advances.sciencemag.org/cgi/content/full/1/10/e1500743/DC1
Supplementary Materials for
The burrowing origin of modern snakes
Hongyu Yi and Mark A. Norell
Published 27 November 2015, Sci. Adv. 1, e1500743 (2015)
DOI: 10.1126/sciadv.1500743
The PDF file includes:
Materials and Methods
Fig. S1. Vestibular shape of all samples.
Fig. S2. Placement of shape variables.
Fig. S3. Regression of centroid size to scores of principal component 1.
Fig. S4. Distribution of shape variables reconstructed for the hypothetical ancestor
of crown snakes.
Fig. S5. Principal components analyses of vestibular shape.
Fig. S6. Missing data in shape variables.
Fig. S7. An alternative phylogeny for all samples.
Table S1. Taxon sampling in the three habitat groups.
Table S2. MANOVA test of Procrustes coordinates among the three habitat
groups.
Table S3. Scores of the first two principal components.
Table S4. Accuracy of the linear discriminant function analysis.
Table S5. Habit predictions for D. patagonica and the hypothetical ancestor of
modern snakes.
Table S6. Habit predictions for D. patagonica and the hypothetical ancestor of
modern snakes.
Table S7. Habit predictions for D. patagonica and the hypothetical ancestor of
modern snakes.
Table S8. CT scanning parameters*.
Reference (32)
Materials and Methods:
X-ray Computed Tomography (CT) was completed at the American Museum of
Natural History. Among the 44 samples, 37 were scanned in a GE v|tome|x dual-tube CT
scanner. We CT scanned Dinilysia patagonica (MACN-RN 1014) at the resolution of 26
μm, and X-ray of 170 kv and 130 mA. Modern samples include dry skeletons and
alcohol-preserved specimens, and were scanned at 70 - 220 kv and 80 - 200 mA (Table
S8). We obtained CT data of seven samples (non-AMNH specimens) from the CT lab of
the University of Texas, Austin. We built virtual models of the inner ear for all samples;
the models in PLY format are deposited in MorphoBank (29) Project 2170.
We used six type-2 landmarks (17) and 22 semilandmarks placed along the lateral
surface of the vestibule and the medial surface of the lateral semicircular canal (Fig. S2).
Vestibule expansion or shrinkage was captured with the curvature of the vestibule, and
the distance between the vestibule and lateral semicircular canal. The six type 2
landmarks are: intersection of the vestibule and lateral semicircular canal (c0-0: [curve
number]-[landmark number]), intersection of the vestibule and utricle (c0-15), medial
intersection of the posterior and lateral semicircular canals (c1-0), intersection of the
lateral semicircular canal and lateral ampulla (c1-9), intersection of the anterior and
lateral ampullae (s0), and intersection of the anterior and posterior semicircular canals
(s1). The semilandmarks are equidistant points along two curves surrounding the
vestibule and lateral semicircular canal, automatically placed in Landmark Editor 3.6 (32)
(Fig. S2D).
The six type-2 landmarks are defined as intersection points between two
homologous structures, but they can move along the sutures without additional position
constraint. To increase the consistency in landmark placement, we incorporated a
reference plane (Fig. S2B) – the plane contains landmarks c0-0, c0-15, and s0, and
intersects as much of the medial line of the lateral semicircular canal as possible.
In some samples, taxon-specific morphologies can render the position of some
landmarks uncertain or inapplicable. We define a landmark as missing when its position
cannot be determined due to separation of two anatomical structures that are fused in
other samples, or vice versa. For instance, the posterior and lateral semicircular canals are
connected in most squamates, but separate in Varanus indicus (Fig. S6). In this case,
landmark c1-0 was first placed at an approximate position on the lateral semicircular
canal, and later estimated using the position of landmark c1-0 in the average Procrustes
shape of all samples. After generalized Procrustes analysis, the dataset contains no
missing data of shape coordinates.
