This is a talk I gave in the 2013 Joint Meeting of Ichtyologist and Herpetologists in Albuquerque, NM, USA. This talk is dirived from a paper we publiched in 2012 in Zoologica Scripta

1. PHYLOGENETIC SIGNAL AND VARIATION OF
VISCERAL PIGMENTATION IN
EIGHT ANURAN FAMILIES
Zoologica Scripta 2012 41(6): 547-556
DIOGO B. PROVETE et al.

2. Biological traits
¨ Models of trait evolution
¤ Plasticity vs. Conservatism => phylogenetic constraints
and the role of environment
¤ Meaning of phylogenetic signal => related species
have similar traits

3. Pigmented cells
Wallin,2002

4. Visceral melanocytes
Differential
occurrence among
organs

5. Goals
¨ Function of visceral pigmentation not yet understood
¤ Hypothesis relate the role of melanin as a protective
agent against toxic agents and also in thermoregulation
¨ Analyse the distribution of visceral
pigmentation among species
¨ Test for a phylogenetic signal using a
comparative method (Pavoine et al. 2010)

6. Methods
¨ Pigmentation categories on the surface of 16 visceral
organs of 35 anuran species, belonging to 13 genera
from 8 families nested within Meridianura (sensu Frost
et al. 2006)

7. Methods
¨ Data collection
¤ Field: We collected calling adult anurans in several
locations in the state of São Paulo, southeastern
Brazil
¤ Literature: Franco-Belussi et al. (2011, 2012)
¤ Museum specimens: DZSJRP, CCLZU, JJ-MNRJ

8. Visceral melanocytes quantification
Franco-Belussi et al., 2009 Anat. Rec.
Figure 2. (Legend on page 179.)

9. category 1, we assigned category 0 to that organ in that ified by Grant et al. (2006) and Hedges et al. (2008), for
Fig. 1 Phylogenetic tree constructed for the analysis of trait diversity showing the higher-order groups (left), families (right) and species

10. Decomposing trait diversity along the
nodes of a phylogeny
FIG. 2. Four different situations that occur in the decomposition of trait diversity among the nodes of a phylogenetic tree: (A
SANDRINE PAVOINE ET AL.490 Ecological Monograp
Vol. 80, No
Pavoine et al. 2010 Ecol. Monog
Phylogenetic signal Plasticity Random

11. Results
¨ Phylogenetic signal in each organ/structure
is significantly biased towards the root in
the heart, testicle, lumbar parietal
peritoneum, lumbar nerve plexus, as
well as considering all organs together.
¨ Analysis was run to 12 out of 16 organs
(4 organs did not have pigmenation)
Lumbar parietal
peritoneum
TQE = 0.179
SN test = 0.01
FN test = 0.01
Ro test = 0.01
TQE = 0.15
SN test = 0.40
FN test = 0.07
Ro test = 0.01
TQE = 0.084
SN test = 0.29
FN test = 0.10
Ro test = 0.32
Lumbar
nerve plexus
All organsVertebral
column
TQE = 0.092
SN test = 0.02
FN test = 0.01
Ro test = 0.01
TQE = 0.096
SN test = 0.82
FN test = 0.32
Ro test = 0.79
TQE = 0.068
SN test = 0.01
FN test = 0.11
Ro test = 0.01
TQE = 0.126
SN test = 0.01
FN test = 0.01
Ro test = 0.01
J K L M
0.002 0.008 0.012
0.002 0.008 0.012
0.018 0.022
0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.0180.022
0.028
ation among the nodes of the anuran phylogenetic tree. (A–L) Variation of

