ISSCA

International Society for the Study

and Conservation of Amphibians

(International Society of Batrachology)

SEAT

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Source : MNHN, Paris

AITTES

INTERNATIONAL JOURNAL OF BATRACHOLOGY

October 1994 Volume 12, N° 3 °

Alytes, 1994, 12 (3): 93-108. Bibliothèque Centrale Muséum 93

3001 00111632 5

Comparative electrophoretic investigation on

Rana balcanica and Rana ridibunda

from northern Greece

Theodora S. SOFIANIDOU*, Hans SCHNEIDER** & Ulrich SINSCH***

* Department of Zoology, University of Thessaloniki, Thessaloniki, Greece

** Zoologisches Institut der Universität, Poppelsdorfer SchloB, 53115 Bonn, Germany

*** Institut für Biologie der Universität, Rheinau 3-4, 56075 Koblenz, Germany

A total of 315 water frogs pertaining to either Rana ridibunda or Rana

balcanica was collected at 28 sites distributed over northem Greece. The intra-

andinterspecific variability of the gene pools was assessed by applving vertical

polyacrylamide gel electrophoresis on samples of blood, liver, skeletal and

heart muscle. We compared the allelic variation of eight enzyme loci and of

two non-enzymatic proteins among populations and species using the

UPGMA,, Fitch-Margoliash- and maximum-likelihood-methods. The variability

between conspecific populations was considerably smaller than the interspe-

cific variability. Moreover, at the ADA locus altematively fixed alleles were

found in R. balcanica and R. ridibunda. Thus, the species status of the

bioacoustically detected R. balcanica was confirmed.

INTRODUCTION

Until recently Rana ridibunda Pallas, 1771 has been considered a monotypic species

with a wide geographical range including northern Africa, Europe and the western regions

of Asia (MERTENS & WERMUTH, 1960; GÜNTHER, 1990). However, intensive bioacoustic

studies on features of the mating calls recorded in populations in Armenia (SCHNEIDER &

EGIASARJAN, 1990), Egypt (AKEF & SCHNEIDER, 1989), Greece (SCHNEIDER & SOFIANIDOU,

1985; SCHNEIDER et al., 1984), Israel (NEVO & SCHNEIDER, 1983), Kazakhstan (SCHNEIDER

& EGiasaRJAN, 1991) and Turkey (JOERMANN et al., 1988) led to the unequivocal

distinction of three different species within this geographical range (SCHNEIDER & SINSCH,

1992): Rana ridibunda in the terra typica restricta in Kazakhstan, and in Armenia, eastern

Greece and Bulgaria, Rana levantina (SCHNEIDER et al., 1992) in Egypt, Israel and western

Source : MNHN, Paris

94 ALYTES 12 (3)

Turkey, and Rana balcanica (SCHNEIDER et al., 1993) in Greece, Albania and Yugoslavia.

Additional morphometric comparisons revealed slight morphological differentiations

between these species (SCHNEIDER et al., 1992, 1993). Analyses of allozyme variation

among the bioacoustically detected taxa corroborated their species status (NEVO &

Ficiucci, 1988; NEVO & YANG, 1982; SiNsCH & EBLENKAMP, 1994; SOFIANIDOU, unpubl.

data).

All previous studies on the allozyme variation between the new species R. balcanica

and R. ridibunda have been performed on a rather large geographical scale and focused on

resolving the taxonomic problems by comparison of bioacoustically classified populations.

In the present investigation, allozyme electrophoresis was used to specify more precisely

the distribution of R. balcanica and R. ridibunda in Greece. Our aims were: (1) to assess

the genetic variability of each species on a small geographical scale by collecting samples

along an east-west transect through northern Greece; (2) to compare the intraspecific and

interspecific genetic variation; (3) to investigate the stability of genotypes at the

neighbouring limits of geographical distribution at the Nestos River; and (4) to compare

the results with those of the bioacoustic studies.

