Taxonomic position of some rana species in a dendrogram from their globin composition

Comp. Biochem. Physiol. Vol. 70B, pp. 421 to 425, 1981

0305-0491/81/I 10421-05102.00/0 Copyright © 1981 Pergamon Press Lid

Printed in Great Britain. All rights reserved

TAXONOMIC POSITION OF SOME RANA SPECIES IN A DENDROGRAM FROM THEIR GLOBIN COMPOSITION* P. CARDELLINI l, G. CASASANDREU2, C. DI BELLO3, R. MARTINO4 and M. SALA 1 qstituto di Biologia Animale, Universit/~ di Padova, Italy, 2Instituto de Biologia, Universidad Nacional Autonoma de Mtxico, aIstituto di Chimica Industriale, Universit/l di Padova, Italy and 4Istituto di Fisiologia Umana, Universit~ di Padova, Italy (Received 10 March 1981)

Abstract--1. The aminoacidic composition of the globin of the genus Rana has been analyzed. 2. The resulting data and those from five other species of Ranidae, from one species of Pipidae and one of Bufonidae having been compared in pairs according to the formula of Harris and Teller gave

origin to a "dissimilarity matrix". 3. In a "Phylo" computer program, consistent with Moore's model, the data of the input matrix have been used to find the mutual taxonomic relations of the 11 anuran species examined.

INTRODUCTION Among the proteins analysed for problems in molecular evolution, haemoglobin is much used because it is widely diffused and easily purified from red cells. The haemoglobin subunits are built with polypeptidic chains relatively short (140-150 residues) whose chemical composition can be analysed in quite good condition with current protein chemistry techniques (Chauvet & Acher, 1971). Haemoglobin is a useful evolutionary index not only for practical reasons. In contrast to most enzymatic proteins, this protein is responsible for an entire physiological function, oxygen transport and therefore it is directly subjected to selective pressure. This function is a phenotypical character on which selection acts, apparently related to a few structural genes and not to many as in the case of a polymorphic function (Simpson, 1964). With the data from the analysis of globins from different species of animals the globin phylogenetic tree has been constructed (Dickerson, 1973). If the genetic similarity, based on protein composition and the organic similarity, resulting from taxonomic position, are correlated (Selander & Johnson, 1973; Avise, 1974), the phylogenetic trees for many species may be constructed on the basis of the likes and unlikes of their haemoglobins. The elaboration by a computer of the data of the variations in the globin and in its genealogic tree, according to a particular program, has been used to find the rate of molecular evolution in different metazoan species. Hence it was found higher in mammals than in lower vertebrates, but of the mammals the lowest rate occurred in the higher primates (Goodman et al., 1971). * This research was financially supported by the Italian Ministry of Education. 421

For their phylogenetic position between aquatic and terrestrial vertebrates, amphibians are of particular interest for research into transitional molecules. In the haemoglobin of amphibians much variation has been found at molecular level even within the same genus (Baldwin & Riggs, 1974); so that their haemoglobin turns out to be excellent material for studying the relationship between the phylogenetic and the taxonomic position on the basis of molecular evolution within small systematic units, where karyological and morphological analysis has so far been ineffective in solving restricted phylogenetic problems (Morescalchi, 1974). Research of this type has been done on various species of the genus Bufo (Guttman, 1972) and of the family Hylidae (Perez et al., 1975). The aim of the present research was to construct a phylogenetic tree of some species of the genus Rana with data from the chemical composition of their globins, mathematically elaborated from a computer according to Moore et al.'s model (1972) for phylogenetic problems. MATERIALS AND METHODS For chemical analysis the haemoglobin from adults of the following species was used: Rana berlandieri (from Chamela, Jalisco, Mexico), Rana latastei (from Padua, Italy), Rana montezumae (from Valte de M~xico, Mexico) and Rana palraipes (from Las Tuxlas, Veracruz, Mexico). Their red cells were separated, purified and haemolysed; then the haemoglobin was purified through a chromatographic column of ultragel ACA 44 and dialyzed. The globin was precipitated in acid acetone at -20°C and dried in vacuum. The quantitative determination of the aminoacids of the globins, after acid hydrolysis under standard conditions (6N HCI in evacuated sealed tubes, at ll0°C for 24, 48 and 72 hr), was obtained by means of an automatic aminoacids analyzer C. Erba Mod. 3A28, equipped with an automatic sampler Mod. 128. For serine and threonine, because of

422

P. CARDEFLINI et al, Table 1. Aminoacid composition of the hemoglobin (residues/mol) of the 11 Anuran species studied in the present paper

~

-~.

