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Pterosaur integumentary structures with complex feather-like branching 1
Zixiao Yang1, Baoyu Jiang1, Maria E. McNamara2, Stuart L. Kearns3, Michael 2
Pittman4, Thomas G. Kaye5, Patrick J. Orr6, Xing Xu7, Michael J. Benton3 3
4
1. Center for Research and Education on Biological Evolution and Environments, 5
School of Earth Sciences and Engineering, Nanjing University, Nanjing 210023, 6
China 7
2. School of Biological, Earth and Environmental Sciences, University College Cork, 8
Cork T23 TK30, Ireland 9
3. Department of Earth Sciences, University of Bristol, Bristol BS8 1RJ, UK 10
4. Vertebrate Palaeontology Laboratory, Department of Earth Sciences, University of 11
Hong Kong, Pokfulam, Hong Kong, China 12
5. Foundation for Scientific Advancement, Sierra Vista, Arizona USA 13
6. UCD School of Earth Sciences, University College Dublin, Belfield, Dublin 4 14
D04V1W8, Ireland 15
7. Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate 16
Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing 100044, 17
China 18
19
Pterosaurs were the first vertebrates to achieve true flapping flight, but in the 20
absence of living representatives, many questions concerning their biology and 21
lifestyle remain unresolved. Pycnofibres, the integumentary coverings of 22
pterosaurs, are particularly enigmatic: although many reconstructions depict 23
2
fur-like coverings composed of pycnofibres, their affinities and function are not 24
fully understood. Here we report the preservation in two anurognathid pterosaur 25
specimens of morphologically diverse pycnofibres that show diagnostic features 26
of feathers, including non-vaned grouped filaments and bilaterally branched 27
filaments, hitherto considered unique to maniraptoran dinosaurs, and preserved 28
melanosomes with diverse geometries. These findings could imply that feathers 29
had deep evolutionary origins in ancestral archosaurs, or that these structures 30
arose independently in pterosaurs. The presence of feather-like structures 31
suggests that anurognathids, and potentially other pterosaurs, possessed a dense 32
filamentous covering that likely functioned in thermoregulation, tactile sensing, 33
signalling, and aerodynamics. 34
Feathers are the most complex integumentary appendages in vertebrates1. Most 35
feathers in modern birds possess an axial shaft from which branch lateral barbs and 36
barbules. Much is known about the anatomy, developmental biology, and genomic 37
regulation of these structures, but their deep evolutionary origin is controversial2-4. 38
Feathers and feather-like integumentary structures have been reported in many 39
theropod dinosaurs (including birds)3,5 and ornithischians such as Psittacosaurus6, 40
Tianyulong7, and Kulindadromeus8. Feather-like or hair-like structures, termed 41
pycnofibres9, have also been reported in several pterosaur specimens9-13, but their 42
nature is not resolved. 43
Here we report remarkably well-preserved pycnofibres in two anurognathid 44
pterosaurs and demonstrate, using evidence from morphology, chemistry and 45
3
macroevolutionary analyses, that the preserved pycnofibres bear key features of 46
feathers: monofilaments, two types of non-vaned grouped filaments, bilaterally 47
branched filaments that were previously considered unique to maniraptoran dinosaurs, 48
and preserved melanosomes with diverse geometries. Both specimens studied are 49
from the Middle–Late Jurassic Yanliao Biota (ca. 165–160 Mya14). NJU–57003 50
(Nanjing University) is a newly excavated specimen from the Mutoudeng locality and 51
CAGS–Z070 (Institute of Geology, Chinese Academy of Geological Sciences), which 52
has been noted previously13, is from the Daohugou locality. Both specimens are 53
near-complete and well-articulated, with extensive soft tissues (Figs. 1 and 2, and 54
Supplementary Figs. 1–5). Both specimens are identified as anurognathids17 (see 55
Supplementary text for osteological descriptions). 56
Preserved soft tissues include structural fibres (actinofibrils) and pycnofibres. 57
