Investigation into the phylogeny of         Odobenus RosmarusA report for Nello Cristianini for the unit EMATM0004    Comp...
IntroductionThis project investigates the evolutionary history of Odobenus rosmarus (The walrus). The evolutionof the Pinn...
Nucleotide density          5000                                                    Nucleotide density   20000.5          ...
Results of CYTB blast:Rank       Latin name                      Common Name                  Total Score(Max 760)1       ...
If we remove the Polar bear to allow us to zoom in we can see five distinct groups. The data showsthe Walrus is about equa...
Four phylogenetic trees were built, one for each Cytochrome from both amino acid and nucleotidesequences’. In order to bui...
Addressing the question of how many species of OdobenusRosmarus there are we utilize a selection of walrus samples fromthe...
ConclusionThe analysis that we have performed present results that stand in contrast to the two papers Ulfureet al (1995) ...
Appendix A                                                             Accession Number                                   ...
Appendix BCLUSTAL 2.1 multiple sequence alignment cytochrome Bgi|115494578|ref|YP_778707.1|       MTNIRKVHPLAKIINSSLIDLPTP...
gi|8038011|gb|AAF71578.1|           HETGSNNPSGIPSDSDKIPFHPYYTIKDILGALLLTLALATLVLFSPDLL   85gi|1122916|gb|AAB50570.1|      ...
gi|37695534|gb|AAR00312.1|       THTSTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK   98gi|115494821|ref|YP_778750.1|    T...
Appendix C bibliographyAndersen et al. (1998). Population Structure and gene flow of the Atlanstic Walrus (Odobenus       ...
Investigation into the phylogeny of odobenus rosmarus
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Investigation into the phylogeny of odobenus rosmarus

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Investigation into the phylogeny of odobenus rosmarus

  1. 1. Investigation into the phylogeny of Odobenus RosmarusA report for Nello Cristianini for the unit EMATM0004 Computational Genomics and Bioinformatics Algorithms By Samuel R Neaves SN0550 November 2011
  2. 2. IntroductionThis project investigates the evolutionary history of Odobenus rosmarus (The walrus). The evolutionof the Pinnipedia (Odobenidae- walruses, Otariidae- eared seals, including sea lions and fur seals &Phocidae- earless seals) is said to be enigmatic with the exact relationships between subspecies indispute. The majority of authors support a monophyletic origin of the pinnipeds from a caniform,however there are others who suggest a diphyletic origin with the phocidae being related to themustelids (The mustelids are themselves a disputed family). Arnason et al (1995).A further dispute is that some authors divide the walrus into three sub species of Odobenusrosmarus + (rosmarus, divergen or laptivai) however recent work by (Lindqvist et al, 2009),concludes that laptivai are not a distinct species from divergen. The aim of this investigation is togather evidence for the true phylogeny.Data DescriptionThe primary species for this investigation will be Odobenus rosmarus rosmarus. The completemitochondrial DNA accession number in genbank is: NC_004029(.2). Odobenus rosmarusrosmarus’s phylogeny will be computed in relation to Erignathus barbatus(Bearded Seal,representing Phocidae ) Zalophus californianus(California Sea Lion, representing Otariidae) Ursusmaritimus (Polar bear, representing Caniformia) and Gulo gulo (Wolverine, representing mustelids).Homo sapiens are used as an out group to root the phylogenetic trees. For the full table of accessionnumbers see appendix A.Sequence statistics.Odobenus rosmarus rosmarus mitochondrial DNA was statistically analyzed with the followinginformation found:The size of the genome is 16565 base pairs.The number of each base:A C G T5401 4310 2414 4440The base count frequency:A C G T0.3260 0.2602 0.1457 0.2680This shows that there are twice as many A’s as G’s, with roughly the same amount of C’s and T’s overthe whole genome. This seems an interesting break from the norm of A and T content being similarand G and C content being similar. To further investigate and in order to consider local fluctuationsin the frequencies of nucleotides we employ sliding windows of size 5000, 2000 and 500 and plot thefrequencies.