Amino acid Lysine Histidine Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Half Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

37 46 21 70 25 28 52 24 38 70 4 44 6 19 83 17 33 6

37 45 17 68 22 40 56 21 40 69 6 48 7 17 83 19 31 4

43 48 17 69 26 35 46 22 38 72 7 50 8 19 83 16 33 5

37 47 18 63 25 32 56 23 41 71 4 42 6 2t 81 15 33 7

36 43 25 52 20 37 46 20 35 63 9 38 5 16 69 20 30 --

38 43 25 56 31 35 46 20 37 63 9 41 4 19 66 20 31 --

41 44 18 61 22 36 45 20 36 63 9 44 3 16 53 18 30 --

41 52 17 58 21 29 44 21 38 74 6 38 4 17 69 20 30 5

41 50 15 62 25 39 38 20 36 75 8 42 4 22 69 19 30 6

45 43 19 59 28 41 44 22 24 59 11 40 3 19 62 16 34 7

46 42 16 66 31 37 36 23 39 69 2 30 13 28 65 25 34 6

* Directly analysed in the present work. f Trader C. D. and Frieden E., 1966. ++Tentori L. et al., 1965, ~Cristomanos A. A., 1967, 1972. ¶ Ferigo E. et al., 1972. their lability, the data were calculated at time zero (Moore & Stein, 1963). The titration of cysteine was done by Ellman's method (1959). The values of tryptophane were calculated according to the modalities suggested by Beaven & Holiday (1952). We compared the resulting data with each other and also with those found in the literature for other species of frogs. These data, elaborated according to the mathematical formula of Harris & Teller (1973) as modified by Ghiretti-Magaldi et al. (1975), gave the "composition divergence" (D) between the globins of the various species. The observed divergence normalized (i,e. OD1) has to be considered the measure of the degree of affinity between two proteins. Values close to zero represent almost perfect homology between two proteins, i.e. taxonomic closeness of the species to which they belong. The reciprocal taxonomic positions of the frog species examined on the basis of their phyletic distance (graphed as a comparative Table called a "dissimilarity matrix") were determined by well defined algorithms according to a model proposed by Moore et al. (1973) for phylogenetic problems. On the basis of Moore's model a program in Fortran IV. H, called "Phylo" was made*. The elaboration of this program by an IBM computer 370/158 gave the most credible phyletic network. By its nature this network is an open one, i.e. without any apparent point of origin. To establish this point the data of the species Xenopus laeris have been inserted in the program together with the data of the frogs (Rana). Xenopus laevis belongs to the Pipidae, generally considered one of the most primitive families (Estes, 1975}. Therefore the dichotomy point from which Xenopus separates from the other species has to be considered the point closest to the origin. The various species of Rana have been located in the oriented tree according to the sequence given by the computer program. * This program was made ex novo by Dr P. Zampirollo.

It was opportune to use also the data concerning the globin of Bufo spinosus of the Bufonidae, generally considered more recent than the Ranidae. Therefore these data are suitable as a control of the validity of the dendrogram: if the phyletic tree is correct, then on it Bufo should be farther from Xenopus than from the Rana species. RESULTS The quantitative values of the residues of 16 aminoacids, obtained from either our analysis or the literature concerning the eleven a n u r a n species, are c o m p a r e d in Table 1. Table 2 reports the numerical data from the binomial comparison of all the quantitative data concerning the globins of each species with those of the other species. The complex of all the resulting global divergencies forms the "dissimilarity matrix" in which the highest values occur in c o m p a r i n g the data of the globin of Xenopus laevis with those of all the other species. Also in the c o m p a r i n g of the globin of Bufo with those of the Rana species the values are high. W i t h i n the species of Rana the lowest coefficient of dissimilarity is obtained from the comparison between Rana grylio a n d R. catesbeiana. Even the comparisons between R. palmipes a n d R. montezumae, R. palmipes a n d R. berlandieri, R. berlandieri a n d R. montezumae also gave low values. The highest values are those from the comparisons between R. berlandieri and R. esculenta, R. berlandieri a n d R. ridibunda, R. montezumae and R. esculenta, R. montezumae a n d R. ridibunda. In Fig. 1 !s reported the d e n d r o g r a m resulting from the computerised elaboration, according to the program Phylo, of the data of .the dissimilarity matrix.

