Investigation into the phylogeny of

Odobenus Rosmarus

A report for Nello Cristianini for the unit EMATM0004

Computational Genomics and Bioinformatics

Algorithms

By Samuel R Neaves SN0550

November 2011

Introduction

This project investigates the evolutionary history of Odobenus rosmarus (The walrus). The evolution

of 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 in

dispute. 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 the

mustelids (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 Odobenus

rosmarus + (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 to

gather evidence for the true phylogeny.

Data Description

The primary species for this investigation will be Odobenus rosmarus rosmarus. The complete

mitochondrial DNA accession number in genbank is: NC_004029(.2). Odobenus rosmarus

rosmarus’s phylogeny will be computed in relation to Erignathus barbatus(Bearded Seal,

representing Phocidae ) Zalophus californianus(California Sea Lion, representing Otariidae) Ursus

maritimus (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 accession

numbers see appendix A.

Sequence statistics.

Odobenus rosmarus rosmarus mitochondrial DNA was statistically analyzed with the following

information found:

The size of the genome is 16565 base pairs.

The number of each base:

A C G T

5401 4310 2414 4440

The base count frequency:

A C G T

0.3260 0.2602 0.1457 0.2680

This shows that there are twice as many A’s as G’s, with roughly the same amount of C’s and T’s over

the whole genome. This seems an interesting break from the norm of A and T content being similar

and G and C content being similar. To further investigate and in order to consider local fluctuations

in the frequencies of nucleotides we employ sliding windows of size 5000, 2000 and 500 and plot the

frequencies.

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.

0 2000 4000 6000 8000 10000 12000 14000 16000 180000.1

0.2

0.3

0.4

0.5

Nucleotide density

A

C

G

T

0 2000 4000 6000 8000 10000 12000 14000 16000 18000

0.4

0.5

0.6

0.7A-T C-G density

A-T

C-G

0 2000 4000 6000 8000 10000 12000 14000 16000 180000.1

0.2

0.3

0.4

0.5

Nucleotide density

0 2000 4000 6000 8000 10000 12000 14000 16000 18000

0.4

0.5

0.6

0.7A-T C-G density

A

C

G

T

A-T

C-G

0 2000 4000 6000 8000 10000 12000 14000 16000 180000

0.2

0.4

0.6

0.8Nucleotide density

0 2000 4000 6000 8000 10000 12000 14000 16000 18000

0.4

0.5

0.6

0.7A-T C-G density

A

C

G

T

A-T

C-G

5000 2000

500

Results of CYTB blast:

Rank Latin name Common Name Total Score(Max 760)

1 Halichoerus grypus Grey Seal 681

2 Gulo gulo Wolverine 680

3 Phoca vitulina stejnegeri Harbour Seal 679

4 Erignathus barbatus Bearded Seal 679

5 Ictonyx libyca Saharan Striped Pole cat 679

These results are interesting because they do not include any Otariidaes, suggesting that Pinnipedia

have a diphyletic origin from the ancient caniform with the Odobenidae, Phocidae and the Mustelids

on one branch and Otariidaes on another.

Results of CYTC blast

Rank Latin Name Common name Total Score

1 Tremarctos ornatus Spectacled bear 447

2 Otaria byroni South American Sea Lion 446

3 Arctocephalus townsendi

Guadalupe fur seal 445

4 Neophoca cinerea Australian Sea Lion 444

5 Callorhinus ursinus Northern fur seal 444

This results is a contrast to the CYTB blast results, this time with many Otariidaes, no Phocidaes a

high ranking Caniformia and no Mustelids. This data appears to support the monophyletic origin

hypothesis or a diphyletic origin but with the Odobenidae on the branch of Otariidaes. To further

the investigation we add the initially selected organisms to the data set and compute the genetic

distances between each pair. We utilize the Jukes-Cantor correction to account for multiple

substitutions that have occurred in the same space.

(

)

This states that the number of substitutions per site between two sequences (K) can be estimated

from the observed fractions that differ (d).

With this applied on cytochrome b it is clear that the Polar bear is very distantly related compared to

the other species. It is interesting that the data suggests that the Spectacled bear is a closer relation

to the pinnipeds than the Polar bear.

