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Supplementary Material

Estimating coalescent events - Model comparison and date randomisation tests

We estimated the timing of coalescent events of E. pachyrhynchus by creating a Bayesian

Skyline Plot (Drummond et al., 2005) in BEAST 2. We used a constant, exponential increase,

Bayesian Skyline coalescent priors as well as an extended Bayesian Skyline coalescent prior.

A three-way comparison, comparing each model produced high substitution rates (>2

s/s/Ma). The Bayes Factor comparison indicated that the most likely model was the Bayesian

Skyline Plot, and the second most likely model was a constant population size. As the model

likelihood values were only within two of each other (which may indicate low information

content of our data [Ho et al., 2011]), we chose the simpler model of constant population size

as recommended by Ho et al., (2011). Nevertheless, we constructed the Bayesian Skyline Plot

(Fig S8) using TRACER v1.6.0.

The mean substitution rates for the Bayesian Skyline Plot were 2.13 s/s/Ma (95% CI 1.14-

4.94 s/s/Ma), and the constant population size were 2.69 s/s/Ma (95% CI 2.71-5.89 s/s/Ma),

which is a similar rate to other seabirds (e.g. Otago shag (L. chalconotus) 3.20 s/s/Ma,

Foveaux shag (L. stewarti) 1.73 s/s/Ma; Rawlence et al., 2015), however faster than Adélie

penguins (Pygoscelis adeliae) (0.29-0.88 s/s/Ma; Millar et al., 2008).

Given the shallow time-depth of our analyses (1151 calibrated calendar years BP, where

‘present’ is 2016), we assessed whether our data contained sufficient temporal structure to

allow reliable estimates of substitution rates, by randomising the dates across samples in 20

independent replicates, and repeating the constant population size model analysis, as

suggested by Ho et al., (2011) for low information content ancient DNA datasets. The 95%

CI’s of estimated substitution rates were visualised in TRACER. If the original mean rate-

1

estimate was contained within the 95% CI of the randomised data sets, there was insufficient

temporal structure for reliable rate estimation (Ho et al., 2011). A more conservative

approach argues that rates are unreliable if the 95% CI of the original rate-estimate and

randomised data sets overlap (Duchene et al., 2015). Caution therefore needs to be taken over

the reliability of our mean substitution rate estimate (despite being similar to those of other

seabirds [see Rawlence et al., 2015]), given the wide confidence intervals and that the

temporal CR dataset failed both randomisation tests (over half the 95% CI’s overlapped with

the mean empirical rate estimate) (cf. Foveaux Shag versus Otago Shag; Rawlence et al.,

2015). This caveat reflects the fact that short mitochondrial CR fragments provide only

limited information for substitution rate estimation (Ho et al., 2007, 2011).

2

Table S1. COI and CR sequences obtained from GenBank and used in this study. Sample number refers to tissue samples of which COI was obtained in Cole et al., (2017) and which CR has been obtained in this study. Note additional COI sequences were obtained in Cole et al., (2017) but were not used in this study. These are referred in Table S2.Species Accession number Sequence Sample numberAptenodytes forsteri EU525299 COIAptenodytes patagonicus EU525303 COIDiomedia exulans DQ137168 COIEudyptes chrysocome AB775513 CREudyptes chrysocome DQ525800 COIEudyptes chrysolophus MF469857 COIEudyptes chrysolophus MF469863 COIEudyptes chrysolophus MF469859 COIEudyptes chrysolophus MF469864 COIEudyptes chrysolophus FJ582595 COIEudyptes filholi DQ525795 COIEudyptes moseleyi DQ525790 COIEudyptes pachyrhynchus MF469874 COI AD 257Eudyptes pachyrhynchus MF469869 COI AD 259Eudyptes pachyrhynchus MF469870 COI AD 260Eudyptes pachyrhynchus MF469873 COI AD 261Eudyptes pachyrhynchus MF469876 COI AD 262Eudyptes pachyrhynchus MF469881 COI AD 264Eudyptes pachyrhynchus MF469879 COI AD 265Eudyptes pachyrhynchus MF469877 COI AD 266Eudyptes pachyrhynchus MF469880 COI AD 267Eudyptes pachyrhynchus EU525345 COIEudyptes robustus MF469856 COI AD 270Eudyptes robustus MF469855 COI AD 272Eudyptes robustus EU525347 COIEudyptes schlegeli MF469865 COIEudyptes schlegeli MF469862 COIEudyptes schlegeli MF469861 COIEudyptes schlegeli MF469858 COIEudyptes schlegeli FJ582599 COIEudyptes sclateri MF469866 COI AD 304Eudyptula minor EU525402 COIEudyptula novaehollandiae EU525396 COIMegadyptes antipodes DQ137184 COIMegadyptes antipodes FJ822140 CRMegadyptes antipodes FJ391944 CRMegadyptes antipodes FJ391945 CRMegadyptes antipodes FJ391946 CRMegadyptes antipodes FJ391947 CRMegadyptes antipodes FJ391948 CR

