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Amphibian epithelial and morphological adaptations to dry habitats: a
preliminary survey of adaptive trait variation among Colombian dry
forest anurans.
Thesis dissertation presented by:
Juan Salvador Mendoza Roldán
Director:
Dr. Andrew J. Crawford.
Universidad de Los Andes, Bogotá, Colombia.
2014.
Resumen: Los anuros poseen una organización dermal simple que ha evolucionado para
solucionar los problemas atribuidos a la terrestrealizacion. La innovación estructural como
la aparición de glándulas con un amplio espectro de secreciones y la presencia de regiones
especializadas, altamente vascularizadas han permitido la supervivencia de los anuros
adultos en ambientes secos, dominados por altas temperaturas y la presencia de sustratos y
corrientes de aire desecantes. Estas especies muestran adaptaciones tegumentarias para la
perdida de agua, que van desde la presencia de osteodermos y co-osificación craneal hasta
el uso de secreciones de origen lipídico. Estas adaptaciones morfológicas se encuentran
acopladas con rasgos etológicos y ecológicos que configuran la estrategia adaptativa de la
especie. La presente contribución se enfoca en la caracterización básica de las estructuras
del tegumento, por medio de microscopia de luz. Se comparó la variación de caracteres
discretos entre poblaciones y en algunos casos especies hermanas presentes en hábitats
húmedos y secos. Se probó el efecto de algunas variables climáticas sobre el tamaño
corporal para establecer el valor adaptativo de las diferencias intra e inter especificas
existentes entre proporciones de la tibia y el cráneo, medidas relacionadas con la relación
superficie y volumen. Las comparaciones realizadas entre poblaciones hermanas de
distintos orígenes geográficos y de hábitat se realizaron para describir la relación existente
entre algunos aspectos de la morfología externa, histología características pluviométricas,
haciendo énfasis en la biota anfibia de uno de los ecosistemas terrestres más amenazados de
Colombia, el bosque seco tropical.
Abstract: Anurans possess a very simplified dermal organization, which has evolved to
solve the basic problems of terrestrialization. Structural innovation, presence of specialized
highly vascularized regions and gland sets with a wide diversity of secretions, have allowed
adult anurans to survive in desiccating environments that are dominated by dry substrates
and air currents, in places with elevated day temperatures; geographically this places may
be generalized by having extended periods with no rainfall thus dominating dry conditions.
These species show interesting integumentary adaptations to avoid water loss that range
from the presence of osteoderms and skin co ossification to the use of lipid based
impermeable secretions, generally these morphological adaptations are coupled with
behavioral traits that together configure the adaptive strategy of the species. The present
contribution focuses on the examination of anatomical components configuring the dermal
organization of some Caribbean dry forest species, and by means of light microscopy
characterize the basic structure of species integument to compare variation of discrete traits
among conspecific populations and in some cases pairs of dry and wet habitat sister
species. Body size variation was tested to establish the adaptive value in water economy
conferred by body proportions related to the total surface to volume ratio. Body proportions
included the analysis of variation between sibling populations, where total lengths (SVL)
were contrasted with Tibial length and Craneal width. Comparisons among conspecific
populations from different geographical and habitat related origin were made in order to
describe the basic relation between external morphology, histology and the habitat rainfall
category (Dry or Wet), focusing on the frog biota from one of Colombia´s most threatened
land ecosystem the Seasonally dry tropical forest.
Introduction:
The colonization of terrestrial habitats by amphibians begun in the end of the Devonian
period 360 million years ago, when freshwater Rhipidista with lobed fins, migrated from
pond to pond during the dry season, behavior that aided in the survival of water dependent
animals, with physiological boundaries for free dwelling on terrestrial ecosystems (Toledo
et al. 1993; Romer, 1959). Dehydration always will be the earliest of amphibian problems;
many fossil species show the presence of scales and bony plates that favored water
retention (Colbert, 1969 in Toledo et al. 1993). Present amphibians are poorly adapted to
strict terrestrial life, their skin is a very simple dermal integument that does not generally
serve as a barrier to the flow of water from and towards the amphibian body, creating two
mayor selective pressures crucial in the evolution of modern amphibians; aquatic species
tend to hydrate and loose inner solutes and terrestrial forms tend to dehydrate by means of
evo transpiration (Porter 1972).
The morpho-physiological interaction of amphibians with their abiotic environment is a
complex and dynamic system of related process, arid habitats such as dry forests, deserts
and open shrub lands and savannas, impose a rigorous environmental filter that has caused
morphological, physiological and behavioral evolution of a wide diverse of adaptive traits
that function in synergy to configure independent overall adaptive strategies for each
member of the anuran community (Toledo et al. 1993; Duellman and Trueb, 1986).
Amphibians don not drink the water required for metabolic function (Ex. Phyllomedusa),
this water penetrates their bodies, principally by the way of the integument , special zones
for rehydration are present in different zones of the amphibian body, reason why skin
permeability to water differs from one part of the animal to the other (Toledo et al. 1993).