MATERIAL AND METHODS

At 28 sites distributed over northern Greece (Table I, fig. 1), we collected a total of

315 individuals of Rana balcanica and Rana ridibunda during three successive breeding

periods. At the Epeiros sites where R. balcanica occurs syntopically with R. epeirotica,

species identification was based on the mating calls. Specimens were measured, sexed and

numbered. Samples of blood (plasma and red cell hemolysates), liver, skeletal and heart

muscle were obtained from each specimen and stored at —35°C until electrophoretic

analysis. The carcasses remaining after tissue sampling were preserved and deposited in the

collection of the Laboratory of Zoology at the University of Thessaloniki.

For electrophoresis, we employed the vertical polyacrylamide gel technique using the

SE 600 Hoefer apparatus. Gels included a stacking and a separating portion and were 1.5

mm or 0.75 mm thick. Electrophoresis was conducted under refrigeration (4-6°C). Mostly,

a discontinuous buffer system was used (stacking gel buffer Tris-HCI, pH 6.8; resolving

buffer Tris-HCI, pH 8.8; reservoir buffer Tris-glycine, pH 8.3), whereas in case of the LDH

we employed a borate pH 8.2 system (PASTEUR et al., 1988). The samples (plasma and

aqueous extracts) of tissues were run in gels with two different percentages of monomer

in the separating gel (7.5% T — 10% T). The power supply was set for constant

amperage (30 mA), and the duration of electrophoresis varied from 3-6 h depending on

the protein.

Enzymes and other proteins were stained using standard procedures (PASTEUR et al.,

1988). Proteins of different individuals were compared side by side on the same gel to

avoid errors due to the slightly varying absolute mobilities in different gels. Enzyme

systems examined were: adenosine deaminase (ADA, EC 3.5.4.4), a-glycerophosphate

dehydrogenase (œ-GPD, EC 1.1.1.8), lactate dehydrogenase (LDH, EC 1.1.1.27), malate

Source : MNHN, Paris

SOFIANIDOU, SCHNEIDER & SINSCH 95

Table I. - List of sampling localities in Northern Greece and number of water frogs

collected for protein analyses. The spatial relations between the sites are given

in fig. 1.

Number of

Locality individuals per

sample

Epeiros

Panvotis Lake (a and b at the same locality)

Louros River

Parakalamos (upper part of the Kalamas River)

Sagiada Wetland

Nea Selefkeia marsh

Kalodiki, swampy lake

Macedonia

Axios River, river side pond

Axios River, river bank

Doirani Lake

Gallikos River, estuary region

Gallikos River, 35 km north of site 10

Thessaloniki, brooklet in the forest Seih-Su

Lankadas, small river

Kerkini Lake

Agion Oros, Moni Zographu

Thasos Island

Nestos River, river bank

Nestos River, river side pond

Thrace

Kompsatos River

Thermes of Echinos

Vistonis Lake

Komotini, small river

Evros Delta

Samothraki Island

Erythropotamos River (near Didymoteicho)

Kufovuno, small branch of Erythropotamos

Valtos, Orestiada, brook (a and b at the same locality)

Ardas River, at Komara (a, b and c at the same locality)

Total

Source : MNHN, Paris

96 ALYTES 12 (3)

Fig. 1. — Map of northern Greece indicating the spatial relations between the sampling sites (dots

and numbers) of water frogs. Table I gives the names of the sampling sites and the number of

individuals collected. 1: Epeirus; Il: Thessalia; III: Macedonia; IV: Thrace.

dehydrogenase (MDH, EC 1.1.1.37), mannose phosphate isomerase (MPI, EC 5.3.1.8),

6-phosphogluconate dehydrogenase (6-PGD, EC 1.1.1. 37), phosphoglucomutase (PGM,

EC 2.7.5.1). Additionally, the electromorphs of plasma albumin (ALB) and of a soluble

muscle protein (MProt) were analyzed.

