1
Supplementary information.
Methods
Sample preparation
The three moa bones were: Emeus crassus, Museum of New Zealand (MNZ) S91, tibiotarsus from Castle Rocks, Otago; Dinornis giganteus, MNZ S34094, phalange from Hodge Ck, Takaka; and Megalapteryx didinus, MNZ S23808, tibiotarsus from Mt Owen, Takaka. The elephant bird specimen was Mullerornis agilis, MNZ S38300, tibiotarsus from Beloha, Madagascar. Dremel tool and disposable carborundum disks were used to first remove approximately 2mm of the exterior surface, and then an approximately 0.1g section of cortical bone. The bone was powdered using an 8mm tungsten ball bearing in a Braun Mikrodismembrator U (B. Braun Biotech International, Germany) at 2000 rpm for 30 sec. in sterilised stainless steel containers. The powder was decalcified overnight in 10-30 vol of 0.5M EDTA (pH8) at room temperature along with a negative control (no bone powder). The sediment was collected by centrifugation, digested with proteinase K/DTT overnight at 50-55'C, and extracted twice with Tris-saturated phenol, and once with chloroform. The DNA was desalted using Centricon-30 (Amicon) devices and concentrated to approximately 140uL. Between 3-5 extractions were performed on each specimen. Modern tissues followed the above protocol from the digestion phase onwards.
Modern samples
Samples were obtained from the tissue collection of Allan Wilson (now held by Svante Pääbo, Max Planck Institute for Molecular Anthropology, Leipzig) for the Elegant Crested tinamou (breast muscle), Tataupa tinamou (toe), and cassowary (muscle). Other samples were kiwi (erythrocytes, supplied by M. Potter, Massey University, New Zealand from individual K86, Northland, New Zealand) and emu (tissue, Louisiana State University Museum of Zoology B-8607, from San Diego Zoo).
PCR/Sequencing
Extinct taxa: PCR amplifications were performed in 25uL PCR reactions using 1uL of extract, 1.25 U Ampli-Taq and 1X buffer (Perkin Elmer), 2mg/ml BSA, 2mM MgCl2, 0.25mM dNTPs, and 1uM each primer. A paraffin-wax barrier was used to separate the primers, MgCl2, and DNA from the other components until the initial denaturation step. The PCR profile was 92'C for 40sec, followed by 40 cycles of 92'C denaturation for 40 sec, annealing for 1 min, and extension at 72'C for 1 minute. The annealing temperature was 55'C for primers in the 12S, 16S or tRNA genes, 52'C for primers in protein-coding regions apart from ND6, and 50'C or less for primers in the ND6 and control region genes. Where sections spanned two such regions the lower annealing temperature was used. Sequences of Mullerornis DNA were obtained for the 12S (primer pairs 1753 12SA/1985 12SH; 1856 12SE2/2020Rb; and 1999 12SJ/2150 12SB5), COII (8861Fb/9036RH), ATP8 (9043Fb/9241RH) and Cyt b (15671FH/15902RH) genes (see Table 3 below). A hi-fidelity enzyme (Platinum Taq Pfu Hi-fi, Gibco-BRL) was used to amplify problematic sections of the moa control region and 12S DNA genes according to manufacturers instructions, with the above conditions.
All PCRs included negative extraction and amplification controls and followed standard ancient DNA protocols26. Half of the amplified products were visualised on a 2% LMP agarose gel (NuSieve GTG) using ethidium bromide, and bands of the correct size were excised and melted in 150uL dH20 at 65'C for 20 min. Re-amplification reactions were performed using 1uL of the melted gelcut and 35 cycles of PCR as above, but with no initial denaturation step. 2uL of the resulting products were visualised and quantified using ethidium bromide, and the remainder purified (Whatman Vectraspin, Millipore Ultrafree-30 or Qiagen Qiaquick or Qiaex II). Sequencing reactions were performed on both strands one to two times, and up to eight times in problematic areas, using an ABI BigDye PRISM kit and ABI 377, according to manufacturers instructions.
Extant taxa: Long range amplifications were performed using a GeneAmp XL-PCR (Perkin-Elmer) kit according to the manufacturers instructions with the primer pairs 03725FH-16SFor.2/10218RH-COIII-LR, and 08171FH-tSer-LR/16120Rb-tPro-LR at an annealing temperature of 57'C. Products were recovered, and 400-600 bp regions were re-amplified and sequenced as above. 12S sequences were generated for each taxon with the primer pair 1753 12SA/2150 12SB5, using DNA extracts and the re-amplification conditions. The rhea (AF090339) and ostrich (NC001953) sequences were obtained from Genbank. All sequences were manually aligned using Sequence Navigator (ABI).
Quantitation
The oligonucleotide 08334FQuant was synthesised to include a 19 bp deletion relative to the moa sequence. This primer was used with 8807Rb-COII to create a construct 540 bp long, which was quantified using ethidium bromide on a 4% GTG NuSieve agarose gel. Competitive PCR was performed after Krings et al. using a dilution series of this construct, the primers 08293FH-tAsp/8807Rb-COII, and constant amounts of moa DNA. Products were visualised using ethidium bromide as above.
Cloning
Approximately 7-10 ng of PCR product was ligated into the Promega pGEM-T vector system (Oxford and London), or Pharmacia SureClone ligation kit (Barcelona) and white colonies detected using LB/ampicillin/IPTG/X-Gal plates according to manufacturers instructions. A sterile pipette tip was touched to the surface of white colonies and transferred to a PCR reaction mixture prior to re-amplification (as above) using either the primers SP6 and T7, or those used in the original PCR amplification. Where homopolymers caused premature sequence termination, dRhodamine dyes and 10% DMSO were used.
Authentication
DNA was extracted from separate bone samples, and amplified, cloned and sequenced as above in laboratories in Barcelona and London, except that a sterilised coffee grinder was used to powder the bone, and Promega Taq (Madison, WI) and EcoTaq (Madrid, Spain) polymerases were used, respectively. Both laboratories amplified the previously reported section of 12S3 using the primer pairs 12S 1753FH/2150RH. Sections of the Emeus 16S (3348Fb/3797RH), COII (8320Fb/8807Rb) genes were sequenced in Barcelona, and Dinornis COI (7807FH/8325Rb), Cyt b (15303Fb/15783Rb) and Mullerornis 12S (1856 12SE2/2020 12SF3) genes were sequenced in London. The Emeus COII and Dinornis COI, 12S and Cyt b products were also cloned.
Numts
A putative nuclear insertion of mitochondrial sequences (numt) was detected at the 5' end of the control region and featured insertions of 82 bp and 12bp with respect to the mtDNA sequences (data not shown). Multiple primers, overlapping sequences, and cloning experiments were used to characterise the numt which appears to exist at least between tRNA Glu and the middle of the control region, but was not detected in other regions of the moa mt genomes, or in any of the other taxa although the control region was not sequenced for these.
Phylogenetics
The maximum likelihood tree for the 10.7Kb dataset was estimated by both heuristic and constrained exhaustive searches using PAUP 4.0b4a27. The general reversible model of substitution was used with rate heterogeneity between sites modelled by a discrete gamma distribution. The heuristic search consisted of 10 random addition sequences followed by branch swapping. For the constrained exhaustive search, the 2 moa, the 2 tinamous and the emu and cassowary were each assumed to form monophyletic pairs. Under these constraints, all 105 possible unrooted trees are considered in the search. Both search strategies produced the same maximum likelihood tree (Figure 1). This tree had a log likelihood of -52849.50 and an estimated gamma shape parameter of 0.26. Nearest neighbour interchange values were calculated using the bootstrap frequency outputs to determine the local stability of the phylogenetic tree4.
