Using barcoding to delineate subtribe, genus and species:
Agrostidinae and Lachnagrostis (Poaceae)
AJ Brown, HB Cross, DJ Murphy, DJ Cantrill and AJ Lowe
The University of Adelaide, National Herbarium of Victoria,
State Herbarium of South Australia
PhD Thesis: Speciation mechanisms in
Australasian Lachnagrostis
Acknowledgments:
 Plant collection access, Herbarium loans and approvals for
DNA testing: staff of
- MEL, AD, HO, PERTH, CANB, BRI, L, AK, CHR, WELT, PRE
 DNA analysis: staff of
– The International Barcode of Life project (iBOL) – Biodiversity
Institute of Ontario (BIO)
– The University of Adelaide and the National Herbarium of Victoria GrassBol project of the Commonwealth Environment Research
Facilities (CERF), Taxonomy Research & Information Network
(TRIN)
 Assistance with data analysis, sequence alignment and
Bayesian Inference (MrBayes): Stuart Gardner and Anna Syme
 Assistance with graphical presentations: Teresa Lebel and
Stuart Gardner
Lachnagrostis in the scheme of things
1970-80’s
1990’s
2000’s
Family:
Poaceae
Poaceae
Poaceae
Subfamily:
Pooideae
Pooideae
Pooideae
Supertribe:
Tribe:
Subtribe:
Poodae
Agrostideae
Aveneae
Alopecurinae
Poeae
Agrostidinae
What’s in a Name?
Common Blown-grass, New Zealand Wind-grass
1786 Avena filiformis
1791 Agrostis avenacea
1809 Agrostis retrofracta
1810 Agrostis debilis
1812 Vilfa retrofracta
1820 Lachnagrostis filiformis
1829 Deyeuxia retrofracta
1833 Calamagrostis retrofracta
1854 Agrostis ligulata
1878 Deyeuxia forsteri
1915 Deyeuxia filiformis
1917 Calamagrostis avenacea
What’s in a Name?
Common or Pacific Blown-grass, New Zealand Wind-grass
1786 Avena filiformis
1791 Agrostis avenacea 1940’s
1809 Agrostis retrofracta
1810 Agrostis debilis
1812 Vilfa retrofracta
1820 Lachnagrostis filiformis
1829 Deyeuxia retrofracta
1833 Calamagrostis retrofracta
1854 Agrostis ligulata
1878 Deyeuxia forsteri
1915 Deyeuxia filiformis
1917 Calamagrostis avenacea
What’s in a Name?
Common or Pacific Blown-grass, New Zealand Wind-grass
1786 Avena filiformis
1791 Agrostis avenacea 1940’s
1809 Agrostis retrofracta
1810 Agrostis debilis
1812 Vilfa retrofracta
1820 Lachnagrostis filiformis NZ 1995, Australia 2001
1829 Deyeuxia retrofracta
1833 Calamagrostis retrofracta
1854 Agrostis ligulata
1878 Deyeuxia forsteri
1915 Deyeuxia filiformis
1917 Calamagrostis avenacea
What’s in a name?
introduced genera
Agrostis
Ammophila
Calamagrostis Gastridium
Polypogon
1753
1809
1763
1812
1789
Agraulus
Arundo
Arundo
Agrostis
Agrostis
Agrestis
Calamagrostis
Lachnagrostis Alopecurus
Apera
Psamma
Milium
Cinna
Decondolia
Milium
Phalaris
Polypogon
Sporobolus
Trichodium
Vilfa
Chaetotropis
What’s in a name?
endemic genera
Deyeuxia
Dichelachne
Echinopogon
Lachnagrostis Pentapogon
1812
1833
1812
1820
1810
Agrostis
Agrostis
Agrostis
Agrostis
Stipa
Avena
Alopecurus
Alopecurus
Avena
Bromidium
Anthoxanthum Cinna
Calamagrostis
Calamagrostis Cinna
Deyeuxia
Cinna
Deyeuxia
Dichelachne
Dichelachne
Muhlenbergia
Didymochaeta
Echinopogon
Stipa
Vilfa
Pentapogon
Vilfa
Vilfa
Agrostidinae genera
Lachnagrostis
Agrostis
Calamagrostis
Deyeuxia
Polypogon
Chaetotropis
Ammophila
Dichelachne
Echinopogon
Pentapogon
Gastridium
Triplachne
BARCODING
•
To confirm Lachnagrostis as a separate genus to Agrostis
•
To delineate the species of Lachnagrostis – (particularly Sth
African candidates)
•
To confirm suspected new ‘pseudocryptic’ but undescribed taxa
– (plasticity or genetic basis?)
•
To establish a phylogeny for Lachnagrostis – (to examine
speciation mechanisms)
•
To delineate the genera of Agrostidinae (particularly the
Australasian taxa)
•
To confirm the subtribe status of Agrostidinae within tribe
Poeae
Agrostidinae DNA analysis
 Sampled over 700 collections
 Representing over 220 taxa
 Analysed for cpDNA: rbcL and matK; nDNA: ITS
rbcL
matK
ITS
Number attempted
582
616
304
Number successful
426
185
152
% success
73
30
50
GenBank sequences
19
67
226*
* 130 Agrostis, 67 Calamagrostis
35
30
Number of samples
Specimen
age and
success
25
20
ITS success
15
ITS failure
10
5
0
1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000
Decade
100
Percent of total samples
90
80
•
70
60
50
ITS success
40
ITS failure
30
20
•
•
•
10
0
1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000
Decade
Obvious degradation