3

Megadyptes antipodes FJ391949 CRMegadyptes antipodes FJ391950 CRMegadyptes antipodes FJ391951 CRMegadyptes antipodes FJ391952 CRMegadyptes antipodes FJ391953 CRMegadyptes antipodes FJ822137 CRMegadyptes antipodes FJ822138 CRMegadyptes antipodes FJ822139 CRMegadyptes antipodes FJ822141 CRMegadyptes antipodes FJ822142 CRMegadyptes antipodes FJ822143 CRMegadyptes waitaha KP644153 CRMegadyptes waitaha FJ391950 CRMegadyptes waitaha FJ391954 CRMegadyptes waitaha FJ391955 CRMegadyptes waitaha FJ391956 CRMegadyptes waitaha FJ391957 CRMegadyptes waitaha FJ391958 CRMegadyptes waitaha FJ391959 CRMegadyptes waitaha FJ391960 CRMegadyptes waitaha FJ391961 CRMegadyptes waitaha FJ391962 CRMegadyptes waitaha FJ391963 CRMegadyptes waitaha FJ391964 CRMegadyptes waitaha FJ391965 CRMegadyptes waitaha FJ391966 CRPygoscelis adeliae DQ137183 COIPygoscelis antarcticus EU525474 COIPygoscelis papua EU525486 COISpheniscus demersus EU525504 COISpheniscus humboldti DQ137180 COISpheniscus magellanicus EU525505 COISpheniscus mendiculus DQ137179 COI

4

Table S2. Samples used in our study, including the number of base pairs obtained for three molecular markers (COI, Eudyptes specific CR and Megadyptes specific CR), the sample types, collection locality and approximate age based on stratigraphic context or radiocarbon years BP (BP where present is 2016). References for these dates are listed in the reference list. ‘X’ indicates that the marker of interest did not amplify, NA means the PCR was not attempted for that sample. GB indicates that COI sequences from that sample has been obtained (Cole et al., 2018), but are not analysed in this study. DNA was extracted twice from samples with two ‘JW’ numbers. *Note XXX Genbank numbers will be added following review.

Sample Number Genus Species

Base pairs COI

Genbank Accession

Base pairs

Eudyptes CR

Genbank Accession

Base pairs Megadyptes

CR

Genbank Accession

Tissue Type

Museum Number Locality Sample Type Age (BP)