Alternative phylogenetic hypotheses for ratites were formulated as constraints and the maximum likelihood tree was obtained for each hypothesis using an exhaustive search. A conservative approach was used such that only the specified basal ratite taxa were constrained in position. These hypotheses were 1) That presented by Sibley and Ahlquist2 and van Tuinen et al.6 with ostrich as an outgroup to all other ratites (chicken, tinamous(ostrich(rhea, kiwi,(emu,cassowary))))), with no constraints on the position of the moas as these were not present in this study. 2) That presented by Lee et al.8 with kiwi and moa as an outgroup to the extant ratites (chicken, tinamous((moas,kiwi)(rhea,ostrich,(emu,cassowary)))). The likelihood ratio (LR) statistic between these alternative hypotheses and the unconstrained maximum likelihood hypothesis was tested using a parametric bootstrap28. For each of these hypotheses, sequences with the same length as the real data were simulated along the alternative tree using Seq-Gen31. These simulated datasets were analysed using the same procedure as the real sequences. Both the unconstrained maximum likelihood tree and the maximum likelihood tree that is compatible with the hypothesis being considered were estimated. The LRs of these two trees were accumulated for each replicate dataset and this distribution was used to test the LR for the real data. This tests whether the alternative hypothesis was correct and the maximum likelihood tree was obtained by chance. For both alternative hypotheses, the LR for the real dataset was larger than that for all the simulated datasets.
The ML position of the elephant-bird Mullerornis within the ratite phylogeny was calculated using the short dataset, with the other taxa constrained to their positions in the ML tree for the entire protein coding dataset. The resulting placement is shown in Fig 1., although likelihood ratio tests showed the support was not strong for this, but no signal linked Mullerornis with any ratite taxon in particular, such as the ostrich (data not shown).
To estimate the divergence dates of the ratite lineages, a molecular clock approach was used. Using the maximum likelihood tree obtained above, rooted using the two tinamou sequences and the chicken, a constant rate of evolution was assumed for the ratites and was calibrated using the geological divergence estimate of 82 Myr for the separation of the moa. The analysis was performed using a simple extension of the quartet method of Rambaut and Bromham32 to allow more than four taxa. The assumption of rate constancy amongst the ratites was tested using a likelihood ratio test of the molecular clock model33,34. The likelihood ratio was 12.68, so rate constancy could be rejected (P<0.01 under a 1/2 2 distribution with 5 degrees of freedom). However the unconstrained tree of Figure 1 suggests that the ostrich may have an elevated rate of substitution. To test this, the molecular clock likelihood ratio test was repeated but with the ostrich allowed a different rate to the rest of the ratites (in effect allowing the ostrich's branch length to be unconstrained). This extra rate reduces the degrees of freedom in the test but with a likelihood ratio of 0.449 (P=0.92) this two rate model is clearly consistent with clock-like behaviour. This model has little effect on the divergence estimates, with ostrich dates becoming younger by 5% the largest change.
Archive reference section.
31. A. Rambaut, N. C. Grassly, Comput. Applic. Biosci., 13, 235-238 (1997).
32. A. Rambaut, L. Bromham, Mol. Biol. Evol., 15, 442-448 (1998).
33. J. Felsenstein, J. Mol. Evol., 17, 368-376 (1981).
34. S. S. Wilks, Ann. Math. Stat., 9, 60-62 (1938).
35. S. Kwok, et al., Nucl. Acids Res.18, 999-1005 (1990).
Table 3.
Primers:
Forward / Taxa / Reverse / Taxa00034 Fdin-CR CCCCACAAAATAAGAGAAGCC* / D / 00176 Rmoa-CR GATGGGTATGAGCCAATGC* / D,Ec
00400 FH-CR GTGATCACTGGTATCAGATGG / Ec / 00441 Rb-CR CGCATACCGGGTCTGTTTATGC / D,Ec
00437 Fb-CR TCACGAGAAATCAGCAACC / D,Ec / 00522 RH-CR TGGCCCTGACTTAGGAACCAG / Ec
00811 Fb-CR GTGGAATCATTCGGACACTG / D,Ec / 00832 Rb-CR GGGTTGAGGTATCATAACC / Ec
00817 Fb-CR GGAATCATTCGGACTCTGATGCAC / D,Ec / 00871 Rb-CR ATGTCCAGCGAGCATTCACTA / D,Ec
01278 FB-tPhe GGCACTGAAGATGCCAAGATGGTA / D,Ec / 00980 Rmoa PolyT-CR GGTGTAAGTATATATACAGGCGAAAAA / D
01753 FH-12SA AAACTGGGATTAGATACCCCACTAT / D,Ec,M / 01301 Rb-12S ACGTAAGGTTAGGACTAAGTC / D
01856 Fb-12SE2 CTTGGCGGTGCCCTAAACCCA / M / 01309 RH-12S GCGAGAACTAACGGTAAGGTTAGG / D,Ec
01999 FB-12SJ CCGCTAACAAGACAGGTC / M / 01805 RH-12SP2 TTAAGCGTTTGTGCTCGTAG / D,Ec
02035 Fb-12SC2 GAGATGGAAGAAATGGGCTAC / D,Ec / 01985 Rb-12SH CCTTGACCTGTCTTGTTAGC / M
02471 Fb-16S AAAAGTTACTTGACGCAATAGAG / D,Ec / 02020 Rb-12SF3 GAAAATGTAGCCCATTTCTTCC / M
02907 Fb-16S CATGTTGGCCTTCAAGCAGC / D,Ec / 02150 RH-12SB5 GTGACGGGCGGTATGTAC / D,Ec,M
