De La Salle University College of Science The Graduate School Biology Department

MORPHOMETRIC ANALYSIS OF SELECTED ANURAN SPECIES AT DIFFERENT GOSNNER STAGES FOUND IN DIFFERENT GEOGRAPHICAL LOCATION
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Genesis Julyus T. Agcaoili , Jean Marixel B. Caspe , Fernando D. Fabia Jr ., Mark Archei O. Javiera, Gian Carlo E. Sorianoa Biology Department, De La Salle University, 2401 Taft Ave., Manila

ABSTRACT

Intraspecific morphometric variations at different larval (Gosnner) stages were studied in a number of population samples of different anuran species relative to their geographical locations. Tadpoles were collected from seventeen (17) areas in the Philippines and Indonesia. Four (4) morphological characters: body length, total length, maximum tail height, and minimum tail height, served as the bases for analysis. Using multiple regression and repeated statistical measures of ANOVA (Analysis of Variance), it was found out that the morphological characters used in the study are reliable bases for identification, classification or characterization of the anuran species. It was also shown that among the four morphological characters studied, body length and maximum tail height as best-fit indicators of the taxon, as correlated to the geographical location of the specimen concerned in the study. It is then surmised that geography and ecology can influence the morphometry of the organisms and thus the morphological characters used in the study are good discriminators for distinguishing one taxon from the other.

Keywords: Anuran, morphometric analysis, gosner stage, body size, biogeography

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1. INTRODUCTION
Anuran amphibian tadpoles have interesting physical attributes: they have a composite head and body and a muscular tail with no vertebrae; pair of eyes and usually external nares; and spiracles that ensure the exit of water pumped through respiratory and food trapping structures (Saidapur, 2001). There are certain morphological variations that are evident in these anuran amphibian tadpoles that cannot only be attributed because of their adaptation to diverse habitats such as puddles, ponds, stagnant or gently flowing water bodies or even to fast- flowing streams but also because of phylogeny (Duellman & Treb, 1986; Mc Diarmid & Altig, 1999; Saidapur, 2001). These anuran tadpoles are easily and heavily influenced and thereby with varying ecological conditions, morphological features, time of the year when they are found in different geographical zones, length of time of metamorphosis, and size at transformation differs within and between species (Saidapur, 2001; Lima & Pederassi, 2012). The growth and development rates in anurans are affected by numerous environmental factors such as temperature, rainfall, photoperiod, pool dessication, food supply and diet quality, environmental iodine levels, pond hydrology, and breeding habitat. Intrinsic factors such as tadpole size or egg size and yolk reservoirs can also influence the growth and development of the anuran (Saha & Gupta, 2011). Generally, these morphological differences are related to ecological conditions. For instance, habitat selection and body form, foraging behavior, and oral armature are interrelated (Saidapur, 2001). Indeed, the plasticity of the anurans development is largely contributed also to their great ecological and geographical diversity (Lima & Pederassi, 2012). It should be then noted that the understanding of the tadpoles environmental occupation or habitat by tadpoles is associated to morphological differences. However, there are inadequate records of tadpole descriptions. Even in some areas of the world, the description of anuran amphibian tadpoles morphology is not greatly exhausted (Mc Diarmid & Altig, 1999; Saidapur, 2001). Tadpoles, especially, if they can be appropriately identified and described, would provide a more efficient means of sampling the occurrence of species at a given site than their adult counterparts (Mc Diarmid & Altig, 1999). There have been some studies conducted that attempted to interpret morphology in the context of tadpole ecology, however, there are mixed results (Mc Diarmid & Altig, 1999, Saidapur, 2001). For these anuran amphibian tadpoles to be distinguished from one another and be evaluated for their ecological or evolutionary significance, morphometric analysis is performed. Morphometric analysis deals with the variation and change of form, specifically size or shape of organisms and has been

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increasingly utilized in developmental contexts (Klingenberg, 2002; Larson,2005). It has been applied in different fields of Biology. An example of information which can be derived from data acquired from morphometrics is whether a specific structure can be used as a feature to discriminate one taxon from another. A structure can be used that way if it is uniquely presented in a certain taxon compared to others (Verbruggen et al. 2005). On the other hand, if a certain structure is similarly presented in many taxa, then it cannot serve as a reliable basis of identification, classification, or characterization of a taxon. One may know whether a structure can serve as a reliable analysis for these purposes or not by performing a variety of statistical analysis. In every study on morphometrics, multivariate statistics has been the chosen statistical method. At present, the new method of morphometrics combine geometric concept of shape with the procedures of multivariate statistics, and compose a powerful and flexible set of tools udes for analysis of morphological variation. (Klingenberg, 2002). In this study, a morphological comparison was done among the specimens collected from different localities wherein each species had different Gosner stages. The present study records the occurrence of anuran tadpoles in the seventeen (17) specific sites in Mindoro, Bukdinon, Cotobato, Palawan, Zambaonga, and Java, Indonesia and describes the morphometry of the tadpoles. The purpose of this study is to have an in-depth analysis of both morphometric and qualitative external morphological characteristics of different larval stages in a number of population samples of these anuran tadpoles found in different localities.

