Neutron tomography of <i>Austrosequoia novae-zeelandia</i>e comb. nov. (Late Cretaceous, Chatham Islands, New Zealand): implications for Sequoioideae phylogeny and biogeography

<p>The Tupuangi Flora of the Chatham Islands, New Zealand, reveals a south polar forest ecosystem, and important biogeographical links between eastern and western Gondwana. We employed neutron tomography (NT) to image fossil Cupressaceae seed cones from the Upper Cretaceous (Cenomanian) strata of the Tupuangi Formation. This technique facilitated the non-destructive ‘virtual extraction’ of three-dimensional, coalified specimens, whilst they were still embedded within a large volume of supporting silicate sedimentary rock. This study is the first reported application of NT in palaeobotanical taxonomy, and the combination of virtual and manual extraction techniques enabled a more complete treatment than would otherwise be possible if taxonomic data were limited to only one of these approaches. The seed cones were identified as <i>Austrosequoia novae-zeelandiae</i> (Ettingshausen) Mays & Cantrill comb. nov. In this case, NT data supplemented the compression fossil data by providing details such as the three-dimensional measurements of the gross morphology, and accurate estimations of bract-scale complex number. Furthermore, this technique appears to show promise in differentiating between organic compounds within an individual specimen. However, anatomical details and fine-scale morphology were indiscernible due to present limitations in spatial resolution. <i>Austrosequoia novae-zeelandiae</i> is interpreted as a stem group of Sequoioideae; it shares synapomorphic seed cone characters with extant sequoioids (e.g. <i>Sequoia</i> and <i>Sequoiadendron</i>), and plesiomorphic stomatal structures and leaf morphology. Abundant epiphyllous fungi (<i>Plochmopeltinites</i> sp.; Microthyriaceae) were also identified on the leaf cuticles of <i>A. novae-zeelandiae</i>. The high abundance of <i>Austrosequoia</i> in the Tupuangi Flora supports a cupressaceous floral province at south polar latitudes during the early Late Cretaceous. Furthermore, this stem group of Sequoioideae in eastern Gondwana during the early Late Cretaceous suggests an alternative, south-to-north dispersal route of sequoioids before the final continental separation of eastern and western Gondwana.</p

Colours of Australian amber.

<p>A) burst piece of yellow amber, some still embedded in matrix, sample THA01 2529.43; B) Top: Partial nodule of orange amber embedded in matrix, sample THA01 2451.7; Bottom left: cross-section through orange amber nodule containing organic matter, sample Minerva 2a 1857.43; Bottom right: cross-section through orange amber droplet, (not collected) Minerva 2a 1923.5; C) fragments from burst piece of dark orange amber sample Minerva-1 1837.5; D) fragments from burst piece of red amber, sample Minerva 2a 1843.2; E) fragments from burst piece of dark brown amber sample Minerva 2a- 1945.2; F) fragments from burst piece of milky brown amber, sample Minerva 2a 1956.7. Scale bar: 4 mm.</p

Types and interpretations of inclusions found in the Otway amber.

<p>Upper bold box indicates bioinclusions; lower bold box indicates pseudoinclusions. Relative abundances are based visual assessments of inclusion types in the entire sample set. For a breakdown of relative abundances per amber colour, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121307#pone.0121307.t003" target="_blank">Table 3</a>.</p><p>*Interpretations based on the pseudo-protist classification scheme of Girard et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121307#pone.0121307.ref030" target="_blank">30</a>].</p><p>Types and interpretations of inclusions found in the Otway amber.</p

Examples of bioinclusions from Otway amber.

<p>(A) Probable specimen of <i>Kraeuselisporites</i> Raine, 2008, Sample THA01 2466.89 2D2-1. (B) <i>Cyathidites minor</i> Couper, 1953, Sample THA01 2466.89 2D2-1. (C) Randomly oriented network of filamentous microorganisms, Sample THA01 2466.89 2D2-2. (D) Mutually aligned filamentous microorganisms pictured with ovoid and fusiform inclusions, Sample THA01 2466.89 2D2-1. (E) Degraded organic matter exhibiting cracks along outer margin, Sample THA01 2466.89 009–1. (F) Degraded organic matter with attached filamentous microorganisms, Sample THA01 2531.07–007. Scale Bars: 100 μm (A–D); 50 μm (E); 200 μm (F).</p

Study area map within the Otway Basin (grey box) off the coast of Victoria, Australia (inset).

<p>White dots indicate amber-bearing well locations including well names. Dark grey shading represents modern land; light grey represents continental crust.</p

Examples of other inclusions from Otway amber.

<p>(A) Spherical inclusions (Type A pseudoinclusions) with internal vesiculation, Sample Minerva-1 1836.9. (B) Concentration of ovoid inclusions (Type B pseudoinclusions) on the left side (dark orange) and no inclusions of the right side (lighter orange), Sample THA01 2451.4. (C) Mutually aligned fusiform inclusions (Type B pseudoinclusions), Sample THA01 2451.7. (D) Ovoid inclusion (Type B pseudoinclusion) with single vesicle; Sample THA01 2466.89 2D2-2. (E) Ovoid inclusions (black arrows; Type B pseudoinclusions); lower ovoid inclusion (Type B pseudoinclusions) is stacked on top of an irregular filamentous inclusion (grey arrow; Type D pseudoinclusion); pictured with filamentous microorganisms, Sample THA01 2466.89 2D2-2. (F) Vesicular ovoid inclusion (Type B pseudoinclusion) with projection, Sample THA01 2466.89 2D2-1. (G) Irregular filamentous inclusion (grey arrow; Type D pseudoinclusion) surrounded by mutually aligned filamentous microorganisms and ovoid inclusions (Type B pseudoinclusions), Sample THA01 2466.89 005. (H) Amorphous inclusion (Type E pseudoinclusion), Sample THA01 2466.89 2D2-2. (I) Amorphous inclusions (Type E pseudoinclusion) surrounded by mutually aligned ovoid inclusions, Sample THA01 2466.89 2D2-2. Scale bars: 50 μm (A, F); 200 μm (B); 100 μm (C–E, G–I).</p

Determining the history of resin exudation and solidification based on the relationship between the relative abundances of pseudoinclusion types and amber colour.

<p>–: not present; +: uncommon; ++: common; +++: abundant, n.o.: not observed (due to opacity), n.d.: not described. Mobility (of resin); inferred from the pseudoinclusion assemblage preserved in each colour of amber (I: immobile; M: mobile). Environment (of solidification); inferred from inclusion assemblage and/or amber morphology. (B: upper tree branches; T; tree trunk; G: ground surface; R: underground (root resin); P: pond). Inclusions were not observed in milky brown amber (sample ID: Minerva-2a 1956.7); therefore, it is not included in the table. Modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121307#pone.0121307.ref030" target="_blank">30</a>].</p><p>*Most common interpretation based on pseudoinclusion abundances</p><p>Determining the history of resin exudation and solidification based on the relationship between the relative abundances of pseudoinclusion types and amber colour.</p

Sample accession numbers.

<p>Sample numbers (given as well location, depth of collection (m)-sample number) and associated accession numbers. All samples are housed at the Melbourne Museum, part of Museum Victoria (MV).</p><p>Sample accession numbers.</p