Friday, 14 April 2017

Balancing drought versus herbivory survival in Spekboom restoration

by Alastair Potts

There are three major stresses that spekboom cuttings need to survive: drought, herbivory and frost. The last stress has been discussed in previous posts (see Rob Duker's "Thicket restoration and frost: the forgotten enemies") and can be overcome quite simply by avoiding areas that experience frosts.

Drought survival was the focus of initial spekboom restoration research. This suggested that smaller cuttings (so-called "fingerlings") had extremely low survival rates, and survival increased with the size of the cutting. This led to the suggestion of the current truncheon size of ~>4 cm stem diameter as part of the restoration protocol. 

These truncheons are planted to a depth of  ~10-20 cm (sometimes deeper) sticking upright out of the ground. In a previous post ("To the root of the problem with Spekboom restoration"), I highlighted that the roots tend to only grow from the cut base, which has been been buried deep in the soil and limiting the access the plant has to soil moisture after light rainfall events. However, an additional problem is that these cuttings take a long time to develop a root-to-shoot ratio that is sufficient to survive drought, but more importantly, withstand herbivory. 

A root-to-shoot ratio describes the amount of plant tissue that has supportive functions (i.e. the roots) relative to the amount of tissue that has growth functions (i.e. above-ground stems and leaves). Roots allow a plant to absorb water and nutrients from the surrounding soil, and thus root-to-shoot ratios are usually discussed in terms of plant health and water/nutrient absorption. This is certainly an important consideration for spekboom truncheons, but what is of greater importance is the effect that this low root-to-shoot ratio has on herbivory survival.

Without an established root system (relative to the above-ground plant), a spekboom truncheon is more susceptible to browsing and catastrophic herbivory-related uprooting. Browsers eating spekboom generally include a pulling motion after the bite; this pulling motion can easily uproot a truncheon. (Note that baboons have also been observed pulling up truncheons). 

Spekboom has evolved to withstand heavy browsing from indigenous game. Even in an area with arguably the highest elephant-browsing density in the world — at Hapoor Dam in Addo Elephant National Park where the elephants have decimated the thicket vegetation surrounding the dam (Landman et al. 2012)  spekboom is one of the first species to return to canopy dominance as you move away from the dam. Also, in relation to its annual rainfall, AENP supports the highest biomass of herbivores anywhere in Africa (Mills et al. 2014), and likely the world. As an aside, an untested hypothesis is that the primary dispersal agents of spekboom are elephants as there habit of walking while munching on a spekboom branch results in a rain of vegetative parts that can propagate new individuals.  

So, why are spekboom truncheons susceptible to indigenous herbivory? The root-to-shoot ratio is wrong. There is not enough going on under the ground to support what is going on above the ground.

We need the truncheons to be large to withstand drought-stress, but being large makes them more apparent in the landscape, take a long time to develop the necessary roots, and ultimately highly susceptible to herbivory.

How can we deal with this catch twenty-two?  

My suggestion is to return to smaller truncheon sizes (the "fingerlings"). The fingerlings:

  • are less apparent in the landscape, and
  • can develop the right root-to-shoot ratio in a shorter time period.
Also, this is how I imagine spekboom has been distributed through the landscape, via propagule-rain driven by elephant browsing. But this rain would have been throughout the year (not a once off planting as in restoration), and so the fingerlings would have been rained down during windows of opportunity and when establishment would not be possible (e.g. in droughts).     

Thus, this still leaves the problem of drought survival. A lone spekboom fingerling out in the open has little chance of survival. However, there are a few things that can be done to improve these chances:

  • Planting spekboom close together in a contoured band has been shown to have outstanding results at the Camdeboo National Park restoration sites established by Peter Burdett (results shared by Bruce Taplin at the Thicket Forum 2015). 
  • The pure mechanical effect of creating a contour line, by rip-ploughing, soil bags, car tyres etc. creates drainage lines of increased soil moisture. If positioned correctly, these lines can also create an ameliorated microclimate for a small plant.
  • Loosening the soil prior to planting has also been reported (reference needed) to improve truncheon survival and growth. 
  • Hydromulch, a woodfibre mulch applied with water and a tackifier (natural glue), traps water and dew and creates an excellent medium for encouraging root development in fingerlings (see the images in my post about the rooting window hypothesis).

