Introduction

Grasses form one of the most important taxonomic groups within the angiosperms in terms of

economic value, with the major food crops such as rice, maize and wheat belonging to the Poaceae.

They form 70% of all food crops in the world as well as being used for fodder for cattle and

bamboos being used for construction (Constable 1985) . Grass fibres are used in the production of

paper as well as the bioethanol derived from the lignins from grasses such as Miscanthus being used

as an alternative fuel source (Faix, Meier et al. 1989).

Along with the economic uses, grasses contribute a huge amount to the biodiversity of this planet.

There are over 9000 species of grasses, found from xeric to aquatic habitats, having colonised most

of the globe, dominating biomes in the form of grasslands as well as being important pioneer

species in primary or secondary successions, meaning they play a vital role in the establishment of

new ecosystems.

Due to the major significance of this important angiosperm group, studying the phylogenetics of it

gives us a deeper understanding on how the group is organised, when and where the major

diversification events took place, and try and elucidate any taxonomic conflicts there might be.

Also, with the changing global climate, dated reconstruction of phylogenies can provide us with

very important information on how major clades reacted to past climate change and how it affected

its diversification rates (Roquet, Sanmartin et al. 2009). With grasses being a major component of

biomes, phylogenetic research on them would be invaluable. However, the phylogeny of Poaceae

isn’t completely resolved as there are still questions pertaining to certain sub-groups within the

family. With the publication of the GPWG (Barker, Clark et al. 2001), some of these questions has

been answered such as the monophyley of the PACCAD clade as well as the earliest diverging

lineages of the Poaceae being the Anomochlooideae, Pharoideae and Peuliodeae. However, some

questions were raised from this report, namely the relationships among the major lineages in the

PACCAD clade and the resolution of the BEP clade (Clark, Zhang et al. 1995).

With this in mind, the project was aimed to gain sequences for 39 species of grasses, out of which

12 were from Phylostachys genera representing the woody bamboos. The rest contained some

Australian herbaceous grasses as well as other Bambusoideae species. The Phylostachys was

selected to shed light and hopefully help resolve the BEP clade whilst the Australian grasses were

chosen to provide resolution in the PACCAD clade, which was alluded to the GPWG report. These

sequences were to be aligned to previously derived sequences and used to create a phylogenetic

tree. The gene regions used for analysis were atpB-rbcL intergenic spacer and trnL intron, trnL-F

intergenic spacer (the names of these two regions are hereafter called trnL-F as they are continuous

tandemly arranged pieces of DNA). These two gene regions found on the cholorplast were chosen

are they are commonly used for phylogenetic study, especially from species to family level (Soltis

and Soltis 1998). This plastid DNA is non-recombining and maternally inherited in most

angiosperms. Hence, different sequences found on the plastid genome should share a similar

evolutionary and provide congruent phylogenetic trees.

Materials and Methods

Plant Materials

Species used in this study were collected by Dr. Trevor R. Hodkinson and Surrey Jacobs. 39

samples were made available for this project, which included fresh samples, samples stored in

silica, samples stored in liquid CTAB as well as already extracted total DNA (See Appendix I for

more details).

Isolation of total genomic DNA

Total genomic DNA (tDNA) was extracted from a range of 0.1-0.7g, depending on how much plant

material was available and state of preservation. tDNA was extracted using the protocol adapted

from Hodkinson et al (Hodkinson, Waldren et al. 2007). For the samples stored in liquid CTAB,

and extra step in the extraction method was followed, so as to remove traces of CTAB on the plant

material. This involved washing the each of the samples in distilled water a minimum of three

times, and then placing the plant material on tissue paper to absorb the water. This was allowed to

dry in a fume hood for 10 mins, and then the dry mass was measured and the extraction process as

outlined in the protocol was conducted.

Following the extraction, the tDNA was run on an agorose gel stained with ethidium bromide so as

to visualise the DNA under UV light. If there was no DNA visible, amount of tDNA used in the gel

was increased or a new gel was created with a lower/higher amount of ethidium bromide. If the gel

was still unsuccessful, the extraction process was carried out once again. 100 l of the tDNA was

then pelleted, washed and purified using a JetQuick PCR purification kit (GENOMED Inc.) and

eluted using TE buffer (10mM Tris-HCl; 1mM EDTA; pH 8.0) and stored at -20°C until use.