If we remove the Polar bear to allow us to zoom in we can see five distinct groups. The data shows

the Walrus is about equally distant from the Otariidaes and the Phocidaes , with the Otariidaes

closer 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 the

Phocidaes are clearly grouped with Mustelids along with the Polar bear. The Spectacled bear is once

again on its own slightly closer to Phocidaes than the Otariidaes. This leaves the Walrus again as an

outliner being roughly equal distances from the two major clusters.

Four phylogenetic trees were built, one for each Cytochrome from both amino acid and nucleotide

sequences’. In order to build the cytochrome C nucleotide tree, a number of animals including

Odobenus rosmarus had to use amino acid to nucleotide transformation due to unavailability of

sequence data, which as this is not a one to one relation results in some random substitutions which

may affect the accuracy of this graph.

The results present a confused picture with many contradictions between the four trees. However if

we discount the Cytochrome C nt tree there appears to be some consensus, all the Otariidae and

Phocidae are consistently grouped together and the Odobenus Rosmarus is seen to first split from

the common ancestor of both the Otariidae and Phocidae which then diverged at a later date, this

stands in contrast to the results in Arnason et al(1995) which show the Phocidae first splitting, with a

later split between the Otariidae and Odobenus Rosmarus. However (Lento et al, 1995) does offer

some evidence for Odobenus Rosmarus being an early divergence from the common pinniped

ancestor which would be consistent with these results. There are major differences in the placing of

Ursus 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 as

the Phocidae, however the cytochrome B tree has the Mustelids and Tremarctos ornatus close to

the Otariidae, with Ursus maritimus being a distance relation. Castresana (2001) presents evidence

that Cytochrome B is more reliable for constructing trees at the genus and family level and therefore

this 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 as

three distinct families within the suborder of Caniformia and does not have any one group as an

ancestor to the other.

Multiple alignments

In order to build multiple alignments and identify polymorphic sites the heuristic CLUSTALW tool was

used to align both the cytochrome B and cytochrome C protein sequences. This was set to use the

BLOSUM Protein weight matrix with a GAP open penalty set to 10, GAP extension penalty set to

0.20, GAP distances set to 5 and No End Gaps set to ‘No’. Too see the full alignments refer to

appendix B. It is clear that both alignments are very good apart from of course the out-group and

the Polar bear in cytochrome B. The majority of polymorphic sites in cytochrome B are consistent

with the groupings of Odobenidae, Phocidae and Otariidae. They include both indels and point

mutations. The sites are fairly sporadic across the sequences which is in contrast to the polymorphic

sites in cytochrome C which mostly lie between the 50th and 100th amino acid with the extremities

remaining constant.

Addressing the question of how many species of Odobenus

Rosmarus there are we utilize a selection of walrus samples from

the (Lindqvist et al, 2009) study. These sequences are ATL25

tRNA-Trp and tRNA-Pro genes from the mtDNA region of the

genomes. We follow the same procedure as earlier computing

the genetic distance between the samples using jukes cantor

correction and plotting these on a graph. We use this

computation to build an unrooted phylogenetic tree. Both the

tree and the distance plot conforms with (Lindqvist et al, 2009)

conclusion that the walruses sampled from the Laptev sea are

indeed just a subgroup of the Pacific walrus because they exist in

a sub branch of Odobenus rosmarus divergens and their genetic

distance is mixed amongst the Pacific samples. This data and

analysis therefore does not justify labeling these as a separate

species.

A further point of note is that the Atlantic walrus genetic data

show signs of going through a genetic bottle neck due to the lack

of diversity compared to the Pacific walrus. This information sits

with the historic fact, that the Atlantic walrus was almost hunted

to extinction by the 1950’s with numbers beginning to recover

since then. Whereas the more remote locations inhabited by the

Pacific walrus protected them from human hunting which has

allowed there numbers to remain much higher throughout the

20th century and therefore accounting for the greater genetic

diversity shown in the samples. If further larger samples are

collected and more detailed analysis’s show the same results

then it may be it will be time to change the current NCBI tax

browser to show only two species of Odobenus Rosmarus.