12. Pigmentation in the urogenital system
Pericardium Cardiac blood vessels
0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.018
0.002 0.008 0.012 0.002 0.008 0.0120.018
0.022 0.028
0.01 0.03 0.05 0.07 0.09
TQE = 0.244
SN test = 0.87
FN test = 0.73
Ro test = 0.84
TQE = 0.121
SN test = 0.03
FN test = 0.07
Ro test = 0.14
Kidney Renal veins
Tes cle Rectum Mesenterium
Lumbar parietal
peritoneum
TQE = 0.179
SN test = 0.01
FN test = 0.01
Ro test = 0.01
TQE = 0.15
SN test = 0.40
FN test = 0.07
Ro test = 0.01
TQE = 0.084
SN test = 0.29
FN test = 0.10
Ro test = 0.32
Lumbar
nerve plexus
All organsVertebral
columnE F J K L M
B C G H I
n anurans d D. B. Provete et al. TQE = 0.130
0.005 0.015 0.025 0.035 0.045 0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.018 0.022 0.028
SNtTest = 0.02
FNtest = 0.01
Ro test = 0.01
TQE = 0.244
SN test = 0.87
FN test = 0.73
Ro test = 0.84
TQE = 0.121
SN test = 0.03
FN test = 0.07
Ro test = 0.14
KidneyLung Renal veins
TQE = 0.078
SN test = 0.72
FN test = 0.23
Ro test = 0.05
TQE = 0.121
SN test = 0.02
FN test = 0.01
Ro test = 0.07
TQE = 0.152
SN test = 0.07
FN test = 0.02
Ro test = 0.14
D E F
0.002 0.008 0.012 0.005 0.015 0.025 0.035 0.005 0.015 0.025
Fig. 6 Decomposition of the diversity of categories of pigmentation
pigmentation categories as measured in a single specified organ. —
pigmentation categories of all organs among the 32 species. Circles
TQE = 0.130
0.005 0.015 0.025 0.035 0.045 0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.018 0.022 0.028
SNtTest = 0.02
FNtest = 0.01
Ro test = 0.01
TQE = 0.244
SN test = 0.87
FN test = 0.73
Ro test = 0.84
TQE = 0.121
SN test = 0.03
FN test = 0.07
Ro test = 0.14
KidneyLung Renal veins
TQE = 0.078
SN test = 0.72
FN test = 0.23
Ro test = 0.05
TQE = 0.121
SN test = 0.02
FN test = 0.01
Ro test = 0.07
TQE = 0.152
SN test = 0.07
FN test = 0.02
Ro test = 0.14
D E F
0.002 0.008 0.012 0.005 0.015 0.025 0.035 0.005 0.015 0.025
0.0
Fig. 6 Decomposition of the diversity of categories of pigmentation a
pigmentation categories as measured in a single specified organ. —M
pigmentation categories of all organs among the 32 species. Circles a
given below each tree. The larger the circle, the larger the trait dive

13. Cardio-respiratory system
The species from Terrarana, Leptodactylidae, Hylodidae and
Proceratophrys had no pigmentation in the pericardium, whereas
Odontophrynus an Leiuperidae had a small amount of pigmentation

14. Cardio-respiratory system
Heart Pericardium Cardiac blood vessels
TQE = 0.130
0.005 0.015 0.025 0.035 0.045 0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.018
0.00
0.022 0.028
0.01 0.03 0.05 0.07 0.09
SNtTest = 0.02
FNtest = 0.01
Ro test = 0.01
TQE = 0.244
SN test = 0.87
FN test = 0.73
Ro test = 0.84
TQE = 0.121
SN test = 0.03
FN test = 0.07
Ro test = 0.14
KidneyLung Renal veins
Tes cle
Lumbar parietal
peritoneum
TQE = 0.179
SN test = 0.01
FN test = 0.01
Ro test = 0.01
Ve
co
A
D E F J K
B C G H
Visceral pigmentation in anurans d D. B. Provete et al. TQE = 0.130
0.005 0.015 0.025 0.035 0.045 0.002 0.00
SNtTest = 0.02
FNtest = 0.01
Ro test = 0.01
TQ
SN
FN
Ro
KidnLung
TQE = 0.078
SN test = 0.72
FN test = 0.23
Ro test = 0.05
TQE = 0
SN test
FN test
Ro test
D E
0.002 0.008 0.012 0.005 0.015 0.0
Fig. 6 Decomposition of the d
pigmentation categories as me
pigmentation categories of all

15. Digestory system
dium Cardiac blood vessels
2 0.018 0.002 0.008 0.012 0.018
0.002 0.008 0.012 0.002 0.008 0.0120.018
0.022 0.028
0.01 0.03 0.05 0.07 0.09
0.244
= 0.87
= 0.73
= 0.84
TQE = 0.121
SN test = 0.03
FN test = 0.07
Ro test = 0.14
Renal veins
Tes cle Rectum Mesenterium
Lumbar parietal
peritoneum
TQE = 0.179
SN test = 0.01
FN test = 0.01
Ro test = 0.01
TQE = 0.15
SN test = 0.40
FN test = 0.07
Ro test = 0.01
TQE = 0.084
SN test = 0.29
FN test = 0.10
Ro test = 0.32
Lumbar
nerve plexus
All orVertebral
columnF J K L M
C G H I
d D. B. Provete et al.