DNA amplification and sequencing

The atpB-rbcL IGS and trnL-F region of the purified DNA was tehn amplified using a polymerase

chain reaction (PCR). Each of the two genen regions were amplified by their respective primer,

namely “c” and “f” for trnL-F and “1R” and “2R” for atpB-rbcL IGS.

Each of the 50 l PCR reactions consisted of several chemicals which is outlined in Table 1 whilst

the parameters used in the PCR reactions is outlined in Table 2.

atpB-rbcL trnL-F

Amount (µl) PCR chemicals

31.75 Sterile distilled H2O Sterile distilled H2O

10 5x colourless GoTaq Flexi buffer

(Promega corp.)

5x colourless GoTaq Flexi buffer

(Promega corp.)

4 25mM MgCl2 25mM MgCl2

0.5 “1R” primer “c” primer

0.5 “2R” primer “f” primer

1 10mM dNTPs 10mM dNTPs

0.25 GoTaq DNA polymerase

(Promega corp.)

GoTaq DNA polymerase

(Promega corp.)

50

Table 1: Chemicals and respective amounts used in the PCR reactions

Step Temperature (oC) Time

Premelt 95 1 min

Denaturation 95 45 sec

x32 cycles Annealing 50 45 secs

Extension 72 2 mins

Final extension 72 7 mins

Soak 4

Table 2: PCR parameters executed on a GeneAmp PCR system 9700 (Applied Biosystems). Soak time is

synonymous with hold time where the PCR products are held for an indefinite period of time.

The products of the PCR reaction was run on an agarose gel stained with ethidium bromide to

determine whether the PCR reaction was successful. If the reaction wasn’t successful, the PCR was

run again, but by changing some of the parameters or concentrations of some of the chemicals. The

cycles could be reduced or the annealing temperature increased to 52°C (as for the case of trnL-F).

The concentrations of the DNA and MgCl2 can be altered accordingly. To optimise the PCR

reaction, the amount of DNA was increased to 3µl and the 25mM MgCl2 was was increased to 5µl.

In this case, the amount of water was reduced so the total reaction mixture was 50 µl.

The successful PCR products were cleaned once again using a JetQuick PCR purification kit

(GENOMED Inc.) but used ultra-pure sterile water instead of TE buffer as the elution buffer. This

cleaned product underwent cycle sequencing on a GeneAmp PCR system 9700 (Applied

Biosystems), similar to the PCR reaction. Each of the cycle sequencing reaction were 10µl in

volume and contained 1.5µl of the PCR product, 2µl of either atpB-rbcL IGS primers (“1R” or

“2R”) or trnL-F primers ( “c” or ”f”), 1.5µl of BigDye Terminator v1.1, v3.1 5x sequencing buffer

(Applied Biosystems), 1µl of BigDye Terminator v3.1 Cycle Sequencing RR-100 (Applied

Biosystems) and 4 µl of sterile distilled H2O. The parameters used for the cycle sequencing reaction

is shown in Table 3:

Step Temperature (oC) Time

Initial

Denaturation

96 1 min

Denaturation 96 10 sec

x28 cycles Annealing 50 5 sec

Extension 60 4 mins

Soak 4

Table 3Cycle sequencing parameters executed on a GeneAmp PCR system 9700 (Applied Biosystems). Soak

time is synonymous with hold time where the cycle sequencing products are held for an indefinite period of

time.

The products of the cycle sequencing went a further purification method by ethanol precipitation.

This is important to remove and residual cycle sequencing chemicals in the DNA that would

interfere with the DNA sequencer. The cleanup involved mixing each of the samples with 52 µl of a

solution of 50 µl 100% ethanol and 2 µl of 3M sodium acetate. This was left at room temperature

away from direct light for 15 mins after which it was left in ice for 30-45 mins away from direct

light again. The samples were then centrifuged for 30 mins at 4000rpm. After immediately spinning

the samples down, the samples were inverted with paper towel replacing the caps and placed upside

down in the centrifuge bucket. This was then centrifuged to a maximum of 180rcf. The tubes were

then turned the right way up and the paper towel was removed, and capped replaced. 20 µl of 70%

ethanol was then added to each sample and then centrifuged at 4000rpm for 20 mins. The samples

were then inverted once again with a paper towel replacing the caps, placed upside down in the

centrifuge bucket and centrifuged at 1000 rpm for 1 min. This is done to remove the excess wash

buffer. The tubes are removed from the centrifuge, placed the right way up and left in a fume hood

away from direct light for 25-30 mins. This is done to remove all the ethanol present in the sample

so as not to interfere with the sequencer.