JW 718 Eudyptes chrysocome NA 131 MK120620 NA Blood 370MNew Island,

Falkland Islands

Wild 4

JW 719 Eudyptes chrysocome NA 131 MK120621 NA Blood 382FNew Island,

Falkland Islands

Wild 4

JW 720 Eudyptes chrysocome NA 131 MK120622 NA Blood 374MNew Island,

Falkland Islands

Wild 4

JW 758 Eudyptes chrysolophus NA 131 MK120623 NA Blood MPJJ 1Jardin

Japonase, Crozet

Wild 13

JW 759 Eudyptes chrysolophus NA 131 MK120624 NA Blood MPJJ 2Jardin

Japonase, Crozet

Wild 13

JW 526 Eudyptes filholi NA 131 MK120625 NA Blood PIT 153746471 GLS #018 Male

Campbell Island Wild 3

JW 527 Eudyptes filholi NA 131 MK120626 NA Blood 0153723532 GLS #010 Male


JW 528 Eudyptes filholi NA 131 MK120627 NA Blood 167856729 GLS #8 Female


AD 107 Eudyptes pachyrhynchus GB 131 MK120628 NA Bone ANWC B21142Hunter Island,

TasmaniaArchaeological 461

AD 108 Eudyptes pachyrhynchus GB 131 MK120629 NA Bone ANWC B21143Hunter Island,

TasmaniaArchaeological 461

AD 155 Eudyptes pachyrhynchus GB 131 MK120630 NA Bone ANWC B21145Louisa River

Cave, Tasmania

Archaeological 541

AD 257 Eudyptes pachyrhynchus GB 131 MK120631 NA Toe-pad OR.26082 Dusky Sound,

Southland Colony 32

5

AD 259 Eudyptes pachyrhynchus GB 131 MK120632 NA Toe-pad OR.15371

Jackson Head,

WestlandColony 47


Jackson Head,

WestlandColony 47


Turnbull River Mouth,

WestlandColony 31


Bathing Beach,

SouthlandColony 28


Jackson Head,

WestlandColony 46


Jackson Head,

WestlandColony 47


Solander Island,

SouthlandColony 68


Jackson Head,

WestlandColony 47

AD 247 Eudyptes pachyrhynchus 499 MK120774 131 MK120640 NA Bone Av-36090 Cannibal Bay, Otago Fossil/Archaeological 829

AD 244 Eudyptes pachyrhynchus 490 MK120775 131 MK120641 NA Bone Av-22264Preservation

Inlet, Southland

Fossil 48

AD 240 Eudyptes pachyrhynchus 467 MK120776 131 MK120642 NA Bone Av-34136 Pounawea, Otago Archaeological 687

AD 295 Eudyptes pachyrhynchus X 131 MK120643 NA Bone S.38968 Mussel Point, Marlborough Archaeological 688



AD 317 Eudyptes pachyrhynchus 356 MK120780 131 MK120646 NA Bone S.36369.7Lake

Grassmere, Marlborough

Fossil 1151

AD 318 Eudyptes pachyrhynchus 356 MK120781 131 MK120647 NA Bone U13-L1Foxton,

Manawatu-Wanganui

Archaeological 651

AD 357 Eudyptes pachyrhynchus 283 MK120782 131 MK120648 NA Bone S.23882 North Cape, Northland Fossil 0-6000

6

AD 340 Eudyptes pachyrhynchus 263 MK120783 131 MK120649 NA Bone Av-11170Lake


Fossil 1151

AD 310 Eudyptes pachyrhynchus 142 MK120784 X NA Bone S.38443Whakataki

Beach, Wellington

Fossil 0-6000

AD 311 Eudyptes pachyrhynchus 142 MK120785 X NA Bone S.38605 Ocean Beach, Hawkes Bay Fossil 0-6000

AD 360 Eudyptes pachyrhynchus 142 MK120786 X NA Bone S.45819Te Kaukau

Point, Wellington

Fossil 0-2000

JW 130 Eudyptes pachyrhynchus 458 MK120787 X NA Tissue FCP1 Westport, Westland Wild 3

AD 263 Eudyptes pachyrhynchus 426 MK120788 131 MK120650 NA Toe-pad OR.15834

Jackson Head,

WestlandColony 46

AD 298 Eudyptes pachyrhynchus 356 MK120789 131 MK120651 NA Toe-pad

12501 (Auckland)

Puysegur Point,

SouthlandColony 82

AD 258 Eudyptes pachyrhynchus 142 MK120790 131 MK120652 NA Toe-pad OR.26083 Dusky Sound,

Southland Colony 32

AD 287 Eudyptes pachyrhynchus X 131 MK120653 NA Bone S.36369.1Lake


Fossil 1151

AD 243 Eudyptes pachyrhynchus X 131 MK120654 NA Bone Av-34866 False Islet, Otago Fossil/Archaeological 829