trend with time
No definitive cut-off age
20-40% success even
after 40 years
Preservation quality
most important
Sampled Plant Part
part
leaf
result
ITS success
ITS fail
inflorescence ITS success
ITS fail
number
40
21
68
32
percent
67
34
68
32
rbcL BOLD TaxonID Tree
rbcL BOLD TaxonID Tree
matK tree (MrBayes)
Lachnagrostis
Polypogon
Agrostis
Lachnagrostis
Agrostis
Podagrostis
Calamagrostis,
Ammophila
Deyeuxia,
Dichelachne,
Echinopogon,
Pentapogon
outgroup
matK tree – Agrostis and Lachnagrostis
0.63
Australasian Lachnagrostis
0.69
Nth hemisphere Polypogon
0.92
0.91
Nth hemisphere Agrostis (incl. A. capillaris)
Polypogon tenellus (WA)
0.99
Sth African Agrostis (syn. Lachnagrostis)
0.88
Lachnagrostis punicea ssp. punicea (SE Aust)
Lachnagrostis punicea ssp. filifolia (SE Aust)
0.92
1.00
Australasian Agrostis &
Nth hemisphere Agrostis
(incl. A. stolonifera)
matK tree – Calamagrostis and Ammophila
0.86
0.92
0.72
Podagrostis
Calamagrostis
0.70
Calamagrostis & Gastridium phleoides
0.92
Calamagrostis epigejos
Calamagrostis purpurea
Calamagrostis canadensis
Ammophila arenaria
Calamagrostis sesquiflora
Ammophila breviligluma
Calamagrostis lapponica
Calamagrostis rubescens
matK – Deyeuxia and Dichelachne
0.86
0.97
Deyeuxia densa
Echinopogon mckiei & E. intermedius
Deyeuxia quadriseta
Deyeuxia monticola
Deyeuxia talariata
Dichelachne crinita
Dichelachne inaequiglumis
0.92
Dichelachne micrantha
Dichelachne rara
Echinopogon caespitosa
Pentapogon quadrifidus
Echinopogon cheelii
Deyeuxia frigida
Deyeuxia brachyanthera
Deyeuxia minor
Deyeuxia scaberula
Amphibromus recurvatus
Outgroup (Alopecurus,
Apera, Phleum)
0.92
ITS Tree (MrBayes)
0.91
Lachnagrostis (Aust & NZ)
1.00
0.98
0.97
Lachnagrostis collicola & L. morrisii (Tas)
0.99
Polypogon monspeliensis
Lachnagrostis punicea
0.64
1.00
Lachnagrostis (NZ)
& Agrostis
Lachnagrostis plebeia (WA)
1.00
Agrostis personata
0.96
Dichelachne & Deyeuxia
0.52
0.99
Calamagrostis
0.71
Deyeuxia talariata
Calamagrostis bolanderi
0.61
Calamagrostis canadensis
Calamagrostis deschampsioides
Gastridium phleoides
0.79
0.92
Ammophila & Calamagrostis epigejos
Outgroup (Briza media,
Poa, Puccinellia, Phalaris)
Briza minor
NZ Lachnagrostis ITS
Taxon
Distribution
Australasian clade New Zealand clade
L. ammobia
restricted
1
L. billardierei
widespread
4
L. elata
widespread
2
L. filiformis
widespread
1
L. glabra
restricted
1
1
L. littoralis ssp. littoralis widespread
5
1
L. littoralis ssp. salaria
restricted
3
L. leptostachys
restricted
3
L. lyallii
widespread
10
L. pilosa ssp. pilosa
widespread
8
L. striata
widespread
4
L. tenuis
restricted
2
L. uda
restricted
1
5
1
4
NZ Lachnagrostis ITS
Taxon
Distribution
Australasian clade New Zealand clade
L. ammobia
restricted
1
L. billardierei
widespread
4
L. elata
widespread
2
L. filiformis
widespread
1
L. glabra
restricted
1
?
?
1
5
?
?
1
L. littoralis ssp. littoralis widespread
5
1
L. littoralis ssp. salaria
restricted
3
L. leptostachys
restricted
3
L. lyallii
widespread
10
L. pilosa ssp. pilosa
widespread
8
L. striata
widespread
4
L. tenuis
restricted
2
L. uda
restricted
1
4
Additional genera required for ITS dataset
• Agrostis from Australia poorly represented though well
sampled
• Agrostis from Asia and South America not represented but
few sampled
• Agrostis/Lachnagrostis from Sth Africa poorly represented
• Some Australian Lachnagrostis spp. poorly represented
• Deyeuxia and Dichelachne from Australia under
represented though well sampled
• Polypogon tenellus, Pentapogon quadrifidis and
Echinopogon from Australia missing though well sampled
• Ammophila from Nth hemisphere with one representative
• Chaetotropis and Podagrostis from Americas and
Zingera from Russia not represented
Other genera can be sourced from GenBank though coverage is
incomplete and some not represented (e.g. Bromidium)
ITS - Neighbour Joining
ITS – Neighbour Joining
ITS – Neighbour Joining
ITS – Neighbour Joining
ITS – Neighbour Joining (Lachnagrostis)
Barcoding Results (to date)
Goal
Confirmation of Agrostidinae
Delineation of Agrostidinae genera
Confirmation of Lachnagrostis
Delineation of Lachnagrostis spp.
New Lachnagrostis species ID
Lachnagrostis phylogeny
rbcL
matK
ITS
Barcoding Results (to date)
Goal
Confirmation of Agrostidinae
rbcL
matK
ITS
P


