Moa bone samples extracted for DNA for this work.

<p>Data for moa bones was obtained from the references indicated in the table notes, or were sourced as described in the Materials and Methods. fr - femur, tbt - tibiotarsus, tmt - tarsometatarsus, NI - North Island.</p

Complex Species Status for Extinct Moa (Aves: Dinornithiformes) from the Genus <i>Euryapteryx</i>

<div><p>The exact species status of New Zealand's extinct moa remains unknown. In particular, moa belonging to the genus <i>Euryapteryx</i> have been difficult to classify. We use the DNA barcoding sequence on a range of <i>Euryapteryx</i> samples in an attempt to resolve the species status for this genus. We obtained mitochondrial control region and the barcoding region from <i>Cytochrome Oxidase Subunit I</i> (<i>COI</i>) from a number of new moa samples and use available sequences from previous moa phylogenies and eggshell data to try and clarify the species status of <i>Euryapteryx</i>. Using the <i>COI</i> barcoding region we show that species status in <i>Euryapteryx</i> is complex with no clear separation between various individuals. Eggshell, soil, and bone data suggests that a <i>Euryapteryx</i> subspecies likely exists on New Zealand's North Island and can be characterized by a single mitochondrial control region SNP. <i>COI</i> divergences between <i>Euryapteryx</i> individuals from the south of New Zealand's South Island and those from the Far North of the North Island exceed 1.6% and are likely to represent separate species. Individuals from other areas of New Zealand were unable to be clearly separated based on <i>COI</i> differences possibly as a result of repeated hybridisation events. Despite the accuracy of the <i>COI</i> barcoding region to determine species status in birds, including that for the other moa genera, for moa from the genus <i>Euryapteryx</i>, <i>COI</i> barcoding fails to provide a clear result, possibly as a consequence of repeated hybridisation events between these moa. A single control region SNP was identified however that segregates with the two general morphological variants determined for <i>Euryapteryx</i>; a smaller subspecies restricted to the North Island of New Zealand, and a larger subspecies, found on both New Zealand's North and South Island.</p></div

COI sequence differences, biogeography, and eggshell thicknesses of <i>Euryapteryx</i>.

<p><b>A</b>. Phylogenetic analysis and grouping of <i>Euryapteryx</i> samples at various levels of COI sequence divergence. A phylogenetic tree was constructed in MEGA 5.05 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Tamura1" target="_blank">[18]</a> using Maximum Likelihood and Tamura-Nei parameters (log likelihood −1581.8; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Tamura2" target="_blank">[20]</a>). Bootstrap values were calculated from 500 replications. Sequence differences were calculated using K2 parameters. Individual <i>Euryapteryx</i> samples are numbered (for museum voucher numbers see supplementary information) and coloured according to location (see B). Samples are grouped according to percent COI divergence (<0.8%, <1.25%, and <1.6%). See supplementary information for divergence tables. Approximate sizes for two genetic variants (557C/T) of <i>Euryapteryx</i> (see text) are shown against that of an adult chicken. <b>B</b>. Biogeography of <i>Euryapteryx</i> populations according to (left) mitochondrial control region sequences from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a> or (right) COI sequences. Samples that form clades are joined by colour. The main COI groups were determined using a <1.25% divergence limit. This limit most closely approximated the clades formed using control region sequences. The complex interactions between individual members of each COI clade (see A) are not shown. Figure numbers refer to moa samples; 1 - AIM B6595ii <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Baker1" target="_blank">[3]</a>, 2 - WO 527 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 3 - AIM B6580 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Baker1" target="_blank">[3]</a>, 4 - AIM B6228 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Baker1" target="_blank">[3]</a>, 5 - MNZ S40891 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 6 - MNZ S465 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 7 - CM Av21330 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Baker1" target="_blank">[3]</a>, 8 - CM Av29440a <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 9 - CM Av8378 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Baker1" target="_blank">[3]</a>, 10 - MNZ S39965 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 11 - CM Av9188 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Baker1" target="_blank">[3]</a>, 12 - AM 6237 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 13 - OU Anthro FB271 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 14 - OM Av4735 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 15 - OM Av5191 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Bunce1" target="_blank">[4]</a>, 16 - AIM B9243, 17 - OM Av9821 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Baker1" target="_blank">[3]</a>, 18 - CM Av38561 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Baker1" target="_blank">[3]</a>. <b>C</b>. Eggshell thicknesses of <i>Euryapteryx</i>. Eggshell thicknesses from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Huynen2" target="_blank">[10]</a> (mm) are grouped according to association with class I (blue) or class II (orange) control region sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Huynen2" target="_blank">[10]</a>. These sequences cover a highly variable ∼30 bp fragment that is capable of distinguishing ‘thin’ <i>Euryapteryx</i> eggshells from ‘thick’. *The association of a class II sequence with this 1.11 mm eggshell may be in doubt as the sequence was obtained from the outer layer of the eggshell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090212#pone.0090212-Huynen2" target="_blank">[10]</a>.</p