AD 268 Eudyptes pachyrhynchus NA 131 MK120655 NA Toe-pad OR.28064

Murphy Beach,

WestlandBeach-wreck 16

AD 320 Eudyptes pachyrhynchus NA 131 MK120656 NA Toe-pad Av-29716

South Beach, Greymouth,

WestlandBeach-wreck 45

AD 321 Eudyptes pachyrhynchus NA 131 MK120657 NA Toe-pad Av-17448 Punakaiki,

Westland Beach-wreck 18

AD 322 Eudyptes pachyrhynchus NA 131 MK120658 NA Toe-pad Av-1363 Greymouth,


AD 325 Eudyptes pachyrhynchus NA 131 MK120659 NA Toe-pad Av-814 Hokitika,


AD 326 Eudyptes pachyrhynchus NA 131 MK120660 NA Toe-pad Av-44

Solander Island,

SouthlandColony 85

AD 334 Eudyptes pachyrhynchus NA 131 MK120661 NA Toe-pad Av-36574 Okarito,


AD 335 Eudyptes pachyrhynchus NA 131 MK120662 NA Toe- Av-22735 Rapahoe Beach-wreck 35

7

pad Beach, Westland

JW 348 Eudyptes pachyrhynchus NA 131 MK120663 NA Tissue FCP2 Westport, Westland Wild 3

JW 349 Eudyptes pachyrhynchus NA 131 MK120664 NA Tissue FCP3 Westport, Westland Wild 3

JW 350 Eudyptes pachyrhynchus NA 131 MK120665 NA Tissue FCP4Murphy’s

Beach, Westland

Wild 3

JW 351 Eudyptes pachyrhynchus NA 131 MK120666 NA Tissue FCP5Murphy’s

Beach, Westland

Wild 3

JW 412 Eudyptes pachyrhynchus NA 131 MK120667 NA Tissue LB-12810 Brighton Beach, Otago Wild 11

JW 414 Eudyptes pachyrhynchus NA 131 MK120668 NA Blood LB-12809 Okarito, Westland Wild 12

AD 323 Eudyptes pachyrhynchus NA 131 MK120669 NA Skin Av-850Preservation

Inlet, Southland

Colony 95

AD 324 Eudyptes pachyrhynchus NA 131 MK120670 NA Feather Av-417

Coal Island, Preservation

Inlet, Southland

Colony 96

JW 319 Eudyptes pachyrhynchus NA 131 MK120671 NA Blood WH31Codfish Island,

SouthlandWild 0

JW 334 Eudyptes pachyrhynchus NA 131 MK120672 NA Blood JH2016Jackson Head,

WestlandWild 0

JW 337 Eudyptes pachyrhynchus NA 131 MK120673 NA Blood MS2Milford Sound,

SouthlandWild 0


SouthlandWild 0


SouthlandWild 0


SouthlandWild 0


SouthlandWild 0

8


SouthlandWild 0


SouthlandWild 0


SouthlandWild 0


SouthlandWild 0


WestlandWild 0


WestlandWild 0


SouthlandWild 0


SouthlandWild 0

JW 645 Eudyptes pachyrhynchus NA 131 MK120686 NA Blood WH11ACodfish Island,

SouthlandWild 0


SouthlandWild 0


SouthlandWild 0


SouthlandWild 0


SouthlandWild 0


SouthlandWild 0


WestlandWild 0

JW 656 Eudyptes pachyrhynchus NA 131 MK120693 NA Blood JH21 Jackson Wild 0

9

Head, Westland

JW 658 Eudyptes pachyrhynchus NA 131 MK120694 NA Blood JH Cave 2016Jackson Head,

WestlandWild 0

JW 659 Eudyptes pachyrhynchus NA 131 MK120695 NA Blood JH 2016Jackson Head,

WestlandWild 0


SouthlandWild 0


SouthlandWild 0


SouthlandWild 0

AD 154 Eudyptes robustus GB 131 MK120699 NA Bone ANWC B21144 Prion Beach, Tasmania Archaeological Unknown

AD 245 Eudyptes robustus 499 MK120793 131 MK120700 NA Bone Av-28286 Native Island, Southland Fossil 1505

AD 294 Eudyptes robustus 356 MK120794 131 MK120701 NA Bone S.36176 Mason Bay, Southland Fossil 0-407

AD 270 Eudyptes robustus GB 131 MK120702 NA Toe-pad OR.19353 The Snares Colony 39

