Pyriglenaleuconotaallsequen

nexus file with all mtDNA ND2 sequences. Please contact the first author for any further details

PyriglenaleuconotaSEQUENCES

Excel file with information about sequences in the file "Pyriglenaleuconota

Pyriglena leuconota

nexus file with sequences of mtDNA ND

EBSD microcharacterization.

<p>(a) An EBSD orientation map of a cross section through the Vs-1 eggshell, with the outer surface of the eggshell at the top of the image and non-indexed regions marked in black. The colour scheme relates to the orientation of the calcite crystal lattice, with red colours indicating an alignment of the c-axis with the eggshell surface normal. High angle grain boundaries (>10° misorientation) are marked by black lines and low angle boundaries (2–10° misorientation) by grey lines. The grain structure can be subdivided into 3 distinct layers, with layer boundary positions highlighted by the black arrows. Numerous low angle boundaries are present in the outermost layer, some of which are marked by blue arrows. (b) A comparable EBSD orientation map of a cross section through <i>P. ruber</i> eggshell, using the same colour scheme as in (a). This shows many of the same features visible in (a), including the presence of 3 distinct layers (marked by black arrows) and an abundance of low angle boundaries in the outermost layer (blue arrows). The boundaries between EBSD grain structure seem to mirror the eggshell structural layer boundaries. Some subtle differences in the crystallographic structure between these extinct and modern eggshell are visible such as the less serrated nature of the grain boundaries in the middle layer and the greater spacing of the individual shell units in the modern specimen. (c) and (d) SEM photomicrographs of fractured cross sections through through the Vs-1 and <i>P. ruber</i> eggshells respectively. These images complement the EBSD orientation maps, highlighting the 3-layered structure of both eggshells but lacking the detailed grain structure information visible in (a) and (b). The scale bars in a, b, and c images are 200 µm.</p

Site geology.

<p>(a) Location and geological sketch map of the Lower-Middle Miocene Tudela Formation in the Bardenas Reales de Navarra Natural Park, with location of the Valdesabina fossil avian nest locality studied in this work. (b) Scheme showing the lithostratigraphy of the Tudela Formation, with the position of the Vasldesabina fossil avian nest locality and the Sisares and Barranco del Fraile sections, and its lateral eastward gradation to the Zaragoza Gypsum Formation. (c) Composite magnetostratigraphic log of the Tudela Formation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046972#pone.0046972-Larrasoaa1" target="_blank">[3]</a> and its correlation to the ATNTS2004 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046972#pone.0046972-Lourens1" target="_blank">[14]</a>. The location of fossil localities within the Tudela Formation is also shown (italics denote fossil localities correlated to, but not placed within, the studied magnetostratigraphic sections). All fossil localities from the Tudela Formation contain avian eggshell fragments.</p

SEM microcharacterizations.

<p>(a) SEM radial sections reveal a three laminated eggshell structure, indicative of an avian eggshell, with an average total thickness of 470 µm, consistent with Kohring <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046972#pone.0046972-Kohring1" target="_blank">[20]</a> observations on modern flamingos. The 163 µm thick layer 1 consists of elongated calcite crystals that radiates mostly outward from nuclei at the eggshell base. The 262 µm thick layer 2 is characterized by a crystallographic arrangement only easily described with EBSD microcharacterization (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046972#pone-0046972-g005" target="_blank">Figures 5</a> a and b). The 63 µm thick layer 3 displays long rectangular crystals with a spongy appearance due to multiple voids in the crystalline structure. (b) Backscattered electron microscopy (BSEM) of the same eggshell fragment. BSEM allows a better observation of the outer most eggshell surface covered with a thin calcium phosphate granular layer that does not appear to be a diagenetic artifact. Note the well-defined basal crystals and obvious large pore canals, which by themselves indicate a high moisture level in the nest, here congruent with the floating nest. (c) SEM of the basal crystals shows clearly well-defined spherulitic crystals that surround the nuclei. (d) BSEM of the same specimen upper eggshell section reveals the spongy appearance of this eggshell, the high concentration of micro size pore canals (black arrows), most of which seem to abut below the outer-most thin covering to indirectly connect with the eggshell surface. The sum of these microscopic features support a flamingo type eggshell and egg. The white arrows point to the granular relief of the surficial layer.</p

Nest and egg descriptions.

<p>(a) The limestone block Vs-1 with nest and eggs <i>in situ</i>. The exposed surface is interpreted as being at 10–15 cm from the upper side of Bed 2 of the Valdesabina Limestone. One egg is exposed in the upper left of the figure a few cm from the main concentration or clutch enclosed in a rectangle. The block is correctly orientated with the eggs on its upper surface. Arrows point to cf. Fabaceae material. (b) Detail of the main concentration of eggs. (c) Detail of the vegetable matter forming the nest material (d) A fragmentary hatchling tibiotarsus exposed in section in the side of Vs-1.</p

Osteological, oological, nesting strategy, and ecological characters mapped onto a phylogenetic tree modified from Mayr [<b>23</b>] by the insertion of the BAS specimen to show hypothesised character transitions on the flamingo lineage.