Atlantic Pacific Laptivai

Conclusion

The analysis that we have performed present results that stand in contrast to the two papers Ulfure

et al (1995) and Lento et al (1995). Proving that the question of pinniped evolution is indeed very

interesting with a variety of hypothesis still in contention. The examination of the question of if

there 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 this

study and Lindqvist et al’s (2009) study. Which when taken with the low numbers of samples and the

use of amplicons, as well as the inherent difficulty of sampling Odobenus Rosmarus potentially

leading to sampling errors, such as close relatives being sampled, leaves the hypothesis very much

still 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 to

protect this charismatic and vulnerable artic creature from the threats of hunting and habitat

destruction that continue to push many creatures to extinction.

A pair of curious Walruses (image from http://www.free-extras.com/images/walrus-8927.htm)

Appendix A

Accession Number

Proteins Nucleotides

Latin Name Common Name

mtDna Cytochrome B Cytochrome C Cytochrome B Cytochrome C

Odobenus Rosmarus Rosmarus

Atlantic Walrus CAD21718 NP_659340.3 NC_004029.2 NA

Zalophus californianus

California sea lion, representing the Otariidae

YP_778707.1 YP_778698.1 D26524.1 AJ616896.1

Erignathus barbatus

Bearded Seal, representing Phocidae

YP_778837.1 YP_778828.1 AY140982.1 FJ839388.1

Ursus maritimus

Polar bear, representing Caniformia

AAF71578.1 NP_597984.1 NC_003428.1 NA

Gulo gulo Wolverine, representing Mustelids

YP_001382271.1 YP_001382262.1 L77960.2 EU544598.1

Homo Sapiens Human, is used as an outgroup

AAA31851.1 NP_061820.1 S88250.1 NM_018947.5

Halichoerus grypus

Grey Seal ACZ28998.1 NP_007072.1 GU167293.1 GU733706.1

Phoca vitulina stejnegeri

Harbor seals BAI60013.1 NP_006931.1 AB510422.1 NA

Ictonyx libyca Saharan Striped Polecat

ABV57060.1 NA EF987739.1 NA

Tremarctos ornatus

spectacled bear AAB50570.1 YP_001542732.1 U23554.1 NA

Otaria byronia South American Sea Lion

AAQ95107.1 AAR00312.1 AY713034.1 AJ891144.1

Arctocephalus townsendi

Guadalupe fur seal

YP_778759.1 YP_778750.1 AF380897.1 NA

Neophoca cinerea

Australian Sea Lion

YP_778746.1 YP_778737.1 AF380915.1 NA

Callorhinus ursinus

Northern fur seal

YP_778694.1 YP_778685.1 HQ895717.1 HM171421.1

Odobenus Rosmarus samples.

