Rehan and Richards 



JESO Volume 139, 2008 



Purification Kit. Manufacturer's instructions were followed with one additional step, in 

 which an extra centrifugation was incorporated after proteinase K. digestion to remove bee 

 fragments that might clog the spin columns. Portions of the mitochondrial gene COI were 

 amplified using primers mtd-8 and 12 (Simon et al. 1994; University of British Columbia 

 Biotechnology Laboratory, Vancouver) in 20 ul reactions with 2.5 mM MgC12, 200 uM 

 dNTPs, 2.0 uM each primer, 0.25 U Sigma Jumpstart Taq polymerase, and 2 ul of Taq 

 reaction buffer (supplied with the enzyme). PCR reactions were as follows: initial heating 

 to 94°C for 5 min, followed by 30 repetitions of 94°C for 1 min. 54°C for 1 min, 72°C for 1 

 min, and final extension at 72°C for 5min. Amplification products were purified by ethanol 

 precipitation and analyzed on 0.8% agarose gels containing lOmg/ml ethidium bromide. 

 DNA sequencing was carried out at McGill University and Genome Quebec Innovation 

 Centre in Montreal, using the same primers. Sequences were edited using BIOED1T (Hall 

 1999) and aligned using CLUSTAL (Thompson et al. 1994) using default settings with 

 the exception of gap open penalties increased to 50. All sequences have been deposited in 

 GenBank under accession numbers EF534228-E1 534247. We used Analysis of Molecular 

 Variance (AMOVA) as implemented in Arlequin 3.11 (Excofifier et al. 2005) to compare 

 genetic variation within and among specimens identified with the revised morphological 

 character. 



Results 



Morphological Differences 



Using light microscopy and SEM images in combination with corresponding COI 

 sequences revealed the subtle yet consistent dimorphism between these species. Ceratina 

 calcarata are less punctate than C. dupla t )USt as Mitchell ( 1 962) describes, but the placement 

 of punctures around the parapsidal and medial lines is better at differentiation than their 

 abundance (key to females, couplet 2). This difference was confirmed by an 87% correct 

 identification rate among the non-entomologists tested. 



FIGURE 2. Hind femurs of male Ceratina. a. C. dupla, hind femur somewhat dilated 

 toward base, but without a median projection, b. C. calcarata, hind femur with a median. 



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