This author also concludes that Inter and intra specific variation among skin traits may be
related to adaptation for a particular environment, and this variation may confer structural
differences in the integuments. Canziani and Cannata (1980) have shown that arid region
Ceratophrys ornata, has a smooth ventral skin except in the pelvic region, where it is
granular, on the other hand Individuals from moist temperate climates have uniformly
granular ventral skin; while dehydrating arid area frogs may lose less water, but moist area
frogs are better rehydrating by the presence of a granular skin.
Skin thickness and the number of epidermal skin layers vary across amphibian species, in
the process of keratinization a process related with the aquatic or terrestrial environment.
Interspecific analyses have shown that species from the African genus Ptcychadena have an
inverse relationship between body size and skin width, with the largest having the thinnest
skin (Le Quang Trong, 1975). Other genus such as the African Phrynobatrachus, show
variable skin thickness related to diversity of habitat, forest species have thinner skins than
do savanna species of the same size, and ubiquitous species have a skin thickness
intermediate to these two (Le Quang Trong, 1971). Skin gland density per square
millimeter of skin is greater in the savanna-dwelling than in the forest dwelling species of
frogs, in savanna species there is a predominance of mucous glands, these produce mucous
secretions that help the animal in its adaptation to high temperatures and low relative
humidity environments (Le Quang Trong, 1975). Mucous production depends directly on
gland density and it has been shown that the mucous protects against desiccation. These
Mucous glands are important in thermal and water economy relationship of the frog and its
environment, mucous discharges aid in the control of body temperature and also maintain
the amphibian skin moist for cutaneous respiration.
The secretions produced by serous cutaneous glands in the order Anura exhibit highly
variable ultrastructural features (Delfino et al. 1992). Serous storage bodies represent a
hetorogenous class of structures ranging from vesicles containing translucent products to
dense membrane bounded aggregates; their morphological variation also includes
accumulations resembling multivesicular bodies. This heterogenity reflects specific
biosynthetic pathways during the post golgian maturation phase which can be easily seen in
the premetamorphic stages of development, mature serous products are consistent for each
species in each genus investigated (Delfino 1991).
Daly et al. (1987) comment that the wide variability in both composition and function of
serous secretions of anuran skin reflects evolution of the survival strategies in the living
families. Species from genus Phyllomedusa are known for exhibiting great polymorphism
in their sereous gland morphology, studies performed by G. Delphino et al. (1998), show
that variation of size and histochemical properties vary among species present in Argentina.
Phyllomedusa species possess at least three serous gland types that have been classified on
base of morphological and histochemical characterization. Skin permeability has been
found to be greatly influenced by cutaneous lipids (Schim and Bardem, 1965). Blaylock et
al. (1976) described peculiar glands in Phyllomedusa. These glands named as lipid
secreting glands, were proved to be related with regulating evaporative water loss through
the skin, frogs from this genus spreads lipids over the body surface using all limbs with a
stereotyped whipping behavior (Blaylock et al.1976).
Arboreal hylids are potentially more exposed to dehydrating conditions, thus some authors
as Yorio and Bentley (1977), have described much of the adaptations favoring water
conservation by the body. Lipid quantity in the ventral skin of Agalychnis dacnicolor is less
than in the ventral skin of other anuran species such as Bufo marinus, Rana pipiens and
Xenopus laevis. In some Phyllomedusa species (ex. P. bicolor), ossified structures appear as
bony spines which project outwards, covered by epidermis and originate from basal
osseous plates in the dermis that possess low vascularized regions aiding in water
conservation.
The ventral pelvic or inguino-femoral region in anurans has a powerful capacity for water
absorption. Habitat and hydration capacity in an anuran can be related to the vascularization
of the integument in the pelvic region (Roth, 1973). The skin of the pelvic region is
morphologically different from that of other parts of the body, being thinner and well
vascularized. The degree of terrestriality of a species seems to be related with the greater
intensity of cutaneous vascularization in the pelvic region, this morphological aspect is
linked with behavioral postures for rehydration, adaptations that favor positive water flow
into the body. Structurally, water absorption pads are configured by small verrucae
hydrophilica, a cutaneous structure provided with specific vascular plexa (Drewes et al.,
1977). Each verruca is usually composed by a central granular gland, surrounded by four to
six mucous glands; Capillary blood vessels of various sizes are distributed over the surface
of the verruca, some of these are placed at the base of the sulci, near the epidermis, these
sulci store water thus preventing evaporation. Kolbelt and Lisenmair (1986) have described
that it is more probable that water absorption is taken place along the sulci than on the
surface of the verruca. In the amphibians the presence of capillaries in a sub epidermal
position is considered as a primitive character, epidermal capillaries are an adaptation of
some terrestrial amphibians to rapid abortion of water (Czopek and Szarsk, 1989 in Toledo
et al., 1993).In this thesis morphological aspects of the amphibian skin are discussed based
on histological observations performed on light microscopy, a preliminary characterization
of dry forest species and comparisons between wet forest populations are shown as a
qualitative approach for trait variation.