Lap 1 EU728526 Pac 8 EU728538 Atlan 4 EU728567 Atlan 14 EU728549

Lap 2 EU728527 Pac 9 EU728539 Atlan 5 EU728568 Atlan 15 EU728550

Lap 3 EU728529 Pac 12 EU728542 Atlan 6 EU728569 Atlan 16 EU728551

Lap 4 EU728530 Pac 13 EU728543 Atlan 7 EU728570 Atlan 17 EU728552

Lap 5 EU728525 Pac 14 EU728562 Atlan 8 EU728571 Atlan 18 EU728553

Pac 1 EU728531 Pac 15 EU728563 Atlan 9 EU728572 Atlan 19 EU728554

Pac 2 EU728532 Pac 16 EU728564 Atlan 10 EU728573 Atlan 20 EU728555

Pac 3 EU728533 Atlan 1 EU728561 Atlan 11 EU728546 Atlan 21 EU728556

Pac 4 EU728534 Atlan 2 EU728565 Atlan 12 EU728547 Atlan 22 EU728557

Pac 5 EU728535 Atlan 3 EU728566 Atlan 13 EU728548 Atlan 23 EU728558

Pac 6 EU728536

Pac 7 EU728537

Appendix B CLUSTAL 2.1 multiple sequence alignment cytochrome B

gi|115494578|ref|YP_778707.1| MTNIRKVHPLAKIINSSLIDLPTPSNISAWWNFGSLLAACLALQILTGLF 50

gi|115494844|ref|YP_778746.1| MTNIRKTHPLAKIINNSLIDLPAPSNISAWWNFGSLLAVCLALQILTGLF 50

gi|37620596|gb|AAQ95107.1| MTNIRKVHPLAKIINNLLIDLPAPSNISAWWNFGSLLAVCLALQILTGLF 50

gi|115494690|ref|YP_778694.1| MTNIRKVHPLAKIINSSLIDLPAPSNISAWWNFGSLLATCLVLQILTGLF 50

gi|115494830|ref|YP_778759.1| MTNIRKTHPLAKIINNSLIDLPAPSNISTWWNFGSLLAACLALQILTGLF 50

gi|269302297|gb|ACZ28998.1| MTNIRKTHPLMKIINNSFIDLPTPSNISAWWNFGSLLGICLILQILTGLF 50

gi|282154709|dbj|BAI60013.1| MTNIRKTHPLMKIINNSFIDLPTPSNISAWWNFGSLLGICLILQILTGLF 50

gi|115494788|ref|YP_778837.1| MTNIRKTHPLIKIINSSFIDLPTPSNISAWWNFGSLLGICLILQILTGLF 50

gi|8038011|gb|AAF71578.1| --------------------------------------------------

gi|1122916|gb|AAB50570.1| MTNIRKTHPLAKIINSSFIDLPTPSNISAWWNFGSLLGVCLILHILTGLF 50

gi|157461069|gb|ABV57060.1| MANIRKTHPLAKIINNSFVDLPTPSSISAWWNFGSLLGICLIIQILTGLF 50

gi|153124668|ref|YP_001382271. MTNIRKTHPLAKIINNSFIDLPTPSNISAWWNFGSLLGICLILQILTGLF 50

gi|21425423|emb|CAD21718.1| MTNIRKTHPLAKIINNTFIDLPTPSNISAWWNFGSLLATCLILQILTGLF 50

gi|552606|gb|AAA31851.1| --------------------------------------------------

gi|115494578|ref|YP_778707.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100

gi|115494844|ref|YP_778746.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100

gi|37620596|gb|AAQ95107.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100

gi|115494690|ref|YP_778694.1| LAMHYTSDTTTAFSSVAHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100

gi|115494830|ref|YP_778759.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100

gi|269302297|gb|ACZ28998.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYLHANGASMFFICLYMHVGR 100

gi|282154709|dbj|BAI60013.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYLHANGASMFFICLYMHVGR 100

gi|115494788|ref|YP_778837.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLYMHVGR 100

gi|8038011|gb|AAF71578.1| --------------------------------------------------

gi|1122916|gb|AAB50570.1| LAMHYTADTTTAFSSVAHICRDVNYGWVIRYMHANGASMFFICLFMHVGR 100

gi|157461069|gb|ABV57060.1| LAMHYTSDTTTAFSSVTHICRDVNYGWIIRYMHANGASMFFICLFLHVGR 100

gi|153124668|ref|YP_001382271. LAMHYTSDTATAFSSVTHICRDVNYGWVIRYMHANGASMFFICLFLHVGR 100

gi|21425423|emb|CAD21718.1| LAMHYTSDTTTAFSSITHICRDVNYGWIIRYMHANGASMFFICLYAHMGR 100

gi|552606|gb|AAA31851.1| --------------------------------------------------

gi|115494578|ref|YP_778707.1| GLYYGSYTLTETWNIGIILLFTIMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|115494844|ref|YP_778746.1| GLYYGSYTLTETWNIGIILLFTIMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|37620596|gb|AAQ95107.1| GLYYGSYTLTETWNIGIILLLTVMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|115494690|ref|YP_778694.1| GLYYGSYTLTETWNIGIILLLTIMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|115494830|ref|YP_778759.1| GLYYGSYTLAETWNIGIILLFTIMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|269302297|gb|ACZ28998.1| GLYYGSYTFTETWNIGIILLFTIMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|282154709|dbj|BAI60013.1| GLYYGSYTFTETWNIGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|115494788|ref|YP_778837.1| GLYYGSYTFMETWNIGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|8038011|gb|AAF71578.1| --------------------------------------------------