AD 272 Eudyptes robustus GB 131 MK120703 NA Toe-pad OR.26830

Penguin Creek, The

SnaresColony 16

AD 275 Eudyptes robustus 169 MK120795 131 MK120704 NA Toe-pad OR.17375

Western Chain, The

SnaresColony 44

AD 273 Eudyptes robustus 168 MK120796 131 MK120705 NA Toe-pad OR.26831

Muttonbird Creek, The

SnaresColony 16

AD 274 Eudyptes robustus 171 MK120797 131 MK120706 NA Toe-pad OR.26832 Sinkhole Flat,

The Snares Colony 16

AD 271 Eudyptes robustus NA 131 MK120707 NA Toe-pad OR.24113 The Snares Colony 29

AD 319 Eudyptes robustus NA 131 MK120708 NA Toe-pad Av-804 Otago Coast,

Otago Beach-wreck 124

JW 127/485 Eudyptes robustus 462 MK120798 131 MK120709 NA Blood SCP8

Toru, Western

ChainWild 3

JW 120/480 Eudyptes robustus 459 MK120799 131 MK120710 NA Blood SCP1 South

Promontory Wild 3

10

JW 121/481 Eudyptes robustus 459 MK120800 131 MK120711 NA Blood SCP2 South

Promontory Wild 3

JW 122/482 Eudyptes robustus 459 MK120801 131 MK120712 NA Blood SCP3 North

Promontory Wild 3

JW 125/713 Eudyptes robustus 459 MK120802 131 MK120713 NA Blood SCP6 Colony 3 Wild 3

JW 126/484 Eudyptes robustus 459 MK120803 131 MK120714 NA Blood SCP7

Toru, Western

ChainWild 3

JW 129 Eudyptes robustus 459 MK120804 131 MK120715 NA Blood SCP10Toru,

Western Chain

Wild 3

JW 128 Eudyptes robustus 458 MK120805 131 MK120716 NA Blood SCP9Toru,

Western Chain

Wild 3

JW 124/712 Eudyptes robustus 457 MK120806 131 MK120717 NA Blood SCP5 Colony 3 Wild 3

JW 180 Eudyptes robustus NA 131 MK120718 NA Blood 68M The Snares Wild 3

JW 181 Eudyptes robustus NA 131 MK120719 NA Blood 38F The Snares Wild 3









JW 456 Eudyptes robustus NA 131 MK120728 NA Blood 46 gvn F The Snares Wild 3


JW 711 Eudyptes robustus NA 131 MK120730 NA Blood SCP4 North Promontory Wild 3

AD 367 Eudyptes schlegeli or chrysolophus 356 MK120807 131 MK120731 NA Bone S.37221 Native Island,

Southland Fossil 1505

JW 869 Eudyptes schlegeli NA 131 MK120732 NA Blood ROPE4461Green Gorge,

Macquarie Island

Wild 10

JW 870 Eudyptes schlegeli NA 131 MK120733 NA Blood ROPE4449 Green Gorge, Macquarie Wild 10

11

Island

AD 344 Eudyptes sclateri 352 MK120808 131 MK120734 NA Bone S.47280Sharks Tooth,

Kaikoura, Canterbury

Fossil 0-6000

AD 389 Eudyptes sclateri 280 MK120809 131 MK120735 NA Bone

4/1/5 Black Rocks Cresent BR4 Box 2a 1

(Bird)

Black Rocks, Wairarapa, Wellington

Archaeological 736-566

AD 393 Eudyptes sclateri 283 MK120810 X NA Bone


(Bird)



AD 304 Eudyptes sclateri GB 131 MK120736 NA Toe-pad OR.21942 Antipodes

Islands Colony 48

AD 302 Eudyptes sclateri 499 MK120811 131 MK120737 NA Toe-pad OR.12646 Campbell

Island Colony 69

AD 303 Eudyptes sclateri NA 131 MK120738 NA Toe-pad OR.12647 Campbell

Island Colony 69

AD 305 Eudyptes sclateri NA 131 MK120739 NA Toe-pad OR.21941 Antipodes

Islands Colony 48

AD 327 Eudyptes sclateri NA 131 MK120740 NA Toe-pad Av-1353 Antipodes

Islands Colony 93


Islands Colony 93


Islands Colony 93

AD 333 Eudyptes sclateri NA 131 MK120743 NA Toe-pad Av-21754 Campbell

Island Colony 67

AD 306 Eudyptes sclateri NA 131 MK120744 NA Skin OR.26573

Anchorage Bay,

Antipodes Islands

Colony 21

AD 307 Eudyptes sclateri NA 131 MK120745 NA Skin OR.21944 Antipodes Islands Colony 38