Figure 1. Strategies often employed by the dry forest community to avoid water stress: A)
posture employed by Hyloscirtus sp. An arboreal hylid during mid-day; B) underground
retreats may be used to aestivate or as a humid refuge by Rhinella humboldti; C) Tree trunk
cavities are used by arboreal hylids.
Part 2. Body size.
The effect of climate on body size proportions has been studied along aridity gradients and
a trend between rainfall, limb length and cranial width has been observed (Lee, 1993).
Environmental heterogeneity and body size has been studied in different latitudes under
distinct level of analysis. Olalla et al. (2009) performed a community assemblage approach
for the variation observed in Brazilian Cerrado anurans, and concluded that water deficit is
the only explanatory variable for the observed pattern which dictates that larger body sizes
are associated with dryer areas. On the contrary Greene et al (2013), based on 23 years of
measurements and skeletochronology on a temperate species found that body size is more
related to abundance than to abiotic factors such as rainfall. So patterns have been
discussed as being more related with phenotypic plasticity than to a real evolutionary
response. As part of this thesis body size was tested among different dry forest related
species and their wet forest sister population. Iterspecific analysis was performed for two
wet forest-dry forest sister species to test for any phylogenetic trend in body size.
Methods:
Part 1. Histology.
96 skin samples from a total of 27 individuals from 10 species in four families were
collected from the Inguinal, ventral and dorsal regions of the frog´s body. Samples were
fixed in 10% formalin, dehydrated in ascending series of ethyl alcohols and embedded in
paraffin. Transverse skin sections of 7 micrometers were hydrated and stained with
Ehrlich´s hematoxylin and Eosine method (1886), this process was carried out by an ICA
institute histopathologist. Analysis was performed using optical microscopy and measures
were obtained using an ocular micrometer. The work was documented with photographs
taken using a digital camera. The examined material belongs to collections performed by
the author in the departments of Guajira, Cesar, Atlántico, Cordoba, Bolivar, Cesar,
Antioquia and Huila. Measurements are presented as descriptive on base of literature
records for cutaneous adaptive structures following Toledo et al. (1993), Mangione et al.
(2009); Delfino et al. (1998); Elias et al. (1957) and Perez et al. (1996), Duellman et al.
(1986). Nomenclature and morphometric methods follow these authors as well.
Figure3. Species analyzed through histology: Left: Arboreal species, family Hylidae A)
Trachycephalus typhonius, B) Hypsiboas crepitans; C) Phyllomedusa venusta. Center:
terrestrial Leptodactylidae A) Leptodactylus fuscus; B) Leptodactylus fragilis; C)
Leptodactylus bolivianus. Right: terrestrial miscellanea A) Ceratophrys calcarata; B)
Rhinella humboldti; C) Pseudopaludicola pusila.
Part 2. Body size.
A total of 427 museum specimens were measured belonging to ten species in four families.
The present study is limited to sexually mature males that were distinguished from females
by secondary sexual characters like nuptial pads, vocal sacs, and spines or by sexing the
individual directly by means of gonad inspection. Only males were selected because of the
certainty of discarding juvenile frogs that can´t be easily distinguished from females in
many cases. Three measures were taken from each individual: SVL (Snout vent Length),
TL (Tibia length) and cranium width (CW), which represents measurements relative to
bone structures, whose dimensions do not change dramatically after fixation and
preservation. All Measurements were performed with dial caliper accurate to 0.1 mm.
A quantitative intraspecific test was performed using the GIS data available for every
measured individual in the museum, this made possible the inclusion of an additional
analysis with BIOCLIM variables (Bio1, Bio 12 and BIO 15) vs. the distribution of body
proportions (TL/SVL; CW/SVL) found in individuals collected from different localities,
that were deposited in the amphibian collection at ICN, Instituto de Ciencias Universidad
Nacional de Colombia. Interspecific analyses were performed among sister species such as
Dendrobates truncatus and D. auratus (Dendrobatidae) and from Trachycephalus typhonius
and T. resinifictrix (Hylidae), the remaining species were analyzed independently by
intraspecific comparisons between sisters populations.
Figure 2. Measures used to test body size relations
Results
Seven morphological adaptations related with water economy were described in the dermal
integument: presence of E-K Layer and calcified layers, Presence of specialized lipid
glands; Elevated mucous gland density; epidermal sculpturing and epidermal grooves;
Iridiophores, interdependency with the lymphatic system. Specialized vascular plexa and
verruca hydrophilica, are differentially distributed in the ventral region within a single
species, following apparent geographical patterns. Most of the structures have an
asymmetrical distribution along the anuran skin conferring differential properties to ventral,
inguinal and dorsal portions of the animal. Terrestrial and arboreal species differ greatly in
tegumentary structures and thus were analyzed separately; a descriptive analysis is
presented for the three regions explored; Dorsal, Ventral and Inguinal portions of the frog´s
skin. Results here presented are from nine species in four families including:
Leptodactylidae (Leptodactylus bolivianus, L. fuscus, L. fragilis Pseudopaludicola pusila);
Hylidae (Hypsiboas crepitans, Phyllomedusa venusta, Trachycephalus typhonius);
Bufonidae (Rhinella humboldti), and Ceratophrydae (Ceratophrys calcarata) a table is
presented with the details of the corresponding measurements for each species and
population (Table 1).