supplementary_info_revised

Supplementary tables and figure

Sequence alignment and haplotype assignments.

<p>Haplotype assignments refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018728#pone-0018728-g003" target="_blank">Figure 3</a>. Numbers refer to position in Genbank reference sequence NC_002784.1. A point (.) refers to the same base as the reference sequence. Abbreviations are: REF – reference sequence, AU – Australian farmed, NZ – New Zealand farmed, KI – King Island Emu, COI – cytochrome <i>c</i> oxidase subunit I.</p

Geographic distribution of emu taxa and historic shoreline reconstructions around Tasmania.

<p>Modern Emu are currently found throughout mainland Australia. Extinct emu taxa were restricted to their respective islands: the Kangaroo Island Emu (purple), the King Island Emu (red) and the Tasmanian Emu. Twenty-five thousand years ago Tasmania, Flinders and King Island were connected to mainland Australia. Approximately 17,500 years ago King Island lost its direct connection with mainland Australia. By 14,000 years ago Tasmania, Flinders and King Island started to disconnect from the mainland, but were still connected to each other. By 11,000 years ago King Island was isolated from Tasmania, while the Tasmania was still connected to Flinders Island. Presently Tasmania, Flinders, King and Kangaroo Island are all isolated and disconnected from mainland Australia (modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018728#pone.0018728-Lambert1" target="_blank">[23]</a>).</p

Modern and extinct emu.

<p>The modern Emu (centre) and King Island Emu (right) with human outline shown approximately to scale. Apart from obvious size differences, there were reports of colour differences between these emu taxa.</p

Comparison of the cranium contour in modern and King Island Emu.

<p>Several (partial) skulls from modern Emu are shown at different stages in their development: A – Adult, B – Immature-Adult, C – Juvenile. Two partial skulls are shown for the King Island Emu D & E <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018728#pone.0018728-Spencer1" target="_blank">[8]</a>. The black lines indicate the contour of the upper/rear surface of the cranium. The adult and immature-adult modern Emu show a frontally flattened cranium, whereas King Island Emu show a more dome shaped cranium. Initially this difference was considered a species level difference, but juvenile modern emu show the same dome shaped cranium in both taxa and therefore appears not to be taxonomically significant.</p

Haplotype network for modern Emu (green) and King Island Emu (red).

<p>Concatenated haplotypes of control and COI regions totalling 2638bp each. The black circle indicates a hypothetical haplotype, the distance between each neighbouring haplotype corresponds to the number of substitutions that separate them. Numbers correspond to positions in the mitochondrial genome as mentioned for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018728#pone-0018728-t001" target="_blank">Table 1</a>, underlined numbers represent substitutions that occurred in the cytochrome <i>c</i> oxidase subunit I gene as supposed to the control region.</p

<i>Coturnix sp</i>. samples used for which location data were available.

<p>Nearest location to samples used are shown. Numbers correspond to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006400#pone-0006400-t001" target="_blank">Table 1</a>. Each square represents a single sample. Colours delineate species and sub-species (see Key).</p