JW 183 Eudyptes sclateri NA 131 MK120746 NA Blood Ant 48 Antipodes Islands Wild 11





JW 499 Eudyptes sclateri NA 131 MK120751 NA Blood Ant 7 Antipodes Wild 11

12

Islands













AD 391 Eudyptes clade X 499 MK120812 111 MK120764 NA Bone


(Bird)



AD 342 Eudyptes clade X 499 MK120813 X NA Bone Av-25895 Le Bons Bay, Canterbury Fossil 0-6000

AD 309 Eudyptes clade X 356 MK120814 X NA Bone3/2/8 Black

Rocks Midden BR3 N168-9/77



AD 248 Eudyptes clade X 286 MK120815 X NA Bone Av-21751

Rakauteru Cave,

Kaikoura Canterbury


AD 381 Eudyptes clade X 282 MK120816 X NA Bone Av.32062Needle Point

North, Marlborough


AD 394 Eudyptes clade X 274 MK120817 X NA Bone


(Bird)



13

AD 282 Eudyptes clade X 251 MK120818 X NA Bone M1/II/4-60 (rust + 5r)

Centennial Inn,

Paekakariki, Wellington


AD 93 Megadyptes antipodes 377 MK120819 NA GB Bone

Otago UF2/L2, LB3 A6 bird L2+Llb Bag

LB/D/F

Long Beach, Otago Archaeological 541-351

AD 96 Megadyptes antipodes 204 MK120820 NA GB Bone Av-10616 Pahia Pa, Southland Archaeological 471-192

AD 94 Megadyptes antipodes 141 MK120821 NA GB Bone Otago UE3/L2 Long Beach, Otago Archaeological 441-151

AD 91 Megadyptes waitaha 141 MK120822 NA GB Bone S.35913 Native Island, Southland Fossil 1507

AD 92 Megadyptes waitaha 138 MK120823 NA GB Bone S.35426 Native Island, Southland Fossil 1507

AD 284 Megadyptes waitaha 213 MK120824 NA 402 MK120765 Bone

Moa hunter midden, Old

area, October 62, Left

humerus small

Sinclair, Paremata, Wellington


AD 286 Megadyptes waitaha 141 MK120825 NA 402 MK120766 Bone Yellow SurfaceMana Island

North, Wellington


AD 378 Megadyptes waitaha 141 MK120826 NA 402 MK120767 Bone S.38481 Delaware Bay, Nelson Fossil 1330-918

AD 283 Megadyptes waitaha 137 MK120827 NA 402 MK120768 Bone

Moa hunter midden, Old

area, October 62, Left

humerus large

Sinclair, Paremata, Wellington


AD 289 Megadyptes waitaha 109 MK120828 NA 402 MK120769 Bone S.42136 Delaware Bay, Nelson Fossil 1330-918

AD 285 Megadyptes waitaha 233 MK120829 NA 255 MK120770 Bone N-160/50Sinclair,

Paremata, Wellington


AD 371 Megadyptes waitaha 141 MK120830 NA 255 MK120771 Bone Av-34054 Pounawea, Otago Archaeological 687

AD 375 Megadyptes waitaha X NA 242 MK120772 Bone Av-34866 False Islet, Otago Fossil/Archaeological 829

AD 276 Megadyptes waitaha 252 MK120831 NA 402 MK120773 Bone AR-83Centennial

Inn, Paekakariki,


14

Wellington

AD 379 Megadyptes sp. 139 MK120832 NA X Bone S.42118Whangamoa River Mouth,

NelsonFossil 0-6000

AD 366 Megadyptes sp. 141 MK120833 NA X Bone Av-36083 False Islet, Otago Fossil/Archaeological 829

AD 358 Megadyptes sp. 130 MK120834 NA X Bone S.41881.1 Mussel Point, Marlborough Archaeological 736-566

