1. Population and Evolutionary Genetic Analyses Using Multiple Nuclear and Mitochondrial Loci in Steller Sea Lions. John W. Bickham, Ryan M. Huebinger, Thomas R. Gelatt, Greg O’Corry-Crowe, and Joseph I. Hoffman

2. ACKNOWLEDGEMENTS Project began in 1992 I thank the following for providing samples: T. Loughlin, T. Gelatt, V. Burkanov and D. Calkins Southeastern Alaska and PWS- ADF&G Gulf of Alaska and Aleutian Is.-NOAA sponsored cruises

3. ACKNOWLEDGEMENTS Russia: Vladimir Burkanov, Nikolay Pavlov, Vladimir Vertynkin, Evgeny Mamaev. The Alaska SeaLife Center, Don Calkins For help in the laboratory and for data analysis I thank Alyson Baker, Robert Trujillo, John Patton, Cole Matson and Dmitri Politov.

4. Population Estimates for Steller Sea Lions (1960-1994) Population Estimates in Thousands 0 20 40 60 80 100 120 140 160 180 200 1960s1970s1980s1990s1994 Eastern Stock East of 144°W Western Stock West of 144°W

5. Population Trends  The total SSL population numbered between 240,000 and 300,000 during the late 1950’s and early 1960’s.  Only 116,000 were estimated range-wide in 1989. -1969--1979--1989-

6. 30° 45° 60° KAM OKH BER PWS NCA ORE SEA COM WALCAL EAL WGA CGA BRC KUR RUSSIAALASKA 135°W 180°135°E Sampled Rookeries JAPAN

7. Previous Genetic Studies Bickham et al. (1996): Discovered high genetic differentiation between populations on either side of 144˚W; subsequently recognized an eastern stock (threatened) and western stock (endangered) Discovered high genetic differentiation between populations on either side of 144˚W; subsequently recognized an eastern stock (threatened) and western stock (endangered) Also found high diversity of “mtDNA haplotypes and a clear pattern of macrogeographic variation” Also found high diversity of “mtDNA haplotypes and a clear pattern of macrogeographic variation”

8. 30° 45° 60° KAM OKH BER PWS NCA ORE SEA COM WALCAL EAL WGA CGA BRC KUR RUSSIAALASKA 135°W 180°135°E Sampled Rookeries JAPAN Cape Suckling 144°W WESTERN STOCK EASTERN STOCK The Western and Eastern Stocks

9. Previous Genetic Studies (cont) Bickham et al. (1998) Found evidence for three possible groups: Asian and central groups within the western stock and an eastern stock Found evidence for three possible groups: Asian and central groups within the western stock and an eastern stock Could have arisen due to two, possibly three, glacial refugia that separated the populations during the Pleistocene era Could have arisen due to two, possibly three, glacial refugia that separated the populations during the Pleistocene era

10. A Third Stock?  Previous research suggests that animals sampled from Russian rookeries are genetically distinct from other western stock sea lions. 30° 45° 60° KAM OKH BER PWS NCA ORE SEA COM WALCAL EAL WGA CGA BRC KUR RUSSIAALASKA Cape Suckling 135°W 180°135°E Sampled Rookeries JAPAN 144°W EASTERN STOCK WESTERN STOCK ASIAN STOCK

11. Previous Genetic Studies (cont) Baker et al. (2005) Subdivided the Western Stock into Asian Stock (OKH, KUR, KAM) and Western Stock (COM east through PWS) Subdivided the Western Stock into Asian Stock (OKH, KUR, KAM) and Western Stock (COM east through PWS)

12. In this presentation we will: Summarize a 14-year range-wide population- genetic analysis based upon the mtDNA control region of Steller sea lions. Present preliminary results from an ongoing complementary study of cytochrome b sequences. Compare phylogeographic and population genetic patterns for control region vs. cyt b sequences. Present data from nuclear microsatellite loci that corroborate some of the findings of stock structure in Steller sea lions.