<p>Characters come directly from the text. Black, blue, and red numbers illustrate osteological, oological/nest, and environmental characters, respectively. OSTEOLOGICAL CHARACTERS (black numbers): Tibiotarsus: 1: distinct notches on the distal rim of <i>condylus lateralis</i> and <i>medialis</i>; 2: a wide <i>sulcus extensorius</i>; 3: a medially located <i>sulcus extensorius</i>; 4: a longer supratendinal bridge (in comparison with Palaelodidae); 5: a distinct projection of the lateral and medial condyles from the shaft; 6: a distinct and conspicuous sulcus proximal to the medial condyle; 7: lack of a medial ridge in the <i>trochlea cartilaginis tibialis</i>; 8: a rounded and prominent tuberosity for the lateral attachment of the <i>Retinaculum Extensorium Tibiotarsi</i> (RET). <i>Retinaculum Extensorium Tibiotarsi</i> separated from the articular facet of the intercotylar eminence by: 9: conspicuous sulcus; 10: deep sulcus; 11: narrow <i>sulcus extensorius</i>; 12: lateral attachment of RET laterally to the <i>sulcus extensorius</i>; 13: deep intercondylar incision; 14: intercondylar incision extends over lateral condyle to the diaphysis; 15: ridge in the medio-distal portion of <i>sulcus extensorius</i>; 16: tuberosity for the RET medial attachment; 17: shallow sulcus cranial to the <i>trochlea cartilaginis tibialis</i>; 18: deep sulcus surrounded by crests. Tarsometatarsus: 19: a conspicuous crest in <i>trochlea metatarsi</i> III, in plantar view. Skull: 20: presence of salt glands. EGGS AND NESTS (blue numbers): 21: trilaminated eggshell structure; 22: thin outer granulose calcium phosphate layer; 23: eggshell thickness of or approximately 466 µm; 24: eggshell thickness of or approximately 258 µm; 25: Layer 3 of or approximately 63 µm; 26: Layer 3 of or approximately 30 µm; 27: thin columns in L2; 28: Pronounced spherulitic crystals in L1; 29: Wide columns in L2; 30: moderately pronounced spherulitic crystals in L1; 31: Large cylindrical columns in L2; 32: Bulky spherulitic crystals in L1; 33: 45×30 mm eggs; 34: 90×53 mm eggs; 35: Single egg per clutch; 36: Two to seven eggs per clutch; 37: Floating nest; 38: Twig-nest; 39: Ground nest; 40: Mud, volcano-shaped nest. NESTING ENVIRONMENTS (red numbers): 41: Nesting in lacustrine environment; 42: Nesting in endorheic lacustrine environment; 43: Nesting in normal-oligohaline lacustrine environment; 44: Nesting in oligohaline to mesohaline lacustrine environment; 45: Nesting in mesohaline to hyperhaline lacustrine environment; 46: Nesting in shallow to extremely shallow lacustrine environment.</p

Geological context of the fossil nest.

<p>(a) Sedimentary log of the Valdesabina locality and its correlation to the neighbouring Sisares and Barranco del Fraile sections, for which magnetostratigraphic data are available. Reconstructed lake level variations are based on sedimentological and paleontological (ostracod) data (see also text). (b) Field examples of some of the normal faults that cut the Valdesabina limestone level (left) and a detailed view of the vertical striation pitches observed on the surface of one of these steeply dipping normal faults (right).</p

Comparison of the Palaelodidae and Phoenicopteridae distal tibiotarsi.

<p>(a) <i>Phoenicopterus ruber</i> (MZUSP 88485); (b) BAS new species; (c) <i>Agnopterus sicki</i> (MHNT 4257); (d) <i>Phoenicopterus croizeti</i> (MHNT 5085); (e) <i>Palaelodus</i> spp. (MHNT 5010). The most diagnostic characters found in BAS are a crest in the <i>Retinaculum Extensorium Tibiotarsi</i> (RET) divided by a deep and conspicuous sulcus from the articular facet for the intercotylar eminence of the tarsometatarsus (b), a deep intercondylar incision extending over the lateral condyle, a tuberosity for the RET medial attachment and a deep sulcus surrounded by crests cranial to the <i>trochlea cartilaginis tibialis</i> (not shown). RET: <i>Retinaculum Extensorium Tibiotarsi</i>. SRET: sulcus between the RET and the articular facet for the intercotylar eminence of the tarsometatarsus in the BAS specimen.</p