Delineation of Agrostidinae genera
Confirmation of Lachnagrostis
Delineation of Lachnagrostis spp.
New Lachnagrostis species ID
Lachnagrostis phylogeny
• Partial resolution of and support for existing taxonomy
Barcoding Results (to date)
Goal
Confirmation of Agrostidinae
Delineation of Agrostidinae genera
rbcL
matK
ITS
P
P

P

P
Confirmation of Lachnagrostis
Delineation of Lachnagrostis spp.
New Lachnagrostis species ID
Lachnagrostis phylogeny
• Partial resolution of and support for existing taxonomy
Barcoding Results (to date)
Goal
Confirmation of Agrostidinae
Delineation of Agrostidinae genera
Confirmation of Lachnagrostis
rbcL
matK
ITS
P
P
P

P


P
P
Delineation of Lachnagrostis spp.
New Lachnagrostis species ID
Lachnagrostis phylogeny
• Partial resolution of and support for existing taxonomy
Barcoding Results (to date)
Goal
Confirmation of Agrostidinae
Delineation of Agrostidinae genera
Confirmation of Lachnagrostis
Delineation of Lachnagrostis spp.
rbcL
matK
ITS
P
P
P


P



P
P
P
New Lachnagrostis species ID
Lachnagrostis phylogeny
• Partial resolution of and support for existing taxonomy
Barcoding Results (to date)
Goal
Confirmation of Agrostidinae
Delineation of Agrostidinae genera
Confirmation of Lachnagrostis
Delineation of Lachnagrostis spp.
New Lachnagrostis species ID
rbcL
matK
ITS
P
P
P



P




P
P
P

Lachnagrostis phylogeny
• Partial resolution of and support for existing taxonomy
Barcoding Results (to date)
Goal
Confirmation of Agrostidinae
Delineation of Agrostidinae genera
Confirmation of Lachnagrostis
Delineation of Lachnagrostis spp.
New Lachnagrostis species ID
Lachnagrostis phylogeny
rbcL
matK
ITS
P
P
P




P





P
P
P


• Partial resolution of and support for existing taxonomy
• Possible new, previously unsuspected relationships