Dorsal skin:
Leptodactylids possess a simple dorsal epithelium rich in dermal and subdermal mucous
glands that resemble mucous gland described for other amphibians (Duellman et al. 1986),
these last glands present in both species possess elongated secretory ducts that differ from
previously described structures in other Leptodactylids (Figure 1A), other structures that
can be observed in Leptodactylids are the presence of pores that interrupt the calcified layer
and extend to the surface of the epidermis (Figure 1B). Serous glands are poorly
represented in species like Leptodactylus fuscus and L. fragilis, but can be abundant and
large in Leptodactylus bolivianus (Table 1.). Mucous glands can be also found related with
dorsal folds in L. fuscus (Fig. 5A) and show an aggregate distribution (220 glands in one
millimeter of skin). Intersticial spaces product of cell apoptosis can be observed beneath the
stratum compactum in L. bolivianus (Fig. 4A) Leptodactylids possess a clearly defined
calcified layer (CL) that can be continuous as in L. fragilis (Fig 5B) or poorly interrupted as
in L. bolivianus (Fig. 4A). Dorsal skin from recently emerged post metamorphic individuals
(Dorsal #55 in Table 1) show the early appearance of the calcified layer in the dorsal skin of
this species. Dry area Leptodactylids have a thicker skin than mesic forms but a
considerably thinner stratum corneum (Table 1.)
Figure 4. A) Leptodactylus bolivianus, dorsal skin showing the presence of large serous
glands, a thick calcified layer and sub dermal mucous glands with elongated secretory
ducts; B) Leptodactylus fragilis, dorsal skin showing pore that interrupts the calcified layer.
Bar = 100 µm.
A B
Figure 5. A) Leptodactylus fuscus, dorsal fold sowing high density of mucous glands; B)
Leptodactylus fragilis, dorsal skin showing the presence of mucous glands and poorly
interrupted calcified layer. Bar = 100 µm.
Hylids show a greater diversity of dorsal glands than other dry forest frogs, skin from
Phyllomedusa venusta has three types of serous glands and one type of mucous glands.
Serous glands are specialized syncythia that differ greatly in morphology and secretory
products which stain differently when running a routine dye. Two types of serous glands are
related to the production of proteinous substances employed in defense against predators
and infections, these are the glands defined as Ia and Ib as seen in figure 6 A. Histological
nomenclature used for Phyllomedusa was based on Delfino et al. (1998). Histologically
type I glands produce large collection of sphaeroidal densely stained granules or
translucentlls vesicles, this glad type has a great morphological variation and is
differentiated from other gland types by having a secretory compartment ensheathed by a
contractile layer of mioepithelial cells, but for its upper pole, several layers of
undifferentiated cells rest in the Syncythium. These are both adenoblasts and myoblasts
involved in the cycling of the secretory unit and myoepithelial layer. The neck is localized
at the boundary between the epidermis and dermis and it is encircled by chromatic units, it
holds a slender cavity joining the exiguous lumen of the secretory unit to the gland duct,
which is entirely contained within the thickness of the epidermis. Specialized glands related
to the production of lipid secretions are defined in the literature as Type II glands, these
manufacture discrete secretory bodies appear weakly stained, type II glands possess a
relatively larger lumen than type I glands. The lumen of these glands varies in width and is
bounded by a thin layer of flat cells with nuclei located at the apex of the secretory unit;
cells with a high nucleo-plasmatic ratio are stratified form the gland neck. This species
additionally possess a great number of Iridiophores that form a layer next to
chromatophores just beneath the epidermis. These are deposit centers of urate salts from the
A B
nitrogen metabolism (Fig. 6 A). Particularly this species lacks a calcified layer, but other
hylids such as Trachycephalus typhonius and Hypsiboas crepitans have the presence of a
poorly interrupted layer, the first species has a greater degree of calcification of the layer
reaching width of up to 24.7 µm (Table 1). T. typhonius shows a great abundance of serous
glands that produce proteinous secretions (Fig 7.)
Figure 6. A) Dorsal section from Phyllomedusa venusta showing gland diversity; serous
glands Ia and Ib, and mucous glands (M), and iridiophores (I). B) Dorsal section from P.
venusta showing a detail of a Type II lipid gland that has a larger lumen and structured
secretory vesicles can be observed. Bar=100 µm.
A B
Figure 7. Dorsal skin from Trachycephalus typhonius, showing the presence of a poorly
interrupted calcified layer (CL) and the presence of large serous proteinous substance
secreting glands (SG). Bar = 100 µm.
Other Terrestrial anurans such as bufonid Rhinella humboldti possess a cornified epidermis
with thickened regions that form worts and spines (Fig. 8B); bufonids show epidermal
sculpturing and regions with low gland density, including mucous glands that are poor and
spaced, thus bufonid skin is very dry. Serous glands produce proteinous secretions that are
more concentrated over the extension of the parotid gland. Calcium layer was only found in
the dorsal skin of humid area individuals and was heavy calcified in some regions of the
dorsum, reaching 24.7 µm in thickness (Figure 8A). Arid area individuals lacked the
calcified layer in every region of the body.
