A workflow for pollen identification.

The reproductive ecology of cactus is not well-studied. A small, side project of mine is to determine the pollinator guild of buckhorn cholla at Sunset Cove, Mojave Desert, and with which plant species, if any, it shares pollinators. The genera Opuntia and Cylindropuntia are known to be insect-pollinated, but I am curious which of the more than 659 species of bees in the Mojave Desert desert are pollinators.

As visitation does not necessarily lead to pollination, I removed the pollen loads from 22 bee visitors I caught during insitu observation periods. I also removed stigma from the cholla to quantify heterospecific pollen deposition i.e. evidence of pollinator sharing. Pollen ID is not easy task and so I have developed a workflow to make it more streamlined.

Prep a reference collection:

  1. Create a reference collection by removing pollen from the anthers of several flowers of every species blooming in the area. Store in ethanol.
  2. Mount and stain the pollen with fushcin jelly.
  3. Image each species of pollen grain at 3 magnifications. Measure the length and width of about 10 grains per species. I calibrated Lumenera’s Infinity Analyze software using a stage micrometer to make this really quick.
  4. Make a reference document to consult. I use a word doc where every page is a species. Add in the photos at several magnifications, the mean size and any notes.
Sample reference page for Echinocereus engelmanni (Hedgehog cactus)

To go through the stigma or bee pollen load samples, I use my Canon EOS 60D dslr with a 60mm macro lens pointed confocally into a light microsite at 100x. I used the remote shooting utility from Canon to control the camera with my computer and display the view onto a second monitor.

Home example of confocal setup
  1. I designate each coverslip on the slide as a zone and do 8 transects through each, counting the grains. Each line in my spreadsheet is a transect, each column is a species. I use 5 columns for buckhorn so I never have to count very high.
  2. I don’t count damaged grains, or grains in air bubbles.
  3. Each slide gets its own folder. I take photos of each heterospecific grain with the file name as the zone + transect + species, which is simple using the photo utility. Knowing where the grain is on the slide and what its surroundings are will be helpful if you need to find it again.
  4. The species can be tentative for now so don’t get too bogged down.
  5. Take photos of unknowns when first encountered and assign them morphospecies ID. I put these in a separate folder as a reference.
  6. Some species are easy to ID. Quite a few are not. The more grains you see the easier it is to spot the differences.
  7. To help ID, we can take a page from entomologists. Sort the photos by their tentative IDs, putting each species in a folder so they are visible all at once (do a bulk rename to append the folder name first). It is difficult to compare grains unless they are side by side, which isn’t realistic with one microscope.
  8. Sort until each folder contains identical grains, then assign them a species from the reference collection. Or assign them to a species group for species that are virtually identical (likely Asteraceae!). Assign any remaining to morphospecies. Update the datasheet with the corrections.  
Buckhorn cholla (larger) and silver cholla (smaller). Thankfully the most abundant grains are simple to differentiate.