Matkin et al,: Ecotypic variation and predatory behavior among Orcinus orca off the eastern Aleutian Islands 



77 



acoustic signals and allows spectrographic representa- 

 tions of the sound frequency versus time and pressure. 

 Spectrograms were produced with an 87.5% overlap of 

 the analyzed time series. Resulting spectrograms had 

 a time resolution of 2.9 milliseconds and a frequency 

 resolution of 43 Hz. 



We categorized calls by ear and by visual inspec- 

 tion of distinct upper and lower frequency components 

 of the sound spectrum (UFC and LFC, respectively), 

 as described by Miller and Bain (2000) and Yurk et 

 al. (2002). When categorizing the calls as distinct, 

 particular attention was given to 1) the existence and 

 contour shapes of UFCs; 2) LFC contour shapes; 3) 

 LFC segmentation (elements separated by silent inter- 

 vals); and 4) the component structure (elements within 

 the LFC arising from abrupt shifts in contour and not 

 separated by silent intervals) of the LFCs (Ford, 1991; 

 Yurk, 2005). 



The three known ecotypes of killer whales inhabit- 

 ing waters off British Columbia and southern Alaska 

 (resident, transient, and offshore) are acoustically dis- 

 tinguishable by 1) vocalization rate; 2) the occurrence 

 of different discrete calls; 3) the syllables used in calls; 

 and 4) the production rate and characteristics of echolo- 

 cation clicks. Transient killer whales, which appear to 

 rely on passive listening to catch their marine mammal 

 prey, vocalize less frequently than resident killer whales 

 (Deecke et al., 2005). Transients rarely use echolocation 

 clicks, in contrast to resident and offshore killer whales 

 (Deecke et al., 2005). All calls of transient killer whales 

 are distinct from the calls of resident whales by 1) an 

 audible quavering of the fundamental sound frequencies 

 (instead of a crisp appearance of these sound frequen- 

 cies that is typical of calls from resident killer whales), 

 and 2 ) a distinctively lower amount of different call syl- 

 lables and a distinct order of these syllables compared 

 to those in calls of resident killer whales (Yurk, 2005). 

 Transient and resident killer whales are distinguish- 

 able from offshore killer whales by their use of unique 

 call types (Yurk, 2005). We determined whether the 

 encountered whales fell into discrete acoustic groups 

 and, if so, whether those acoustic groups were similar to 

 any of the acoustic groups observed in British Columbia 

 and southern Alaska. Analysis was completed by Yurk 

 (2005), independent of knowledge of genetic differences 

 and social associations among groups. Call rates were 

 estimated from field estimates of killer whale group 

 sizes for each encounter. 



Genetics DNA was extracted from the skin portion of 

 the biopsies using proteinase K digestion, phenol and 

 chloroform purification, and ethanol precipitation using 

 standard procedures (Sambrook et al., 1989) 



We obtained mtDNA sequences using the following 

 procedure; 1) the entire D-loop region was PCR-ampli- 

 fied by using custom-designed primers that annealed to 

 the flanking tRNA-Thr and 12s-rRNA regions (Barrett- 

 Lennard, 2000); 2) the PCR product was purified with 

 QIAQuick® spin columns (Qiagen, Valencia, CA) follow- 

 ing protocols supplied by Qiagen, Ltd. (Valencia, CA); 3) 



a sequencing reaction was performed with Fs-Taq® (Ap- 

 plied Biosystems, Foster City, CA) system reagents and 

 protocols supplied by Applied Biosystems, Ltd. (Foster 

 City, CA); and 4) the sequence was resolved on an Ap- 

 plied Biosystems 377 (Applied Biosystems, Foster City, 

 CA) automated DNA sequencer. Because the sequence 

 was too long (950 bases) to be entirely resolved in one 

 direction, sequencing reactions were run from each 

 end of the amplified fragment. We visually checked the 

 output graphs from the automated sequencer and cor- 

 rected the computer-generated sequences accordingly. 

 We also used the approximately 400-base overlap in the 

 sequences of opposite directions to check for errors. As 

 a final check of accuracy, we overlaid each output graph 

 with a reference graph on a transparent sheet, and 

 scanned the two graphs for differences. We then aligned 

 unique sequences using the program CLUSTAL-W (Eu- 

 ropean Bioinformatics, Cambridge, UK) (Thompson et 

 al., 1994). 



Results 



Summary of survey effort and encounters 

 with killer whales 



On 250 occasions, groups of one or more killer whales 

 were encountered during the surveys that covered a total 

 of 22,491 miles in 421 days in the eastern Aleutians and 

 False Pass-Unimak Island area (Table 1). The majority 

 of survey effort and encounters occurred west of Unimak 

 Pass during summer; surveys in False Pass-Unimak 

 Island area were not initiated until 2003. From approxi- 

 mately half of our encounters with groups of killer 

 whales in both regions, we obtained genetic samples 

 or acoustic recordings (Table 1). Killer whales of the 

 offshore ecotype were encountered only once (in 2003) 

 and both acoustic and genetic samples were obtained 

 during this encounter. 



Use of acoustic data, genetic analysis, 

 and group association to infer lineage 



Genetic and acoustic analyses revealed the presence of 

 three killer whale populations. As described in more 

 detail below, one population clustered genetically and 

 acoustically with resident killer whales ranging from 

 Puget Sound, Washington to Kenai Fjords, Alaska, 

 and a second population clustered with transient killer 

 whales from the same general area. Accordingly, those 

 two groups were provisionally classified as a resident 

 killer whale group and a transient killer whale group, 

 respectively. The third population clustered genetically 

 with offshore killer whales sampled off British Columbia, 

 and were provisionally classified as an offshore killer 

 whale group. Acoustic comparison was not possible in 

 the case of offshore killer whales because of a scarcity 

 of recordings. 



Resident, transient, and offshore killer whales have 

 never been observed interacting socially in the ar- 