Generalized Procrustes Analysis aligns the 28 landmarks and semilandmarks of
all samples. We used the semilandmark sliding algorithm that minimizes bending energy
in the R program Geomorph V 1.1-5 (30). Phylogenetic signal is insignificant in the
dataset (p = 0.28, iteration = 10), tested by permuting Procrustes shape coordinates of
terminal taxa on the snake cladogram (Fig. 1G). In contrast, signal for function (habitat)
is significant. MANOVA test using the Procrustes coordinates returned significant
difference among the three habitat groups (p = 0.01, Table S2).
Principal component analysis extracts major shape variations as principal
components (PCs) in the order of percent total variance explained. The first six PCs
explain 87 percent of the total variance, of which 47 percent is explained by PC1. Linear
relationship between centroid size of Procrustes shapes and PC1 score was insignificant
in a regression test (p = 0.09 for slope = 0; Fig. S3).
Linear discriminant analysis was performed among the three habit groups to
describe between-group shape separation. The group memberships of Dinilysia
patagonica and the hypothetical ancestor were both assigned as unknown in the input
data. The linear discriminant analysis then predicted both taxa to belong to the burrowing
group. We used standard statistical software (Minitab 17) for the linear discriminant
function analysis.
We mapped the shape coordinates on the phylogenies using squared-change
parsimony, implemented in MorphoJ (31). For the hypothetical ancestor of crown snakes,
shape coordinates of the inner ear were reconstructed with and without Dinilysia
patagonica. For both reconstructions, the hypothetical ancestor was predicted as a
burrower using principal component analysis (Fig. 3; Fig. S5) and linear discriminant
analysis (Table S5 to 7).
Fig. S1. Vestibular shape of all samples. (A) Specimen (1) to (12). (B) Specimen (13)
to (24). (C) Specimen (25) to (36). (D) Specimen (37) to (44). Not to scale.
Fig. S2. Placement of shape variables. (A) General anatomy of the snake bony labyrinth
represented by Ptyas mucosa. (B) Virtual model of the bony labyrinth and the reference
plane. (C) Landmarks s0 and s1, and two surface curves consisting of semilandmarks. (D)
Landmarks: c0-0, c0-15, c1-0, and c1-9. Semilandmarks: c0-1 to c0-14, and c1-1 to c1-8.
Not to scale.
Fig. S3. Regression of centroid size to scores of principal component 1. Blue squares
are aquatic species; black triangles are generalists; red dots are burrowing species. The
cross is Dinilysia patagonica.
Fig. S4. Distribution of shape variables reconstructed for the hypothetical ancestor
of crown snakes. (A) the hypothetical ancestor reconstructed with Dinilysia patagonica.
(B) The hypothetical ancestor reconstructed without Dinilysia patagonica.
Fig. S5. Principal components analyses of vestibular shape. (A) The hypothetical
ancestor of crown snakes (45) was reconstructed with Dinilysia patagonica (44). (B) The
hypothetical ancestor of crown snakes (45) was reconstructed without Dinilysia
patagonica (44). Taxon list: 1. Laticauda colubrina, 2. Aipysurus laevis, 3. Laticauda
semifasciata, 4. Acrochordus javanicus, 5. Platecarpus coryphaeus, 6. Hydrophis
caerulescens, 7. Heloderma suspectum, 8. Lampropeltis getulus, 9. Anguis fragilis, 10.
Varanus indicus, 11. Pareas hamptoni, 12. Lamprophis lineatus, 13. Corallus caninus,
14. Python molurus, 15. Naja naja, 16. Eunectes murinus, 17. Vermicella annulata, 18.
Trimeresurus stejnegeri, 19. Rhinobothryum lentiginosum, 20. Echiopsis curta, 21. Ptyas
mucosa, 22. Boiga irregularis, 23. Pygopus nigriceps, 24. Chrysopelea ornata, 25.
Aprasia pulchella, 26. Cylindrophis maculatus, 27. Uropeltis ceylanica, 28. Anniella
pulchra, 29. Typhlops jamaicensis, 30. Eryx colubrinus, 31. Anilius scytale, 32.
Loxocemus bicolor, 33. Heterodon platirhinos, 34. Simoselaps bertholdi, 35.
