Amphibian epithelial and morphological adaptations to dry ...

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




T. typhonius 0.2957 0.13319

T. typhonius and T. resinifictrix0.129039 0.07097




Precipitación Estacionalidad

Estacionalidad LTEstacionalidad Craneo

Valor P Valor P




T. typhonius and T. resinifictrix 0.711 0.027

T. typhonius 0.687552 0.47




Amphibian epithelial and morphological adaptations to dry ...

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