Terrestrial burrower, Ceratophrys calacarata possess a unique character in its dorsal skin,
the presence of a thick calcified structure called E-K layer, structure associated with
adjacent living cells (Fig 8C), and this tissue may be 7.49-49.4 µm thick. Skin in this
species is also very thick (128-350 µm). The E-K layer is poorly interrupted and is only
present in the dorsal skin.
Figure 8. A) Dorsal Skin from Rhinella humboldti showing calcified layer (CL) and the
presence of large serous glands (SG); B) Rhinella humboldti dorsal skin showing a spine
formed in the outer epidermis by keratinocytes. C) Dorsal skin from Ceratophrys
calcarata, showing the presence of an E-K layer and mucous gland (M). Bar = 100 µm.
Ventral skin:
Different structures may be found in amphibian ventral skin, some species like
Leptodactylus bolivianus show the presence of a poorly structured ventral region with large
blood vessels (L) Fig 9B. The ventral lymph system lies just beneath the dermis.
Phyllomedusa venusta water absorption pads are located only in the ventral skin, verruca
hydrophilica are present along the venter and Type II lipid glands are completely absent
(Fig 9A). Species such as Hypsiboas crepitans have a specialized ventral water absorption
pad and it is configured by numerous pronounced and vascularized verrucae (Fig 9C.).
Species such as Ceratophrys calacarata lack absorption structures in the ventral skin.
Ventral skin in Pseudopaludicola pusilla differs little from the dorsal skin and has no
specialization to facilitate water absorption. Rhinella humboldti possess well defined and
vascularized ventral verruca.
A B C
Figure9. A) Ventral skin from Phyllomedusa venusta showing the presence of water
absorption verrucae, B) ventral skin from Leptodactylus bolivianus showing a well
extended vascular plexa (L), related to the ventral lymphatic sac; C) ventral skin from
Hypsiboas crepitans showing the presence of well vascularized sulci (S).
Inguinal skin:
Inguinal skin shows fairly specialized water absorbtion pads in most of the species. The
ventral region of the thigh In Leptodactylus fuscus (Fig. 10A.),and most of leptodactylids
show the distribution of vascular plexa formed by epidermal capillaries (EC), that configure
pronounced and heavy vascularized verruca, epidermal capillaries are located in the base of
the verruca and large mucous glands occupy most of the stratum spongiosum (Fig 10A).
Verrucae from arboreal species such as Trachycephalus typhonius differ by having all
epidermal cappilaries in the distal lining of the sulcus (Fig. 10B) and by having a greater
degree of vascularization on the verruca as shown for Trachycephalus resinifictrix in figure
10 D. Terrestrial, Ceratophrys calacarata possess a complex vascular plexa, distributed
along the whole sulcus, some mucous glands (Fig. 10A) are also present. This type of
verruca is the only type of water absorption structure found in the complete ventral region
of this frog. Arboreal Hypsiboas crepitans possess small verruca hydrophilica along the
underside of the thigh, but are not as pronounced and specialized as the ventral structures;
Phyllomedusa venusta, another arboreal species, lacks inguinal verruca and has an
important number of type II lipid glands in this section of the skin, indicating that this is not
a water absorption zone. The bufonid, Rhinella humboldti also show the presence of
inguino-femoral verruca.
A B C
Figure 10. A) Verrucae hydrophilica in Leptodactylus fragilis, showing the presence of
vascular plexa (EC) and large mucous glands; B) Verruca hydrophilica in Trachycephalus
typhonius, showing the presence of vascular plexa in the distal portion of the sulcus (EC);
C) Verruca in Ceratophrys calacarata showing an organized structure for water absorption,
configured by ascending epidermal capillaries along the inner portion of the sulcus (EC)
and the presence of few mucous glands (M). D) Verruca in arboreal Trachycephalus
resinifictrix showing important vascular plexa distributed along the verruca (EC). Bar = 100
µm.
