Fully fledged enantiornithine hatchling revealed by Laser-Stimulated Fluorescence supports precocial nesting behavior

Laser-Stimulated Fluorescence (LSF) is used to identify fully fledged feathering in the hatchling enantiornithine bird specimen MPCM-LH-26189, supporting precocial nesting behavior in this extinct group. The LSF results include the detection of a long pennaceous wing feather as well as cover feathers around the body. The LSF technique showed improved detection limits over and above synchrotron and UV imaging which had both been performed on this specimen. The findings underscore the value of using a wide range of analytical techniques.

www.nature.com/scientificreports www.nature.com/scientificreports/ The brown-colored feather patches appear across the entire body (Fig. 1B) and show the best detail on the left side of the neck and next to the left wing. The patch on the left side of the neck (Figs 1 and 2A,B) comprises elongate structures banded with lighter and darker color, with a 'bushy' appearance. In the right wing, there are two patches associated with the ulna and radius (Figs 2C and 3). Interestingly, multiple thin filaments stem obliquely from the ulna shaft (Figs 2C and 3). However, the most noteworthy feather is associated with the left wing and is anatomically displaced, close to the sacrum (Figs 1B and 2E). This feather is ~3 cm long and barbs are well-preserved along different portions of the hollow rachis with those at the distal end forming an angle of ~30°. This wing feather is very similar in both anatomy, location and relative size to an enantiornithine embryo specimen from China 9 .

Discussion
The preservation of feathers in MPCM-LH-26189, as revealed by LSF, indicates that this hatchling was fully fledged and not largely featherless. This supports precociality as a nesting behavior in enantiornithines. It is noteworthy that the long wing feather of MPCM-LH-26189 is extremely similar to the Chinese enantiornithine embryo specimen that was first used to propose precociality in this group 9 . MPCM-LH-26189 is highly articulated, which is congruent with the body fossil and soft tissue preservation in the enantiornithines of Las Hoyas 3 . According to the LSF results, there are two types of feathers in MPCM-LH-26189, raising the question of whether this confirms both remigial and cover feathers. Such an assertion is important because cover feathers have never been documented in enantiornithines. In this specimen, some preserved feathers are located near the neck (Fig. 2A), a body region of modern birds that is only known to have cover feathers 8 . The fact that the specimen is an unambiguous hatchling means that such cover feathers would be the enantiornithine-equivalent of down feathers in crown birds. If so, this first evidence could serve as a clue to identify adult enantiornithine cover feathers, which remain elusive.
Synchrotron generated x-ray beams were applied to MPCM-LH-26189 1 , which has become a popular method of analyzing fossils [17][18][19][20][21] . Due to micron scale beam size they are exquisite at resolving the smallest details and fine scale differences in matrix-fossil density 18 . Combined with Energy Dispersive Spectroscopy, they are capable of detailed and sensitive spectral analysis resulting in previously unseen elemental maps of soft tissue residues 20 . However, synchrotron hutches are typically open air or employ helium tents because of their inherent lack of sensitivity to elements at the lower end of the atomic scale 22 . Unless operating in a high vacuum, lower energy x-rays are absorbed by the intervening gas 23 . Silicon is a typical cut off point, so carbon and other lighter elements would not normally be in the detectable range 23 , potentially overlooking morphologies that they preserve. Typically, fossil feathers are easily visible in white light as carbon films 24 . Carbon is a very low fluorescence element 25 , so under LSF, feathers are typically black and show up in high contrast by the fluorescence of the background matrix 5,7,26,27 . However, the Las Hoyas hatchling showed no evidence of carbon films under white light or in previous UV and synchrotron imaging 1 (Fig. 1A; Fig. 1 of 1 ). This suggests the possibility that there is too low a percentage of carbon to be easily visible in the matrix, but under LSF, the residual carbon (or possibly the detected iron 1 ) quenches the fluorescence 25 in the local area revealing the feather filaments.
In bridging detection limits in synchrotron and UV analytical techniques, Laser-Stimulated Fluorescence (LSF), a rapid, low-cost technique 5 , has helped to clarify the developmental strategy of MPCM-LH-26189 and of enantiornithines more generally. This example underscores the range of data that remains undiscovered in important fossils, and the value of adopting a broader analytical repertoire.

Methods
Laser-Stimulated Fluorescence (LSF) imaging followed the protocol of Kaye et al. 5 , which was developed in 7,26,28,29 . Thus, only an abbreviated version is provided here. A 405 nm laser diode was used to fluoresce the specimen follow standard laser safety protocol. 30 second time-exposed images were taken with a Nikon D810 DSLR camera and a 425 nm laser blocking filter. Post processing applied uniformly across entire images (equalization, saturation and color balance) was performed in Photoshop CS6 software.