2. OBJECTIVES
The general objective of our study is to examine and analyze patterns and relationships of morphometric variations of different anuran species at various Gosnner stages found in different geographical location. The study specifically aims to attain the following objectives:

1.

To identify the relationship between the independent morphometric indicators of different

anuran species namely; body length (mm), total length (mm), minimum tail height (mm), max tail height (mm), and Gosnner stages found in different geographical locations in Philippines and Indonesia. 2. To recognize the level of significance existing between and within this group parameters using the repeated statistical measure of ANOVA.

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3. MATERIALS AND METHODS

Samples. Tadpoles were collected from seventeen (17) localities found in the Philippines and Indonesia namely: Siburan forest, Sablayan, Mindoro; Brgy. Silipon: So. Mahayahay: Mt Kitanglad, Libona, Bukidnon, Brgy. Tagabinet: Kayasan, Puerto Princesa, Palawan; Mt. Baco Range: Alangsa Creek, Baco, Oriental Mindoro; Lake Sebu, South Cotabato; Estrella Falls, Brgy. Estrella,Narra, Palawan; Brookes Point, Palawan; West Java, Indonesia; Zamboanga; Mt. Apo, South Cotabato; Aborlan, Palawan; Mt. Halcon, Oriental Mindoro; Davao del Sur; Pasonanka National Park , Zamboanga; Agcaoayan Creek, Puerto Galera, Oriental Mindoro; and Barrio Lamlahak: Mt. Three Kings: Foi Creek, Lake Sebu, South Cotabato, Mindanao. Tadpoles were caught by a dip net and were preserved in 70% ethanol (Mc Diarmid & Altig, 1999). Classification and Development Stage. Developmental changes were determined according to Gosner (1960). In this analysis, three distinct stages of tadpole development (Etkin, 1968) can be observed from the data: premetamorphic (growth of feeding tadpoles; Gosner stage 25-35), prometamorphosis (growth and differentiation of hind limbs; Gosner 36-41), metamorphic climax (forelimb development and tail resorption; Gosner 42-46).

Characters studied. Morphometric measurements were made with a vernier calliper, a mm ruler, and/ or an ocular micrometer fitted to a dissecting microscope. Measurement of tadpoles were based on existing studies by Tyler (1968), Altig (1970), Mc Donald & Davies (1990) and Meyer (1991). Analyzed larvae were in various stages of development: from 25 to 44 (according to Gosner 1960). A total of four developmental stages were measured: tadpoles without limbs (Gosner Stage 21-25), with limbs emerging (Gosner stage 26-29); two fully grown limbs (Gosner stage 30- 34); and with four limbs (Gosner stage 3539). The following characteristics were then measured: body length (from the snout to the anterior margin of the hindlimb at insertion), total length (from the snout to the tail tip), maximum tail length, and minimum tail length. Statistical Analysis. Using the data provided, it can be analysed using repeated measures of Analysis of Variance (ANOVA). ANOVA was performed on untransformed values of the data to verify whether the sample means of allopatric groups are derived from the same population. It basically identify if there are significant differences among the population of tadpoles found in varied locations. Data were also

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subjected in multiple regression analysis since the data have dependent and independent variables. A post-hoc analysis using Tukey test was administered in order to evidently state the significance among localities and different dependent variables of the study. Data were analyzed using the software STATISCA 10.0 version.