A B
C D
Table 1. Measurements of integument and structures in seven species of seasonally dry forest frogs
and comparisons among sister populations. Leptodactylus bolivianus : Leptodactylidae
Locality
skin Region
and code
TOTAL
Thickness
µm
Stratum
corneum Epidermis
Stratum
spongiosum
Stratum
compactum
Calcified
layer
Continous
or
interrupted
Mucuous
gland
density
Granular
gland
density
Mucous
gland
dimensions
Granular
gland
dimensions
Location of
verruca
hydrophilic
a
Dorsal.#1 333.45 0.247 19.76 24.7 106.21 17.29 continous 18/1000 3/1000 93.1X196 34.3X132.3
Ventral.#2 419.8 1235 24.7 0 296.4 0 0 0 0 0 0
Inguinal. #3 209.95 3705 29.64 49.4 86.45 4.94 Interrupted 13/1000 0 61.75X34.58 0
Dorsal.#37 218.85 4.94 49.4 86.45 79.04 29.64 Continous 53/9,015.5 68/9,015.5 49.4X49.4 74.1X98.8
Ventral.#38 326.04 7.41 49.4 128.44 148.2 25 Interrupted 75/5,693.35 2/10000 56.81X46.93 86.45X83.98
Inguinal.#39 296.4 2.47 24.7 25 244 0 0 35/1000 0 37.05X49.4 0
Dorsal# 55 121.14 0.98 11.76 34.3 74.1 3.93 Continous 74/1000 0 24.7x24.7 0
Ventral#56 148.2 9.88 37.05 37.05 51.87 0 0 85.5/1000 0 49.4X37.05 0
Inguinal#57 83.98 2.47 24.7 24.7 32.11 0 0 42/1000 0 19.76x17.29 0
Leptodactylus fuscus : Leptodactylidae
Arauca Dorsal.#48 153 3.7 24.7 49.4 106.21 2.47 Interrupted 54.34 /
Ventral. #47 116.09 4.94 29.64 37.05 44.46 0 0 64/1000 0 /
Inguinal# 46 160.55 2.47 14.82 49.4 98.8 0 0 /
Dorsal. #49 106.21 24.7 61.75 0 0 40/1000 1/1000 /
Ventral #50 125.97 2.47 29.64 61.75 32.11 4.94 Interrupted 83/1000 1/1000 54.34x61.75 /
Inguinal #
51 / /
Dorsal. #94 271.7 7.41 34.58 148.2 86.45 7.41 Continous 220/1000 / /
Ventral. #95 210.76 0.81 49.4 61.75 98.8 2.47 Interrupted / 49.4x49.4 /
Inguinal #96 106.21 2.47 29.64 69.16 49.4 0 0 / /
Dorsal. #97 169.84 7.41 29.64 71.63 69.16 9.88 Continous 73/1000 / 61.75x74.1 /
Ventral.#98 123.95 7.41 37.05 24.7 54.3 12.35 Interrupted / 49.4x24.7 /
Inguinal.
#99 113.62 2.47 44.46 24.7 66.69 4.94 Interrupted / /
Trachycephalus typhonius : Hylidae
Dorsal.#7 419.9 7.41 37.05 148.2 217.7 24.7 continous 49/1,000 17/1,000 86.45X98.8 358.15X382.85 0
Ventral.#8 359.2 2.47 37.05 358.15 227.24 12.35 Interrupted 21/1,000 7/1,000 74X37.05 358X148.2 Ventral
Inguinal. #9 560.69 2.47 61.75 135.85 407.55 9.88 Interrupted 16/1,000 6/1,000 81.51X61.75 234.65X111.15Inguinal
Phyllomedusa venusta : Hylidae
Dorsal. #19 259 7 37 111 136 / / / / / /
Ventral. #20 252 10 37 86 124 / / / / / /
Inguinal.
#21 254 7 49 136 62 / / / / / /
Dorsal . #25 345.8 4.94 24.7 160.65 135.85 0 0 30/1000 10/1000 79.09x61.75 296.4x239.59
Ventral. #26 358.15 2.47 37.05 197.6 123.5 0 0 47/1000 74.1x209.95 135.85x185.25
Inguinal.
#27 247 4.94 24.70 37.05 49.4 0 0 15/1000 6/1000 / /
Pseudopaludicola pusilla : Leptodactylidae / 0
Dorsal. #40 135.85 2.47 12.35 41.99 86.45 0 0 122/1000 12/1000 37.05x24.7 / 0
Cesar, Dry
forest Ventral. #41 88.92 2.47 24.7 24.7 37.05 0 0 84/1000 2/1000 / / 0
Inguinal.
#42 54.34 4.94 13/1000 / / / 0
Dorsal. # 13 350.74 4.94 49.4 140.79 155.61 7.41 Interrupted 21/1000 18/1000 106.21x123.5136x148 0
Ventral. #14 422.37 2.47 74.1 247 98.8 0 0 22/1000 1/1000 / / Ventral
Inguinal.
#15 340.86 7.41 111.15 123.5 98.8 0 0 15/1000 2/1000 / / Inguinal
Dorsal.#58 229.71 4.94 37.05 98.8 88.92 4.94 Interrupted 44/1000 24/1000 / / 0
Ventral. #59 380.6 4.94 79.04 74.1 0 0 32/1000 8/1000 61.75x61.75 83.98x98.8 Ventral
Inguinal.
#60 2.47 17.29 81.51 49.4 0 0 31/1000 21/1000 49.4x61.75 123.5x61.76 Inguinal
Rhinella humboldti : Bufonidae 0 / / / /
Dorsal # 43 412 12 49 178 173 0 / / / /
Ventral #44 191 5 25 25 136 0 / / / /
Inguinal # 45 210 2 25 49 148 0 / / / /
Dorsal. # 64 340.86 14.82 37.05 135.85 153.18 24.7 0 / / / /
Ventral. # 65 271.7 2.47 24.7 123.5 123.5 0 0 / / / /
Inguinal.