03348 Fb-16S GGGACTTGTATGAATGGCTA / D,Ec / 02577 Rb-16S GCTTAAATTCATTTTGCTTGG / D,Ec
03725 FH-16S For2 AATAGGGTTTACGACCTCGATGTTGGATCAGG / E,C,K,ET,TT / 02919 Rb-16S GTTGAGCTTTGACGCACTC / D,Ec
03787 Fb-16S CGATTAACAGTCCTACGTG / D,Ec,E,C,K,ET,TT / 03420 Rb-16S CAGGGTCTTCTCGTCTTATG / D,Ec
04168 Fb-ND1 CCTAAGCTACATACAAGC / K,ET,TT / 03797 RH-16S CGACCTGGATTTCTCCGGTCTG / D,Ec
04307 Fb-ND1 GCAACCCCAATCCTAGCCCTA / D,Ec,E,C,K / 04311 Rb-ND1 GGGATTCAAATGGTGATGGCTAG / D,Ec,E,C,K,ET
04315 FH-ND1 CCCAATCCTAGCCCTATTACTAGC / E / 04480 Rb-ND1 CATATGAGATGGTCTGTG / TT
04747 FH-ND1 CCATTCGCCCTATTCTTCCTAGC / D,Ec,E,C,K,ET,TT / 04783 Rb-ND1 GGGTTGAGGAATAGGATAGTGG / K
05139 FH-tGln CGAACCTACACAAAGGAGATC / TT / 04791 Rb-ND1 GAGCTTGGGTTGAGGAATAG / D,Ec,E,C
05197 FH-tMet GGTCAGCTAATTAAGCTATCG / D,Ec,E,C,K,ET / 04905 RH-ND1 TTGGTCGTAGCGGAATCG / E,C,K,ET,TT
05575 FH-ND2 CTAGGCCTAGCACCATTCCA / D,Ec,E,C,K,ET,TT / 05201 RH-tMet CCATCATTTTCGGGGTATGG / D,Ec,E,C,K,ET,TT
05976 Fb-ND2 CCAGTACTAAATGCCACCCTAATAC / E,C,K / 05583 Rb-ND2 CCTTGGAGGACTTCTGGGAA / D,Ec,E,C,K,ET,TT
06016 FH-ND2 GGACTCCCTCCATTAACAGG / D,Ec,E / 05791 RTTin GCTATTCAGCCTAGGTGGGAGA / TT
06045 FH-ND2 CCTAAATGACTAATCCTCCAAG / E,K,ET,TT / 06067 Rb-ND2 GATGATTAGGGCTGTAGTTG / D,Ec
06335 Fb-tTrp GGCCTTCAAAGCCTTAAATAAGAG / D,Ec,E,C,K,ET,TT / 06120 RH-ND2 GCATAGTATGCGAGGCGTAG / E,C,K,ET,TT
06598 Fb-tTyr GAAGAGGAATTGAACCTCTG / E,K / 06367 Rb-tAla GATACAGGTTAATGTCCTGTTGG / D,Ec,E,C,ET,TT
06620 FH-tTyr AAGGACTACAGCCTAACG / D,Ec,E,C,K,ET,TT / 06437 RH tAsn GGGATCGAGGCCCATCTGTC / TT
06634 Fb-tTyr CCTAACGCTTGAACACTCAGC / Ec / 06631 RH-tTyr GTCACAGGTAAGATGGCTG / E,C,K,ET
07023 Fb-COI AAGCCGGAGCAGGAACTGGA / D,Ec,E,C,K / 06642 Rb-COI CGGGTAATGAAGGTCACAGG / D,Ec,E,C,K,ET,TT
07027 FH-COI GGAGCAGGAACAGGATGAAC / TT / 07047 Rb-COI CTGCATGAGCCAGGTTTCC / D,Ec,E,K,ET
07056 Fb-COI CCCTTTAGCAGGTAACCTCG / K,ET / 07101 RH-COI CCTAGGATAGAGGAAACACCTGC / D,E,C
07459 FH-COI GAACCATTCGGATACATAGG / D,Ec,E,C,ET,TT / 07111 RH-COI GATTGCTCCTAGGATGGAGG / C,ET,TT
07546 FH-COI GTCGGAATAGACGTAGACAC / E,K / 07490 Rb-COI CCCATACAATGAATCCCAGG / D,Ec,E,C,K
07744 FH-COI CACGACACATACTACGTAG / E,K,ET,TT / 07553 Rb-COI GTGGCTGATGTGAAGTAG / E,TT
07807 FH-COI CTCTCTATAGGTGCCGTATTTGC / D,Ec,K,ET / 07561 Rb-COI GGCGATGATTATTGTGGCTGATG / Ec,E,K,ET
07812 FH-COI GGAGCAGTATTTGCCATTC / D,C,ET / 07816 Rb-COI GGGAATCAGTGTGTAAATCCTG / D,Ec,E,C,K,ET,TT
08171 FH-COII-LR CGAACCTCCACACGCTGGTTTCAAGCC / E,C,K,ET,TT / 08323 Rb-COII GGTTGGCCACATGAGATG / D,Ec,E,C,K,ET
08293 FH-tAsp CCTATTACATAGCCTTGTC / D,Ec / 08358 RH-COII GGGGGATGATGCGTCTTG / E,K,ET,TT
08320 Fb-tAsp TCACAGGTCAAAACCCTG / D,Ec,E,C,K,ET,TT / 08508 RH-COII GGATGGTTCAGATTAGTTCGAC / D,Ec,ET
08334 FQuant CCTATTACATAGCCTTGTCCCCTGTACATCTCATGTGG / D,Ec / 08796 Rb-COII CATGAGTGGAGAACATCTCC / E,C,K,ET
08500 Fb-COII CTCCTCTAACACAGTAGATGC / E,C / 08807 Rb-COII GGAACTGCTCATGAGTGG / D,Ec,E,TT
08728 FH-COII CTACCATCAGGCTACTTCCG / D,E,C,K,ET,TT / 09025 Rb-tLys GGCTAGTGCTGTTCCATAGC / D,Ec,C,ET
08735 FQuant GGTCACTTCCGACTACTACGAGTTGTAGTCCCAATAGA / Ec,E / 09036 RH-tLys CTCTAGCTTAAAAGGCTAGTGC / M
08861 Fb-COII GATGCAATCCCAGGACGA / D,Ec,M,E,C,ET / 09241 Rb-ATP8 TGGTCGAAGAAGCTTAGGTTCA / M
08945 FH-COII GGGCAAATCATAGCTAC / TT / 09284 Rb-ATP6 GTAGGATTAGTGGGATTCC / E,K
09043 Fb-tLys GCTATGGAACAGCACTAGC / D,Ec,M,E,C,ET,TT / 09349 Rb-ATP6 GTCGGTTAGTGATTCAGC / D,Ec,C,ET,TT
09246 Fb-ATP8 CTGAACCTGACCATGAACC / D,Ec,E,C,K,ET / 09738 RH-ATP6 GTTGAATTAGAAGATGGCCGGC / D,Ec,E,K,TT
09251 FLR-ATP6 CCCCCTGAACCTGACCATGAACCTAAGC / C,K / 09755 Rb-ATP6 GTGGCAGTAGAGATGAGTTGG / E,C,K,ET
09619 Fb ATP6 CAACCGTCCATCTCTCTAGG / TT / 10116 RLR-COIII GGGGTGTGGTGGCCCTGGAAGGTGC / E,ET
09712 Fb-ATP6 TTCGACCATTAGCCCTAGG / D,Ec,E,C,K,ET,TT / 10218 RH-COIII-LR GGGGCTAGGCTTGAGTGGAAGAAGGCTC / E,C,K,ET,TT
09732 FH-ATP6 GGTGTCCGACTTACAGCTAACC / D,Ec,ET / 10227 Rb-COIII GGGGCTAGGCTTGAGTGGA / D,Ec,E,C,K,ET
10161 FH-COIII CAGTCCAAAAAGGACTACG / D,Ec,E,C,K,ET,TT / 10743 Rb-tGly GGGGTCACACCAGATTTTAGAG / D,Ec,E,C,K,ET,TT
10695 FH COIII CCTCTACATATCCATCTACTG / TT / 11152 Rb-tArg ATTTGCTGAGCCGAAATCAG / D,Ec
10716 FH-tGly GAGGATCCTGCTCTTCTAG / D,Ec,E,C,K,ET / 11184 RH tArg GGGACATAAGAAAGTTATAG / TT
11095 FH-ND3 CTAGGACTAGTCTATGAATG / TT / 11442 Rb-ND4L TGGTCTGAGCCGTGTGTTCG / E,C,K,ET
11120 Fb-ND3 CAAGGAGGCCTCGAATGAGCA / D,Ec,E,C,K,ET / 11589 Rb-ND4 GTCATTGCAGGCTGATGGTGG / E,C,K,ET
11407 FH-ND4L GCCTTCTCTGCATGCGAAGC / D,Ec,ET / 11598 Rb-ND4 GGGTTAAGTCATTGCAGGC / D,Ec,TT
11461 Fb-ND4L CGAACACACGGCTCCGACCA / K / 11873 Rb-ND4 CCTCATCGGGTGATTAGGATTAG / Ec
11472 FH-ND4L GGCTCAGACCACCTCCACAACC / E,C,K / 11886 RH-ND4 GGTTGATTTCCTCATCGGG / D,E,ET,TT
11823 FH-ND4 TCCTAGCCTTCTCAGCCAC / TT / 11909 RH-ND4 TAGATTCCGGCGCTAAGTCG / E,C,K
11868 FH-ND4 CATTTGAGGCAACCCTAATTCC / D,Ec,E,C,K,ET / 12356 Rb-ND4 ACTAGGCCCATGTGGCTTAC / E,C,K,ET
11904 FH-ND4 CCCGTTGAGGAAATCAACC / E,C,K,ET / 12364 Rb-ND4 GCAGCAATGACTAGGCCTATG / D,Ec,E,C,K,ET,TT