  3. 3. Nucleotide density 5000 Nucleotide density 20000.5 0.5 A A0.4 C 0.4 C G G0.3 T 0.3 T0.2 0.20.1 0.1 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 A-T C-G density A-T C-G density0.7 0.7 A-T A-T0.6 C-G 0.6 C-G0.5 0.50.4 0.4 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 Nucleotide density 500 0.8 A 0.6 C G 0.4 T 0.2 0 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 A-T C-G density 0.7 A-T 0.6 C-G 0.5 0.4 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 A sliding window of size 5000 does not show a great deal of variation amongst the composition however a smaller windows clearly show peaks and troughs, which shows that the nucleotides are not drawn from a independent and identically distributed probability distribution as the distribution changes along the genome. With a caveat of caution because of the apparent violation of the aggregate frequencies, the GC content is also plotted; at the smallest window size this seems to show six distinct waves of variation in both AT and CG content. Next we employ an ab initio method to find protein encoding genes. The single-nucleotide permutation test calculates the significance of Open Reading Frames(ORFs) with a threshold set to be longer than all ORFs in a random sequence and it finds 1 gene. If we set α to 5% then we get a larger value of 12 genes found. We are careful to set the correct genetic code for vertebrate Mitochondrial. We translate these genes into protein sequences and identify cytochrome B and cytochrome C by translating into amino acid sequences and blasting. Once identified we run further protein blasts using both cytochromes to identify the nearest other species.
  4. 4. Results of CYTB blast:Rank Latin name Common Name Total Score(Max 760)1 Halichoerus grypus Grey Seal 6812 Gulo gulo Wolverine 6803 Phoca vitulina stejnegeri Harbour Seal 6794 Erignathus barbatus Bearded Seal 6795 Ictonyx libyca Saharan Striped Pole cat 679These results are interesting because they do not include any Otariidaes, suggesting that Pinnipediahave a diphyletic origin from the ancient caniform with the Odobenidae, Phocidae and the Mustelidson one branch and Otariidaes on another.Results of CYTC blastRank Latin Name Common name Total Score1 Tremarctos ornatus Spectacled bear 4472 Otaria byroni South American Sea Lion 4463 Arctocephalus Guadalupe fur seal 445 townsendi4 Neophoca cinerea Australian Sea Lion 4445 Callorhinus ursinus Northern fur seal 444This results is a contrast to the CYTB blast results, this time with many Otariidaes, no Phocidaes ahigh ranking Caniformia and no Mustelids. This data appears to support the monophyletic originhypothesis or a diphyletic origin but with the Odobenidae on the branch of Otariidaes. To furtherthe investigation we add the initially selected organisms to the data set and compute the geneticdistances between each pair. We utilize the Jukes-Cantor correction to account for multiplesubstitutions that have occurred in the same space. ( )This states that the number of substitutions per site between two sequences (K) can be estimatedfrom the observed fractions that differ (d).With this applied on cytochrome b it is clear that the Polar bear is very distantly related compared tothe other species. It is interesting that the data suggests that the Spectacled bear is a closer relationto the pinnipeds than the Polar bear.
  5. 5. If we remove the Polar bear to allow us to zoom in we can see five distinct groups. The data showsthe Walrus is about equally distant from the Otariidaes and the Phocidaes , with the Otariidaescloser to Mustelids and as far from the Phocidaes as it is from the Spectacled bear.Performing the same procedure for cytochrome C we get similar results however, this time thePhocidaes are clearly grouped with Mustelids along with the Polar bear. The Spectacled bear is onceagain on its own slightly closer to Phocidaes than the Otariidaes. This leaves the Walrus again as anoutliner being roughly equal distances from the two major clusters.
  6. 6. Four phylogenetic trees were built, one for each Cytochrome from both amino acid and nucleotidesequences’. In order to build the cytochrome C nucleotide tree, a number of animals includingOdobenus rosmarus had to use amino acid to nucleotide transformation due to unavailability ofsequence data, which as this is not a one to one relation results in some random substitutions whichmay affect the accuracy of this graph.The results present a confused picture with many contradictions between the four trees. However ifwe discount the Cytochrome C nt tree there appears to be some consensus, all the Otariidae andPhocidae are consistently grouped together and the Odobenus Rosmarus is seen to first split fromthe common ancestor of both the Otariidae and Phocidae which then diverged at a later date, thisstands in contrast to the results in Arnason et al(1995) which show the Phocidae first splitting, with alater split between the Otariidae and Odobenus Rosmarus. However (Lento et al, 1995) does offersome evidence for Odobenus Rosmarus being an early divergence from the common pinnipedancestor which would be consistent with these results. There are major differences in the placing ofUrsus maritimus, Tremarctos ornatus and Gulo gulo between the cytochrome b and c trees,cytochrome c puts the mustelids, Ursus maritimus and Tremarctos ornatus on the same branch asthe Phocidae, however the cytochrome B tree has the Mustelids and Tremarctos ornatus close tothe Otariidae, with Ursus maritimus being a distance relation. Castresana (2001) presents evidencethat Cytochrome B is more reliable for constructing trees at the genus and family level and thereforethis tree may be taken as a more reliable indicator to the true phylogeny.The online resource tax browser collated by NCBI has the Odobenidae, Phocidae and Otariidae asthree distinct families within the suborder of Caniformia and does not have any one group as anancestor to the other.Multiple alignmentsIn order to build multiple alignments and identify polymorphic sites the heuristic CLUSTALW tool wasused to align both the cytochrome B and cytochrome C protein sequences. This was set to use theBLOSUM Protein weight matrix with a GAP open penalty set to 10, GAP extension penalty set to0.20, GAP distances set to 5 and No End Gaps set to ‘No’. Too see the full alignments refer toappendix B. It is clear that both alignments are very good apart from of course the out-group andthe Polar bear in cytochrome B. The majority of polymorphic sites in cytochrome B are consistentwith the groupings of Odobenidae, Phocidae and Otariidae. They include both indels and pointmutations. The sites are fairly sporadic across the sequences which is in contrast to the polymorphicsites in cytochrome C which mostly lie between the 50th and 100th amino acid with the extremitiesremaining constant.