4. RESULTS AND DISCUSSION

A total of two hundred fifty five specimens, at various Gosnner stages, were collected and sampled from seventeen different localities in the Philippines and nearby country. Multiple regression was employed on this study to identify the relationship between the independent morphometric indicators of Anuran species namely; body length (mm), total length (mm), minimum tail height (mm), max tail height (mm), and Gosnner stages to its dependent criterion which is the geographical location. This was carried out with the response variable Y (the geographical location of the specimens collected) depending on five explanatory variables (x1, x2, x3, x4 and x5), namely the independent morphometric indicators of Anuran species, the body length (mm), total length (mm), minimum tail height (mm), maximum tail height (mm), and Gosnner stages. The mean responses depend on these explanatory variables according to the linear function: y = 0 + 1x1 + 2x2 + ... pxp Table below presents the summary of values between the several explanatory variables (morphometric measures of Anuran specimens from various geographical locations where specimens where collected, together with its residual plots. Table 1. Summary Table of Morphometric measures of Anurans Observed Value Predicted Value Residual Standard pred.value Standard residual Standard error (Pred.Value) Minimum Maximum Mean Median 25.00000 44.00000 31.83607 32.00000 28.79448 33.27875 31.83607 31.87742 -8.27875 11.84232 0.00000 -0.99848 -2.71075 1.28577 0.00000 0.03685 -1.36170 1.94784 0.00000 -0.16423 0.778967 2.265572 1.055527 0.958483

This further explains there are indeed relationships existing between the response and explanatory variables. The residual values in table 1 determine that this multiple regression model is appropriate for the data. It is illustrated in Figure 1 below.

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Normal Probability Plot of Residuals 3

Expected Normal Value

-1

-2

-3 -10

-8

-6

-4

-2

2 Residuals

10

12

14

Figure 1. Normal graph showing the scatter plot matrix of the samples.

The plots all appear to show more or less random relationships around the center value. To recognize the level of significance existing between and within these group parameters, the repeated statistical measure of ANOVA was carried out. Results are summarized in Table 2 and illustrated in Figure 2. TABLE 2. Tukey HSD Test among the explanatory and response variables (Correlation is significant at the .05 level {2-tailed}) Locations 1 2 3 4 5 6 Gosnner Stage / / / / / / Body length (mm) / / / / / / Total length (mm) / / / / / / Minimum Tail Height (mm) / / / / / / Maximum Tail Height (mm) / / / *** / /

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7 8 9 10 11 12 13 14 15 16 17
*** denotes significance / denotes no significance

/ / / / / / / / / / /

*** / *** *** *** *** / / / *** /

/ / / *** *** / *** / *** *** /

/ / / / *** *** / / / / /

*** / / *** *** *** *** *** / *** /

Note: Coding manual for the location is provided in the appendices

Locality; LS Means Wilks lambda=.01346, F(80, 196.88)=3.5617, p=.00000 Effective hypothesis decomposition Vertical bars denote 0.95 confidence intervals

120 100 80 60 40 20 0 -20 1 3 5 7 9 Locality 11 13 15 17 Gosnner Stage Body Length (mm) Total Length (mm) Min Tail Height (mm) Max Tail Height (mm)

Figure 2. Level of Significance existing between and within Variables The harmonic mean of all the explanatory variables relative to the locations where the species were found was shown in (Figure 2). The figure shows that a 95% confidence intervals, the variable total body length (mm) covers the widest range of deviation of mean values as

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compared to the least deviation of mean values for the variable minimum tail height (mm). Tukey HSD test was utilized by the researchers in order to evaluate if the values of the possible pairs of explanatory means are equal, assuming that the samples were all of the same.

5. CONCLUSIONS

With the assumptions that tadpoles collected from the same place where classified under the same species or taxon, different morphological features were treated separately to indicate the significance to the response variable. It is in the assumption that one feature may be similar for different taxa but another feature may be presented differently in different taxa. In the context of the data given to us, after treatment with the appropriate statistical analysis, we conclude that morphological features of different Anuran species can be a good basis of its identification, classification or characterization. Body length and maximum tail heights of tadpoles can be considered as best indicative of their taxon, as significantly correlated to the geographical location of the specimen concerned in the study.

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LITERATURES CITED

Klingenberg CP. (2001). Morphometrics and the role of the phenotype in studies of the evolution of developmental mechanisms. Elsevier Larson PM. (2005). Ontogeny, Phylogeny, and Morphology in Anuran Larvae: Morphometric analysis of cranial development in Rana tadpoles (Anura: Ranidae). JMorphol. Lima MSCS, Pederassi, J. (2011). Morphometrics and ratio of body proportionality of tadpoles of Rhinella icterica (Anura, Bufonidae) at different developmental stages. Brazilian Journal of Biology Vol. 72 Issue 3, p 623 Mc Diarmid RW, Altig R. (1999). Tadpoles: The Biology of Anuran Larvae. University of Chicago Press Saha BK, Gupta BBP. (2011). The Development and Methamorphosis of an Endangered Frog, Rana Leptoglossa (COPE, 1868). International Journal of Advanced Biological Research Saidapur SK. Behavioral Ecology of Anuran Tadpoles: The Indian Scenario. PINSA

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