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.

Field sample processing

This fall I have been processing the insects and pollen samples that I collected this spring from my fieldwork in the Mojave Desert. The insects were primarily caught using pantraps, and were transferred into 90% isopropyl alcohol for preservation. With the help of our lab’s two undergraduate practicum students, Shobika and Shima, we are gradually getting them nicely organized into collection boxes.

I pinned many, many bees and wasps when I worked on a pollinator census during my undergrad in West Hamilton. These are the steps I use for processing insect samples:

  • Remove insects from alcohol.
  • Give the bees a rinse in water to fluff out their body hairs (this step works variably well, we may need to give some of the larger specimens a spa day in the future)
  • Gently dry with a paper towel, this causes the wings to uncurl. Wing venation is very important for identification.
  • Under a dissecting microscope, pin from top to bottom through the upper right-hand side of the insect’s thorax into a stryofoam block. You want the insect to be completely horizontal.
  • Gently uncurl the legs from the body and unfurl 1 antenna.
  • Affix an insect identification label underneath the insect with the text readable from the left side of the insect. These labels should have date and location of collection, unique identifier and the name of the collector.
  • Place into foam lined box.
  • Repeat!
  • Very small insects get pointed rather than pinned. The right side of the thorax is glued to a triangle cut out of cardstock, and the triangle based is pinned instead.

I have also been mounting pollen samples whenever I can squeeze the time in. I collected stigmas from the field and have been storing them in ethanol-filled small tubes.

Process:

  • Let slide warmer heat up
  • Using a transfer pipette, remove the pollen-ethanol suspension and transfer drop by drop onto warm slide, letting the alcohol evaporate and ensuring it does not run over the edges.
  • Place stigma onto slide.
  • Rub the inside of the centrifuge tube that was storing the sample with a bit of fushcin jelly, place  onto slide as well. Cut out 2 more small cubes of jelly, place over drop locations. Cover with slide cover and leave on warmer to melt jelly. Label slide.

For a different experiment that I have not yet processed, I will put the tubes into a centrifuge, spin down and pipette out the pellet to save time and labour. Quite a few tubes from the current experiment are extremely small and I am concerned about their ability to hold up under the force of a centrifuge. I need a less labour intensive process to make slides for my upcoming field season. I can think of two main options right now – use sturdy tubes that I can centrifuge, or collect into small tubes without adding ethanol, and mount each evening while at the research station. This will cut down the need to let the alcohol evaporate.