  7. 7. Addressing the question of how many species of OdobenusRosmarus there are we utilize a selection of walrus samples fromthe (Lindqvist et al, 2009) study. These sequences are ATL25tRNA-Trp and tRNA-Pro genes from the mtDNA region of thegenomes. We follow the same procedure as earlier computingthe genetic distance between the samples using jukes cantorcorrection and plotting these on a graph. We use thiscomputation to build an unrooted phylogenetic tree. Both thetree and the distance plot conforms with (Lindqvist et al, 2009)conclusion that the walruses sampled from the Laptev sea areindeed just a subgroup of the Pacific walrus because they exist ina sub branch of Odobenus rosmarus divergens and their geneticdistance is mixed amongst the Pacific samples. This data andanalysis therefore does not justify labeling these as a separatespecies.A further point of note is that the Atlantic walrus genetic datashow signs of going through a genetic bottle neck due to the lackof diversity compared to the Pacific walrus. This information sitswith the historic fact, that the Atlantic walrus was almost huntedto extinction by the 1950’s with numbers beginning to recoversince then. Whereas the more remote locations inhabited by thePacific walrus protected them from human hunting which hasallowed there numbers to remain much higher throughout the20th century and therefore accounting for the greater geneticdiversity shown in the samples. If further larger samples arecollected and more detailed analysis’s show the same resultsthen it may be it will be time to change the current NCBI taxbrowser to show only two species of Odobenus Rosmarus. Atlantic Pacific Laptivai
  8. 8. ConclusionThe analysis that we have performed present results that stand in contrast to the two papers Ulfureet al (1995) and Lento et al (1995). Proving that the question of pinniped evolution is indeed veryinteresting with a variety of hypothesis still in contention. The examination of the question of ifthere are two or three walruses species came to the same conclusion as (Lindqvist et al, 2009)despite using different techniques and methods. It must be said that the same data was used for thisstudy and Lindqvist et al’s (2009) study. Which when taken with the low numbers of samples and theuse of amplicons, as well as the inherent difficulty of sampling Odobenus Rosmarus potentiallyleading to sampling errors, such as close relatives being sampled, leaves the hypothesis very muchstill open to refutation.While the evolution of pinnipeds remain inconclusive there remains the need for further more in-depth studies to allow for reliable conclusions to be drawn so that wise actions can be taken toprotect this charismatic and vulnerable artic creature from the threats of hunting and habitatdestruction that continue to push many creatures to extinction. A pair of curious Walruses (image from http://www.free-extras.com/images/walrus-8927.htm)
  9. 9. Appendix A Accession Number Proteins NucleotidesLatin Name Common mtDna Cytochrome B Cytochrome C Cytochrome B Cytochrome C NameOdobenus Atlantic Walrus CAD21718 NP_659340.3 NC_004029.2 NARosmarusRosmarusZalophus California sea YP_778707.1 YP_778698.1 D26524.1 AJ616896.1californianus lion, representing the OtariidaeErignathus Bearded Seal, YP_778837.1 YP_778828.1 AY140982.1 FJ839388.1barbatus representing PhocidaeUrsus Polar bear, AAF71578.1 NP_597984.1 NC_003428.1 NAmaritimus representing CaniformiaGulo gulo Wolverine, YP_001382271.1 