Rhinotyphlops caecus, 36. Sonora semiannulata, 37. Exiliboa placata, 38. Achalinus
spinalis, 39. Typhlosaurus lineatus, 40. Rhinophis philippinus, 41. Xenopeltis unicolor,
42. Dibamus novaeguineae, 43. Bipes canaliculatus, 44. Dinilysia patagonica, and 45.
the hypothetical ancestor of crown snakes.
Fig. S6. Missing data in shape variables. (A) The inner ear of Ptyas mucosa in lateral
view; the posterior and lateral semicircular canals are connected. (B) The inner ear of
Varanus indicus in lateral view; the posterior and lateral semicircular canals are separate.
(C) Close-up view of the lateral semicircular canal in Varanus indicus. Not to scale.
Fig. S7. An alternative phylogeny for all samples. Mosasauria is the sister group to all
snakes in this phylogeny adapted from refs No. 7 and 22.
Table S1. Taxon sampling in the three habitat groups. Snakes are listed separately
from lizards and amphisbaenians. Daggers denote fossils.
SNAKES
AQUATIC
GENERALIST
BURROWING
Laticauda colubrina
Lampropeltis getulus
Cylindrophis maculatus
Aipysurus laevis
Pareas hamptoni
Uropeltis ceylanica
Laticauda semifasciata
Lamprophis lineatus
Typhlops jamaicensis
Acrochordus javanicus
Corallus caninus
Eryx colubrinus
Hydrophis caerulescens
Python molurus
Anilius scytale
Naja naja
Loxocemus bicolor
Eunectes murinus
Heterodon platirhinos
Vermicella annulata
Simoselaps bertholdi
Trimeresurus stejnegeri
Rhinotyphlops caecus
Rhinobothryum lentiginosum
Sonora semiannulata
Echiopsis curta
Exiliboa placata
Ptyas mucosa
Achalinus spinalis
Boiga irregularis
Rhinophis philippinus
Chrysopelea ornata
Xenopeltis unicolor
Dinilysia patagonica†
NON-SNAKE
SQUAMATES
Platecarpus coryphaeus†
Heloderma suspectum
Aprasia pulchella
Anguis fragilis
Anniella pulchra
Varanus indicus
Typhlosaurus lineatus
Pygopus nigriceps
Dibamus novaeguineae
Bipes canaliculatus
Table S2. MANOVA test of Procrustes coordinates among the three habitat groups.
df
SS.obs
MS
P.val
Habitat groups
2
0.604
0.302
0.01
Total
43
2.106
0.049
NA
Table S3. Scores of the first two principal components.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Taxon
Aprasia pulchella
Cylindrophis maculatus
Heloderma suspectum
Lampropeltis getulus
Laticauda colubrina
Uropeltis ceylanica
Aipysurus laevis
Anguis fragilis
Anniella pulchra
Laticauda semifasciata
Varanus indicus
Pareas hamptoni
Lamprophis lineatus
Corallus caninus
Typhlops jamaicensis
Python molurus
Naja naja
Eryx colubrinus
Anilius scytale
Loxocemus bicolor
Heterodon platirhinos
Eunectes murinus
Vermicella annulata
Trimeresurus stejnegeri
Simoselaps bertholdi
Rhinotyphlops caecus
Rhinobothryum lentiginosum
Sonora semiannulata
Exiliboa placata
Echiopsis curta
Acrochordus javanicus
Achalinus spinalis
Ptyas mucosa
Platecarpus coryphaeus
Typhlosaurus lineatus
Rhinophis philippinus
Hydrophis caerulescens
Boiga irregularis
Pygopus nigriceps
Xenopeltis unicolor
Dibamus novaeguineae
PC1 (47%)
-0.0621
-0.1818
0.1355
0.0431
0.2549
-0.1908
0.1678
-0.0682
-0.1309
0.3777
0.0865
-0.0223
-0.0187
-0.0566
-0.0360
-0.0982
0.1541
-0.0745
-0.1915
-0.1950
0.1318
-0.0783
0.0182
0.0921
-0.1152
0.0583
0.0743
0.1295
-0.0045
0.2133
0.0481
-0.0771
0.1276
0.2130
-0.1381
-0.2028
0.2110
0.1985
-0.0681
-0.2348
-0.1660
PC2 (17%)
0.0003
0.0264
-0.0929
0.0529
0.1077
-0.0201
-0.0251
-0.0475
-0.0226
0.0314
-0.0657
-0.0752
0.0753
-0.1576
-0.1864
-0.0758
0.1080
0.0182
-0.0002
0.0854
-0.1306
-0.1279
-0.0281
-0.0344
0.0969
-0.1534
0.0199
-0.0325
-0.0243
0.0368
-0.0422
-0.1537
-0.0160
0.2191
0.0498
-0.0215
0.0373
0.0205
0.0031
0.1877
0.0833
42
43
44
45
Chrysopelea ornata
Bipes canaliculatus
Dinilysia patagonica
Hypothetical ancestor of crown snakes
(Reconstructed with Dinilysia
patagonica)
0.1483
-0.1592
-0.2265
-0.0865
0.0900
0.0978
0.1195
-0.0337
Table S4. Accuracy of the linear discriminant function analysis.