12312 FH-ND4 GGTGCACTAATAACCAGCTCAATCTG / D,Ec,E,C,K / 12800 Rb-ND4 GTATGGGGAGAATGTGGAG / D,Ec,E,C,K
12326 FH-ND4 GCTCCATCTGCTTACGCCAAACTG / D,C,ET,TT / 12955 RH-tSer GGCTCAGATGCAAGAATTAGCAGTTC / C,ET,TT
12651 FH-ND4 TCGCACTCTTCAACTGATC / E,C,K,ET / 13200 Rb-ND5 GGGATGAGGCTGAGAAAGAAGGC / D,Ec,E,C,ET
12788 FH-ND4 CTCAAACACACGAGAACACC / D,Ec,E,C,ET,TT / 13235 Rb-ND5 GTTTCTGTTCCTGAGTAGATG / E,C,K,TT
13052 FH-ND5 GGTCTTAGGAACCACCCATCTTGGTG / D,Ec,E,C,K,ET,TT / 13592 Rb-ND5 GCCTACGTCTCCAATACGGTTGTAG / D,Ec,E,C,K,ET,TT
13558 FH-ND5 GGCTGATGACACGGACGAGC / ET,TT / 14013 RH-ND5 GGGAGGTTAAGGCCAATAGTAAC / D,Ec
13563 Fb-ND5 GATGACACGGACGAGCAGAAG / E,C,K,ET / 14037 RH-ND5 GAGATGTGAAAGAATGCCAGTTG / ET,TT
13575 FH-ND5 CAGACGCCAACACAGCTGC / D,Ec / 14040 RH-ND5 GATATGGAGGAAGGCTAG / Ec,C,K
13939 Fb-ND5 CCTCCATATTCGCCGCCAC / TT / 14050 RH-ND5 GAAGGCATGGGTTGAGATATGG / E,K
14002 FH-ND5 CCACATCAAGCCAACTAGG / D,Ec,E,C,K,ET / 14461 Rb-ND5 GATGTGATGAGAAGGCCAG / D,Ec,E
14452 FH-ND5 CCCGACTCGCACTAGGCAG / D,Ec,E,C,K,ET,TT / 14476 Rb-ND5 GTGGGTAGGATGTTGGATG / C,K,ET
14817 Fb-ND5 CCTCTACCTCACTACACACCG / D,E,K,ET / 14515 Rb ND5 CCATTTTGGTTAGAGTAGGTA / TT
14848 Fb-ND5 CCTACCTGGGTTCCTTCGC / TT / 14539 RH-ND5 CCGAGGATTGTGACGACGATGG / E
14859 FH-ND5 GGAACATTCGCCCTATCCATCC / Ec,E,C,K / 14893 Rb-ND5 GGGATTTTCGAATGTTAGGGGCCAT / E,C,TT
14987 FH-Cytb CCCCTCCAACATCTCTGCTTGATG / D,Ec,E / 14946 Rb-Cytb AGGGGGAGGGGAGGTCAATTAGG / D,Ec,ET
15303 Fb-Cytb GGTTATGTCCTCCCATGAGGA / D,Ec,E,C,K,ET,TT / 15035 RH-Cytb GGCTAGTAGTAGTCCTG / D,E,C,K
15671 FH-Cytb CCCAGAAAACTTCACACCAGC / D,Ec,M,E,C,K,ET / 15345 Rb-Cytb CCGATGTAGGGGATGGCTGAG / D,Ec,E,C,K,ET,TT
15928 Fb GCAGCCAACCAGTAGAAC / D,Ec / 15709 Rb-Cytb GCGTATGCGAATAGGAAATATCA / K
16152 FH-tPro CTCCAGCTCCCAAAGCTG / D,Ec / 15783 Rb-Cytb GGAATAGGATTAGGACGGAG / D,Ec,E,C,ET,TT
16653 Fb-ND6 GGAGAAGGATTGGATGCCACTG / D,Ec / 15902 RH GGCTGACTTCCGATTCATG / M
16733 Fb-tGlu CCTACTTGGTTCTACCCAAGG / D,Ec / 16060 Rp-tThr CTTCGGTTTACAAGACCGATG / E,K,ET,TT
16075 Rb-tThr GGGTGTGGTCTTCATTCTTTGG / C,ET
16120 Rb-tPro-LR CAGCTTTGGGAGCTGGAGGTAGAGGTTTAAGTCC / E,C,K,ET,TT
16140 RH-tPro GAGAATATCAGCTTTGGGAGC / D,Ec,E
16219 Rb-ND6 GTGGTGTTGGAGCTTGTAC / D,Ec
16740 Rb-tGlu ACAACGACGGTTTTTCAGAC / D,Ec
Primer pairs used with taxa in the study. The numbers refer to the position of the 3' base in the chicken sequence (Genbank X52392), except for * which is numbered according to the Dinornis moa sequence. F and R are forward and reverse, and the target gene is given using the conventional acronym. H refers to a primer compatible with human mtDNA while b refers to a bird specific primer, according to Kwok et al.35. The taxa amplified with each primer are shown by D, Dinornis; Em, Emeus; M, Mullerornis; E, emu; C, cassowary; K, kiwi; ET, Elegant Crested tinamou; TT, Tataupa tinamou.
Figure 3
Cloned sequences of ratites and tinamous. Sequence positions (bold titles) are numbered according to the chicken whole mitochondrial sequence (Genbank X52392). The results are summarised in Table 1. Dots represent identity to the direct PCR sequence (above), dashes represent missing data.
Tataupa tinamou, ND1 4747-5201
Tataupa4 CCATTTGCCC TATTCTTCCT AGCAGAATAT GCCAACATTA TACTAATAAA CATAATAACC 60
clone1R ...... 37
clone_1F ...... 37
clone_2R ...... 37
clone2F 0
clone_3F 0
clone4R ...... 37
clone_4F 0
clone_5R ...... 37
clone5F 0
clone_6R ...... 37
clone6F 0
clone7R ...... 37
clone_7R 0
clone_8F 0
clone_11 ...... 37
clone_12 ...... 37
clone_13 ...... 37
clone_14 ...... 37
clone_15 ...... 37
clone_16 ...... 37
clone_17 ...... 37
clone_18 ...... 37
clone_19 ...... 37
clone_20 ...... 37
Tataupa4 ACTATTTTAT TTATTAACCC AAGCTCACTA AACCTACCTC CCCAAGTCCT TCCATTAGCA 120
clone1R ...... 97
clone_1F ...... 97
clone_2R ...... 97
clone2F 0
clone_3F ...... 58
clone4R ...... 97
clone_4F 0
clone_5R ...... 97
clone5F 0
clone_6R ...... C...... 97
clone6F 0
clone7R ...... T ...... 97
clone_7R 0
clone_8F ...... 58
clone_11 ...... 97
clone_12 ...... 97
clone_13 ...... 97
clone_14 ...... 97
clone_15 ...... 97
clone_16 ...... 97
clone_17 ...... 97
clone_18 ...... 97
clone_19 ...... 97
clone_20 ...... 97
Tataupa4 CTGGCCACAA AGACCCTCCT ATTATCCTCA GGGTTTCTAT GAATCCGGGC CTCTTACCCA 180
clone1R ...... 157
clone_1F ...... 157
clone_2R ...... C ...... 157
clone2F ..C ...... 43
clone_3F ...... 118
clone4R ...... 157
clone_4F 0
clone_5R ...... 157
clone5F ...... 33
clone_6R ...... 157
clone6F ...... 41
clone7R ...... 157
clone_7R ...... 44
clone_8F ...... 118
clone_11 ...... 157
clone_12 ...... 157
clone_13 ...... 157
clone_14 ...... 157
clone_15 ...... 157
clone_16 ...... 157
clone_17 ...... C...... 157
clone_18 ...... 157
clone_19 ...... 157
clone_20 ...... 157
Tataupa4 CGATTTCGTT ACGACCAACT TATACAGTTA CTATGAAAAA ACTTCCTCCC ACTTACCCTA 240
clone1R ...... 217
clone_1F ...... 217
clone_2R ...... 217
clone2F ...... 103
clone_3F ...... 178
clone4R ...... 217
clone_4F 0
clone_5R ...... 217