Several other loci such as MDH-2 or MProt-2 and non-enzymatic muscle and plasma

proteins were scored in preliminary analyses and proved to be monomorphic. Since this

study focused on taxonomic distinction between the two species and not on an estimate

of their phylogenetic distance, we refrained from including the presumptive monomorphic

loci into the main study.

Multiple loci were numbered according to the mobility of their products from anode

to cathode. Stainable bands corresponding to the alleles of one presumptive locus were

assigned letters according to their mobility, beginning with the band closest to the anode.

Average heterozygosity per locus (H, = observed frequency; H, = expected frequency),

proportion of polymorphic loci (P %), and the mean number of alleles per locus (A) were

calculated for each sample. We used the G-test to detect deviations of the observed

heterozygosity from the Hardy-Weinberg equilibrium, and the non-parametric Wilcoxon-

Mann-Whitney U test (two-tailed) for the interspecific comparisons of H, and P %.

CAVALLI-SFORZA’S chord distance (CAVALLI-SFORZA & EDWARDS, 1967) was calculated for

all pairwise combinations of samples using the program GENDIST 3.4 of the package

PHYLIP, version 3.4 (FELSENSTEIN, 1985). Although we did not consider presumably

monomorphic loci, these estimates of genetic distance are useful to resolve the relative

Source : MNHN, Paris

SOFIANIDOU, SCHNEIDER & SINSCH 97

genetic relationships between the examined populations by computing rooted and

unrooted trees based on four algorithms: (1) UPGMA method (program NEIGHBOR

3.41); (2) FITCH-MARGOLIASH method assuming equal rates of evolutionary change in all

lineages (KITSCH 3.41); (3) FircH-MARGOLIASH method without evolutionary clock

(FITCH 3.41); (4) maximum-likelihood method (CONTML 4.42). AI calculations are

based on the cited programs of the package PHYLIP (FELSENSTEIN, 1985).

RESULTS

ALLELIC VARIATIONS OF PROTEINS

A total of 10 presumptive loci (enzymes: ADA, «-GPD, LDH-1, LDH-2, MDH-I,

MPI, 6-PGD, PGM-2; non-enzymatic proteins: ALB, MProt) were scored in 32 samples

of frogs from 28 sites (Table II). Except for one (MDH-1), all loci were polymorphic,

producing two to four bands of distinct electrophoretic mobility which we consider the

results of the activity of different alleles of the same locus. The corresponding frequencies

of the alleles are listed in Table II. Observed heterozygosity did not deviate significantly

from expectations (H,) in all but two populations (Vistonis Lake and Komotini River,

significant deficit of heterozygotes). However, the average observed heterozygosity H, (P

= 0.00388, Wilcoxon U test), the proportion of polymorphic loci P % (P = 0.000256, U

test), and the mean number of alleles per locus A (P = 0.01723, U test) were significantly

greater in R. ridibunda populations than in R. balcanica populations.

The following account of the polymorphic loci demonstrates that (1) alternatively

fixed alleles at the ADA locus permitted an unequivocal distinction between R. balcanica

and R. ridibunda populations, and (2) there was a clear geographical variation of allele

frequencies at other loci, which distinguished the R. balcanica populations of Epeiros from

their conspecifics in Macedonia. We refrained from speculations on presumptive

homologies of electromorphs detected in the present study with those in studies of other

authors because different electrophoretic conditions render such comparisons unreliable.

Allozymes

Adenosine deaminase

The two alleles permitted an unequivocal electrophoretic discrimination between

samples of R. balcanica from Epeiros and Macedonia and R. ridibunda from Thrace. Rana

balcanica populations were fixed for the a allele, R. ridibunda populations for the b allele.

«-glycerophosphate dehydrogenase

AIl samples collected from R. balcanica populations were monomorphic for the a

allele. The only exception was one of the two samples from the Nestos River, in which we

also detected the b allele at a frequency of 25 %. In contrast, the b allele predominated

in the R. ridibunda populations.