13. StockRegionSample Size EASTERN STOCKNCA (1 ROOKERY)51 ORE (1 ROOKERY)85 BRC (2 ROOKERIES)23 SEA (3 ROOKERIES)76 TOTAL235 WESTERN STOCKPWS (2 ROOKERIES)153 BER (1 ROOKERY)42 CGA (6 ROOKERIES)197 WGA (5 ROOKERIES)108 EAL (6 ROOKERIES)275 CAL (10 ROOKERIES)247 WAL (3 ROOKERIES)98 COM (1 ROOKERY)126 TOTAL1246 ASIAN STOCKKAM (2 ROOKERIES)71 KUR (5 ROOKERIES)207 OKH (2 ROOKERIES)237 TOTAL515 GRAND TOTAL1,710 Control Region Sample Sizes

14. StockRegionSample Size EASTERN STOCKNCA (1 ROOKERY)27 ORE (1 ROOKERY)32 BRC (2 ROOKERIES)15 SEA (3 ROOKERIES)341 TOTAL415 WESTERN STOCKPWS (2 ROOKERIES)27 BER (1 ROOKERY)21 CGA (7 ROOKERIES)114 WGA (5 ROOKERIES)32 EAL (6 ROOKERIES)84 CAL (9 ROOKERIES)73 WAL (3 ROOKERIES)51 COM (1 ROOKERY)46 TOTAL448 ASIAN STOCKKAM (2 ROOKERIES)63 KUR (5 ROOKERIES)74 OKH (2 ROOKERIES)133 TOTAL165 GRAND TOTAL878 Cytochrome b Sample Sizes

15. StockRegionSample Size EASTERN STOCKNCA (1 ROOKERY)25 ORE (1 ROOKERY)25 BRC (2 ROOKERIES)18 SEA (3 ROOKERIES)45 TOTAL113 WESTERN STOCKPWS (2 ROOKERIES)75 BER (1 ROOKERY)13 CGA (3 ROOKERIES)43 WGA (3 ROOKERIES)30 EAL (4 ROOKERIES)172 CAL (3 ROOKERIES)70 WAL (1 ROOKERY)12 COM (1 ROOKERY)25 TOTAL440 ASIAN STOCKKAM (1 ROOKERY)25 KUR (4 ROOKERIES)40 OKH (2 ROOKERIES)50 TOTAL115 GRAND TOTAL668 Microsatellite Sample Sizes

16. L-shaped distribution of control-region haplotypes No evidence of a significant bottleneck or reduction in variation

17. Haplotype LLL OKH KUR KAM COM WAL CAL EAL BER WGA CGA PWS SEA BRC ORE NCA Haplotype Frequency 0.00 0.02 0.04 0.06 0.08 0.10 Haplotype E OKH KUR KAM COM WAL CAL EAL BER WGA CGA PWS SEA BRC ORE NCA Haplotype Frequency 0.00 0.02 0.04 0.06 0.08 0.10 0.12 Haplotype H OKH KUR KAM COM WAL CAL EAL BER WGA CGA PWS SEA BRC ORE NCA Haplotype Frequency 0.00 0.10 0.20 0.30 0.40 0.50 Haplotype BB Haplotype Frequency 0.00 0.10 0.20 0.30 0.40 0.50 OKH KUR KAM COM WAL CAL EAL BER WGA CGA PWS SEA BRC ORE NCA Control Region Haplotype Distributions

18. Haplotype 1 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 Haplotype Frequency Haplotype 4 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 Haplotype Frequency Haplotype 2 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 Haplotype Frequency Haplotype 10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 Haplotype Frequency OKH KUR KAM COM WAL CAL EAL BER WGA CGA PWS SEA BRC ORE NCA OKH KUR KAM COM WAL CAL EAL BER WGA CGA PWS SEA BRC ORE NCA OKH KUR KAM COM WAL CAL EAL BER WGA CGA PWS SEA BRC ORE NCA OKH KUR KAM COM WAL CAL EAL BER WGA CGA PWS SEA BRC ORE NCA Cytochrome b Haplotype Distributions

19. Adak Island CAL Akun Island EAL Seal Rocks PWS Clubbing Rocks WGA Yunaska Island CAL Ugamak Island EAL Chirikof Island CGA Amak Island EAL Bogoslof Island EAL Pinnacle Rock WGA Chowiet Island CGA Marmot Island CGA Adugak Island EAL Attu Island WAL Amchitka Island CAL Seguam Island CAL Medney Island COM Kasatoshi Island CAL Sugarloaf Island CGA Akutan Island EAL Agattu Island WAL Kozlova Cape RUS Walrus Island BER Fish Island PWS Kiska Island Lief Cove CAL Kiska Island Cape St. Stephen CAL Buldir Island WAL Gramp Rock CAL Atkins Island WGA Tag Island CAL Ogchul Island EAL Antsiferova Island KUR Zheleznaya Bay KAM Raykoke Island KUR Iony Island OKH Tuleniy Island OKH Srednego Island KUR Yamsky Island OKH Lovushki Island KUR Chernyye Bratya KUR Forrester Island SEA North Danger Rocks BRC St. George Reef NCA Triangle Island BRC Hazy Islands SEA Rogue Reef ORE White Sisters Islands SEA 0.005 changes Legend Asian Stock Western Stock Eastern Stock NJ Tree for Rookeries Based on Fst Estimates Control Region