Taxonomic position of Rana species

423

Table 2. Dissimilarity matrix between nine species of the genus Rana (Ranidae), one species of the genus Bufo (Bufonidae) and one of the genus Xenopus (Pipidae), from elaboration of the data reported in Table 1 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (i) R. palmipes (1) 0.00000 0.00556 0.00691 0.00447 0.00850 0.00860 0.01005 0.01243 0.01162 0.01453 0.01985 R. berlandieri (2) 0.00000 0.00773 0.00642 0.00812 0.00905 0.01069 0.01406 0.01298 0.01592 0.01991 0.00000 0.00796 0.00963 0.00974 0.00953 0.01199 0.01009 0.01301 0.01712 R. latastei (3) R. raontezumae (4) 0.00000 0.00829 0.00874 0.01058 0.01207 0.01165 0.01482 0.02047 0.0000 0.00384 0.00808 0.00984 0.00979 0.01262 0.01869 R. grylio (5) 0.00000 0.00570 0.00964 0.00888 0.01097 0.01902 R. catesbeiana (6) 0.00000 0.00904 0.00793 0.00871 0.02079 R. pipiens (7) 0.00000 0.00633 0.01396 0.02107 R. esculenta (8) 0.00000 0.01218 0.01943 R. ridibunda (9) 0.00000 0.02098 Bufo spinosus (10) Xenopus laevis (11) 0.00000

The dichotomy starts with the divergence of Xenopus and, through internal points of dichotomy, ends with the terminal branches corresponding to the species of anurans examined. The intermediate points represent only different steps in the molecular evolution. The dichotomy is based on the supposition that the evolutionary mechanism follows a line of binary choice arising from the mutations at molecular level. The type of dendrogram resulting, considered "open", is an indication of a fast developing evolutionary process (Sneath& Sokal, 1973). DISCUSSION AND CONCLUSIONS

The comparative analysis of the aminoacid composition of proteins, a "basis for investigations on their origin and evolution" (Dayoff et al., 1972) is regarded as a technique useful in studying the phylogenetic relationships of organisms (Dickerson, 1971). This analysis may be used even to find the speed of evolution typical for each biological group and to supply data for comparing molecular and organic evolution (Wilson, 1976). The quantitative variations of the aminoacids may be useful also for finding the direction of the evolution and the systematic position of different groups of organisms (Calhoon & Aaronson, 1979). From the analysis of the aminoacid composition it is possible to evaluate the homology between the members of one family of proteins (Fitch, 1973), and, 11 4

!

2

3

6

5

8

9

7

I0

R. p a ~ m i ~ e s

(atastei ~!u,nac cat¢~Bet=~a pipic~t~ c$cu(. ta ~(d~bu,c*lEda (acv(~ R.

R. R, R, R.

XeJ~,pus

8 9

II

Fig. 1. Dendrogram of the eleven Anuran species from the computerised elaboration of the dissimilarity matrix reported in Table 2.

in the absence of data on the sequence of the primary structure, it is meanwhile possible to establish reliably the degree of homology of different organisms, using the quantitative data of the aminoacid residues elaborated according to the formula of Harris & Teller (1973). The results obtained from this elaboration are similar to those from comparing the aminoacid sequence of different species, particularly when the structural differences between the compared proteins are relatively small (Harris & Teller, 1973). The degree of similarity increases when introducing in the formula the number of codons concerning each aminoacid (Ghiretti-Magaldi et al., 1975). In this type of quantitative analysis it is impossible to evaluate the reciprocal substitution which may occur in the primary sequence in the different proteins. However, the fact that such reciprocal substitution has a very low frequency signifies that it can cause little error, in the results (Van Drunten et al., 1978). The dissimilarity matrix resulting from the application of the Harris & Teller formula has to be considered therefore quite reliable. The composition divergence reflects then the phylogenetic distance of the species to which belong these proteins. The reciprocal composition divergence of the globins between all the Rana species considered is remarkably lower numerically than the values obtained from comparing the globin of Xenopus laevis and that of Bufo spinosus, though at minor level. Thus from these results all the species of Rana constitute a group intermediate between Xenopus and Bufo. Within these Rana species the divergence, at its lowest level between grylio and catesbeiana, progressively increases in comparing palmipes and catesbeiana, palmipes and berlandieri, catesbeiana and pipiens, ridibunda and esculenta. Recently it has been proved that the species esculenta is really a hybrid from crossing the two species, lessonae and ridibunda (Dubois, 1977). It seems, therefore, that, between esculenta and ridibunda, affinity should be the highest compared with the other Rana species. Karyological investigations indicate in the extant R. esculenta different populations in which the tendency to form one species more and more differentiated from their ancestors is variably pronounced. The data of the esculenta (Tentori et al., 1965) in comparison with those of the ridibunda (Christomannos, 1971) do not confirm the expected very close affinity between the two species. Presumably the described specimens of esculenta, from which