Structural fibres, common in the pterosaur wing membrane9,12,18, are observed only in 58
the posterior portion of the uropatagium in CAGS–Z070 (Fig. 1o–p). As reported 59
elsewhere, they are parallel to subparallel and closely packed. Individual fibres are 60
0.08–0.11 mm wide (ca. 5 fibres per mm) and at least 1.9 mm long. Pycnofibres are 61
preserved extensively in both pterosaur specimens (especially CAGS–Z070; Figs. 1 62
and 2, and Supplementary Figs. 1, 4 and 5) and are discriminated from structural 63
fibres based on their curved morphology and overlapping arrangement. In the 64
posterior portion of the uropatagium in CAGS–Z070, pycnofibres co-occur with 65
structural fibres; oblique intersections reflect superposition of these features during 66
decay (Fig. 1o–p). 67
4
Pycnofibres are categorized here into four types. Type 1 occurs around the head, 68
neck, shoulder, torso, all four limbs and tail of both specimens (Figs. 1c–e, o–p, 2b–c 69
and f). It comprises curved monofilaments that are 3.5–12.8 mm long and 70–430 μm 70
wide. Some short, distally tapering examples discriminate between dark-toned lateral 71
margins and light-toned axial regions, especially near the filament base where the 72
light-toned axis is wider, suggesting a tube-like morphology (Fig. 1c–e). Type 2 is 73
preserved in the neck, proximal forelimb, plantar metatarsus and proximal tail regions 74
of CAGS–Z070. It consists of bundles of curved filaments of similar length that 75
appear to form brush-like structures at the distal ends of thicker filaments (2.0–13.8 76
mm long and 80–180 μm wide) (Fig. 1f–h). The latter may represent individual thick 77
filaments or fused proximal regions of thinner distal filaments. Type 3 occurs around 78
the head of CAGS–Z070. It comprises straight to slightly curved, distally tapered, 79
central filaments (4.5–7.0 mm long and 50–450 μm wide) with short lateral branches 80
that diverge from the central filament near the midpoint (Fig. 1i–k). There are five 81
Type 3 filaments identified on the head, next to five similar filaments likely of the 82
same nature but obscured by overlapping filaments (Supplementary Fig. 5b). Type 4 83
occurs on the wing membrane of both specimens. It comprises tufts of curved 84
filaments (2.5–8.0 mm long and 70–130 μm wide) that diverge proximally (Figs. 1l–n 85
and 2d–e), in contrast to the clear separation between Type 1 filaments (Fig. 1o–p). 86
Filamentous integumentary structures in extant and fossil vertebrates commonly 87
contain melanin-bearing organelles (melanosomes). Scanning electron microscopy 88
(SEM) of the filamentous structures of NJU–57003 reveals densely packed 89
5
microbodies 0.70 ± 0.11 μm long and 0.32 ± 0.05 μm wide (Fig. 2g–h, Supplementary 90
Figs. 4a–f, 6 and 7, and Supplementary Table 2). As with most melanosome-rich 91
fossil feathers19-21, energy dispersive X-ray spectroscopy (EDS) spectra of the 92
filaments are dominated by a major peak for carbon (Supplementary Fig. 8). These 93
carbonaceous microbodies resemble fossil melanosomes in terms of their geometry, 94
dense packing, parallel alignment relative to the long axis of the integumentary 95
structure (i.e. barbules in Paraves), and preservation within the matrix of the filament 96
(see Supplementary text). Most of the microbodies are oblate and morphologically 97
similar to those that are usually interpreted as phaeomelanosomes in fossils19 (Fig. 2h). 98
Rod-shaped examples, usually interpreted as eumelanosomes in fossils19 (Fig. 2g), are 99
rare. 100
Fourier transform infrared spectroscopy (FTIR) of samples of pterosaur filaments 101
shows four major peaks unique to the filaments (Fig. 2i). These peaks are consistent 102
with the absorption regions of amide I at ca. 1650 cm−1 (principally the C=O 103
asymmetric stretching vibration with some C–N bending), amide II at ca. 1540 cm−1 104
(a combination of N–H in-plane bending and C–N and C–C stretching as in indole 105
and pyrrole in melanin and amino acids), and aliphatic C–H stretching at 2850 cm−1 106