#66 338.39 2.47 29.64 148.2 160.55 0 0 / / / /
Antioquia,
Wet forest
Ventro-
Inguinal
Ventro-
Inguinal
Hypsiboas crepitans: Hylidae
Ventral
Cesar, Dry
Forest ventral
Cesar, Dry
forest
Cundinamar
ca, Wet
Forest
Huila, Dry
forest
Tubará/ Dry
forest
Antioquia,
Dry forest
Inguinal
Huila,
Garzon Inguinal
Cesar Inguinal
Antioquia Inguinal
Cesar, Dry
Forest Inguinal
Huila,
Garzon, Dry
forest Inguinal
Cesar, Dry
Forest Inguinal
Part 2. Body size:
Results for the test of linearity show a direct response for two of the three Bioclim variables
tested Bio 15 and Bio12, (Precipitation seasonality and Mean annual precipitation), Bio 1
(Maximal temperature) showed no clear trend between the species analyzed. Only two
species showed a linear response: The arboreal hylid Trachycephalus typhonius and the
diurnal dendrobatid Dendrobates truncatus. T. typhonius shows a positive linear relation
between cranium width proportions and precipitation seasonality (Figure 11, Table 2). D.
truncatus shows an inverse linear relation between increased precipitation seasonality and
tibial length proportions and a positive relation between increased annual precipitation and
tibial length proportions (Fig12, Table 2). None of the remaining species showed a
significant linear relationship between body proportions and precipitation variables.
Table 2. P value for each test of linearity performed between bioclim variables, Bio 15 and
Bio 12 and considered body proportion (Tibial length and Cranium width)
BIO 12/ TL Bio 12 /Cr Bio 15/ LT Bio 15/Cr
P value P value P value P value
Hypsiboas_pugnax 0.589 0.187 0.7878 0.134
Leptodactylus_fuscu
s 0.898 0.189 0.208 0.222
Leptodactylus_insula
rum 0.293 0.785 0.565 0.357
T. typhonius 0.2957 0.13319 0.711 0.027
T. typhonius and T.
resinifictrix 0.129039 0.07097 0.687552 0.47
Dendrobates_trunca
tus 0.05589 0.683 0.4702 0.523
D. truncatus and D.
Auratus 0.0488 0.689 0.0394 0.519
Pleurodema_brachyo
ps 0.129039 0.07097 0.2138 0.729
Species
Figure 11. linearity test for bioclim variable BIO 15 and Cranium width proportion (Cr).
Figure 12. Linear regression for annual precipitation and seasonality vs Tibial length
proportions (TL) in the diurnal species Dendrobates truncatus.
Discussion
Arboreal and terrestrial species differ in dermal tegument configuration and structure;
terrestrial species show specialized ventral and inguinal patches to actively absorb water
from the substrate, specialized vascular plexa are differently distributed in each species,
showing anatomical differences in the structure of verruca Hydrophilica. In terrestrial
species like frogs from genus Leptodactylus, ventral skin may not have specialized
rehydration structures, this was also evidenced for Pseudopaludicola and Ceratophrys, in
these genera verrucas are restricted only to the inguinal portion of the animals. Some
leptodactylids such as L. bolivianus show a simplified ventral skin associated with the
ventral lymph sac, this interaction between systems may be related to water conservation,
as the lymphatic system serves as a reservoir of water for the blood in case of dehydration
(Hillman et al. 2005). Other terrestrial species as bufonids have drinking water pads in
both ventral and inguinal position. Terrestrial species may possess a thicker and more
keratinized epidermis than arboreal species, especially those burrowing species. Species
like Rhinella humboldti show thicker ventral and dorsal skin in wet forest populations but
similarly thinner ventral and dorsal skin in individuals from the dry forest. Navas (2004),
has proposed that this condition enhances facultative water absorption, even from the dorsal
portion, while the animal is underground (Fig 1B). Burrowing species as Ceratophrys
calcarata possess a wide calcified layer (E-K) that has been argued to be a physical barrier
that reduces water evaporation, but it has also been defined as a plesiomorphic character
within the genus, with no clear function in water conservation (Mangione et al. 2011),
Calcified layers are continuous in Leptodactylus species, with little interruptions except for
gland ducts and pores (Fig. 4A). The calcified layer appears early in the development of L.
bolivianus, being present in recently emerged metamorphs, the calcified layer only appears
to be continuous and thick in the dorsal portion off all species that present it (Table 1).
Mucous gland density can be greater in terrestrial frogs from dry zones and has a function
in both keeping the skin moist for cutaneous gas exchange and in the case of glandular
tissue found in dorsal folds, as a predator deterrent. Mucous glands are also associated with
verruca hydrophilica in most of the species; these glands carry an important function as
secretory units that prevent water loss when the frog is placed in dehydrating substrates.
Arboreal species possess a greater diversity of serous glands and some species have
evolved specialized lipid secreting units (Type II glands). Phyllomedusa venusta shows an
increased abundance of type II glands in the dorsal skin that diminishes when
approximating the ventral skin, the presence of type II glands in the inguinal skin and the
presence of smaller poorly vascularized verruca show that the inguinal portion of the
animal is not specialized in water absorption, suggesting that the venter fulfills rehydration
needs. Phyllomedusa venusta shows large and abundant iridiophores in the dorsal skin,
structures that store urate salts and are thus related with the physiology of water economy.