20. OKH KUR KAM COM CAL EAL CGA WGA PWS BER WAL SEA BRC ORE NCA 0.005 changes NJ Tree Regions Based on Fst Estimates Control Region Legend Eastern Stock Western Stock Asian Stock

21. Zalophus A XXXX UUUUU IIII NNN EEEEE TTTTT E ZZZ F XX VV AAAAA FFFF B R FFFFF RRRR UU XXXXX P SSSS III Q C YYYYY D H GGGG SS BBBBB I YY J IIIII LL GGGGG L CCCCC M ZZ BBBBBB CCC N VVVV DDDDD K O WWWW UUUU OO WW Z QQ JJJ FFF DDD QQQQQ AA PP KKK YYY RRRRR BB GGG YYYY LLL TTT BBBB MMM JJJJ RRR SSS VVVVV AAAA HHHH RR S MMMMM OOOOO Y KKKKK NNNNN QQQ OOOO LLLLL KKKK PPPPP LLLL V W BBB PPP X HH MMMM TTTT II GG TT JJ MM PPPP KK OOO VVV CC HHHHH DD HHH NNNN AAAAAA JJJJJ T NN U EE UUU ZZZZ AAA EEE FF CCCC WWWWW ZZZZZ QQQQ G XXX WWW EEEE DDDD SSSSS 0.001 substitutions/site NJ NJ tree of control region haplotypes shows two distinct eastern stock haplotype lineages.

23. 2 4 5 6 10 7 8 9 11 12 13 14 15 17 16 23 18 19 20 21 22 24 25 26 27 ? 33 30 29 28 34 31 32 ? 1 3 Minimum Spanning Network of Steller Sea Lion Cytochrome b haplotypes n = 878, 1040 bp 29 Variable Positions Ti/Tv = 26/3 13 Silent, 13 Nonsynonymous Transitions 1 Silent, 2 Nonsynonymous Transversions 1 st Position: 10 (9 AAS) 2 nd Position: 6 (6 AAS) 3 rd Position: 13 (0 AAS) 14 Total Variable Amino Acids 200+ 100-200 50-100 25-50 10-25 2-10 1 ALL EASTWEST ASIAN White Line: Silent Mutation Red Line: Amino Acid Shift

24. Regional Cytochrome b Slatkin’s Linearized F ST Midpoint Rooted NJ Phylogram OKH KUR KAM COM CAL CGA WAL WGA BER EAL PWS BRC OR E SEA NCA 0.05 changes Legend Eastern Stock Western Stock Asian Stock

25. Asian Stock Western Stock NJ Phylogram of Rookeries Based on Microsatellite Data Legend Eastern Stock Western Stock Asian Stock

26. OKH KAM KUR WAL CAL Eastern Stock Western Stock Asian Stock COM EAL BER CGA WGA PWS NCA ORE SEA BRC 0.01 NJ Phylogram of Regions Based on Microsatellite Data. Legend Eastern Stock Western Stock Asian Stock

27.  Despite the generally high sample sizes in this study, resolution of population substructure increased when rookeries were grouped into their respective regions. Control region sequences clearly support the recognition of three stocks. Conclusions Control Region Phylogeography Cytochrome b Phylogeography Clearly differentiates the eastern and western stocks but does not resolve the Asian stock. Microsatellite Analysis Nuclear microsatellites show a pattern nearly the same as for the cytochrome b gene. Clearly differentiates the eastern and western stocks but does not resolve the Asian stock.

28. The Asian Stock  The recognition of three management units may be appropriate for Steller sea lion conservation. 30° 45° 60° KAM OKH BER PWS NCA ORE SEA COM WAL CAL EAL WGA CGA BRC KUR RUSSIAALASKA Cape Suckling 135°W 180°135°E Sampled Rookeries JAPAN 144°W EASTERN STOCK WESTERN STOCK ASIAN STOCK