424

P. CARDELLINIet al.

the data derived, belong to a population considerably differentiated from the ridibunda. It is very difficult to explain the remarkable dissimilarity between berlandieri and pipiens: the former is considered the more adapted terrestrial form in the "complex" pipiens (Conant, 1975). The dendrogram from the analysis of any type of real input cannot be a perfect dendrogram, i.e. the search for an absolute minimum in the function of Moore is an excessive demand because the real matrix of dissimilarity only partially satisfies the mathematical conditions by which the model is strictly bound. The mathematical model chiefly used reacts too sensitively to any fluctuation even small. The model of Moore was conceived essentially for the elaboration of the data obtained from a direct comparison between the primary structures of homologues proteins, which data contain more reliable information than that given by the application of Harris & Teller's formula to the quantitative data of the aminoacid residues. From these errors in the quantitative evaluation may rise, which, even small, may reduce the strength of the model, bounded by very strict working rules. Despite such inconveniences the model represents the most rational and objective instrument for the specification of a reliable phylogenetic tree. The computer puts out the best non-oriented network according to the entering data. The orientation of the net, i.e. the definition of the phylogenetic tree, is one extramathematical operation, depending upon purely biological considerations and hypotheses. The choice of the root is the most delicate moment in this operation and the species fit for the role of the ancestral point should be a species recognised objectively as the oldest among all the species considered. The dendrogram supplies graphically the phylogenetic relationship of the species, each having a particular point on the dendrogram, equivalent to its position on the evolutionary line of the globin. The dendrogram confirms the precocious separation from the Pipidae, considered primitive and ancestral, of a branch which gradually gave rise first to a primitive group of frogs, of which some neoarctic species extant and finally to the genus Bufo. The position in the dendrogram stems from the chronological succession of the genera Xenopus (Pipidae), Rana (Ranidae) and Bufo (Bufonidae) corresponding, at family level, to the succession found by means of the simultaneous evaluation of many morphological parameters (Lynch, 1973; Duellman, 1975). At this level, however, using one parameter alone, the analysis of the globin, gave the same units as got by using many parameters. Hence, monothetic and polythetic units are coincident (Sneath& Sokal, 1973). Within the genus Rana the species montezumae, palmipes, berlandieri, catesbeiana and grylio present a position farther from ridibunda and esculenta than from Xenopus laevis. Therefore they should be considered older than these two species. The position of latastei in the dendrogram between the neoarctic species confirms what has been observed in mathematical research as applied to biology, namely the possibility that dendrograms built on the basis of one parameter alone with very simple and clear results, could give,

however, some unexpected results owing to the particular evolution of the chosen parameter in one species, evolved differently compared with the other species considered (Sneath & Sokal, 1973). Inside the group of frogs, in the group of primitive species, appear strictly correlated palmipes, montezumae, and berlandieri; in the intermediate group grylio and catesbeiana, and in the most recent, esculenta and ridibunda. These relationships are coherent with the systematic position assigned to them in the current taxonomy, whereas the position of pipiens in relation to berlandieri, systematically close to each other, is very distant in the dendrogram. It is very interesting to note that the systematic affinity of the two palearctic species, esculenta and ridibunda, does not appear so close in the dissimilarity matrix, but it is close in the dendrogram as in the dichotomy keys of the systematics. Finally, the position of latastei, a species adapted to a terrestrial life, appears, in both the dendrogram and taxonomy, far from the water species, esculenta and ridibunda. There does not appear to be a parallelism between phyletic position and adaptation to terrestrial life, at least in the species examined. Acknowledgements--We thank Professor G. W. Moore for his suggestions and the sending of his program "Itera" which we compared with our "Phylo" program; Professor R. Mitchell for help with the manuscript and Dr A. R. Salvemini, Mr U. Arezzini and Mr C. Friso for technical assistance. REFERENCES

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