A generalist tymovirus of the tallgrass prairie Michael Palmer 1 Andrew Doust 1 Rick Nelson 2 Min...

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

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 bursa-pastoris 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 crus-galli 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

Plants of the Tallgrass Prairie Preserve

Forest cover

Aci

dity

, sa

nd

Sandstone

Limestone or

bottomland

Tallgrass Prairie

mesic forest

savannas

Shortgrass prairie / limestone outcrops

crosstimberscrosstimbersSandstone outcrops

PVBE schema

Sample plants

Extract nucleic acids and sequence

Mine data and interpret

Propose and test ecological

hypotheses

Current status

Plants are sampled without regard for symptoms

PVBE schema

Broad survey




hypotheses

“target plant species”

Ambrosia psilostachyaAsclepias viridis

Panicum virgatumSorghastrum nutansVernonia baldwinii

Ruellia humilis

Ambrosia psilostachya, western ragweed

Asclepias viridis, antelope-horn milkweed Panicum virgatum, switchgrass

Sorghastrum nutans, Indiangrass Ruellia humilis, wild/prairie petunia Vernonia baldwini, western ironweed

Selected target speciesSelected target species

10m10m

Vegetation monitoring

plots

PVBE schema

Broad survey




hypotheses




Ruellia humilis

dsRNA isolation with 454 pyrosequencing

Virus-like particle isolation

PVBE schema

Broad survey




hypotheses




Ruellia humilis

dsRNA isolation with 454 pyrosequencing

Virus-like particle isolation

Intense study of Asclepias viridis

by RT-PCR

Young leaves (5 g)

Grind in liquid

nitrogenTransfer into 50 ml tube

containing 10 ml extraction buffer and 10

ml Ph:Ch

Mix vigorously to form

emulsionCentrifuge

Transfer top phase into new tube

Repeat Ph:Ch extraction

Final aqueous phase

Total NA (for bar coding and making

hybridization target)

Add absolute proof ethanol

(16,5% of aqueous volume)

Pass through enocolumn containing

CF11powder cellulose binding

dsRNA (if ethanol concentration is

16,5%)

Wash in 6 time with application

buffer

Add elution buffer

Transfer eluate to a 15 ml tube

Precipitate with NaOAc and EtOH overnight at -20 ْ C

Centrifuge to pellet dsRNA

Decant and resuspend in 0,1 mM EDTA / 0.3 M NaOAC

Transfer to a microcentrifuge tube and fill with cold ethanol to

reprecipitate

Resuspend in 50 mkl 0.1 mM EDTA

Check the dsRNA by electrophoresis on a 1.5% agarose gel in

0.5X TBE II

1 2 3 4 Lad 1 kb

506,5

1

1,6212,2

396

Extraction buffer: 0.1 M NaCl50 mV Tris, pH 81 mV EDNA, pH 81% SDS

Application buffer:0.1 M NaCl50 mM Tris, pH80.5 mV EDTA, pH816.5% Ethanol

Elution buffer:0.1 M NaCl50 mV EDTA, pH 8

Buffers

Laboratory method for double-stranded RNA purificationLaboratory method for double-stranded RNA purification

344298

bp

Vernonia baldwinii (line 1) and Flavoparmelia sp. (line 4) have no dsRNA.

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) ResultsPreliminary (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 Results2007-2008 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

From ICTVdB

Tymovirus

http://virology.wisc.edu

Icosahedral virion

ssRNA

Genome 6000-7000nt

Infects dicots

Symptoms none to severe; appear in leaves

Transmitted by beetles

Positive, 9Negative,

7

Asclepias viridis Other speciesV

irus-

like

part

icle

dsR

NA

(45

4)

Positive, 5

Negative, 74

Positive, 10

Negative, 7

Positive, 8

Negative, 612

Tymovirus in Asclepias viridis

Negative No data Positive Grand TotalNegative 3 3 1 7

No data 4 5 9

Positive 7 3 10

Grand Total 7 10 9 26

VLP

dsRNA

Species # of positivesAmbrosia psilostachya 1Amorpha fruticosa 1Asclepias viridis 36Asplenium rhizophyllum 1Cephalanthus occidentalis 1Croton monanthogynus 1Cyperus acuminatus 1Desmanthus illinoensis 1Lythrum alatum 1Panicum virgatum 2Ruellia humilis 2Solanum rostratum 1Vernonia baldwinii 1