and 2918 cm−1 22. These peaks also occur in spectra obtained from extant feathers21,23, 107
fossil feathers of the paravian Anchiornis20, and melanosomes isolated from human 108
hair24. Further, spectra of the pterosaur filaments more closely resemble those of 109
pheomelanin-rich red human hair in the stronger absorption regions at ca. 2850 cm−1
110
and 2918 cm−1 and higher resolution in the region ca. 1500–1700 cm−1 than those 111
6
from eumelanin-rich black human hair and the ink sac of cuttlefish24. This, together 112
with the SEM results, suggests that the densely packed microbodies in the pterosaur 113
filaments are preserved melanosomes. The amide I peak at 1650 cm−1 is more 114
consistent with α-keratin (characteristic of extant mammal hair25) than β-keratin (the 115
primary keratin in extant avian feathers22,26). This signal may be original or diagenetic; 116
the molecular configuration of keratin26 and other proteins27 can alter under 117
mechanical stress and changes in hydration levels. 118
The ultrastructural and chemical features of the pterosaur filaments confirm that 119
they are hair-like or feather-like integumentary structures. The four types of filaments 120
described here show distinct distributions and morphologies. They are separated 121
clearly from the sedimentary matrix by sharp boundaries (Supplementary Fig. 4g–i). 122
There is no evidence that one or more filament type(s) were generated taphonomically, 123
e.g. through selective degradation or fossilization, or superimposition of filaments. 124
For instance, although Type 1 and 4 filaments occur widely in both specimens, Type 4 125
occurs only in the wings, while Type 1 occupies the remaining body regions. Type 1 126
filaments are thus not degraded products of Type 4, and Type 4 filaments do not 127
represent superimposed clusters of Type 1 filaments. Filament types 2 and 3 occur 128
only in CAGS–Z070. Type 3 occurs only in the facial area and is associated with Type 129
1, where Types 2 and 4 are not evident. Type 3 filaments are thus not degraded Type 2 130
or 4 filaments. Central filaments of Type 3 are morphologically identical to the short, 131
distally tapering filaments of Type 1, but the branching filaments are much thinner (< 132
40 μm (Type 3) versus >70 μm (Type 1) wide) and shorter (< 0.6 mm vs. > 3.5 mm 133
7
long) than the latter. The branching filaments are thus unlikely to reflect 134
superimposition of clusters of Type 1 filaments. In contrast, the distal ends of Type 2 135
filaments are similar, and have a similar distribution pattern to, Type 1 filaments. An 136
alternative interpretation, that Type 2 filaments might represent superimposition of 137
Type 1 filaments at their proximal ends, is unlikely (see detailed discussion in 138
Supplementary text). 139
Feathers and feather-like integumentary structures have been reported in 140
non-avian dinosaurs, although debate continues about their true nature2. These 141
structures have been ascribed to several morphotypes, some absent in living birds3,5, 142
and provide a basis to analyse the evolutionary significance of pterosaur pycnofibres. 143
The pterosaur Type 1 filaments resemble monofilaments in the ornithischian 144
dinosaurs Tianyulong and Psittacosaurus and the coelurosaur Beipiaosaurus: 145
unbranched, cylindrical structures with a midline groove that widens towards the base 146
(presumed in Beipiaosaurus)3,5. The pterosaur Type 2 filaments resemble the 147
brush-like bundles of filaments in the coelurosaurs Epidexipteryx and Yi3,5,28: both 148
comprise parallel filaments that unite proximally. The morphology and circum-cranial 149
distribution of pterosaur Type 3 filaments resemble bristles in modern birds1, but 150
surprisingly do not correspond to any reported morphotype in non-avian dinosaurs. 151
The Type 3 filaments recall bilaterally branched filaments in Sinornithosaurus, 152
Anchiornis, and Dilong, but the latter filaments branch throughout their length rather 153
than halfway along the central filament(s), as in the pterosaur structure3,5. The 154