YP_001382262.1 L77960.2 EU544598.1 representing MustelidsHomo Sapiens Human, is used AAA31851.1 NP_061820.1 S88250.1 NM_018947.5 as an outgroupHalichoerus Grey Seal ACZ28998.1 NP_007072.1 GU167293.1 GU733706.1grypusPhoca vitulina Harbor seals BAI60013.1 NP_006931.1 AB510422.1 NAstejnegeriIctonyx libyca Saharan Striped ABV57060.1 NA EF987739.1 NA PolecatTremarctos spectacled bear AAB50570.1 YP_001542732.1 U23554.1 NAornatusOtaria byronia South American AAQ95107.1 AAR00312.1 AY713034.1 AJ891144.1 Sea LionArctocephalus Guadalupe fur YP_778759.1 YP_778750.1 AF380897.1 NAtownsendi sealNeophoca Australian Sea YP_778746.1 YP_778737.1 AF380915.1 NAcinerea LionCallorhinus Northern fur YP_778694.1 YP_778685.1 HQ895717.1 HM171421.1ursinus sealOdobenus Rosmarus samples.Lap 1 EU728526 Pac 8 EU728538 Atlan 4 EU728567 Atlan 14 EU728549Lap 2 EU728527 Pac 9 EU728539 Atlan 5 EU728568 Atlan 15 EU728550Lap 3 EU728529 Pac 12 EU728542 Atlan 6 EU728569 Atlan 16 EU728551Lap 4 EU728530 Pac 13 EU728543 Atlan 7 EU728570 Atlan 17 EU728552Lap 5 EU728525 Pac 14 EU728562 Atlan 8 EU728571 Atlan 18 EU728553Pac 1 EU728531 Pac 15 EU728563 Atlan 9 EU728572 Atlan 19 EU728554Pac 2 EU728532 Pac 16 EU728564 Atlan 10 EU728573 Atlan 20 EU728555Pac 3 EU728533 Atlan 1 EU728561 Atlan 11 EU728546 Atlan 21 EU728556Pac 4 EU728534 Atlan 2 EU728565 Atlan 12 EU728547 Atlan 22 EU728557Pac 5 EU728535 Atlan 3 EU728566 Atlan 13 EU728548 Atlan 23 EU728558Pac 6 EU728536Pac 7 EU728537
  10. 10. Appendix BCLUSTAL 2.1 multiple sequence alignment cytochrome Bgi|115494578|ref|YP_778707.1| MTNIRKVHPLAKIINSSLIDLPTPSNISAWWNFGSLLAACLALQILTGLF 50gi|115494844|ref|YP_778746.1| MTNIRKTHPLAKIINNSLIDLPAPSNISAWWNFGSLLAVCLALQILTGLF 50gi|37620596|gb|AAQ95107.1| MTNIRKVHPLAKIINNLLIDLPAPSNISAWWNFGSLLAVCLALQILTGLF 50gi|115494690|ref|YP_778694.1| MTNIRKVHPLAKIINSSLIDLPAPSNISAWWNFGSLLATCLVLQILTGLF 50gi|115494830|ref|YP_778759.1| MTNIRKTHPLAKIINNSLIDLPAPSNISTWWNFGSLLAACLALQILTGLF 50gi|269302297|gb|ACZ28998.1| MTNIRKTHPLMKIINNSFIDLPTPSNISAWWNFGSLLGICLILQILTGLF 50gi|282154709|dbj|BAI60013.1| MTNIRKTHPLMKIINNSFIDLPTPSNISAWWNFGSLLGICLILQILTGLF 50gi|115494788|ref|YP_778837.1| MTNIRKTHPLIKIINSSFIDLPTPSNISAWWNFGSLLGICLILQILTGLF 50gi|8038011|gb|AAF71578.1| --------------------------------------------------gi|1122916|gb|AAB50570.1| MTNIRKTHPLAKIINSSFIDLPTPSNISAWWNFGSLLGVCLILHILTGLF 50gi|157461069|gb|ABV57060.1| MANIRKTHPLAKIINNSFVDLPTPSSISAWWNFGSLLGICLIIQILTGLF 50gi|153124668|ref|YP_001382271. MTNIRKTHPLAKIINNSFIDLPTPSNISAWWNFGSLLGICLILQILTGLF 50gi|21425423|emb|CAD21718.1| MTNIRKTHPLAKIINNTFIDLPTPSNISAWWNFGSLLATCLILQILTGLF 50gi|552606|gb|AAA31851.1| --------------------------------------------------gi|115494578|ref|YP_778707.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100gi|115494844|ref|YP_778746.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100gi|37620596|gb|AAQ95107.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100gi|115494690|ref|YP_778694.1| LAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100gi|115494830|ref|YP_778759.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100gi|269302297|gb|ACZ28998.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYLHANGASMFFICLYMHVGR 100gi|282154709|dbj|BAI60013.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYLHANGASMFFICLYMHVGR 100gi|115494788|ref|YP_778837.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100gi|8038011|gb|AAF71578.1| --------------------------------------------------gi|1122916|gb|AAB50570.1| LAMHYTADTTTAFSSVAHICRDVNYGWVIRYMHANGASMFFICLFMHVGR 100gi|157461069|gb|ABV57060.