Plant Molecular Systematics

Spring 2011

“Problems” with morphologicaldata…

• Convergence and parallelisms

• Reduction and character loss

• Phenotypic vs. genotypic differences

• Evaluation of homology

• Misinterpretation of change or polarity

• Limitation on number of characters

• Phenotypic plasticity

Always searching for new types of characters…

Is molecular data intrinsically better than morphological data?

Central Dogma

Lipid pigments: chlorophyll lycopenes xanthophylls carotene

Phenolics: flavonols flavones tannins anthocyanins

Iridoid compounds

Alkaloids(N-containing)e.g. nicotine caffeine morphine betalains

Secondary Metabolites

Terpenes

Development of Molecular (Chemical) Systematic Methods

“Chemosystematics”

• Early methods relied on chromatography to separate complex mixtures of secondary metabolites, detect them, and then compare between taxa “spot botanists” – very phenetic

• Better separation and identification methods developed (HPLC, MS) – used pathway stages as cladistic characters - phytochemistry

• Move away from secondary metabolites to proteins• Early protein studies used immunological reactions• Development of improved electrophoretic methods – permitted

direct protein comparisons between taxa• Comparison of seed storage proteins• Development of allozyme techniques – direct estimates of genetic

relationships based on allele frequency

Molecular (DNA) Systematics

• Next step was to examine DNA directly through examination and comparison of restriction fragments (RFLP bands)

• Technology evolved to make it feasible to sequence DNA directly

• Initially limited to single genes or non-coding regions

• Now feasible to sequence large numbers of genes or regions or even whole genomes relatively quickly

Molecular Systematics- Can obtain phylogenetically informative characters from any genome of the organism- Assumes that genomes accumulate molecular changes by lineage, as morphological characters do- Possibly greater assurance of homology with molecular data (less likely to misinterpret characters) but homoplasy happens!- Principal advantages are the much greater number of molecular characters available & greater comparability across lineages

Genomes of the Plant Cell

Nuclear

Plastid

Mitochondrial

Table 5.1 from the text

Selection of DNA region to compare:

• Should be present in all taxa to be compared

• Must have some knowledge of the gene or other genomic region to develop primers, etc.

• Evolutionary rate of sequence changes must be appropriate to the taxonomic level(s) being investigated; “slow” genes versus “fast” genes

• It is desirable that sequences can be readily aligned

• The biology of the gene (or other DNA sequence) must be understood to assure homology

Genes frequently used for phylogenetic studies of plants:

• Mitochondrial genome – uniparentally inherited, but genes evolve very slowly and structural rearrangements happen very frequently, so generally not useful in studying relationships

• Plastid genome – uniparentally inherited- rbcL – ribulose-bisphosphate carboxylase large subunit- ndhF – NADH dehydrogenase subunit F- atpB – ATP synthetase subunit B- matK – maturase subunit K- rpl16 intron – ribosomal protein L16 intron

• Nuclear genome – biparentally inherited- ITS region – internal transcribed spacers ITS1 and ITS2- 18S, 26S ribosomal nuclear DNA repeat- adh – alcohol dehydrogenase

Plastid Genome

-Circular, derived from endosymbio- sis of cyanobacteria

-Three zones: LSC (large single copy region) SSC (small single copy region) IR (inverted repeats)

- Genes related to PS and protein synthesis Marchantia polymorpha - liverwort

Figure 5.1 from the text

The Polymerase Chain Reaction (PCR)

Figure 5.2 from the text

Automated Sequencing

Scanning of gel to detect fluorescently-labeled DNAs; data fed directly to computer.

How do we analyze molecular variation?

- DNA nucleotide sequences (point

mutations) - - Structural rearrangements

-insertions and deletions (indels)-inversions

Partial sequence of rbcL (plastid gene coding for Rubisco) in Poaceae

Figure 5.3 from the text

Insertion-Deletion Events

- Can occur as single nucleotide gains or losses or as lengths of 2-many base pairs- Can also be “chunks” of DNA (i.e., losses of introns)

A molecular synapomorphy for Subfamily Cactoideae (Cactaceae) – deletion of the plastid rpoC1 intron…

(Wallace & Cota, Current Genetics, 1995)

Cactaceae: trnL Intron Deletions

North American Clades

South American Clades

Pachycereeae

Corryocactus

“Browningieae I”*

“Browningieae II”*

Cereeae

Trichocereeae

Leptocereeae

Hylocereeae

Shared Deletion 2

- 268 bp

trnL intron deletions – Columnar Cacti

(*Tribe Browningieae polyphyletic)

23 kb inversion in all Asteraceae except for members of Tribe Barnadesieae (now Subfamily Barnadesioideae)

Chloroplast DNA Inversion

Comparative DNA Sequencing

• Obtain DNA samples from representative organisms (try to represent morphological diversity) and outgroups

• Identify DNA region(s) for comparison

• Use PCR to amplify targeted region

• Carry out sequencing reactions

• Run sequencing procedures (automated)

• Align sequences

• Use aligned sequences for phylogenetic analysis (various programs using various algorithms)

• Evaluate data in context of taxonomy and morphology

Panicgrasses(Panicoideae)

Rices(Ehrhartoideae)

Bluegrasses(Pooideae)

Bamboos(Bambusoideae)

Puelioideae

PharoideaeAnomochlooideae

Needlegrasses(Aristidoideae)

Lovegrasses(Chloridoideae)

Micrairoideae

Reeds(Arundinoideae)

Oatgrasses(Danthonioideae)

Major radiationin Oligocene-Miocene epochsinto open habitats

Origin of grassesca. 70-80 myain forests

+

Stamensreduced to 3

Phylogeny ofPooideae(multipledata sets)

Figure 5.5. from the text

Figure 5.7 from the text

Figure 5.4 from the text