clone5F ...... 93
clone_6R ...... G...... C...... G..... 217
clone6F ...... G...... C...... G..... 101
clone7R ...... 217
clone_7R ...... 104
clone_8F ...... 178
clone_11 ...... 217
clone_12 ...... 217
clone_13 ...... 217
clone_14 ...... 217
clone_15 ...... C. 217
clone_16 ...... C...... 217
clone_17 ...... 217
clone_18 ...... 217
clone_19 ...... 217
clone_20 ...... 217
Tataupa4 GCCTTCTGCT TATGACATAC AAGTCTGCCA ATCTCACTTG CAGGTCTTCC CCCGTACTTA 300
clone1R ...... 277
clone_1F ...... 277
clone_2R ...... 277
clone2F ...... 163
clone_3F ...... 238
clone4R ...... 277
clone_4F ...... 14
clone_5R ...... 277
clone5F ...... 153
clone_6R ...... 277
clone6F ...... 161
clone7R ...... 277
clone_7R ...... 164
clone_8F ...... 238
clone_11 ...... 277
clone_12 ...... 277
clone_13 ...... 277
clone_14 ...... 277
clone_15 ...... 277
clone_16 ...... 277
clone_17 ...... 277
clone_18 ...... 277
clone_19 ...... 277
clone_20 ...... 277
Tataupa4 AGAAACCGCA CCCAAAATGG AAATGTGCCT GAAATCTAAG GGTCACTATG ATAAAGTGAA 360
clone1R ...... 337
clone_1F ...... 337
clone_2R ...... 337
clone2F ...... 223
clone_3F ...... C...... 298
clone4R ...... 337
clone_4F ...... 74
clone_5R ...... 337
clone5F ...... 213
clone_6R ...... 337
clone6F ...... 221
clone7R ...... 337
clone_7R ...... 224
clone_8F ...... 298
clone_11 ...... 337
clone_12 ...... 337
clone_13 ...... 337
clone_14 ...... 337
clone_15 ...... A...... 337
clone_16 ...... 337
clone_17 ...... 337
clone_18 ...... 337
clone_19 ...... 337
clone_20 ...... 337
Tataupa4 TATAGAGGTG CACCAACCCT CTCATTTCCT TACTAGATTA GAAAAGTAGG AATCGAACCT 420
clone1R ...... 364
clone_1F ...... 397
clone_2R ...... 364
clone2F ...... 283
clone_3F ...... 358
clone4R ...... 364
clone_4F ...... 134
clone_5R ...... 364
clone5F ...... 273
clone_6R ...... 364
clone6F ...... C ...... 281
clone7R ...... 364
clone_7R ...... 284
clone_8F ...... 358
clone_11 ...... 397
clone_12 ...... 397
clone_13 ...... 397
clone_14 ...... 397
clone_15 ...... 397
clone_16 ...... A 397
clone_17 ...... 397
clone_18 ...... 397
clone_19 ...... 397
clone_20 ...... 397
Tataupa4 ACACAAAGGA GATCAAAACC CCTCATACTT CCTCTATATT ATTTCCTAGT AAGGTCAGCT 480
clone_1F ...... 457
clone2F ...... G ...... 343
clone_3F ...... 418
clone_4F ...... 194
clone5F ....G...... 333
clone6F ...... 341
clone_7R ...... 344
clone_8F ...... 418
clone_11 ...... 446
clone_12 ...... 446
clone_13 ...... 446
clone_14 ...... 446
clone_15 ...... 446
clone_16 ...... 446
clone_17 ...... 446
clone_18 ...... 446
clone_19 ...... 446
clone_20 ...... 446
Kiwi, ND4L 11120-11598
Kiwi_111 CAAGGAGGCC TCTGAATGAG CAGAGTAACA GAAAGTTAGT CTAACTAAGA TAGCTGATTT 60
Clone_5 ...... 38
Clone_1 ...... 38
Clone_9 ...... 38
Clone_7 ...... 38
Clone_10 ...... 38
Clone_12 ...... 38
Clone_13 ...... 38
Clone_8 ...... 38
Clone_18 ...... 38
Clone_6 ...... G...... 38
Clone_14 ...... 38
Kiwi_111 CAGGCTCAGC AGATTATAGT CACCCCTATA ACTTTCTTAT CGACATTACG TACGACCTAA 120
Clone_5 ...... 98
Clone_1 ...... 98
Clone_9 ...... 98
Clone_7 ...... 98
Clone_10 ...... 98
Clone_12 ...... 98
Clone_13 ...... A...... 98
Clone_8 ...... 98
Clone_18 ...... 98
Clone_6 ...... A...... 98
Clone_14 ...... 98
Kiwi_111 GCTTCGTACT GCAGCTTTCA CCCTAAGTGG CCTAGGCCTG AGCCTTTCAC CGAACCCATC 180
Clone_5 ...... 158
Clone_1 ...... 158
Clone_9 ...... 158
Clone_7 ...... 158
Clone_10 ...... 158
Clone_12 ...... 158
Clone_13 ...... 158
Clone_8 ...... 158
Clone_18 ...... 158
Clone_6 ...... A...... 158
Clone_14 ...... 158
Kiwi_111 TTGCATCTCT GCTCCTATTA TGCCTAGAAA GCATAATACT CTCTCTATAC ATCTCACCTC 240
Clone_5 ...... 218
Clone_1 ...... 218
Clone_9 ...... 218
Clone_7 ...... 218
Clone_10 ...... 218
Clone_12 ...... 218
Clone_13 ...... 218
Clone_8 ...... 218
Clone_18 ...... C...... 218
Clone_6 ...... 218
Clone_14 ...... 218
Kiwi_111 TCGATCTGAC CTATCCAAAC CCAAACCCCC ACATTCTCCC TAATCCCAGT ACTCATACTA 300
Clone_5 ...... G...... 278
Clone_1 ...... 278
Clone_9 ...... 278
Clone_7 ...... 278
Clone_10 ...... 278
Clone_12 ...... 278
Clone_13 ...... 278
Clone_8 ...... C. 278
Clone_18 ...... 278
Clone_6 ...... 278
Clone_14 ...... G..... 278
Kiwi_111 GCCTTTTCCG CATGCGAAGC AGGAGCAGGA CTAGCTATAT TAGTAGCCTC TACTCGCACA 360
Clone_5 ...... 338
Clone_1 ...... 338
Clone_9 ...... 338
Clone_7 ...... 338
Clone_10 ...... 338
Clone_12 ...... 338
Clone_13 ...C...... 338
Clone_8 A...... 338
Clone_18 ...... T...... G...... 338
Clone_6 ...... 338
Clone_14 ...... C...... 338
Kiwi_111 CATGGCTCCG ATCACCTACA TAACTTAAAC CTCCTACAAT GCTAAAAATT ATTTTTCCCA 420
Clone_5 ...... 398
Clone_1 ...... 398
Clone_9 ...... 398
Clone_7 ...... 398
Clone_10 ...... 398
Clone_12 ...... 395
Clone_13 ...... 398
Clone_8 ...... 398
Clone_18 ...... 398
Clone_6 ...... 393
Clone_14 ...... 398
Kiwi_111 CAGCCATGCT TTTA 434
Clone_5 ...... 412
Clone_1 ...... 412
Clone_9 ...... 412
Clone_7 ...... 412
Clone_10 ...... 412
Clone_12 395
Clone_13 ...... 412
Clone_8 ...... 412
Clone_18 ...... 412
Clone_6 393
Clone_14 ......
