like Particles (positives only)
Few reads mean that the genome is
poorly sampled
RT-PCR
from
capsid
Non-Asclepias
Asclepias
only
Interspecific
comparison
Capsid
comparison
Analysis
Principal Components Analysis (PCA), with
each position represented by four dummy
variables (the four nucleotides).
AACAAGGACTACGGAGAACCCGTGGAA
The realized dimensionality is much less
than sequence length x 4, because most
positions are not variable.
Common to all PCA results
~50% of variation can be explained by the
first four axes
Axes higher than 2 do not add anything to
the interpretation
3.0
Tymovirus from Asclepias
viridis only
A group from plot 343 in two
different years
PCA Axis 2
A group from many locations,
including most of plot 307 but
one from plot 343, and using
three methods
SAMPLES
vlp
ds
7.307
-3.0
7.343
8.343
-1.5
2.0
PCA Axis 1
Two outlier groups
3.0
Capsid comparison
Location effects still evident
PCA Axis 2
Most non-Asclepias samples
are similar to those from plot
307 Asclepias.
SAMPLES
Ambrosia
Amorpha
Ruellia
av
-3.0
307
343
-1.5
2.0
PCA Axis 1
4.0
Interspecific comparisons
PCA Axis 2
Some Non-Asclepias might be
outliers, but this might be a
methodological artifact, and the
variation within Asclepias is huge
relative to interspecific variation.
SAMPLES
Asplenium
Ambrosia
Desmanthus
Panicum
-2.0
Artemisia
Asclepias viridis
-1.0
PCA Axis 1
3.0
•There are relatively few wellsupported clades
•Non-Asclepias samples are all
nested within well-supported
clades of Asclepias samples
•Does this imply other nonAsclepias hosts are sinks?
Outgroup
Confusions:
Why does tymovirus reach high level only in Asclepias?
Are non-Asclepias ‘sink’ populations? (note: there are several orders of
magnitude more non-Asclepias than Asclepias in the TGPP.)
Are there two different strains?
How does transmission within and between species occur? (milkweed
beetles are specialists)
Are the sequence differences phylogenetic?
Are there relevant structural differences in the RNA?
Is the relationship parasitic, commensal, or mutualistic?