pterosaur Type 4 filaments are identical to the radially branched, downy feather-like 155
8
morphotype found widely in coelurosaurs such as Sinornithosaurus, Beipiaosaurus, 156
Protarchaeopteryx, Caudipteryx, and Dilong3,5. 157
The filamentous integumentary structures in our anurognathid pterosaurs are thus 158
remarkably similar to feathers and feather-like structures in non-avian dinosaurs. 159
Intriguingly, cylindrical (Type 1), radially symmetrical branched (Types 2 and 4) and 160
bilaterally symmetrical branched (Type 3) filaments clearly coexisted in individual 161
animals; these structures may represent transitional forms in the evolution of feathers, 162
as revealed by developmental studies3,5. 163
These new findings warrant revision of the origin of complex feather-like 164
branching integumentary structures from Dinosauria to Avemetatarsalia, the wider 165
clade that includes dinosaurs, pterosaurs, and close relatives4,29. The early 166
evolutionary history of bird feathers and homologous structures in dinosaurs, and the 167
multiple complex pycnofibres of pterosaurs, is enigmatic. A previous study concluded 168
that the common ancestor of these clades bore scales and not filamentous 169
integumentary appendages2, but this result emerged only when the filaments of 170
pterosaurs were coded as non-homologous with those of dinosaurs. There are no 171
morphological criteria, however, for such a determination. The presence of multiple 172
pycnofibre types and their morphological, ultrastructural and chemical similarity to 173
feathers and feather-like structures in various dinosaurian clades, confirms their likely 174
homology with filamentous structures in non-avian dinosaurs and birds. Comparative 175
phylogenetic analysis produces equivocal results: maximum likelihood modelling of 176
plausible ancestral states, against various combinations of branch length and character 177
9
transition models (Supplementary text and Supplementary Fig. 9, Table 3), reveals 178
various potential solutions. The statistically most likely result (Fig. 3 and 179
Supplementary Table 3, highest log-likelihood value) shows that the avemetatarsalian 180
ancestors of dinosaurs and pterosaurs possessed integumentary filaments, with highest 181
likelihood of possessing monofilaments; tufts of filaments, and, especially, brush-type 182
filaments, are less likely ancestral states. This confirms that feather-like structures 183
arose in the Early or Middle Triassic. The alternative tree for Dinosauria, with 184
Ornithischia and Theropoda paired as Ornithoscelida30, produces an identical result. 185
We present these modelling data with caution, however, for two reasons: (1) the 186
tree rooting method can influence the result (Supplementary Table 3), favouring 187
results in which either scales are the basal condition or where non-theropod 188
feather-like structures and feathers evolved independently (Supplementary Figure 9, 189
Table 3), and (2) there is no adequate way to model probabilities of evolution of all 190
six feather types, or to model probabilities of transitions between the six different 191
feather types. 192
The discovery of multiple types of feather-like structures in pterosaurs has broad 193
implications for our understanding of pterosaur biology and the functional origin of 194
feather-like structures in Avemetatarsalia31,32. Potential functions of these structures 195
include insulation, tactile sensing, streamlining and coloration (primarily for 196
camouflage and signalling), as for bristles, down feathers and mammalian hairs31-34. 197
Type 1, 2 and 4 filaments could shape a filamentous covering around the body and 198
wings (Fig. 4) that might have functioned in streamlining the body surface in order to 199
10
reduce drag during flight, as for modern bat fur or avian covert feathers33,35. Type 1 200
and 2 filaments occur in considerably high densities, particularly around the neck, 201
shoulder, hindlimb and tail regions where the high degree of superposition prevents 202