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLFLHVGR 100gi|153124668|ref|YP_001382271. LAMHYTSDTATAFSSVTHICRDVNYGWVIRYMHANGASMFFICLFLHVGR 100gi|21425423|emb|CAD21718.1| LAMHYTSDTTTAFSSITHICRDVNYGWIIRYMHANGASMFFICLYAHMGR 100gi|552606|gb|AAA31851.1| --------------------------------------------------gi|115494578|ref|YP_778707.1| GLYYGSYTLTETWNIGIILLFTIMATAFMGYVLPWGQMSFWGATVITNLL 150gi|115494844|ref|YP_778746.1| GLYYGSYTLTETWNIGIILLFTIMATAFMGYVLPWGQMSFWGATVITNLL 150gi|37620596|gb|AAQ95107.1| GLYYGSYTLTETWNIGIILLLTVMATAFMGYVLPWGQMSFWGATVITNLL 150gi|115494690|ref|YP_778694.1| GLYYGSYTLTETWNIGIILLLTIMATAFMGYVLPWGQMSFWGATVITNLL 150gi|115494830|ref|YP_778759.1| GLYYGSYTLAETWNIGIILLFTIMATAFMGYVLPWGQMSFWGATVITNLL 150gi|269302297|gb|ACZ28998.1| GLYYGSYTFTETWNIGIILLFTIMATAFMGYVLPWGQMSFWGATVITNLL 150gi|282154709|dbj|BAI60013.1| GLYYGSYTFTETWNIGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLL 150gi|115494788|ref|YP_778837.1| GLYYGSYTFMETWNIGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLL 150gi|8038011|gb|AAF71578.1| --------------------------------------------------gi|1122916|gb|AAB50570.1| GLYYGSYLFSETWNIGIILLLTIMATAFMGYVLPWGQMSFWGATVITNLL 150gi|157461069|gb|ABV57060.1| GLYYGSYLFPETWNIGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLL 150gi|153124668|ref|YP_001382271. GLYYGSYTYSETWNIGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLL 150gi|21425423|emb|CAD21718.1| GIYYGSYTLAETWNIGIVLLLTIMATAFMGYVLPWGQMSFWGATVITNLL 150gi|552606|gb|AAA31851.1| --------------------------------------------------gi|115494578|ref|YP_778707.1| SAVPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFMASALVMVHLLFL 200gi|115494844|ref|YP_778746.1| SAVPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFMASALVMVHLLFL 200gi|37620596|gb|AAQ95107.1| SAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFVVSALVMVHLLFL 200gi|115494690|ref|YP_778694.1| SAIPYIGANLVEWIWGGFSVDKATLTRFFAFHFILPFMVSALVMVHLLFL 200gi|115494830|ref|YP_778759.1| SAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFVASALVMVHLLFL 200gi|269302297|gb|ACZ28998.1| SAIPYIGTDLVQWIWGGFSVDKATLTRFFAFHFILPFVVLALAAVHLLFL 200gi|282154709|dbj|BAI60013.1| SAIPYIGTDLVQWIWGGFSVDKATLTRFFAFHFILPFVVSALAAVHLLFL 200gi|115494788|ref|YP_778837.1| SAIPYIGTDLVQWIWGGFSVDKATLTRFFAFHFILPFVVLALAAVHLLFL 200gi|8038011|gb|AAF71578.1| ---------------GGFSVDKATLTRFFAFHFILPFIILALAAVHLLFL 35gi|1122916|gb|AAB50570.1| SAIPYIGTDLVEWIWGGFSVDKATLTRFFAFHFILPFIILALAMVHLLFL 200gi|157461069|gb|ABV57060.1| SAIPYIGNNLVEWIWGGFSVDKATLTRFFAFHFILPFIISALAAVHLLFL 200gi|153124668|ref|YP_001382271. SAIPYIGTSLVEWIWGGFSVDKATLTRFFAFHFILPFIILALAAIHLLFL 200gi|21425423|emb|CAD21718.1| SAIPYVGTDLVEWVWGGFSVDKATLTRFLALHFVLPFMALALTAVHLLFL 200gi|552606|gb|AAA31851.1| --------------------------------------------------gi|115494578|ref|YP_778707.1| HETGSNNPSGISSDSDKIPFHPYYTIKDILGTLLLILTLMLLVMFSPDLL 250gi|115494844|ref|YP_778746.1| HETGSNNPSGISSDSDKIPFHPYYTIKDILGTLFLILILMLLVMFSPDLL 250gi|37620596|gb|AAQ95107.1| HETGSNNPSGISSDSDKIPFHPYYTIKDILGTLLLILILMLLVMFSPDLL 250gi|115494690|ref|YP_778694.1| HETGSNNPSGVSSDSDKIPFHPYYTIKDILGTLLLILILMLLVMFSPDLL 250gi|115494830|ref|YP_778759.1| HETGSNNPSGVSSDSDKIPFHPYYTIKDILGALLLILILMLLVMFSPDLL 250gi|269302297|gb|ACZ28998.1| HETGSNNPSGIMSDSDKIPFHPYYTIKDILGALLLILVLTLLVLFSPDLL 250gi|282154709|dbj|BAI60013.1| HETGSNNPSGIMSDSDKIPFHPYYTIKDILGALLFILVLTLLVLFSPDLL 250gi|115494788|ref|YP_778837.1| HETGSNNPSGISSDSDKIPFHPYYTIKDILGALLLILVLMLLVLFSPDLL 250
