Nine undergraduate students, my graduate student John Sproul, and me.  Over the next ten weeks we will go into the field, find beetles, look at them under the microscope, do dissections, photograph them, make predictions about species boundaries, extract their DNA, PCR some genes, get them sequenced, see gene trees that result, database and blog the results, generate new ideas about species boundaries, decide on more field work, and repeat.  This is a new course, Z499 (Discovering Insect Species), which I will be teaching starting this Tuesday.

I’m very excited about this course.  As part of my project to revise the Bembidiina of North America (funded by NSF), I thought it would be cool to wrap a course around the revision of one subgenus.  I’ve chosen Bembidion subgenus Trepanedoris for the class, and have purposefully avoided working on them for the last few years.  We’ve collected specimens, but for the most part have not sequenced their DNA, and have not looked at genitalic dissections.

Here’s what a member of the subgenus Trepanedoris looks like:

Bembidion (Trepanedoris) pseudocautum

I’m pretty sure there will be undescribed species in this group, and I am very much looking forward to analyzing the data for the first time in class, so that the students make the discoveries along with me.  Some of those discoveries will be made by looking under the microscope, but some when we get DNA sequence data back from the sequencing facility.  My plan is to download and analyze, in class, the chromatograms we will get from the University of Arizona sequencing facility, and have the class do phylogenetic analyses immediately thereafter.  This will be done using Mesquite, Chromaseq (with Phred and Phrap), and Zephyr (with RAxML).  I will see the results for the first time with the students – and given how excited I get when hunches get confirmed, or surprises are revealed, I think it will be great fun for all of us.

Holding back on doing research on this group has been very difficult for me. Before the idea for the class had hatched, I had dabbled a bit in Trepanedoris, enough to sequence 1-4 genes (28S rDNA, COI, CAD, and Topoisomerase I) from 43 specimens.  This showed some interesting patterns, and hinted at a lot of complexity in the group that I really wanted to explore more.  Since then we have collected many more specimens from key localities, and a quick glance at the specimens while sorting under the scope suggests that there are most likely some undescribed species, but the patterns are complex enough that I just don’t trust my first guesses.  I so want to look at the genitalia, and sequence the DNA.  But that will all have to wait, as I want to be surprised along with the whole class.

Here’s a tentative outline and schedule of the course.  We meet for three hours on Tuesday afternoons and three hours on Thursday afternoons.

Week 1

• Tuesday: Introduction.  Exercises with tamarin pictures (26 pictures of different species of tamarins; students have to as a group hypothesize species boundaries). Similar exercise with a subgenus of Bembidion (subgenus Liocosmius pictures and results).
• Thursday: field trip to local site in Corvallis where we will surely find at least one species of Trepanedoris, and possibly four.

Week 2

• Tuesday: prepare specimens from Thursday’s field trip, and mount specimens from Arroyo Seco in California.  Use Lindroth’s key to try to identify specimens.  Sort pictures of 43 already-extracted specimens; have students predict species boundaries based upon pictures.
• Thursday: mini-lecture (current status of studies of Trepanedoris, including history of group; show DNA phylogenies; compare to student’s predictions from Tuesday). Look at Trepanedoris specimens under the scope and try to see morphological characters correlated with gene tree shapes.  Examine ethanol-preserved specimens from local field trip and Arroyo Seco; students choose three specimens each for DNA extraction, and they make predictions
• Saturday-Sunday: weekend field trip to Klamath Marsh NWR

Week 3

• Tuesday: start DNA extraction of local and Arroyo Seco specimens. After incubation has started, choose specimens to extract from weekend field trip.  Finish first DNA extraction, and start extraction of specimens from weekend field trip. I finish the second DNA extraction.
• Thursday:PCR 28S and COI.  While samples are on thermal cycler, mount DNA vouchers from Tuesday’s extractions, and mount some ethyl acetate specimens.
• Outside of class:  John and I will PCR CAD and Topo from the student’s extractions, and re-PCR 28S and COI if needed.

Week 4

• Tuesday: Run PCR gels, prepare products for DNA sequencing.  Examine all second-round DNA vouchers, and make predictions about species boundaries.  Discussion:  how do models work in science?
• Thursday: Re-PCR as needed.  Mini-lecture and discussion:  intro to the ToL, phylogenetic analyses.  Discussion: hypothesis testing vs estimation

Week 5

• Monday: send plates to GATC sequencing facility
• Tuesday: Lecture and discussion about gene trees and species trees; coalescence, dice-based exercises, Mesquite-based simulations.
• Thursday: If sequences are available, then process chromatograms in class and do preliminary analyses. Discuss results.  Otherwise, discussion species boundaries in the context of Mesquite-based simulations.

Week 6

• Tuesday:  See previous Thursday (do what ever is left over from that).
• Thursday: Taking stock:  what have we learned?  What data do we need to gather?  What field work should we do next?
• Saturday and Sunday: overnight field trip to destination of choice (Malheur NWR?)

Week 7

• Tuesday: processing specimens from weekend field trip, including choosing specimens for DNA extraction.  Mini-lecture on how nomenclature works.
• Thursday: DNA extraction of specimens from field trip.  Intro to genitalic preps while waiting; students KOH previously extracted specimens (Arroyo Seco and .

Week 8

• Tuesday: PCRs from second field trip.  Mount specimens from second field trip. Genitalic preps 2: students KOH specimens from second field trip.
• Thursday: PCR gels, prepare products for sequencing.   Help prepare plate.  Genitalic mounts 3: dissection and mounting.
• Outside of class:  John and I will PCR CAD and Topo from the student’s extractions, and re-PCR 28S and COI if needed.

Week 9

• Monday: send plates to GATC sequencing facility
• Tuesday: Imaging: whole body, head, and genitalia.
• Thursday: Imaging, and putting data into Morphbank and Scratchpads.

Week 10

• Tuesday: process chromatograms obtained from GATC.  Conduct “final” gene tree analyses.  Discussion.
• Thursday: Where are we?  What should the next steps be?

We will be blogging about the course as it goes along.