Source : MNHN, Paris

98 ALYTES 12 (3)

Table II. - Allele frequencies at ten presumptive loci in samples of water frogs from

25 localities through northern Greece. Numbers refer to the sites listed in Table

I. Replicate samples are identified by, e.g., la and 1b for site 1. P %: relative

frequency of polymorphic loci; A: average number of alleles per locus; H.:

relative frequency of expected heterozygosity; H,: relative frequency of

observed heterozygosity.

la AD 25/3. 4 5.6 7, 8, 910: 11 12 13, 14 “15

1.00 1.00 1.00 1.00 1.00 1.0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

1.00 1.00 1.00 1.00 1.00 1.0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00]

1.00 1.00 1.00 1.00 1.00 1.0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.67

CR SAS CARS AA LS SE 2053

1.00 1.00 1.00 1.00 1.00 1.0 1.00 1.00 1.00 1.00 1.00 - 1.00 1.00 1.00 1.00

M NE ne Cr ENS ES 100 RES QUE. bre

1.00 1.00 1.00 1.00 1.00 1.0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00)

0.70 0.70 0.80 0.83 0.70 0.7 0.65 0.95 1.00 1.00 0.90 0.92 1.00 0.95 0.92 1.00)

C.30 0.30 0.20 0.17 0.30 0.3 0.35 0.05 - - 0.100.08 - 0.050.08 -

2 2 2 2 + = = 0050.08 - 010 - - 0050.08 -

1.00 1.00 1.00 1.00 1.00 1.0 1.00 0.90 0.92 1.00 0.85 1.00 1.00 0.95 0.92 1.00

ARE 5 CO SES OS A 008 OT ONE

0.35 0.40 0.30 0.33 0.30 0.17 0.05 0.10 - - 0.05 - - O0.050.08 -

0.65 0.60 0.70 0.67 0.70 0.83 0.95 0.90 1.00 1.00 0.95 1.00 1.00 0.95 0.92 1.00

RS CR RUE CN 10

0.85 0.90 0.70 0.67 0.60 0.67 0.60 0.47 0.50 0.50 0.40 0.40 0.50 0.40 0.42 0.50

0.15 0.10 0.30 0.33 0.30 0.33 0.30 0.50 0.50 0.50 0.43 0.60 0.50 0.60 0.50 0.50)

HONOR. ent RME N LUE

DAMON ROUTE

1.00 1.00 1.00 1.00 1.00 1.0 1.00 0.60 0.67 0.62 0.63 0.65 0.50 0.60 0.50 0.50

- + + + - - 0.37 0.33 0.38 0.37 0.35 0.50 0.40 0.50 0.50

.3 0.3 0.3 0.3 0.5 0.3 0.2 0.5 0.3 0.2 0.5 0.5 0.3

14.13 14418153 412 17 143 12 15 16 13

.12 0.14 0.11 0.11 0.15 0.10 0.10 0.16 0.11 0.10 0.12 0.15 0.14

.10 0.13°0.08 0.10 0.11 0.10 0.10 0.16 0.10 0.10 0.11 0.11 0.15

Source : MNHN, Paris

SOFIANIDOU, SCHNEIDER & SINSCH 99

Table II. - Continuation.

16 18 19 20 21 22 23 24 25 26 27a 27b 28a 28b 28c

1.00 1.00 1.00 - - - - - - - - - - -

- = - 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

1.00 0.75 1.00 0.25 0.33 0.40 0.29 0.38 0.50 0.25 0.30 0.30 0.20 0.27 0.34 0.50}

- 0.25 - 0.75 0.67 0.60 0.71 0.62 0.50 0.75 0.70 0.70 0.80 0.73 0.66 0.50}

1.00 0.83 0.67 - - 0.450.31 0.13 0.50 - - 0.10 - 0.27 0.43 0.44]

roger 403 0252100620 NC ee en er ee ES 017

- 0.17 - 0.75 - 0.55 0.69 0.87 0.50 1.00 1.00 0.90 1.00 0.73 0.57 0.39

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00!