  11. 11. gi|8038011|gb|AAF71578.1| HETGSNNPSGIPSDSDKIPFHPYYTIKDILGALLLTLALATLVLFSPDLL 85gi|1122916|gb|AAB50570.1| HETGSNNPSGISSNSDKIPFHPYYTIKDILGVLLLLLALVTLVLFSPDLL 250gi|157461069|gb|ABV57060.1| HETGSNNPSGIPSNSDKIPFHPYYTIKDILGVLLLIITLMTLVLFSPDLL 250gi|153124668|ref|YP_001382271. HETGSNNPSGIPSDSDKIPFHPYYTIKDILGALFLALVLMMLVLFSPDLL 250gi|21425423|emb|CAD21718.1| HETGSNNPSGILSDSDKIPFHPYYTIKDILGLIILILILMLLVLFSPDLL 250gi|552606|gb|AAA31851.1| --------------------------------------------------gi|115494578|ref|YP_778707.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILI 300gi|115494844|ref|YP_778746.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILI 300gi|37620596|gb|AAQ95107.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILI 300gi|115494690|ref|YP_778694.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVVALLLSILV 300gi|115494830|ref|YP_778759.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILV 300gi|269302297|gb|ACZ28998.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALVLSILI 300gi|282154709|dbj|BAI60013.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALVLSILI 300gi|115494788|ref|YP_778837.1| GDPDNYTPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALVLSILI 300gi|8038011|gb|AAF71578.1| GDPDNYIPAN---------------------------------------- 95gi|1122916|gb|AAB50570.1| GDPDNYTPANPVSTPLHIKPEWYFLFAYAILRSIPNKLGGVLALIFSILI 300gi|157461069|gb|ABV57060.1| GDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIPNKLGGVLALILSILV 300gi|153124668|ref|YP_001382271. GDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIPNKLGGVLALVLSILV 300gi|21425423|emb|CAD21718.1| GDPDNYTPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILV 300gi|552606|gb|AAA31851.1| ------------------KPEWYFLFAYTILRSVPNKLGGVLALLLSILI 32gi|115494578|ref|YP_778707.1| LAIIPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEHPFITI 350gi|115494844|ref|YP_778746.1| LAIVPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEHPFITI 350gi|37620596|gb|AAQ95107.1| LAIIPLLHTSKQRGMMFRPISQCLFWLLAADLLTLTWIGGQPVEHPFITI 350gi|115494690|ref|YP_778694.1| LAIIPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEYPFIAI 350gi|115494830|ref|YP_778759.1| LAIIPLLHTSKQRGMMFRPISQFLFWLLVADLLTLTWIGGQPVEYPFITI 350gi|269302297|gb|ACZ28998.1| LAIVPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEHPYITI 350gi|282154709|dbj|BAI60013.1| LAIVPLLHTSKQRGMMFRPISQCLFWFLVADLLTLTWIGGQPVEHPYITI 350gi|115494788|ref|YP_778837.1| LAIAPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEHPYITI 350gi|8038011|gb|AAF71578.1| --------------------------------------------------gi|1122916|gb|AAB50570.1| LAIIPLLHTSKQRGMMFRPLSQCLFWLLAADLLTLTWIGGQPVEHPLVII 350gi|157461069|gb|ABV57060.1| LAIIPLLHTSKQRSMMFRPLSQCLFWLLVADLLTLTWIGGQPVEHPFIII 350gi|153124668|ref|YP_001382271. LAIIPLLHTSKQRGMMFRPLSQCLFWLLVADLLTLTWIGGQPVEHPFITI 350gi|21425423|emb|CAD21718.1| LAIVPSLHTSKQRSMMFRPISQCLFWLLVADLITLTWIGGQPVEHPFIII 350gi|552606|gb|AAA31851.1| LAMIPILHMSKQQSMMFRPLSQSLYWLLAADLLILTWIGGQPVSYPFTII 82gi|115494578|ref|YP_778707.1| GQLASILYFTILLVFMPIAGIIENNILKW- 379gi|115494844|ref|YP_778746.1| GQLASILYFAILLILMPIAGIIENNILKW- 379gi|37620596|gb|AAQ95107.1| GQLASILYFTILLVLMPIAGIIENNILKW- 379gi|115494690|ref|YP_778694.1| GQLASILYFMILLVLMPMAGIIENNILKW- 379gi|115494830|ref|YP_778759.1| GQLASILYFTILLILMPVAGIIENNILKW- 379gi|269302297|gb|ACZ28998.1| GQLASILYFMILLVLMPIASIIENNILKW- 379gi|282154709|dbj|BAI60013.1| GQLASILYFMILLVLMPIASIIENNILKW- 379gi|115494788|ref|YP_778837.1| GQLASILYFAILLVFMPIASIIENNILKW- 