The samples are resuspended in 10 µl hi-di formamide and vortexed. The samples are then heated at

95°C for 5mins after which it is immediately placed in ice for 3-4 mins. The samples are then

transferred to the sequencing plate and briefly spun to remove any bubbles. The plate was then

loaded into a 3130XL Genetic Analyser (Applied Biosystems) for separation of the fragments.

DNA sequence editing, assembly, and phylogenetic analysis

The DNA sequences obtained from the geneteic sequencer was evaluated in Sequence Analysis

v5.3.1 (Applied BioSystems) software, where the corresponding peaks were seen for each of the

bases. This was then analysed, edited and assembled using Auto Assembler v2.1 (Applied

BioSystems) software and concensus sequences were obtained for each of the samples. This was

then imported into PAUP (Sinauer Associates) computer programme where it was aligned by eye

alongside 62 other sequences made available by Dr, Trevor Hodkinson. Once the sequences were

aligned they were subjected to maximum-parsimony analysis using heuristic search options in

PAUP (Sinauer Associates) computer programme. The heuristic searches included 1000 replicates

of random stepwise addition with no more than 75 trees saved per replicate. An exclusion set of 168

characters were used when running the search. The branch swapping algorithm used was tree-

bisection-reconnection (TBR) and model for distance measured was the HKY85 model (Hasegawa,

Kishino et al. 1985) as the concentrations of the bases in the sequences are unequal. Bootstrapping

analysis was then carried out using 1000 replicates using TBR algorithm. Sequences from the

Panicoideae subfamily were used as an outgroup to root the tree.

Results

When the extracted DNA was run on the agarose gel, it could be seen that the tDNA quantity was

very low, except for the 4 freshly collected samples of 2 species of Fargesia, Pleoblastys

pygmeansis and, Phylostachys sp. Along with the freshly collected samples, the tDNA already

extracted that was provided by Dr. Trevor Hodkinson of the 11 Phylostachys species, showed high

amounts of DNA when run in the gel. All these samples amplified well during PCR with the atpB-

rbcL IGS primers, but none of the samples that were stored in the liquid CTAB amplified. The PCR

reactions with the trnL-F were unsuccessful, with no amplifications seen in any of the samples (See

Appendix I for more details).When the successful atpB-rbcL gene region PCR products were taken

to the cycle sequencing stage, all of the products were sequenced successfully.

The aligned matrix was 856 characters long of which 168 characters were excluded. Of the

remaining 758 characters 502 characters were constant. 118 were variable but parsimony-

uninformative and 138 were included parsimony-informative. The tree search using maximum

parsimony yielded 200 equally parsimonious trees with 398 steps (Figure 1) with the consistency

index (CI) being 0.76 and the Retention index (RI) being 0.82. Bootstrap (BS) percentages are

described as low (50-74%), moderate (75-84%) and high (85-100%) (Sungkaew S, Stapleton CM et

al. 2009).

The BEP clade was highly supported (100% BS) as being monophyletic. The monophyly of the

subfamilies however isn’t strongly supported, with a low BS value for the Erhartoideae (53%).

Pooideae is sister to Bambusoideae s.s with 99% BS.

Figure 1: One parsimonious tree obtained from heuristic search with distance measured using HKY85 model. BS values

seen above the branches and the species names in bold and italics were sequenced for this project.

The Bambusoideae s.s seems to be monophyletic (88% BS) including neotropical, paleotropical and

temperate bamboos in the same clade. Monophyly of the Olyreae (99% BS) and Bambuseae (96%)

is highly supported. The sequenced species resolved moderately well in the Arundinaieae tribe

Fargesia 1 P. incernata 9

Pleioblastus 2 Fargesia 4

P monii 10 P arca 15

P nidul 17 P hum 18

Pseudosasa cantorii1 Phylostachys 3 Chimonobambusa quadrangularis 105

P. pub 107 Chimonocalamus pallens 1340 Chimonocalamus sp

Oligostachyum glabrescens 1302 Fargesia 5 Fargesia 8

P anvea 16 Borinda sp.1347 P bambus 13 P. nig SS106 P biseti 14 P virid 12 Menstruocalamus sichuanensis1319

Neohouzeaea kerrii3 Schizostachyum jaculans 307

Bambusa bambos 3 16 Bambusa beecheyana 1313

Bambusa malingensis 1332 Bambusa oldhamii1321 Dendrocalamus asper BAM1WKM Dendrocalamus BAM45WKM