gi|1122916|gb|AAB50570.1| GLYYGSYLFSETWNIGIILLLTIMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|157461069|gb|ABV57060.1| GLYYGSYLFPETWNIGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|153124668|ref|YP_001382271. GLYYGSYTYSETWNIGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|21425423|emb|CAD21718.1| GIYYGSYTLAETWNIGIVLLLTIMATAFMGYVLPWGQMSFWGATVITNLL 150

gi|552606|gb|AAA31851.1| --------------------------------------------------

gi|115494578|ref|YP_778707.1| SAVPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFMASALVMVHLLFL 200

gi|115494844|ref|YP_778746.1| SAVPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFMASALVMVHLLFL 200

gi|37620596|gb|AAQ95107.1| SAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFVVSALVMVHLLFL 200

gi|115494690|ref|YP_778694.1| SAIPYIGANLVEWIWGGFSVDKATLTRFFAFHFILPFMVSALVMVHLLFL 200

gi|115494830|ref|YP_778759.1| SAIPYIGTNLVEWIWGGFSVDKATLTRFFAFHFILPFVASALVMVHLLFL 200

gi|269302297|gb|ACZ28998.1| SAIPYIGTDLVQWIWGGFSVDKATLTRFFAFHFILPFVVLALAAVHLLFL 200

gi|282154709|dbj|BAI60013.1| SAIPYIGTDLVQWIWGGFSVDKATLTRFFAFHFILPFVVSALAAVHLLFL 200

gi|115494788|ref|YP_778837.1| SAIPYIGTDLVQWIWGGFSVDKATLTRFFAFHFILPFVVLALAAVHLLFL 200

gi|8038011|gb|AAF71578.1| ---------------GGFSVDKATLTRFFAFHFILPFIILALAAVHLLFL 35

gi|1122916|gb|AAB50570.1| SAIPYIGTDLVEWIWGGFSVDKATLTRFFAFHFILPFIILALAMVHLLFL 200

gi|157461069|gb|ABV57060.1| SAIPYIGNNLVEWIWGGFSVDKATLTRFFAFHFILPFIISALAAVHLLFL 200

gi|153124668|ref|YP_001382271. SAIPYIGTSLVEWIWGGFSVDKATLTRFFAFHFILPFIILALAAIHLLFL 200

gi|21425423|emb|CAD21718.1| SAIPYVGTDLVEWVWGGFSVDKATLTRFLALHFVLPFMALALTAVHLLFL 200

gi|552606|gb|AAA31851.1| --------------------------------------------------

gi|115494578|ref|YP_778707.1| HETGSNNPSGISSDSDKIPFHPYYTIKDILGTLLLILTLMLLVMFSPDLL 250

gi|115494844|ref|YP_778746.1| HETGSNNPSGISSDSDKIPFHPYYTIKDILGTLFLILILMLLVMFSPDLL 250

gi|37620596|gb|AAQ95107.1| HETGSNNPSGISSDSDKIPFHPYYTIKDILGTLLLILILMLLVMFSPDLL 250

gi|115494690|ref|YP_778694.1| HETGSNNPSGVSSDSDKIPFHPYYTIKDILGTLLLILILMLLVMFSPDLL 250

gi|115494830|ref|YP_778759.1| HETGSNNPSGVSSDSDKIPFHPYYTIKDILGALLLILILMLLVMFSPDLL 250

gi|269302297|gb|ACZ28998.1| HETGSNNPSGIMSDSDKIPFHPYYTIKDILGALLLILVLTLLVLFSPDLL 250

gi|282154709|dbj|BAI60013.1| HETGSNNPSGIMSDSDKIPFHPYYTIKDILGALLFILVLTLLVLFSPDLL 250

gi|115494788|ref|YP_778837.1| HETGSNNPSGISSDSDKIPFHPYYTIKDILGALLLILVLMLLVLFSPDLL 250