379gi|8038011|gb|AAF71578.1| ------------------------------gi|1122916|gb|AAB50570.1| GQLASILYFTILLVLMPIAGIIENNLSKW- 379gi|157461069|gb|ABV57060.1| GQLASILYFMILLVFMPIASIAENNLLKW- 379gi|153124668|ref|YP_001382271. GQLASILYFAILLIFMPVASIVENNLLKW- 379gi|21425423|emb|CAD21718.1| GQLASILYFMILLVFMPIAGMIENSILKW- 379gi|552606|gb|AAA31851.1| GQVASVLYFTTILILMPTISLIENKMLKWA 112CLUSTAL 2.1 multiple sequence alignment Cytochrome Cgi|115494569|ref|YP_778698.1| MAYPFQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50gi|115494681|ref|YP_778685.1| MAYPFQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50gi|37695534|gb|AAR00312.1| MAYPLQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50gi|115494821|ref|YP_778750.1| MAYPFQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50gi|115494835|ref|YP_778737.1| MAYPFQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50gi|21717327|ref|NP_659340.3| MAYPLQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISTMLTTKL 50gi|159524414|ref|YP_001542732. MAYPFQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISTMLTTKL 50gi|19343520|ref|NP_597984.1| MACPFQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISTMLTTKL 50gi|115494779|ref|YP_778828.1| MAYPLQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKL 50gi|5835013|ref|NP_007072.1|COX MAYPLQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKL 50gi|5834861|ref|NP_006931.1|COX MAYPLQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKL 50gi|153124659|ref|YP_001382262. MAYPFQLGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKL 50gi|11128019|ref|NP_061820.1| -------------------------------------------MGDVEKG 7 . *gi|115494569|ref|YP_778698.1| THTNTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98gi|115494681|ref|YP_778685.1| THTSTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98
  12. 12. gi|37695534|gb|AAR00312.1| THTSTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98gi|115494821|ref|YP_778750.1| THTSTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98gi|115494835|ref|YP_778737.1| THTCTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98gi|21717327|ref|NP_659340.3| THTNTMDAQEVETVWTILPAIILIMIALPSLRILYMMDEINSP--FLTVK 98gi|159524414|ref|YP_001542732. THTNTMDAQEVETVWTILPAIILVLIALPSLRILYMMDEINNP--LLTVK 98gi|19343520|ref|NP_597984.1| THTSTMDAQEVETVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98gi|115494779|ref|YP_778828.1| THTSTMDAQEVETVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98gi|5835013|ref|NP_007072.1|COX THTSTMDAQEVETVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98gi|5834861|ref|NP_006931.1|COX THTSTMDAQEVETVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98gi|153124659|ref|YP_001382262. THTSTMDAQEVQTVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98gi|11128019|ref|NP_061820.1| KKIFIMKCSQCHTVEKGG-----KHKTGPNLHGLFGRKTGQAPGYSYTAA 52 .: *...: .** . : *.*: *: . : * *.gi|115494569|ref|YP_778698.1| TMGHQWYWTYEYTDYEDLSFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148gi|115494681|ref|YP_778685.1| TMGHQWYWTYEYTDYEDLSFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148gi|37695534|gb|AAR00312.1| TMGHQWYWTYEYTDYEDLSFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148gi|115494821|ref|YP_778750.1| TMGHQWYWTYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148gi|115494835|ref|YP_778737.1| TMGHQWYWTYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148gi|21717327|ref|NP_659340.3| TMGHQWYWSYEYTDYEDLSFDSYMVPTQELKPGELRLLEVDNRMVLPMEM 148gi|159524414|ref|YP_001542732. TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRAVLPMEM 148gi|19343520|ref|NP_597984.1| TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPVEM 148gi|115494779|ref|YP_778828.1| TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148gi|5835013|ref|NP_007072.1|COX TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148gi|5834861|ref|NP_006931.1|COX TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148gi|153124659|ref|YP_001382262. TMGHQWYWSYEYTDYEDLNFDSYMVPTQELKPGELRLLEVDNRVVLPMEM 148gi|11128019|ref|NP_061820.1| NKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKA 102 . .: * : . ..*: *: : * : * : . :gi|115494569|ref|YP_778698.1| TVRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|115494681|ref|YP_778685.1| TVRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|37695534|gb|AAR00312.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|115494821|ref|YP_778750.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|115494835|ref|YP_778737.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|21717327|ref|NP_659340.3| TVRMLISSEDVLHSWTVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|159524414|ref|YP_001542732. TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|19343520|ref|NP_597984.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|115494779|ref|YP_778828.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|5835013|ref|NP_007072.1|COX TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|5834861|ref|NP_006931.1|COX TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMTMRPGLYYGQCSE 198gi|153124659|ref|YP_001382262. TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198gi|11128019|ref|NP_061820.1| TNE----------------------------------------------- 105 * .gi|115494569|ref|YP_778698.1| ICGSNHSFMPIVIESVPLSCFEKWSASML- 227gi|115494681|ref|YP_778685.1| ICGSNHSFMPIVIESVPLSCFEKWSASMLQ 228gi|37695534|gb|AAR00312.1| ICGSNHSFMPIVIESVPLSYFEKWSTSMLQ 228gi|115494821|ref|YP_778750.1| ICGSNHSFMPIVIESVPLSYFEKWSASMLQ 228gi|115494835|ref|YP_778737.1| ICGSNHSFMPIVIESVPLSYFEKWSASMLQ 228gi|21717327|ref|NP_659340.3| ICGSNHSFMPIVLESVPLSYFEKWSASILQ 228gi|159524414|ref|YP_001542732. ICGSNHSFMPIVLELVPLSYFEKWSASML- 227gi|19343520|ref|NP_597984.1| ICGSNHSFMPIVLELVPLSYFEEWSASML- 227gi|115494779|ref|YP_778828.1| ICGSNHSFMPIVLELVPLSHFEKWSTSML- 227gi|5835013|ref|NP_007072.1|COX ICGSNHSFMPIVLELVPLSHFEKWSTSML- 227gi|5834861|ref|NP_006931.1|COX ICGSNHSFMPIVLELVPLSHFEKWSTSML- 227gi|153124659|ref|YP_001382262. ICGSNHSFMPIVLELVPLSHFEKWSASML- 227gi|11128019|ref|NP_061820.1| ------------------------------
  13. 13. Appendix C bibliographyAndersen et al. (1998). Population Structure and gene flow of the Atlanstic Walrus (Odobenus rosmarus rosmarus) in the eastern Atlantic Artic based on mitochondiral DNA and microsatellite variation. Molecular Ecology(7), 1323-1336.Castresana J. (2001). Molecular biology and Evolution(18), 465-471.Castresana J. (2001). Cytochrome b Phylogeny and the Taxonomy of Great Apes and Mammals. Molecular biology and Evolution(18), 465-471.Lento et al. (1995). Use of Spectral Anaylsis to test hypotheses on the orign of pinnipeds. Molecular Biology and Evolution(12), 28-52.Lindqvist et al. (2009). The Laptev Sea Walrus Odobenus rosmarus laptevi: an engima revisited. Zoologica Scripta(38), 113-127.Ulfure, A., bodin, K., Gullberg, A., Ledge, C., & Mouchaty, S. (1995). A Molecular View of Pinniped Relationships with Particular Emphasis on the True Seals. Journal of molecular Evolution(40), 78-85.

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