Cassowary, COIII 10161-10743
CASS_101 CAGTCCAAAA AGGACTACGA TATGGAATAA TCCTATTCAT TACATCTGAA GCATTCTTCT 60
Clone_1 ...... 11
Clone_2 ...... 60
Clone_3 ...... 41
Clone_4 ...... 41
Clone_6 ...... 41
Clone_7 ...... 41
Clone_8 ...... 11
Clone_9 ...... 41
Clone_11 0
Clone_12 0
Clone_14 0
Clone_15 0
Clone_16 0
Clone_17 0
Clone_19 0
Clone_20 0
CASS_101 TCCTAGGCTT CTTCTGAGCA TTCTTCCATT CAAGCCTAGC ACCAACACCA GAACTAGGAG 120
Clone_1 ...... 71
Clone_2 ...... 120
Clone_3 ...... 101
Clone_4 ...... 101
Clone_6 ...... 101
Clone_7 ...... 101
Clone_8 ...... G ...... 71
Clone_9 ...... T...... 101
Clone_11 0
Clone_12 0
Clone_14 0
Clone_15 0
Clone_16 0
Clone_17 0
Clone_19 0
Clone_20 0
CASS_101 GACTATGACC TCCCACAGGT ATCAAACCTC TTAACCCATT AGAAGTTCCC CTACTCAATA 180
Clone_1 ...... 131
Clone_2 ...... 180
Clone_3 ...... G...... 161
Clone_4 ...... 161
Clone_6 ...... 161
Clone_7 ...... 161
Clone_8 ...... C ...... 131
Clone_9 ...... 161
Clone_11 0
Clone_12 0
Clone_14 0
Clone_15 0
Clone_16 0
Clone_17 0
Clone_19 0
Clone_20 0
CASS_101 CAGCCATTCT CCTAGCATCA GGTGTTACTG TTACATGAGC CCATCATAGA ATCACAGAAG 240
Clone_1 ...... 171
Clone_2 ...... 240
Clone_3 ...... 221
Clone_4 ...... 221
Clone_6 ...... 221
Clone_7 ...... 221
Clone_8 ...... 191
Clone_9 ...... C...... 221
Clone_11 0
Clone_12 0
Clone_14 0
Clone_15 0
Clone_16 0
Clone_17 0
Clone_19 0
Clone_20 0
CASS_101 GTAATCTGAA AACAAGCAAT CCATGCTCTA ACTCTAACAA TCCTGTTAGG GTTCTACTTT 300
Clone_2 ...... 300
Clone_3 ...... C ...... 281
Clone_4 ...... 281
Clone_6 ...... 281
Clone_7 ...... 281
Clone_8 ...... 225
Clone_9 ...... 281
Clone_11 0
Clone_12 0
Clone_14 ...... 27
Clone_15 ...... 14
Clone_16 0
Clone_17 ...... 34
Clone_19 ...... 34
Clone_20 0
CASS_101 ACTGCCCTAC AAGCCATAGA GTATCATGAA GCCTCATTCT CAATCGCCGA CGGAGTCTAT 360
Clone_2 ...... 315
Clone_3 ...... 341
Clone_4 ...... 296
Clone_6 ...... 296
Clone_7 ...... 341
Clone_9 ...... T...... 341
Clone_11 ...... 31
Clone_12 ...... 58
Clone_14 ...... 87
Clone_15 ...... 74
Clone_16 ...... 31
Clone_17 ...... 94
Clone_19 ...... 94
Clone_20 ...... 58
CASS_101 GGTTCCACCT TCTTTGTAGA TACAGGATTC CGCGGCCTAC ATGTAACTCA TTGGGTCATC 420
Clone_3 ...... 353
Clone_7 ...... 352
Clone_9 .. 343
Clone_11 ...... 91
Clone_12 ...... 118
Clone_14 ...... 147
Clone_15 ...... 134
Clone_16 ...... 91
Clone_17 ...... 154
Clone_19 ...... 154
Clone_20 ...... 118
CASS_101 TTTCCTCACC GTATGCCTAC TACGACTAAT CAAATTCCAC TTTACATCCA ACCACCATTT 480
Clone_11 ...... 151
Clone_12 ...... G ...... 178
Clone_14 ...... 207
Clone_15 ...... 194
Clone_16 ...... 151
Clone_17 ...... 214
Clone_19 ...... 214
Clone_20 ...... 178
CASS_101 TGGGTTCGAG GCAGCTGCCA TGATATTGAC ATTTTGTAGG ACGTTATGTG ATTGTTCCTC 540
Clone_11 ...... 211
Clone_12 ...... 238
Clone_14 ...... G...... 267
Clone_15 ...... 254
Clone_16 ...... 211
Clone_17 ...... 274
Clone_19 ...... 274
Clone_20 ...... 238
CASS_101 TACATAACCA TCTACTGATG AGGATCAATG CTGCTTCATA GTATAGCTAA TGTGACAAGA 600
Clone_11 ...... 271
Clone_12 ...... 298
Clone_14 ...... 327
Clone_15 C...... G...... 314
Clone_16 ...... 271
Clone_17 ...... 334
Clone_19 ...... A ...... 334
Clone_20 ...... 298
CASS_101 TTGACTTCCA ATCTCTAAAA TCTGGTGTGA CCCC 634
Clone_11 ...... 305
Clone_12 ...... 332
Clone_14 ...... 361
Clone_15 ...... 348
Clone_16 ...... 305
Clone_17 .A...... 368
Clone_19 ...... 368
Clone_20 ...... 332
Dinornis, Control region 16733-00441
Digi1673 TCCCACTTGG TTCTACCCAA GGCCAGCGGT CTGAAAAACC ACCGTTGTCC TCAACTATGG 60
Clone_2 ...... 38
Clone_3 0
Clone_4 0
Clone_6 ...... 38
Clone_8 ...... 38
Clone_11 ...... 38
Clone_12 ...... 38
Clone_13 ...... 38
Clone_15 ...... 38
Clone_16 ...... 38
Clone_20 ...... 38
Clone_4a ...... 31
Clone_6a ...... 31
Clone_7a ...... 31
Clone_9a ...... 38
Clone_10 ...... 38
Clone_12 ...... 38
Digi1673 GAACTAATTA AACGTATCCC CACAAAATAA GAGAAGCCCC AAGAATCAAT GAGACATCTC 120
Clone_2 ...... 98
Clone_3 ...... T...... 56
Clone_4 ...... 30
Clone_6 ...... 98
Clone_8 ...... 98
Clone_11 ...... 98
Clone_12 ...... 98
Clone_13 ...... 98
Clone_15 ...... 98
Clone_16 ...... 98
Clone_20 ...... 98
Clone_4a ...... 91
Clone_6a ...... 91
Clone_7a ...... 91
Clone_9a ...... 98
Clone_10 ...... 98
Clone_12 ...... 98
Digi1673 TACTAAAGAA GCCCCCCCCT ACCCCCCCGT ACTATAATCA TACTATGTAT TATTGTACAT 180
Clone_2 ...... 158
Clone_3 ...... 116
Clone_4 ...... 90
Clone_6 ...... 158
Clone_8 ...... 158
Clone_11 ...... 158
Clone_12 ...... 158
Clone_13 ...... 158
Clone_15 ...... 158
Clone_16 ...... 158
Clone_20 ...... T...... 158
Clone_4a ...... 151
Clone_6a ...... 151
Clone_7a ...... 151
Clone_9a ...... 158
Clone_10 ...... A...... 158
Clone_12 ...... 158
Digi1673 TAATTGCTAT AGTACATTAT TCATCATGAC ATTAAATGTA TTATAGTACA TTCCCTGCAT 240
Clone_2 ...... 218
Clone_3 ...... 176
Clone_4 ...... 150
Clone_6 ...... 218
Clone_8 ...... 218
Clone_11 ...... 218
Clone_12 ...... 218
Clone_13 ...... 218
Clone_15 ...... 218
Clone_16 ...... 218
Clone_20 ...... 218
Clone_4a ...... 211
Clone_6a ...... 211
Clone_7a ...... 211
Clone_9a ...... 218
Clone_10 ...... 218
Clone_12 ...... 218
Digi1673 TGGCTCATAC CCATCCAACC ATTCAGTGCA ATCAGACACG TTTCCCTTGT TCCCCTCTCG 300
Clone_2 ...... 278
Clone_3 ...... 236
Clone_4 ...... 210
Clone_6 ...... 278
Clone_8 ...... 278
Clone_11 ...... 278
Clone_12 ...... 278
Clone_13 ...... 278