AD 249 X X X X X Bone Av27268

NE Bay, Solander

Island, Southland

Fossil n/a

AD 279 X X X X X Bone M1/11/J/L4 Black

Centennial Inn,



AD 280 X X X X X Bone M1/2/23/Lens 2B

Centennial Inn,



AD 281 X X X X X Bone M1/2/9 Lens B

Centennial Inn,



AD 288 X X X X X Bone S.36369.2Lake


Fossil 1151

AD 312 X X X X X Bone S.45814 White Rock, Wellington Fossil 0-2000

AD 313 X X X X X Bone S.45821Te Kaukau

Point, Wellington

Fossil 0-2000



Fossil 1151



Fossil 1151



Fossil 1151

AD 337 X X X X X Bone Av-14445Cave,

Punakaiki, Westland

Fossil 93

AD 338 X X X X X Bone Av-14444 Limestone Fossil n/a

15

Cave, Punakaiki, Westland

AD 339 X X X X X Bone Av-11568Marfells Beach,

MarlboroughFossil 1151

AD 353 X X X X X Bone Av-34090 Pounawea, Otago Archaeological 687


AD 356 X X X X X Bone

4/1/6 Black Rocks Crescent

BR4 Box 2a1 (bird)



AD 359 X X X X X Bone S.45820Te Kaukau

Point, Wellington

Fossil 0-2000

AD 361 X X X X X Bone S.24724 Paremata, Wellington Archaeological 736-566

AD 368 X X X X X Bone S.35912 Native Island, Southland Fossil 1505

AD 369 X X X X X Bone Av-21257Cape

Wanbrow, Otago

Fossil >10,000



AD 374 X X X X X Bone Av-32062North Needle

Point, Marlborough



AD 382 X X X X X Bone S.41881.2 Mussel Point, Marlborough Archaeological 736-566

AD 383 X X X X X Bone Av-14214Marfells Beach,

MarlboroughFossil 1151

AD 384 X X X X X Bone S.38549 Delaware Bay, Nelson Fossil 1330-918

AD 386 X X X X X Bone Av-190 Paremata, Wellington Archaeological 736-566

AD 387 X X X X X Bone 4/3/8 Black Rocks Cresent

Black Rocks, Wairarapa, Archaeological 736-566

16

BR4 Box 2a 1 (Bird) Wellington



(Bird)




3/1/4 Black Rocks Black Midden BR3 N168-9/77





(Bird)





(Bird)




3/1/8 Black Rocks Black Midden BR3 N168-9/77



17

Table S3. Prior and posterior distributions of parameters for a model of constant population size since the LGM. Timing of events in the case of this model is irrelevant because Nef remained constant through time. However, these events were kept in the model for consistency with the alternative models that included population declines. Timing of events is in number of generations, assuming a generation time of 5 years (Otley et al., 2017).

Parameter Prior distribution Posterior mode 5% 95%

Nef-pre-glaciation and Nef-pre-human * Uniform [10 - 106] NA NA NANef Uniform [10 - 106] 5.81X104 4.28X104 8.38X105

thum Uniform [2 - 160] 1.45X102 1.23X101 1.54X102

tpreglac Uniform [2000 - 3000] 2.87X103 2.06X103 2.96X103

μ rate (COI) Uniform [10-9- 5X10-7] 4.57X10-8 3.10X10-8 4.45X10-7

* the same priors were used for pre-glaciation and pre-human Nef in alternative models tested.

Conditions of the ABC analysis: the pre-glaciation and pre-human Nef is larger than the

modern Nef in all the scenarios (constrains applied to population effective size: Nef

modern<Nef pre-glaciation, and Nef modern<Nef pre-human).

18

Table S4. Comparison of scenarios using the logistic regression approach on the closest 1%, 0.1% and 0.01% of data sets to the observed data.