gi|8038011|gb|AAF71578.1| HETGSNNPSGIPSDSDKIPFHPYYTIKDILGALLLTLALATLVLFSPDLL 85

gi|1122916|gb|AAB50570.1| HETGSNNPSGISSNSDKIPFHPYYTIKDILGVLLLLLALVTLVLFSPDLL 250

gi|157461069|gb|ABV57060.1| HETGSNNPSGIPSNSDKIPFHPYYTIKDILGVLLLIITLMTLVLFSPDLL 250

gi|153124668|ref|YP_001382271. HETGSNNPSGIPSDSDKIPFHPYYTIKDILGALFLALVLMMLVLFSPDLL 250

gi|21425423|emb|CAD21718.1| HETGSNNPSGILSDSDKIPFHPYYTIKDILGLIILILILMLLVLFSPDLL 250

gi|552606|gb|AAA31851.1| --------------------------------------------------

gi|115494578|ref|YP_778707.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILI 300

gi|115494844|ref|YP_778746.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILI 300

gi|37620596|gb|AAQ95107.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILI 300

gi|115494690|ref|YP_778694.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVVALLLSILV 300

gi|115494830|ref|YP_778759.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILV 300

gi|269302297|gb|ACZ28998.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALVLSILI 300

gi|282154709|dbj|BAI60013.1| GDPDNYIPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALVLSILI 300

gi|115494788|ref|YP_778837.1| GDPDNYTPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALVLSILI 300

gi|8038011|gb|AAF71578.1| GDPDNYIPAN---------------------------------------- 95

gi|1122916|gb|AAB50570.1| GDPDNYTPANPVSTPLHIKPEWYFLFAYAILRSIPNKLGGVLALIFSILI 300

gi|157461069|gb|ABV57060.1| GDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIPNKLGGVLALILSILV 300

gi|153124668|ref|YP_001382271. GDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIPNKLGGVLALVLSILV 300

gi|21425423|emb|CAD21718.1| GDPDNYTPANPLSTPPHIKPEWYFLFAYAILRSIPNKLGGVLALLLSILV 300

gi|552606|gb|AAA31851.1| ------------------KPEWYFLFAYTILRSVPNKLGGVLALLLSILI 32

gi|115494578|ref|YP_778707.1| LAIIPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEHPFITI 350

gi|115494844|ref|YP_778746.1| LAIVPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEHPFITI 350

gi|37620596|gb|AAQ95107.1| LAIIPLLHTSKQRGMMFRPISQCLFWLLAADLLTLTWIGGQPVEHPFITI 350

gi|115494690|ref|YP_778694.1| LAIIPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEYPFIAI 350

gi|115494830|ref|YP_778759.1| LAIIPLLHTSKQRGMMFRPISQFLFWLLVADLLTLTWIGGQPVEYPFITI 350

gi|269302297|gb|ACZ28998.1| LAIVPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEHPYITI 350