easy discrimination of adjacent fibres. This, along with the wide distribution and 203
frayed appearance, resembles mammalian underfur adapted for thermal insulation36,35. 204
Despite the less dense packing of Type 4 filaments on the wings, the morphology of 205
the structures is consistent with a thermoregulatory function: down feathers can 206
achieve similar insulation as mammalian hair with only about half the mass, due to 207
their air-trapping properties and high mechanical resilience, effective in retaining an 208
insulating layer of still air38. This may optimize the encumbrance of the large wing 209
area to wing locomotion18. Type 3 filaments around the jaw (Fig. 4) may have had 210
tactile functions in e.g. prey handling, information gathering during flight, navigating 211
in nest cavities and on the ground at night, similar to bristles in birds39. 212
213
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Supplementary Information is available in the online version of the paper. 309
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310
Acknowledgements We thank Qiang Ji, Shu’an Ji and Hao Huang for access to the 311
specimen CAGS–Z070, as well as Simon C. Kohn, Yan Fang, Chunzhao Wang and 312
Tong He for laboratory assistance. This work was supported by the National Science 313
Foundation of China (41672010; 41688103) and the Strategic Priority Research 314
Program (B) of the Chinese Academy of Sciences (XDB26000000) to B.Y.J., the 315
Research Grant Council of Hong Kong-General Research Fund (17103315) to M.P., 316
ERC-StG-2014-637691-ANICOLEVO to M.E.M., and Natural Environment 317
Research Council Standard Grant NE/1027630/1 to M.J.B. 318
319
Author Contributions B.Y.J. and M.J.B. designed the research, Z.X.Y., B.Y.J. and 320
X.X. systematically studied the specimens, Z.X.Y., S.L.K., M.E.M, and P.J.O. did the 321
SEM analysis, Z.X.Y. and B.Y.J. did the FTIR analysis, M.P. and T.G.K. did the LSF 322
imaging, data reduction and interpretation, M.J.B. did the maximum likelihood 323
analyses, and Z.X.Y., B.Y.J., M.J.B., M.E.M, X.X. and P.J.O. wrote the paper; all 324
authors approved the final draft of the paper. 325
326
Author Information Reprints and permissions information is available at 327
www.nature.com/reprints. The authors declare no competing financial interests. 328
Correspondence and requests for materials should be addressed to B.Y.J. 329
(byjiang@nju.edu.cn) or M.J.B. (mike.benton@bristol.ac.uk). 330
331
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332
Figure 1 | Integumentary filamentous structures in CAGS–Z070. a, Overview 333
shows extensive preservation of soft tissues. b–p, Details of the integumentary 334
filaments in the regions indicated in a on the head and neck (b–d, i–j), forelimb (f–g), 335
wing (l–m) and tail (o–p), and illustrated reconstructions of the filaments (e: Type 1 336
filament; h: Type 2 filament; k: Type 3 filament; n: Type 4 filament). Scale bars: 20 337
mm in a; 10 mm in b; 500 µm in c and i; 100 µm in d; 1 mm in f, l, m and p; 200 µm 338
in g and j; 5 mm in o. 339
340
Figure 2 | Preservation, microstructure and chemistry of the integumentary 341
filamentous structures in NJU–57003. a, Laser-stimulated fluorescence6,15,16 image 342
highlights extensive preservation of soft tissues (black areas). b–f, Details of the 343
integumentary filaments in the regions indicated in A on the head and neck (b–c), 344
wing (d–e) and tail (f). g–h, Scanning electron micrographs of the monofilaments on 345
the neck and hindlimb of NJU–57003 (samples 10 and 39, respectively, 346
Supplementary Fig. 1a) show densely packed, elongate and oblate melanosomes. i, 347
FTIR absorbance spectra of the monofilaments, monofilaments with sediment matrix, 348
and sediment matrix in NJU–57003 (Sample 15, Supplementary Fig. 1a) compared 349
with spectra from a feather of Anchiornis (from ref. 20), extant Marabou stork feather 350
(from ref. 21) and black and red human hair melanosomes (from ref. 24). Scale bars: 20 351
mm in a; 1 mm in b, c and e; 5 mm in d and f; 1 μm in g and h. 352