Dendrocalamus latiflorus SS113 Dendrocalamus minor1317

Dendrocalamus membranaceus SS02 04 Dendrocalamus strictus18 Dendrocalamas valida 625 Gigantochloa scortechinii SS309 Melocalamus compactiflorus 175

Bambusa tulda 1328 Dendrocalamus hil BAM24WKM Gigantochloa ligulata SS09 04

Neosinuscalamus affinis SS624 Thy rsostachys siamensis SS02 03 Gen nov SS191 Vietnamosasa ciliata SS208 Vie pus SD1466

Oxybra Stapleton1307 TemO lil SS10 15

Dinochloa malayanaSD1412 Guacha Stapleton1308

Arthrostylidium glabrum 572 Rhipidocladum racemiflorum 76

Temburongia simplex 21774 Cepper SD1435

Neohouzeaua fimbriata SSRP12 Pseudostachyum polymorphunm SS176 Sch izostachyum grande SS10 06 Schizostachyum zollingeri SS09 01

Chu squea patens 571 Cryptochloa granulifera 54

Lithachne pauciflora 48 Piresia sp 601

Oly ra latifolia 614 Brachypodium 22

Lolium 29 Arrhenatherum 27

Alopecurus 30 Nardus 5

Lygeum 18 Leersia 636

Oryza rufipogon SS164 Oryza sativa 46

Ehrharta calycna G25 Panicum 120

Saccharum 104 Miscanthus 5

5 changes

Olyreae

Bambuseae

Pooideae

Ehrhartoideae

Arundinarieae

100

53 100

100

100

99

92

61 57

84

99 85

100

88

55

96

86

76 64

65

66

88

64

Bambusoideae s.s

BEP clade (100)

(76% BS). The Phylostachys and Fargesia are polyphyletic. Figure 2 illustrates the separation of

Figure 3: Bambuseae tribe, separated along the lines of geography, the plant species grouping under Temperate,

Paleotropical and Neotropical.

the Bambuseae tribe into geography, with a moderate support for Temperate bamboos (76% BS),

high support for paleotropical (88% BS) and a low support for neotropical (55% BS).

Discussion

The resolution of the BEP clade in Fig.1 is in congruence with the recent study by Sungkaew et. al

(Sungkaew S, Stapleton CM et al. 2009). They found Erhartoideae were a sister to the lineage

consisting of Pooideae and Bambusoideae. We arrived at the same conclusion with 100% BS value.

However, within the BEP clade, we discovered some incongruence with some of the recent studies.

According to GPWG (2001), we were expected to see Pooideae as a sister group to Bambusoideae,

which is confirmed in our studies as well (Bouchenak-Khelladi, N. et al. 2008). However, this

wasn’t the case as we see Pooideae being a sister group to Olyreae, which is a sister group to

Bambuseae.

This is however in congruence taxonomically, as the monophyletic Olyreae represent herbaceous

bamboos, whilst the monophyletic Bambuseae represent woody bamboos. This means the

phylogenetic analysis of the Bambusoideae is synonymous with the taxanomic separation of the

woody and herbaceous species. It could be hypothesised that the ancestor of the bamboos were

herbaceous and the Bambuseae evolved woodiness from it whilst the Olyreae kept the herbaceous

stem. However, this conclusion should be considered with caution as the previous studies have used

multi-gene analysis, whilst only one gene region was analysed in this study. As only three species

from the Olyreae were sequenced, increasing the sampling set would provide us with a clearer

picture.

Sungkaew et. al (Sungkaew S, Stapleton CM et al. 2009) used multi-gene region phylogenetic

analysis to elucidate the Bambusoideae sub-family, but the dataset with regards to the temperate

bamboos was low. In this study, we increased the sampling of temperate bamboos by incorportating

11 species of Phylostachys, 2 species of Fargesia and 1 species of Pleoblastus. Hence we see a

greater resolution of the Arundinareae tribe (Fig.1).

None of the plant material that was stored in liquid CTAB worked. This might’ve been due to major

degradation of the DNA. The fresh samples that were extracted worked well, as well as the total

DNA that was provided for this project worked as well, as it was extracted from fresh samples. This

highlights the importance of proper storage of specimens.

The samples that were successful in the PCR using the atpB-rbcL gene region, were unsuccessful

under trnL-F. This was due bad stock “c” and “f” primers.