10m

bu

ffer

252

627

282

9 31

30 3

2333

4

35

-5

buffer

1

2

34

5

6

7

8

9

10

11

12

14

13 1

5

16

17

18

19

20 2

122 2

3

24

36

37

38

403

9

41

42

43

45

464

4

47

48

49

50

51

Plot 343 (Stadia)

-15

0

5

10

15

20

25

-35 -25 -15 -5 5 15

Distance (m)

Dis

tan

ce (

m)

Red- Tymovirus infectionBlack – no Tymovirus

Plot 307 (Sand creek)

-15

-10

-5

0

35

40

45

60m -25 -15 -5 5 15

Distance (m)

Dis

tan

ce (

m)

14

15

1617

19

20

21 2

250 2

423

491

23

4

5698

710

40m

1213

11

50m

25

26 2

7 282931

323

3

35

36

30m

70m

34

37

38

39

41

42

43

40

44

45

30

25m

46

47

48

25

30

Plot 208 (Swale)

12

1

32

45

6

7811

10

913

14

15

16

17

18

20

21

22

23

19

24

49

50

37

38

39

40

41

42

46

434

4

45

48

47

28

32

353

634

33

31

29

30

27

25 2

6

-15

-10

-5

0

5

10

15

20

-15 -10 -5 0 5 10 15 20

Distance (m)

Dis

tan

ce (

m)

(+,+,+)

(+,-,+)

(-, -,+)2005, 2006, 2007

Probability of equal infection in each transect <0.01

Asclepias viridis intensely sampled from 3 plots, (and sequenced from selections of two)

Plot 2005 2006 2007

208 Negative NegativePositive

(36%,18/50)

307 Positive PositivePositive

(66%, 33/50)

343 Positive NegativePositive

(82%, 41/51)

Presence of tymovirus in Asclepias viridis over time and space

343 (+,-)

307 (+,+)

208 (-, -)

208 remote

343 remote

RT-PCR results for Asclepias viridis

0% 20% 40% 60% 80% 100%

2008 not TGPP

2008 TGPP

2008 plots

2007 plots

So

urc

e o

f sa

mp

les

% positive

positive negative

Plot 208 (Swale)

Dis

tan

ce (

m)

buffe

r

12

1

32

45

6

7811

109

1314

1516 17

18 20

21

22

23

1924

49

50

37

383940

41

42

4643

44

45

4847

28

32

3536 34

33

31

2930

2725

26

-15

-10

-5

0

5

10

15

20

-15 -10 -5 0 5 10 15 20

Distance (m)

buffer

Tymovirus presence in A. viridis tubers in plot 208 (RT-PCR)

(-/-)

(-/-)

(+/+)

(+/+)

(Plant/ tuber)

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

Most viruses have relatively low proportion of reads in VLPs

Exception: a new tymovirus, but only in Asclepias viridis.

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

-1.5 2.0

-3.0

3.0

SAMPLES

vlp

ds

7.307

7.343

8.343

Tymovirus from Asclepias viridis only

A group from plot 343 in two different years

A group from many locations, including most of plot 307 but one from plot 343, and using three methods

Two outlier groups

PCA Axis 1

PC

A A

xis

2

-1.5 2.0

-3.0

3.0

SAMPLES

Ambrosia

Amorpha

Ruellia

av

307

343

Capsid comparison

PCA Axis 1

PC

A A

xis

2 Plo

t 343

gro

up

Plot

307

gro

upLocation effects still evident

Most non-Asclepias samples are similar to those from plot 307 Asclepias.

-1.0 3.0

-2.0

4.0

SAMPLES

Asplenium

Ambrosia

Desmanthus

Panicum

Artemisia

Asclepias viridis

PCA Axis 1

PC

A A

xis

2Interspecific comparisons

Some Non-Asclepias might be outliers, but this might be a methodological artifact, and the variation within Asclepias is huge relative to interspecific variation.

Outgroup

•There are relatively few well-supported clades

•Non-Asclepias samples are all nested within well-supported clades of Asclepias samples

•Does this imply other non-Asclepias hosts are sinks?

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?

A generalist tymovirus of the tallgrass prairie Michael Palmer 1 Andrew Doust 1 Rick Nelson 2 Min...

Documents

Transcript of A generalist tymovirus of the tallgrass prairie Michael Palmer 1 Andrew Doust 1 Rick Nelson 2 Min...