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00}

0.90 0.60 0.50 0.50 0.33 0.25 0.31 0.25 0.50 0.25 0.30 0.30 0.27

0.10 0.40 0.50 0.50 0.67 0.75 0.69 0.75 0.50 0.75 0.70 0.70 0.73

1.00 0.83 0.87 - - - - - - - - - - (0.140.33 -

- 0.17 0.13 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.86 0.67 1.00)

0.08 - 0.62 0.67 0.52 0.69 0.69 0.67 0.75 0.75 0.70 0.80 0.75

1.00 0.92 1.00 0.38 0.33 0.48 0.31 0.31 0.33 0.25 0.25 0.30 0.20 0.25

0.40 0.43 0.50 0.38 0.50 0.35 0.37 0.38 0.33 0.50 0.30 0.34 0.30 0.39 - 0.30]

0.60 0.54 0.50 0.62 0.50 0.65 0.63 0.62 0.67 0.50 0.70 0.63 0.70 0.61 0.67 0.40]

= 0085 0e 2 De + ur, à - - 0.33 0.20

0.60 0.25 0.13 - - 0.10 - 0.17 - - -

0.40 0.75 0.87 1.00 1.00 0.90 1.00 1.00 0.83 1.00 1.00 1.00 1.00 0.95 1.00

0.3 0.7 0.5 0.5 0.4 0.6 0.5 0.5 0.6 0.5 0.4 0.5 0.7

13 1.8 1.5 1.5 1.4 1.6 1.5 1.5 1.6 1.4 1.4 1.6 1.7

0.11 0.22 0.19 0.22 0.18 0.27 0.22 0.20 0.21 0.16 0.16 0.20 0.26

0.10 0.22 0.17 0.15 0.20 0.19 0.10 0.13 0.20 0.13 0.15 0.17 0.20

Lactate dehydrogenase

Three alleles were detected at the LDH-1 locus. However, in 16 of the 18 R. balcanica

populations this locus was monomorphic for the a allele. Only at Agion Oros did the c

allele, and at the Nestos River the b and c alleles, also occur. In contrast, the c allele

dominated over the a and b alleles in all R. ridibunda populations, except of (1)

Samothraki where all 6 individuals were heterozygous (a/c) and of (2) Komara (replicate

sample 28c) where the a allele dominated. Heterozygotes of the a/c constitution (6 at

Samothraki, 2 at Nestos River, 2 at Lake Vistonis, 1 at Valtos, 5 at Komara) showed the

Source : MNHN, Paris

100 ALYTES 12 (3)

typical five-banded pattern, whereas, due to the small difference in electrophoretic mobility

of a and b gene products, a/b heterozygotes (1 at Nestos River, 1 at Komara) showed only

two readily distinguishable bands.

The LDH-2 locus was monomorphic in all but one populations. The extremely rare

b allele was detected during a repeated sampling in six individuals from the Gallikos River.

Mannose phosphate isomerase

There were conspicuous geographical differences in the frequencies of the two

detected alleles at this locus. The R. balcanica populations of Macedonia were almost

monomorphic for the a allele, whereas the frequency of the b allele increased to about

30% in the conspecific populations of Epeiros. In the R. ridibunda populations, the

frequency of the b allele ranged between 50 and 76 %. Notable exceptions to this general

pattern were the R. balcanica samples from the Nestos River, with allele frequencies

resembling those of R. ridibunda populations.

6-phosphogluconate dehydrogenase

The three alleles detected at the 6-PGD locus again showed a clear geographical

segregation. The R. balcanica populations of Epeiros were monomorphic for the b allele,

whereas low frequencies of the a and c alleles were present in the conspecific populations

of Macedonia besides the dominant b allele. In contrast, the R. ridibunda populations of

Thrace were monomorphic for the c allele (except for Komara: low frequencies of the b

allele).