Hylids Hypsiboas pugnax and species of Trachycephalus show the presence of both ventral
and inguinal verruca, but both differ structurally; specialized organization of the vascular
plexa can be seen in inguinal verruca. Calcified layers appear in some arboreal hylids such
as Trachycephalus and Hypsiboans but are very interrupted, Phyllomedusa venusta, lacks a
calcified layer.
Many features of the dermal organization vary geographically between populations and
may account for variation induced by environmental conditions imposed through ontogeny,
description of this variation still remains preliminary, and thus no sufficient evidence exists
to support an adaptationist hypothesis that accounts for the observed variation. The
analyzed species show a close relation between dermal organization and their natural
history; following three discrete eco types: Arboreal species, fossorial and terrestrial species
with some degree of variation within each ecotype. Tests performed over body size
proportions and environmental variables show no evidence to support Bergmann´s rule,
body proportions are not necessarily related with environmental variables such as
temperature, Precipitation or seasonality, thus few species and population follow a clear
trend. Only two species showed a significant linear relation influenced by precipitation or
seasonality in rainfall. The one that best shows a pattern is Dendrobates truncatus a diurnal
species, who principally inhabits stream forest. Populations from this species inhabiting
places with very low values of mean annual precipitation, have smaller tibia proportions
compared to those populations in places with high rainfall (Fig 12A). A similar pattern
occurs when testing against precipitation seasonality; were tibial lengths are shorter with
increased values of variation (Fig 12 B). Cranium width proportions showed no relationship
for this species, suggesting that the width of the animal´s body is not affected by the
patterns in rainfall. Diurnality can be an important aspect of this frog´s life history, and it
could explain intraspecific variation in body size, as it implies exposure to higher
temperatures and greater water loss as evotranspiration is greater, when compared to the
conditions experimented by species with enhanced nocturnality (Navas, 2004). A second
species that showed a positive relationship between body proportions and precipitation
variables is Trachycephalus typhonius, a positive relation between the coefficient of
variation on the precipitation seasonality and cranium width proportions exists, showing
greater proportions of the skull related to extremely temporal sites with prolonged dry
seasons. A possible explanation for this last trend is difficult to interpret because of the
evident lack of co-osification found on this specie´s head. Further research must consider
that though much of the variability is generated by phenotypic response to environmental
extremes a possible evolutionary trend may exist when comparing sister species, much
more information on this direction is needed.
Acknowledgements: Very grateful with Andrew J. Crawford for his patience and for his
direction in the development of this thesis, this project began from scrap, and evolved
slowly thanks to conversations with him during many years; also with my potential co
director Santiago Madriñan who encouraged part of this work. Special thanks to Dr. Emilio
Realpe, for his help, advice and observations; professor Jhon D. Lynch at ICN for letting
me measure frogs and for his very constructive advice that saved many aspects of this
work; Carlos Navas also has inspired this work and his comments have been important
pillars of this research and special thanks to Fercho at ICA for his wonderful work on the
slides. Fish vet Miguel Mendoza was very helpful with his ideas and advice during the
exploratory phase of the histological work and to my folks Hugo and Luz Mery for helping
me in every step of the way
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Supplementary information:
Significance (p values) obtained in testing of linearity between body proportions versus 3
different bioclim variables: maximal temperature, annual precipitation and seasonality in
precipitation.
Temp Máxima
Temperatura LT Temperatura Craneo
Valor P Valor P
Hypsiboas_pugnax 0.95033 0.19386
Leptodactylus_fuscus 0.7018 0.9424
Leptodactylus_insularum 0.6777 0.6892
T. typhonius and T. resinifictrix 0.5639 0.1658
T. typhonius 0.88 0.2648
Dendrobates_truncatus 0.98 0.64832
D. truncatus and D. Auratus 0.9151 0.7652
Pleurodema_brachyops 0.7219 0.7399
Precipitación Anual
Precipitación LT Precipitación Craneo
Valor P Valor P
Hypsiboas_pugnax 0.589 0.187
Leptodactylus_fuscus 0.898 0.189
Leptodactylus_insularum 0.293 0.785
T. typhonius 0.2957 0.13319
T. typhonius and T. resinifictrix0.129039 0.07097
Dendrobates_truncatus 0.05589 0.683
D. truncatus and D. Auratus 0.0488 0.689
Pleurodema_brachyops 0.129039 0.07097
Precipitación Estacionalidad
Estacionalidad LTEstacionalidad Craneo
Valor P Valor P
Hypsiboas_pugnax 0.7878 0.134
Leptodactylus_fuscus 0.208 0.222
Leptodactylus_insularum 0.565 0.357
T. typhonius and T. resinifictrix 0.711 0.027
T. typhonius 0.687552 0.47
Dendrobates_truncatus 0.4702 0.523
D. truncatus and D. Auratus 0.0394 0.519
Pleurodema_brachyops 0.2138 0.729