17
Figure 3 | Phylogenetic comparative analysis of integumentary filament and 353
feather evolution in pterosaurs and archosaurs. The phylogeny is scaled to 354
geological time, with recorded terminal character states for each species, and 355
estimated ancestral character states at the lower nodes. The model is the most likely of 356
the maximum likelihood models, based on minimum-branch lengths (mbl) and 357
transitions occurring as all-rates-different (ARD), but other results with lower 358
likelihoods show scales as ancestral. The ancestral state reconstruction shows a 359
combination of monofilaments, tuft-like filaments, and brush-type filaments as the 360
ancestral state for Avemetatarsalia and for Dinosauria. The estimated ancestral state 361
for Theropoda comprises all five feather states. Numbered small vertical arrows 362
indicate earliest occurrences of feather types 2–6. Two hypotheses for timing of avian 363
feather origins are indicated: A, early origin, at the base of Avemetatarsalia in the 364
Early Triassic, or B, late origin, at the base of Maniraptora in the Early–Middle 365
Jurassic. 366
367
Figure 4 | Reconstruction of one of the studied anurognathid pterosaurs, 368
exhibiting diverse types of pycnofibres distributed in different body parts. 369
Scales
Monofilaments
Brush-like filaments
Tufts of filaments
Open pennaceous feathers (no secondary branching)
Closed pennaceous feathers (with secondary branching)
THEROPODA
SAUROPODOMORPHA
ORNITHISCHIA
PTEROSAURIA
AVES
MANIRAPTORA
Sordes pilosus
Yanliao anurognathid
Jeholopterus ningchengensis
Tianyulong confuciusi
Scelidosaurus harrisoniiGigantspinosaurus sichuanensis
Hesperosaurus mjosi
cf Stegosaurus sp. Liaoningosaurus paradoxus Tarchia kielanae
Euoplocephalus tutus
Dyoplosaurus acutosquameus
Scolosaurus cutleri
Kulindadromeus zabaikalicus Psittacosaurus sp. Centrosaurus apertus
Chasmosaurus belli
Triceratops horridus
Tenontosaurus sp.
Mantellisaurus atherfieldensis Parasaurolophus walkeri
Hypacrosaurus altispinus
Magnipaulia laticaudus
Corythosaurus casuarius
Corythosaurus intermedius
Lambeosaurus clavinitialis
Lambeosaurus magnicristatus
Brachylophosaurus canadensis
Maiasaura peeblesorum
Edmontosaurus annectens
Edmontosaurus regalis
Gryposaurus monumentensis
Gryposaurus notabilis
Prosaurolophus maximus
Saurolophus angustirostris
Saurolophus osborni
Tehuelchesaurus benitezii
Mamenchisaurus youngi
Barosaurus lentus
Diplodocus sp.
Camarasaurus sp. Auca Maheuvo titanosaur
Pelorosaurus becklesii Carnotaurus sastrei
Sciurumimus albersodoefri
Allosaurus fragilis Concavenator corcovatus
Dilong paradoxus
Yutyrannus huali Tarbosaurus bataar
Tyrannosaurus rex
Sinocalliopteryx gigas
Compsognathus longipes
Juravenator starkiiSinosauropteryx prima Ornithomimus edmontonicus
Shuvuuia deserti
Beipiaosaurus inexpectus
Similicaudipteryx yixianensis
Protarchaeopteryx robusta
Caudipteryx dongi
Caudipteryx zoui
Pedopenna daohugouensis
Epidexipteryx hui
Scansoriopteryx heilmanni
Eosinopteryx brevipenna Velociraptor mongoliensis
Sinornithosaurus millenii
Microraptor gui
Microraptor zhaoianus
Jinfengopteryx elegans
Aurornis xui
Anchiornis huxleyi
Archaeopteryx lithographica
Xiaotingia zhengi Confuciusornis sanctus Rahonavis ostromi
DINOSAURIA
AVEMETATARSALIA
COELUROSAURIA
SAURISCHIA
6
5
4
4
4
3
3
2
2
2
1
1
PSEUDOSUCHIA (’crocodile-line archosaurs’)
A B
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250 Induan
Olenekian
Anisian
Ladinian
Carnian
Norian
Rhaetian
Hettangian
Sinemurian
Pliensbachian
Toarcian
Aalenian
Bajocian
Bathonian
Callovian
Oxfordian
Kimmeridgian
Tithonian
Berriasian
Valanginian
Hauterivian
Barremian
Aptian
Albian
Cenomanian
Turonian
Coniacian
Santonian
Campanian
Maastrichtian
Danian
Selandian
Thanetian
Ypresian
Lutetian
Bartonian
Priabonian
Rupelian
Chattian
Lower
Middle
Upper
Lower
Middle
Upper
Lower
Upper
Paleocene
Eocene
Oligocene
Triassic Jurassic Cretaceous Paleogene
early origin of feathers late origin of feathers
1
2
3
4
5
6
2,3
4
5
6
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