N simulations retained scenario 1 scenario 2 scenario 3 scenario 440000 (1%) 0.6719

[0.6587,0.6851]0.1020 [0.0938,0.1102]

0.1357 [0.1259,0.1454]

0.0904 [0.0822,0.0985]

4000 (0.1%) 0.7150 [0.6897,0.7404]

0.0925 [0.0775,0.1075]

0.1341 [0.1142,0.1540]

0.0583 [0.0456,0.0710]

400 (0.01%) 0.6307 [0.2877,0.9738]

0.2188 [0.0000,0.5502]

0.1101 [0.0000,0.3129]

0.0404 [0.0000,0.1695]

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Fig. S1. Map of sample collection sites used in this study. Prehistoric=prehistoric sub/fossil/archaeological bones, Historic=historical museum skins, Modern=contemporary blood. Historical museum skins that do not have locality collection details associated with them (Table S2) are not shown. Modern breeding sites of Eudyptes penguins are shown (green=E. pachyrhynchus, yellow = E. robustus, orange=E. sclateri, blue=E. schlegeli). Samples from subantarctic islands outside the Australian and New Zealand region are not shown, but can referred to in Table S2. Historic Macquarie Island samples were obtained from Cole et al., (2017). Note E. chrysolophus breeds outside of this region.

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Fig. S2. Bayesian phylogenetic tree of Eudyptes pachyrhynchus based on 131bp mitochondrial CR. Coloured ovals represent the regional district that the sample originated. Posterior probabilities of 0.91 or higher are shown.

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Fig. S3. Demographic models tested in Eudyptes pachyrhynchus associated with post-glacial climate change, and human arrival in New Zealand. Time 0, 20 and 200 correspond to approximate sampling times.

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Fig. S4. Bayesian phylogenetic tree of up to 500 bp mitochondrial COI gene, of all extant sphenisciform taxa. Eudyptes robustus samples from the Western Chain are indicated with WC. AD numbers with the species name indicated were from historical museum skins, whereas samples without the species name were prehistoric subfossil or archaeological bone. Posterior probabilities for main clades >0.76 are shown.

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Fig. S5. Bayesian phylogenetic tree of up to 402 bp mitochondrial CR, of seven Megadyptes prehistoric sub-fossil/archaeological bone samples identified in Fig S2 (shown in bold). Posterior probabilities for the main M. antipodes/M.waitaha clades are shown.

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Fig. S6. Temporal haplotype network for three Eudyptes species based on CR sequences. Coloured circles represent different haplotypes, with the size of the circle and number within the circle corresponding to the number of samples that had that haplotype. Small white circles represent those haplotypes that are present in one or two-time periods, but are absent in the time period displayed. Black circles represent haplotypes that were absent from the dataset. Each layer represents a different time period, based on the sample type used (Prehistoric=sub-fossil/archaeological bone, Historic=historical museum skins, Modern=contemporary blood). The coloured cylinders connect those haplotypes that are present through multiple time periods. Green circles are E. pachyrhynchus, yellow circles are E. robustus and orange circles are E. sclateri. The single prehistoric pink circle is Eudyptes clade X.

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Fig. S7. Temporal haplotype network for three Eudyptes species based on CR sequences. Coloured circles represent different haplotypes, with the size of the circle and number within the circle corresponding to the number of samples that had that haplotype. Small white circles represent those haplotypes that are present in one or two-time periods, but are absent in the time period displayed. Black circles represent haplotypes that were absent from the dataset. Each layer represents a different time period, based on the sample type used (Prehistoric=sub-fossil/archaeological bone, Historic=historical museum skins, Modern=contemporary blood). The coloured cylinders connect those haplotypes that are present through multiple time periods. Green circles are E. pachyrhynchus, yellow circles are E. robustus, orange circles are E. sclateri, light blue circles are E. schlegeli, mauve circles are E. chrysolophus, dark blue circles are E. filholi, the purple circle is E. chrysocome. The single prehistoric pink circle is Eudyptes clade X.

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Fig. S8. Bayesian Skyline Plot representing the coalescent history of Eudyptes pachyrhynchus. The thick solid lines represent the median estimate of effective population size, while the shaded green areas represent the 95% credible intervals. The period from Polynesian colonization of New Zealand ca. 1280 AD to 1450 AD during which NewZealand’s megafauna was hunted to extinction (thick orange bar), and European arrival 1769 AD (thin orange bar) are shown.

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Fig. S9. Model checking using a principal component analysis in the space of summary statistics using datasets simulated with the prior distributions of parameters (orange circles), observed dataset (yellow circle) and posterior distribution datasets (red circles). The model fits well the observed data because the observed dataset (yellow circle) is in the middle of datasets from the posterior predictive distribution. A) is PC1 and PC2, and B) is PC3 and PC4.

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