Predicted group
True group
Aquatic
Generalists
Burrowing
Aquatic
4
2
0
Generalists
2
10
4
Burrowing
0
6
15
Total N
6
18
19
N correct
4
10
15
0.667
0.556
0.789
N correct proportion
N = 43
N correct = 29
Proportion correct = 0.674
Table S5. Habit predictions for D. patagonica and the hypothetical ancestor of
modern snakes. The hypothetical ancestor was reconstructed with D. patagonica.
Observation
Dinilysia
patagonica
Hypothetical
ancestor
Predicted From Group
Groups
Burrowing Aquatic
Generalists
Burrowing
Squared
Distance
Probability
21.046
10.092
4.780
0.000
0.066
0.934
Burrowing Aquatic
Generalists
Burrowing
13.506
3.466
1.749
0.002
0.297
0.701
Table S6. Habit predictions for D. patagonica and the hypothetical ancestor of
modern snakes. The hypothetical ancestor was reconstructed without D. patagonica).
Observation
Dinilysia
patagonica
Hypothetical
ancestor
Predicted From
Groups
Group
Burrowing Aquatic
Generalists
Burrowing
Squared
Distance
17.119
8.341
3.142
Probability
Burrowing Aquatic
Generalists
Burrowing
12.183
2.851
2.077
0.004
0.403
0.593
0.001
0.069
0.930
Table S7. Habit predictions for D. patagonica and the hypothetical ancestor of
modern snakes. The hypothetical ancestor was reconstructed with D. patagonica and an
alternative phylogeny in fig. S7.
Observation
Dinilysia
patagonica
Hypothetical
ancestor
Predicted From Group
Groups
Burrowing Aquatic
Generalists
Burrowing
Squared
Distance
Probability
17.130
0.001
0.069
0.931
Burrowing Aquatic
Generalists
Burrowing
8.425
1.298
8.354
3.141
0.092
0.010
0.350
0.640
Table S8. CT scanning parameters*.