Thus, the combination of mechanical contour ploughing that loosens the soil (or some other water-trapping technique) and creates microclimates (on south-facing micro-slopes in the ditch and in the soil), hydromulch and a high density of spekboom fingerlings could very well be the recipe for seeding the landscape with spekboom resilient to drought and herbivory. As the mechanical structure of the contour breaks down from erosion, the spekboom will likely take over as a biological contour, filtering water and erosion and increasing water infiltration. 

Saturday, 1 April 2017

Proteinase K, can it improve the quality of DNA extracted from Sulfhydryl rich leaves ?

By Timothy Macqueen

I have recently begun work on attempting to extract DNA from the leaves of Protea susannae and P. eximia hybrids using the Doyle&Doyle CTAB method. There have been several set-backs in the quality of the DNA I have extracted. In many of the DNA gels I have run, only one or two of my samples have shown successful amplification. 
An early gel run. The only amplified DNA sample was the second to right. The rest were PCR and gel positives

 The 260/230 ratios of my samples has been consistently low. I wanted to determine the reason for this, so I posted my problem on the scientific community website, Researchgate. On it, H. Ayyez of the University of Al-Qadisiyah suggested that this was because I was experiencing organic contamination due to not adding Proteinase K as a part of my extraction. 

Before I continue, I'd better explain the importance of the 260/280 and 260/230 values to DNA amplification and analysis. A Nano-drop is used to measure these values. The 260/280 value of a DNA sample indicates the purity of the DNA extracted. A value around 1.8 is considered to be pure. 

Low 260/280 ratios may be caused by: 
• Presence of residual phenol or other reagents associated with the extraction protocol
• A very low concentration( > 10 ng/ul).of nucleic acid

The 260/230 ratio is important as a second measure of DNA purity after the 260/280 ratio. An optimum value for this ratio is between 2.0-2.2. 

Low 260/230 ratios may be the result of:
 • Carbohydrate carryover (often a problem with plants). 
• Residual phenol from nucleic acid extraction.  OR most importantly-

  • The presence of organic contaminants, such as (but not limited to): phenol, TRIzol, chaotropic salts and other aromatic compounds.                                                                                                                                                                                                       Samples with 260/230 ratios below 1.8 are considered to have a significant amount of these contaminants that will interfere with downstream applications. This is especially true for reverse transcription.
    Protea hybrid at Van Staadens nature reserve. It contains many of the distinct morphological traits of  P. susannae. That distinct sulphurous odor, distinctive to leaves containing sulfhydryl compounds. Could these organic compounds be the cause for all my troubles trying to amplify the DNA of my hybrid proteas?    

One such compound is a Thiol. This is a organosulphur compound that contains carbon bonded sulfhydryl group. This is known to occur in my samples because the leaves of P. susannae and its hybrids when crushed, produce a distinct sulfurous odor. This odor is distinct  to Thiols. 

The 260/280 values in my samples were very close to or at 'pure' ratio values while my 260/230 (yellow highlighting) ratios were very low. 
So what can Proteinase K do to improve the quality of my 260/230 ratios? Well, Proteinase K is a serine proteinase which is able neutralise a wide variety of contaminating proteins from organic compounds. It is also used to inhibit nucleases from degrading the nucleic acids to be extracted. 

Will it work? Well, the only way I can know for certain is by testing this hypothesis. Currently our lab is out of stock of Proteinase K. We have just ordered a new batch though. Once it is received I will use it in my extraction method and report back to this blog on whether or not it improved the quality and purity of my DNA extractions. 


Cremlyn, R.J. 1996. An Introduction to Organosulfur Chemistry. Chichester: John Wiley and         Sons. ISBN 0-471-95512-4.
Patai, S. 1974. The chemistry of the thiol group. London: Wiley. ISBN 0-471-66949-0.

Links to website references (All accessed on 01/04/2017)