Phosphoglucomutase

As in the allelic distribution in the MPI, the frequencies of the two alleles at the

PGM:-2 locus varied geographically. The R. balcanica populations of Macedonia were

almost monomorphic for the b allele, whereas the frequency of the a allele increased to

about 30 % in the conspecific populations of Epeiros. In the R. ridibunda populations, the

frequency of the a allele increased even more, to about 70 %. The a/b heterozygotes

showed a broad band ranging from the positions of the homozygote a and b bands.

Non-enzymatic proteins

Albumin

The frequencies of the most common alleles, b and c, varied clinally along an

east-west axis in R. balcanica and in R. ridibunda. Allele d was frequent only at Komara

and at Gallikos River, whereas it was rare at Nestos River and at Valtos and absent in

all other samples. Allele a occurred in low frequencies at some localities in Epeiros and

Macedonia, i.e. exclusively in populations of R. balcanica.

Muscle protein

The three different stainable bands found in the fraction of soluble muscle protein

showed geographically and interspecifically varying frequencies. In the R. balcanica

Source : MNHN, Paris

SOFIANIDOU, SCHNEIDER & SINSCH 101

populations of Epeiros, only the b band was detected, whereas in the populations of

Macedonia the c band reached frequencies ranging from 33 to 87 %. The rare a band was

found only once, at the Axios River. Finally, the c band was often the only one detected

in the R. ridibunda populations, but at some sites low frequencies of the b bands were also

found. Frequent two-banded heterozygotes of the b/c type indicated that the three bands

are probably due to allelic variation of the same locus.

GENETIC DIFFERENTIATION

The genetic variation between samples collected at the same or nearby sites (< 1 km),

and between samples from different localities, was assessed by calculating CAVALLI-

SFoRzA’s chord distance (Table III). However, we excluded the replicate samples from

Valtos and Komara (sites 27 and 28 in Thrace) because of missing data which might bias

the comparison.

Random effects of sampling

The chord distances between samples collected at the same site (Panvotis Lake), and

between those collected in close vicinity (Axios River, Nestos River), ranged between 0.001

and 0.133. These three sites were inhabited by R. balcanica populations.

Intraspecific variation

The chord distances between the 18 R. balcanica populations ranged from 0.005

between the Louros and Kalamas Rivers to 0.462 between the geographically distant

localities Panvotis Lake and Gallikos River. Within the major geographical regions

Epeiros and Macedonia, genetic variation was considerably smaller. The six populations

from Epeiros were genetically very similar, with chord distances between 0.005 and 0.056

(median: 0.018). The twelve populations from Macedonia were less uniform, with chord

distances ranging between 0.012 and 0.438 (median: 0.119). Finally, the intraspecific

genetic variation among the ten R. ridibunda populations from Thrace varied with chord

distances of 0.006-0.324 (median: 0.072) within the range of the R. balcanica populations

from Macedonia.

Interspecific variation

Finally, the comparison between the populations of R. balcanica and R. ridibunda

yielded chord distances in the range of 0.553 to 1.625 (median: 1.197). Even the smallest

chord distance between any interspecific pair was still greater than the greatest distance

between pairs of conspecific populations.

Grouping of populations

AII populations examined were grouped with respect to their genetic similarity using

four algorithms which are commonly applied in the reconstruction of phylogenetic

relationships. All algorithms led to identical or similar groupings of populations.