Taxon
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Specimen number
Voxel
size (x=y,
mm)
0.048
Voxel Filter
size (z,
mm)
0.048
N
Scanning
lab
0.047
0.047
AMNH
AMNH R-161779
0.044
0.044
AMNH R-92269
0.081
0.081
0.1 mm
Cu**
0.1 mm
Cu
N
AMNH FM 1645
0.054
0.054
AMNH
AMNH R-86181
0.042
0.042
AMNH R-161167
0.082
0.082
0.1 mm
Cu
0.1 mm
Cu
N
AMNH R-95965
0.025
0.025
AMNH
AMNH R-21745
AMNH R-58389
AMNH R-153711
0.027
0.032
0.016
0.027
0.032
0.016
0.1 mm
Cu
N
N
N
AMNH R-50646
0.040
0.040
N
AMNH
AMNH R-55910
0.039
0.039
AMNH
TNHC, to be
accessioned
FMNH 22468
AMNH R-29349
0.044
0.106
0.1 mm
Cu
N
0.044
0.067
0.114
0.067
N
N
UTCT
AMNH
AMNH R-161747
0.015
0.015
N
AMNH
AMNH R-21057
0.040
0.040
N
AMNH
AMNH R-52395
0.028
0.028
N
AMNH
AMNH R-115397
0.033
0.033
N
AMNH
AMNH R-33243
AMNH R-69292
0.037
0.021
0.037
0.021
N
0.1 mm
AMNH
AMNH
Laticauda
AMNH R-28996
colubrina
Aipysurus laevis AMNH R-5087
Laticauda
semifasciata
Acrochordus
javanicus
Platecarpus
coryphaeus †
Hydrophis
caerulescens
Heloderma
suspectum
Lampropeltis
getulus
Anguis fragilis
Varanus indicus
Pareas
hamptoni
Lamprophis
lineatus
Corallus
caninus
Python molurus
15 Naja naja
16 Eunectes
murinus
17 Vermicella
annulata
18 Trimeresurus
stejnegeri
19 Rhinobothryum
lentiginosum
20 Echiopsis
curta
21 Ptyas mucosa
22 Boiga
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
AMNH
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
irregularis
Pygopus
nigriceps
Chrysopelea
ornata
Aprasia
pulchella
Cylindrophis
maculatus
Uropeltis
ceylanica
Anniella
pulchra
Typhlops
jamaicensis
Eryx colubrinus
Anilius scytale
Loxocemus
bicolor
Heterodon
platirhinos
Simoselaps
bertholdi
Rhinotyphlops
caecus
Sonora
semiannulata
Exiliboa
placata
Achalinus
spinalis
Typhlosaurus
lineatus
Rhinophis
philippinus
Xenopeltis
unicolor
Dibamus
novaeguineae
Bipes
canaliculatus
Dinilysia
patagonica†
AMNH R-161427
0.030
0.030
Cu
N
AMNH R-161965
0.031
0.031
N
AMNH
AMNH R-99706
0.010
0.010
N
AMNH
AMNH R-126605
0.016
0.016
N
AMNH
AMNH R-43344
0.015
0.015
N
AMNH
AMNH R-12851
0.020
0.020
N
AMNH
USNM 12378
0.005
0.016
N
UTCT
FMNH 63117
USNM 204078
FMNH 104800
0.018
0.018
0.021
0.048
0.038
0.053
N
N
N
UTCT
UTCT
UTCT
FMNH 194529
0.029
0.067
N
UTCT
AMNH R-115427
0.016
0.016
N
AMNH
AMNH R-05844
0.023
0.023
N
AMNH
AMNH R-170522
0.016
0.016
N
AMNH
AMNH R-102892
0.014
0.014
N
AMNH
AMNH R-34620
0.014
0.014
N
AMNH
AMNH R-98481
0.018
0.018
N
AMNH
AMNH R-24674
0.010
0.010
N
AMNH
AMNH R-161693
0.044
0.044
N
AMNH
AMNH R-86710
0.006
0.006
N
AMNH
AMNH R-113487
0.018
0.018
N
AMNH
MACN-RN 1014
0.026
0.026
0.1 mm
Cu
AMNH
AMNH
* All AMNH specimens were scanned on site, using loans from the herpetological and
paleontological collections of the American Museum of Natural History. The specimen of
Dinilysia patagonica is non-AMNH, but it was scanned at the American Museum of the
Natural History.
**For AMNH specimens, all scanning parameters follow the output of a GE v|tome|x
dual-tube CT scanner. Copper filter is generally not recommended for recent specimens.
For the recent specimens listed with a 0.1 mm Cu filter, it may have been an operational
error: a “filter” may have been selected for data output when it was not actually in place.
In these cases, we recommend scanning with and without a filter for optimal results.
Institutional abbreviations: American Museum of Natural History (AMNH),
Field Museum of Natural History (FMNH), Museo Argentino de Ciencias Narutales,
Argentina (MACN), Texas Natural History Collections, University of Texas at Austin
(TNHC), and United States National Museum, Smithsonian Institution (USNM).