Acknowledgements

I would like to thank Dr. Trevor Hodkinson for supervising my project as well as all the

postgraduates in the Botany Department for their invaluable assistance during the 10 weeks. I

would also like to thank Dr. Daniel Kelly, Fiona Molloney and Dr. Martyn Linnie for co-ordinating

the UREKA programme. Finally I would like to thank Science Foundation Ireland for funding the

project,

References

Barker, N. P., L. G. Clark, et al. (2001). "Phylogeny and subfamilial classification of the

grasses (Poaceae)." Annals of the Missouri Botanical Garden 88(3): 373-457.

Bouchenak-Khelladi, S. N., et al. (2008). "Large multi-gene phylogenetic trees of the

grasses (Poaceae): Progress towards complete tribal and generic level sampling." Molecular

Phylogenetics and Evolution 47(2): 488-505.

Clark, L. G., W. Zhang, et al. (1995). "A phylogeny of the grass family (Poaceae) based on

ndhF sequence data." American Journal of Botany 82(6 SUPPL.): 120-121.

Constable, G. (1985). Grasslands and Tundra.

Faix, O., D. Meier, et al. (1989). "Analysis of lignocelluloses and lignins from Arundo

donax L. and Miscanthus sinensis Anderss., and hydroliquefaction of Miscanthus." Biomass

18(2): 109-126.

Hasegawa, M., H. Kishino, et al. (1985). "Dating of the human-ape splitting by a molecular

clock of mitochondrial DNA." Journal of Molecular Evolution 22(2): 160-174.

Hodkinson, T., S. Waldren, et al. (2007). "DNA banking for plant breeding, biotechnology

and biodiversity evaluation." Journal of Plant Research 120(1): 17-29.

Roquet, C., I. Sanmartin, et al. (2009). "Reconstructing the history of Campanulaceae with a

Bayesian approach to molecular dating and dispersal-vicariance analyses." Molecular

Phylogenetics and Evolution 52(3): 575-587.

Soltis, D. E. and P. S. Soltis (1998). Choosing and approach and an appropriate gene for

phylogenetic analysis. Molecular systematics of plants II, DNA sequencing. Dordrect,

Kluwer Academic: 1-41.

Sungkaew S, Stapleton CM, et al. (2009). "Non-monophyly of the woody bamboos

(Bambuseae; Poaceae): a multi-gene region phylogenetic analysis of Bambusoideae s.s." J

Plant Res 122: 95-108.

Appendix I: List of Samples and PCR results

Sample No. Species Collection No. PCR atpB-rbcL PCR TrnL-F

1 Fargesia sp. 1 + -

2 Pleoblastus pygmeansis 2 + -

3 Phylostachys sp 3 + -

4 Fargesia sp. 4 + -

5 Fargesia sp. 5 + -

6 Phylostachys angustofolium 6 + -

7 Phylostachys sp. 7 + -

8 Fargesia sp. 8 + -

9 Phylostachys incernata 9 + -

10 Phylostachys monii 10 + -

11 Phylostachys dulcis 11 + -

12 Phylostachys virid 12 + -

13 Phylostachys bambusa 13 + -

14 Phylostachys biseti 14 + -

15 Phylostachys arca 15 + -

16 Phylostachys anvea 16 + -

17 Phylostachys nidul 17 + -

18 Phylostachys hum 18 + -

19 Micraira subulifolia SJ9301 - -

20 Theda chloa 20 - -

21 Pheidochloa gracilis SJ9923 - -

22 Xerochloa laniflora SJ9859 - -

23 Pheidochloa gracilis SJ9921 - -

24 Hygrochloa aquatica SJ9892 - -

25 Austrostipa petraea SJ9696 - -

26 A. flavescens SJ9684 - -

27 A. puberula SJ9775 - -

28 A. platychaeta SJ9700 - -

29 Pseudochaetochloa australiensis SJ9872 - -

30 Hygrochloa aquatic SJ9911 - -

31 A. nodosa SJ9799 - -

32 Eriachne stipacea SJ9918 - -

33 A. drummondii SJ9776 - -

34 A. eremophila SJ9739 - -

35 Aristida behriana SJ9786 - -

36 A. setacea SJ9781 - -

37 A. semibarbata SJ9709 - -

38 A. muelleri SJ 9791 - -

39 Austrostipa sp. SJ 9765 - -

40 Nassella trichotoma SJ 9727 - -

41 A. bladii SJ 9748 - -

42 A. velutina SJ 9758 - -