16. Digestory system
Heart Pericardium Cardiac blood vessels
TQE = 0.130
0.005 0.015 0.025 0.035 0.045 0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.018
0.002 0.008 0.012 0.002 0.008 0.0120.018
0.022 0.028
0.01 0.03 0.05 0.07 0.09
SNtTest = 0.02
FNtest = 0.01
Ro test = 0.01
TQE = 0.244
SN test = 0.87
FN test = 0.73
Ro test = 0.84
TQE = 0.121
SN test = 0.03
FN test = 0.07
Ro test = 0.14
KidneyLung Renal veins
Tes cle Rectum Mesenterium
Lumbar parietal
peritoneum
TQE = 0.179
SN test = 0.01
FN test = 0.01
Ro test = 0.01
TQE = 0.15
SN test = 0.40
FN test = 0.07
Ro test = 0.01
TQE = 0.084
SN test = 0.29
FN test = 0.10
Ro test = 0.32
Lumbar
nerve plexus
All organsVertebral
column
A
D E F J K L M
B C G H I
0.002 0.008 0.012 0.005 0.015 0.025 0.035 0.005 0.015 0.025
0.002 0.008 0.012
0.002 0.008 0.012
0.018 0.022
0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.0180.022
0.028
Visceral pigmentation in anurans d D. B. Provete et al.
influence melanocyte dynamics and the occurrence of pig-
mentation on testes. However, experimental studies on the
pigmentation may vary according to physiological (e.g. age,
nutritional status and diseases; Agius & Agbede 1984)
TQE = 0.130
0.005 0.015 0.025 0.035 0.045 0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.018
0.002 0.008 0.012 0.002 0.008 0.0120.018
0.022 0.028
0.01 0.03 0.05 0.07 0.09
SNtTest = 0.02
FNtest = 0.01
Ro test = 0.01
TQE = 0.244
SN test = 0.87
FN test = 0.73
Ro test = 0.84
TQE = 0.121
SN test = 0.03
FN test = 0.07
Ro test = 0.14
KidneyLung Renal veins
TQE = 0.078
SN test = 0.72
FN test = 0.23
Ro test = 0.05
TQE = 0.121
SN test = 0.02
FN test = 0.01
Ro test = 0.07
TQE = 0.152
SN test = 0.07
FN test = 0.02
Ro test = 0.14
Lumbar parietal
peritoneum
TQE = 0.179
SN test = 0.01
FN test = 0.01
Ro test = 0.01
TQE = 0.15
SN test = 0.40
FN test = 0.07
Ro test = 0.01
TQE = 0.084
SN test = 0.29
FN test = 0.10
Ro test = 0.32
Lumbar
nerve plexus
All organsVertebral
column
TQE = 0.092
SN test = 0.02
FN test = 0.01
Ro test = 0.01
TQE = 0.096
SN test = 0.82
FN test = 0.32
Ro test = 0.79
TQE = 0.068
SN test = 0.01
FN test = 0.11
Ro test = 0.01
TQE = 0.126
SN test = 0.01
FN test = 0.01
Ro test = 0.01
D E F J K L M
0.002 0.008 0.012 0.005 0.015 0.025 0.035 0.005 0.015 0.025
0.002 0.008 0.012
0.002 0.008 0.012
0.018 0.022
0.002 0.008 0.012 0.018 0.002 0.008 0.012 0.0180.022
0.028
Fig. 6 Decomposition of the diversity of categories of pigmentation among the nodes of the anuran phylogenetic tree. (A–L) Variation of
pigmentation categories as measured in a single specified organ. —M. Variation of pigmentation categories as measured considering the
pigmentation categories of all organs among the 32 species. Circles at nodes provide the contribution of nodes to trait diversity, scale are
given below each tree. The larger the circle, the larger the trait diversity in that node. Results of the permutation tests are given at the
bottom of each tree: SN, single-node skewness test; FN, few-nodes skewness test; Ro, root ⁄ tips skewness test (two sided). Total quadratic
entropy (TQE) represents the overall value of trait diversity, the higher the TQE, the higher the diversity of pigmentation categories in a
given organ. See Fig. 1 for node labels. [Correction added on 8 October 2012, after first online publication: Fig. 6 was replaced with the
correct test values for section K].

17. Discussion
¨ Pigmentation protecting against bacterial infections
in testicles (Franco-Belussi & Oliveira 2011) =>
increase fitness
¤ Clade Calamitophrynia
¨ Role of environment in dictating plasticity
¤ Temperature => increase pigmentation => species that
lives in areas with high temperature
¨ Physiological factors (e.g., disease, age, MSH
hormone)

18. Conclusion
¨ Overall, the pigmentation of organs is a very conserved
trait.
¨ Future studies
¤ Histological analyses
¤ Experiments manipulating factors that influence melanin
production and melanocyte migration (Franco-Belussi et al. in
press)
¨ More pictures in MorphoBank http://bit.ly/phylosignal

19. Shortcommings
¨ Dependency of the topology
¤ Incorporate Bayesian methods to account for
phylogenetic uncertainty
¨ Qualitative methods to describe pigmentation
¤ Incorporate quantitative methods
¨ Expand taxon sampling to other speciose anuran
families

20. New directions
¨ Incorporate intraspecific
variation
¨ How pigmentation varies
along space
¨ Climatic and topographic correlates (Extended
RLQ analysis)
¤ Which environmental factors influence the expression and
variation of pigmentation

21. Funding

22. Webite bit.ly/DiogoBrazil
Email: dbprovete@utexas.edu
Thank you!!