Clone_15 ...... 278
Clone_16 ...... 278
Clone_20 ...... C...... 278
Clone_4a ...... 271
Clone_6a ...... 271
Clone_7a ...... 271
Clone_9a ...... 278
Clone_10 ...... T...... 278
Clone_12 ...... 278
Digi1673 TAACACTAAA GTATCCACAA TCTCAGGGTG ATCGAAAGCC ATAACTCTAT CACTTATACA 360
Clone_2 ...... 338
Clone_3 ...... 296
Clone_4 ...... 270
Clone_6 ...... 338
Clone_8 ...... 338
Clone_11 ...... 338
Clone_12 ...... 338
Clone_13 ...... 338
Clone_15 ...... 338
Clone_16 ...... 338
Clone_20 ...... 338
Clone_4a ...... 331
Clone_6a ...... 331
Clone_7a ...... 331
Clone_9a ...... 338
Clone_10 ...... C...... 338
Clone_12 ...... C...... 338
Digi1673 CCTAAAGTGC ACACAGAACG GGGTAAGGTA CTAAGGCCCT TAGCGAAGAC TGACTAGAAG 420
Clone_2 ...... 398
Clone_3 ...... 356
Clone_4 ...... C...... 330
Clone_6 ...... G...... 398
Clone_8 ...... 398
Clone_11 ...... 398
Clone_12 ...... 398
Clone_13 ...... 398
Clone_15 ...... 398
Clone_16 ...... 398
Clone_20 ...... 398
Clone_4a ...... 391
Clone_6a ...... G.....A...... 391
Clone_7a ...... G.....A...... 391
Clone_9a ...... G...... 398
Clone_10 ...... C...... G...... 398
Clone_12 ...... G...... 398
Digi1673 CATAAATTTA TAGCTCGGAC ATAATTCTAA CCCGTACTGT TCAAATCTCG CTTAATCCCT 480
Clone_2 ...... 458
Clone_3 ...... 416
Clone_4 ...... T.T...... 390
Clone_6 ...... 458
Clone_8 ...... 458
Clone_11 ...... 458
Clone_12 ...... 458
Clone_13 ...... 458
Clone_15 ...... 458
Clone_16 ...... 458
Clone_20 ...... 458
Clone_4a ...... 451
Clone_6a ...... 451
Clone_7a ...... 451
Clone_9a ...... 458
Clone_10 ...... 458
Clone_12 ...... 458
Digi1673 ATTTCTCGTA TACAAAATTC CTATGCAGTG CTCTAAGGTC TAAACCTCTA AGTCCCCCAT 540
Clone_2 ...... 518
Clone_3 ...... G. 476
Clone_4 ...... 450
Clone_6 ...... 518
Clone_8 ...... 518
Clone_11 ...... 518
Clone_12 ...... 518
Clone_13 ...... 518
Clone_15 ...... 518
Clone_16 ...... 518
Clone_20 ...... 518
Clone_4a ...... 511
Clone_6a ...... 511
Clone_7a ...... 511
Clone_9a ...... 518
Clone_10 ...... 518
Clone_12 ...... 518
Digi1673 CCATGCTCTT CCACGGGGAT ATGATCCCTG GTATCAGATG GATTTCTTGA TTCCCCCCCT 600
Clone_2 ...... 578
Clone_3 ...... 536
Clone_4 ...... A...... 510
Clone_6 ...... 578
Clone_8 ...... 578
Clone_11 ...... 578
Clone_12 ...... 578
Clone_13 ...... 578
Clone_15 ...... 578
Clone_16 ...... 578
Clone_20 ...... 578
Clone_4a ...... 571
Clone_6a ...... 571
Clone_7a ...... 571
Clone_9a ...... 578
Clone_10 ...... 578
Clone_12 ...... 578
Digi1673 CACGAGAAAC CAGCAACCCC GTGCATAAAC AGACCCGGTA TGCG 644
Clone_2 ...... C 600
Clone_3 ...... 558
Clone_4 ...... 532
Clone_6 ...... 600
Clone_8 ...... 600
Clone_11 ... 581
Clone_12 ...... 600
Clone_13 ...... C 600
Clone_15 ...... 600
Clone_16 ...... 600
Clone_20 ...... C 600
Clone_4a ...... 593
Clone_6a ... 574
Clone_7a ... 574
Clone_9a ...... 600
Clone_10 ...... 600
Clone_12 ...... A...... 600
Dinornis, ND1 4747-5201
DIGI4747 CCATTCGCCC TATTCTTCCT AGCCGAATAC GCTAATATCA TACTAATAAA CATATTAACC 60
Clone_1 ...... 37
Clone_2 ...... 37
Clone_3 ...... 37
Clone_4 ...... 37
Clone_5 ...... 37
Clone_8 ...... 37
Clone_9 ...... 37
Clone_10 ...... 37
Clone_11 ...... 37
Clone_12 ...... 37
DIGI4747 ACCATCCTAT TCATCAACCC TAGCTCACTT AACCTTCCAC CCGAACTGCT CCCAATAGTC 120
Clone_1 ...... 97
Clone_2 ...... T...... 97
Clone_3 ...... T...... 97
Clone_4 ...... 97
Clone_5 ...... 97
Clone_8 ...... 97
Clone_9 ...... 97
Clone_10 ...... 97
Clone_11 ...... 97
Clone_12 ...... 97
DIGI4747 CTAGCCACAA AAACCTTACT ACTATCCTCA GGATTCCTAT GAGTACGAGC CTCATACCCA 180
Clone_1 ...... T ...... 157
Clone_2 ...... 157
Clone_3 ...... 157
Clone_4 ...... 157
Clone_5 ...... 157
Clone_8 ...... 157
Clone_9 ...... G...... 157
Clone_10 ...... 157
Clone_11 ...... 157
Clone_12 ...... 157
DIGI4747 CGATTCCGCT ATGACCAACT AATACACCTC CTCTGAAAAA ACTTCCTACC CCTTACGCTG 240
Clone_1 ...... 217
Clone_2 ...... 217
Clone_3 ...... 217
Clone_4 ...... 217
Clone_5 ...... G...... 217
Clone_8 ...... 217
Clone_9 ...... 217
Clone_10 ...... 217
Clone_11 ...... 217
Clone_12 ...... 217
DIGI4747 GCGCTATGCT TATGACATAC AAGCCTACCA ATTTCCTACG CAGGCCTTCC TCCCTATTAA 300
Clone_1 ...... 277
Clone_2 ...... 277
Clone_3 ...... 277
Clone_4 ...... 277
Clone_5 ...... 277
Clone_8 ..C...... 277
Clone_9 ...... 277
Clone_10 ...... 277
Clone_11 ...... 277
Clone_12 ...... 277
DIGI4747 AGGAAATGTG CCTGAACTAA AGGATCACTA TGATAAAGTG AACATAGAGG TTTACCAGTC 360
Clone_1 ...... 337
Clone_2 ...... 337
Clone_3 ...... 337
Clone_4 ...... 337
Clone_5 ...... 337
Clone_8 ...... 337
Clone_9 ...... 337
Clone_10 ...... 337
Clone_11 ...... 337
Clone_12 ...... 337
DIGI4747 CTCTCATTTC CTAACAGCCC TTAGAAAAGT AGGAATCGAA CCTACACAAA AGAGATCAAA 420
Clone_1 ...... 397
Clone_2 ...... 397
Clone_3 ...... 397
Clone_4 ...... 397
Clone_5 ...... 397
Clone_8 ...... 397
Clone_9 ...... 397
Clone_10 ...... 397
Clone_11 ...... 397
Clone_12 ...... 397
DIGI4747 ACCCTTCATA CTTCCTCTAT ATTATTTTCT AGTAAGGTCA GCTAACAAAG CTATCGGGCC 480
Clone_1 ...... 456
Clone_2 ...... 456
Clone_3 ...... 456
Clone_4 ...... 456
Clone_5 ...... 456
Clone_8 ...... 456
Clone_9 ...... 456
Clone_10 ...... 456
Clone_11 ...... T.... 456
Clone_12 ...... 456
Dinornis, COII/ATP6 8861-9349
DiGi8861 TACGGTCAAT GCTCAGAAAT CTGCGGGGCA AATCACAGCT ACATACCTAT CGTAGTAGAA 120
Clone_1 ...... 45
Clone_2 ...... 45
Clone_3 ...... 45
Clone_4 ...... 45