gi|282154709|dbj|BAI60013.1| LAIVPLLHTSKQRGMMFRPISQCLFWFLVADLLTLTWIGGQPVEHPYITI 350

gi|115494788|ref|YP_778837.1| LAIAPLLHTSKQRGMMFRPISQCLFWLLVADLLTLTWIGGQPVEHPYITI 350

gi|8038011|gb|AAF71578.1| --------------------------------------------------

gi|1122916|gb|AAB50570.1| LAIIPLLHTSKQRGMMFRPLSQCLFWLLAADLLTLTWIGGQPVEHPLVII 350

gi|157461069|gb|ABV57060.1| LAIIPLLHTSKQRSMMFRPLSQCLFWLLVADLLTLTWIGGQPVEHPFIII 350

gi|153124668|ref|YP_001382271. LAIIPLLHTSKQRGMMFRPLSQCLFWLLVADLLTLTWIGGQPVEHPFITI 350

gi|21425423|emb|CAD21718.1| LAIVPSLHTSKQRSMMFRPISQCLFWLLVADLITLTWIGGQPVEHPFIII 350

gi|552606|gb|AAA31851.1| LAMIPILHMSKQQSMMFRPLSQSLYWLLAADLLILTWIGGQPVSYPFTII 82

gi|115494578|ref|YP_778707.1| GQLASILYFTILLVFMPIAGIIENNILKW- 379

gi|115494844|ref|YP_778746.1| GQLASILYFAILLILMPIAGIIENNILKW- 379

gi|37620596|gb|AAQ95107.1| GQLASILYFTILLVLMPIAGIIENNILKW- 379

gi|115494690|ref|YP_778694.1| GQLASILYFMILLVLMPMAGIIENNILKW- 379

gi|115494830|ref|YP_778759.1| GQLASILYFTILLILMPVAGIIENNILKW- 379

gi|269302297|gb|ACZ28998.1| GQLASILYFMILLVLMPIASIIENNILKW- 379

gi|282154709|dbj|BAI60013.1| GQLASILYFMILLVLMPIASIIENNILKW- 379

gi|115494788|ref|YP_778837.1| GQLASILYFAILLVFMPIASIIENNILKW- 379

gi|8038011|gb|AAF71578.1| ------------------------------

gi|1122916|gb|AAB50570.1| GQLASILYFTILLVLMPIAGIIENNLSKW- 379

gi|157461069|gb|ABV57060.1| GQLASILYFMILLVFMPIASIAENNLLKW- 379

gi|153124668|ref|YP_001382271. GQLASILYFAILLIFMPVASIVENNLLKW- 379

gi|21425423|emb|CAD21718.1| GQLASILYFMILLVFMPIAGMIENSILKW- 379

gi|552606|gb|AAA31851.1| GQVASVLYFTTILILMPTISLIENKMLKWA 112

CLUSTAL 2.1 multiple sequence alignment Cytochrome C

gi|115494569|ref|YP_778698.1| MAYPFQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50

gi|115494681|ref|YP_778685.1| MAYPFQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50

gi|37695534|gb|AAR00312.1| MAYPLQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50

gi|115494821|ref|YP_778750.1| MAYPFQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50

gi|115494835|ref|YP_778737.1| MAYPFQMGLQDATSPIMEELTHFHDHTLMIVFLISSLVLYIISTMLTTKL 50

gi|21717327|ref|NP_659340.3| MAYPLQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISTMLTTKL 50

gi|159524414|ref|YP_001542732. MAYPFQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISTMLTTKL 50

gi|19343520|ref|NP_597984.1| MACPFQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISTMLTTKL 50

gi|115494779|ref|YP_778828.1| MAYPLQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKL 50

gi|5835013|ref|NP_007072.1|COX MAYPLQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKL 50

gi|5834861|ref|NP_006931.1|COX MAYPLQMGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKL 50

gi|153124659|ref|YP_001382262. MAYPFQLGLQDATSPIMEELLHFHDHTLMIVFLISSLVLYIISLMLTTKL 50

gi|11128019|ref|NP_061820.1| -------------------------------------------MGDVEKG 7

. *

gi|115494569|ref|YP_778698.1| THTNTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98

gi|115494681|ref|YP_778685.1| THTSTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98

gi|37695534|gb|AAR00312.1| THTSTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98

gi|115494821|ref|YP_778750.1| THTSTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98

gi|115494835|ref|YP_778737.1| THTCTMDAQEVETVWTILPAIILIMIALPSLRILYIMDEINNP--SLTVK 98

gi|21717327|ref|NP_659340.3| THTNTMDAQEVETVWTILPAIILIMIALPSLRILYMMDEINSP--FLTVK 98

gi|159524414|ref|YP_001542732. THTNTMDAQEVETVWTILPAIILVLIALPSLRILYMMDEINNP--LLTVK 98

gi|19343520|ref|NP_597984.1| THTSTMDAQEVETVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98

gi|115494779|ref|YP_778828.1| THTSTMDAQEVETVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98

gi|5835013|ref|NP_007072.1|COX THTSTMDAQEVETVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98

gi|5834861|ref|NP_006931.1|COX THTSTMDAQEVETVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98

gi|153124659|ref|YP_001382262. THTSTMDAQEVQTVWTILPAIILILIALPSLRILYMMDEINNP--SLTVK 98

gi|11128019|ref|NP_061820.1| KKIFIMKCSQCHTVEKGG-----KHKTGPNLHGLFGRKTGQAPGYSYTAA 52

.: *...: .** . : *.*: *: . : * *.

gi|115494569|ref|YP_778698.1| TMGHQWYWTYEYTDYEDLSFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148

gi|115494681|ref|YP_778685.1| TMGHQWYWTYEYTDYEDLSFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148

gi|37695534|gb|AAR00312.1| TMGHQWYWTYEYTDYEDLSFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148

gi|115494821|ref|YP_778750.1| TMGHQWYWTYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148

gi|115494835|ref|YP_778737.1| TMGHQWYWTYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148

gi|21717327|ref|NP_659340.3| TMGHQWYWSYEYTDYEDLSFDSYMVPTQELKPGELRLLEVDNRMVLPMEM 148

gi|159524414|ref|YP_001542732. TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRAVLPMEM 148

gi|19343520|ref|NP_597984.1| TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPVEM 148

gi|115494779|ref|YP_778828.1| TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148

gi|5835013|ref|NP_007072.1|COX TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148

gi|5834861|ref|NP_006931.1|COX TMGHQWYWSYEYTDYEDLNFDSYMIPTQELKPGELRLLEVDNRVVLPMEM 148

gi|153124659|ref|YP_001382262. TMGHQWYWSYEYTDYEDLNFDSYMVPTQELKPGELRLLEVDNRVVLPMEM 148

gi|11128019|ref|NP_061820.1| NKNKGIIWGEDTLMEYLENPKKYIPGTKMIFVGIKKKEERADLIAYLKKA 102

. .: * : . ..*: *: : * : * : . :

gi|115494569|ref|YP_778698.1| TVRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|115494681|ref|YP_778685.1| TVRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|37695534|gb|AAR00312.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|115494821|ref|YP_778750.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|115494835|ref|YP_778737.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|21717327|ref|NP_659340.3| TVRMLISSEDVLHSWTVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|159524414|ref|YP_001542732. TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|19343520|ref|NP_597984.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|115494779|ref|YP_778828.1| TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|5835013|ref|NP_007072.1|COX TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|5834861|ref|NP_006931.1|COX TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMTMRPGLYYGQCSE 198

gi|153124659|ref|YP_001382262. TIRMLISSEDVLHSWAVPSLGLKTDAIPGRLNQTTLMAMRPGLYYGQCSE 198

gi|11128019|ref|NP_061820.1| TNE----------------------------------------------- 105

* .

gi|115494569|ref|YP_778698.1| ICGSNHSFMPIVIESVPLSCFEKWSASML- 227

gi|115494681|ref|YP_778685.1| ICGSNHSFMPIVIESVPLSCFEKWSASMLQ 228

gi|37695534|gb|AAR00312.1| ICGSNHSFMPIVIESVPLSYFEKWSTSMLQ 228

gi|115494821|ref|YP_778750.1| ICGSNHSFMPIVIESVPLSYFEKWSASMLQ 228

gi|115494835|ref|YP_778737.1| ICGSNHSFMPIVIESVPLSYFEKWSASMLQ 228

gi|21717327|ref|NP_659340.3| ICGSNHSFMPIVLESVPLSYFEKWSASILQ 228

gi|159524414|ref|YP_001542732. ICGSNHSFMPIVLELVPLSYFEKWSASML- 227

gi|19343520|ref|NP_597984.1| ICGSNHSFMPIVLELVPLSYFEEWSASML- 227

gi|115494779|ref|YP_778828.1| ICGSNHSFMPIVLELVPLSHFEKWSTSML- 227

gi|5835013|ref|NP_007072.1|COX ICGSNHSFMPIVLELVPLSHFEKWSTSML- 227

gi|5834861|ref|NP_006931.1|COX ICGSNHSFMPIVLELVPLSHFEKWSTSML- 227

gi|153124659|ref|YP_001382262. ICGSNHSFMPIVLELVPLSHFEKWSASML- 227

gi|11128019|ref|NP_061820.1| ------------------------------

Appendix C bibliography

Andersen 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.