Clone_6 ...... 45
Clone_7 ...... 45
Clone_8 ...... 45
Clone_10 ...... 45
Clone_12 ...... T...... 45
Clone_13 ...... 45
DiGi8861 TCAACCCCAC TCACACATTT TGAGAACTGA TCATCACTAC TCTCCTCCTC ATCATTAAGA 180
Clone_1 ...... 105
Clone_2 ...... 105
Clone_3 ...... 105
Clone_4 ...... 105
Clone_6 ...... 105
Clone_7 ...... 105
Clone_8 ...... 105
Clone_10 ...... 105
Clone_12 ...... 105
Clone_13 ...... 105
DiGi8861 AGCTATGGAA CAGCACTAGC CTTTTAAGCT AGAGACAGAG GCACACCCGT CCTCCTTAAT 240
Clone_1 ...... 165
Clone_2 ...... 165
Clone_3 ...... 165
Clone_4 ...... 165
Clone_6 ...... 165
Clone_7 ...... 165
Clone_8 ...... 165
Clone_10 ...... 165
Clone_12 ...... 165
Clone_13 ...... T...... 165
DiGi8861 GATATGCCTC AACTAAATCC GTACCCATGA TTCTCCATCT TACTTATAGT ATGATTCATC 300
Clone_1 ...... 225
Clone_2 ...... 225
Clone_3 ...... 225
Clone_4 ...... 225
Clone_6 ...... 225
Clone_7 ...... 225
Clone_8 ...... 225
Clone_10 ...... 225
Clone_12 ...... 225
Clone_13 ...... 225
DiGi8861 CTCCTACTTC TCATCCAACC TAAATTCCTT TCTTTTACTC ACACAAACCC CCCATCTAAC 360
Clone_1 ...... 285
Clone_2 ...... 285
Clone_3 ...... 285
Clone_4 ...... 285
Clone_6 ...... 285
Clone_7 ...... C..... 285
Clone_8 ...... 285
Clone_10 ...... 285
Clone_12 ...... 285
Clone_13 ...... 285
DiGi8861 AAGACCAAAA TAGCCCCCAT CCCCCCTACA TGAACCTGAC CATGAACCTA ACTCTCTTCG 420
Clone_1 ...... 345
Clone_2 ...... 345
Clone_3 ...... 345
Clone_4 ...... 345
Clone_6 ...... T...... 345
Clone_7 ...... 345
Clone_8 ...... 345
Clone_10 ...... 345
Clone_12 ...... 345
Clone_13 ...... 345
DiGi8861 ACCAATTCTC CAGTCCTCAA CTTCTAGGTG TCCCCCTCAT CCTACTCTCC ACGCTATTCC 480
Clone_1 ...... 405
Clone_2 ...... 405
Clone_3 ...... 405
Clone_4 ...... 405
Clone_6 ...... 405
Clone_7 ...... 405
Clone_8 ...... 405
Clone_10 ...... A...... 405
Clone_12 ...... 405
Clone_13 ...... 405
DiGi8861 CCGCCCTTCT CCTACCATCC CCTAATAGCC GCTGAATCAC TAACCGAC 528
Clone_1 ...... 431
Clone_2 ...... 431
Clone_3 ...... 431
Clone_4 ...... 431
Clone_6 ...... 431
Clone_7 ...... T...... 431
Clone_8 ...... 431
Clone_10 ...... 431
Clone_12 ...... 431
Clone_13 ...... 431
Dinornis, 11120-11958
Di 11120 CAAGGAGGCC TAGAATGAGC AGAATAACAG AAAGTTAGTC TAAACAAGAT AGCTGATTTC 60
Clone_1 ...... 20
Clone_2 ...... 20
Clone_3 ...... 20
Clone_4 ...... 20
Clone_5 ...... 20
Clone_6 ...... 20
Clone_7 ...... 20
Clone_8 ...... 20
Clone_9 ...... 20
Clone_11 ...... 20
Di 11120 GGCTCAGCAG ATTATAGAAA ATGCTATAAC TTTCTTATGT CCGCCCTACA TCTAAGCTTC 120
Clone_1 ...... 80
Clone_2 ...... 80
Clone_3 ...... 80
Clone_4 ...... 80
Clone_5 ...... 80
Clone_6 ...... 80
Clone_7 ...... 80
Clone_8 ...... 80
Clone_9 ...... 80
Clone_11 ...... 80
Di 11120 TACTCAGCCT TCACCTTAAG CGCCCTAGGC CTAGCCTTCC ACCGAACCCA CTTTATCTCC 180
Clone_1 ...... 140
Clone_2 ...... A...... 140
Clone_3 ...... 140
Clone_4 ...... 140
Clone_5 ...... 140
Clone_6 ...... 140
Clone_7 ...... T...... 140
Clone_8 ...... 140
Clone_9 ...... 140
Clone_11 ...... 140
Di 11120 GCCCTACTAT GCTTAGAGAG CATAATACTC TCCATATACA TCGCACTCTC AGTCTGACCA 240
Clone_1 ...... 200
Clone_2 ...... 200
Clone_3 ...... 200
Clone_4 ...... 200
Clone_5 ...... 200
Clone_6 ...... 200
Clone_7 ...... 200
Clone_8 ...... 200
Clone_9 ...... 200
Clone_11 ...... 200
Di 11120 ATCCAAACCC AAACCCCCTC ATTTACCCTA ATCCCACTAC TCATATTAAC ATTCTCCGCA 300
Clone_1 ...... T...... 260
Clone_2 ...... A...... 260
Clone_3 ...... T...... 260
Clone_4 ....G...... 260
Clone_5 ...... 260
Clone_6 ...... 260
Clone_7 ...... 260
Clone_8 ...... 260
Clone_9 ...... 260
Clone_11 ...... 260
Di 11120 TGCGAAGCAG GCACAGGACT GGCCATACTA GTAGCATCCA CCCGAACTCA CGGTTCAGAC 360
Clone_1 ...... G...... 320
Clone_2 ...... 320
Clone_3 ...... G...... 320
Clone_4 ...... 320
Clone_5 ...... 320
Clone_6 ...... 320
Clone_7 ...... 320
Clone_8 ...... 320
Clone_9 ...... 320
Clone_11 ...... 320
Di 11120 CATCTACACA ACCTAAGTCT CCTACAATGC TAAAAATCAT TATCCCCACA ATCATACTCC 420
Clone_1 ...... 380
Clone_2 ...... 380
Clone_3 ...... 380
Clone_4 ...... 380
Clone_5 ...... 380
Clone_6 ...... 380
Clone_7 ...... 380
Clone_8 ...... 380
Clone_9 ...... 380
Clone_11 ...... 380
Di 11120 TACCCACAGC CCTCCTATCC CCACAAAACC TCATCTGAAC TAATACAACC ACCCACAGCC 480
Clone_1 ...... 440
Clone_2 ...... 440
Clone_3 ...... 440
Clone_4 ...... A...... 440
Clone_5 ...... T...... 440
Clone_6 ...... 440
Clone_7 ...... 440
Clone_8 ...... 440
Clone_9 ...... 440
Clone_11 ...... 440
Di 11120 TCCTAATTGC CACTATCAGC CTGCAATGAC TCCACCCA 518
Clone_1 ...... 456
Clone_2 ...... 456
Clone_3 ...... 456
Clone_4 ...... 456
Clone_5 C...... 456
Clone_6 ...... 456
Clone_7 ...... 456
Clone_8 ...... 456
Clone_9 ...... 456
Clone_11 ...... 456
Emeus, 16S 3787-4311
Emeus3787 ATCTGAGTTC AGACCGGAGT AATCCAGGTC GGTTTCTATC TATGCTACAC TCTTCCTAGT 60
Clone_3 ...... 41
Clone_2 ...... 41
Clone_1 ...... 41
Clone_4 ...... 41
Clone_5 ...... 41
Clone_12 ...... 41
Clone_16 ...... T ....T..... 41
Clone_17 ...... C...... 41
Clone_18 ...... 41
Clone_19 ...... 41
Emeus3787 ACGAAAGGAC CGGAAAAGTG AGGCCAATAC TAAACAGCAT GCCTCCTCTC TAATTAGTGA 120
Clone_3 ...... 101
Clone_2 ...... 101
Clone_1 ...... 101
Clone_4 ...... 101
Clone_5 ...... 101
Clone_12 ...... 101
Clone_16 ...... 101
Clone_17 ...... 101
Clone_18 ...... 101
Clone_19 ...... 101
Emeus3787 AACCAACTTA ACTAATAAGA AAACACCCAA CCCTCATCAC ATCCTAGAAA AGGATAGCTA 180