Source : MNHN, Paris

TOI

Table III. - CAVALLI-SFORZA's chord distances between the water frog populations of 25 sampling sites. The replicate samples 27b, 28b and 28c were

excluded because not all loci were scored

la Ib 2 3 4 5 6 7 8 9 10 11 12 13 4 15 16 17 18 19 20 21 22 23 24 25 26 21a 2%a

la | - oo 0007 0010 002 0013 0045 0137 0187 0184 O161 0442 0207 O146 O6 0259 0163 0282 0336 1.339 132 LO4 1193 1233 0972 1.362 1.369 1268 104

1b + 0013 0016 0028 0022 00$6 O1$1 020$ 0202 G17S 0462 0225 0163 OI7 0277 0183 0298 0354 1346 1326 1OS1 1200 1240 0980 1365 1376 1275 1053

2 - 000$ 0017 0006 0037 O108 146 0144 0134 0406 0167 O1 0192 0218 0127 02%S 032 1331 1325 LOW 1192 1235 0962 1.370 1365 1267 1050

3 + 0018 0008 0042 0106 Q14S Q142 0134 0406 0165 10 131 217 0128 0268 0328 1329 1326 1040 LI9I 1235 0959 1370 1.364 1266 104

4 + 001$ 0018 0114 017$ 017 0152 0424 019 0132 0126 0247 0145 0271 0325 1333 1327 1032 LIS8 1229 0964 1366 1360 1262 1045

5 = O1 0108 0140 0137 0127 038 0160 0106 0128 0212 O1IO 0248 020 1357 1329 1032 LI9S 1235 0970 1374 1370 1269 1OS4

6 +012 014$ 0142 0138 039 016$ 0120 0119 027 Q1IO 0259 023 139 1372 LOGS 1237 1277 LOIS 1420 141$ 1.312 1059

7 +003 004$ 0058 0325 0047 OOI8 0017 00% 0040 013 OIGE LI4S 1161 0888 1022 LOGO 0819 1216 LI 1097 O916 >

8 +001? 0055 0311 0016 0017 0040 0068 002 0207 214 1317 1.344 LOG1 1207 1.257 0988 1407 136 1.284 1.059 ÊË

9 - 0070 029 0021 0023 004 0054 0016 GISI O19S 130$ 1332 LOS2 1195 1245 098 1395 1374 1272 1085 <

10 - 0342 0072 0037 0047 0124 00% 0133 0174 LI69 1182 0902 1042 LOBS 0841 1237 1217 1097 0939 n

il + 0302 0301 032 0354 0286 0438 0438 1.547 LS6 1276 1425 1472 1218 1625 L6O1 1.500 1311 nm

mn - 002$ 0047 0052 0018 180 180 127 1306 103$ 1169 1219 0967 1369 1348 1246 1.065 Le

E + 001$ 007 0016 0149 O168 1212 1232 093 100 LI38 0887 1249 1264 1165 0979 ue

14 - 0099 0037 0148 0165 LIS4 1199 0930 LOSS 1105 0868 1253 1232 1133 0952 ©

15 - 0069 0159 0222 1137 1306 0948 1062 108$ 0887 1205 1184 1.106 0951 es)

16 + 0143 O0 1245 1263 097% LI2 1167 0921 1321 138 1197 LOI S

17 + 0193 0773 0882 0564 0650 0684 0.553 0300 0.70 0697 0.562

18 + 0946 0857 0803 0910 0937 07% 1069 1058 0971 0833

19 + O1S1 0132 0087 0068 0147 00$3 004 0063 0113

Source : MNHN, Paris

SOFIANIDOU, SCHNEIDER & SINSCH 103

Therefore, we present the unrooted FircH-MARGOLIAsH-tree (fig. 2) as an example of the

three algorithms based on CAVALLI-SFORZA’sS chord distance matrix and the unrooted

maximum-likelihood-tree (fig. 3) directly calculated from the allele frequencies. The best

FITCH-MARGOLIASH dendrogram with contemporary tips out of 4394 examined had a sum

of squares of 50.023 with a corresponding average percent standard deviation of 24.851.

The best unrooted FITCH-MARGOLIASH tree out of 7093 examined showed a better fit (sum

of squares: 14.611; average percent standard deviation: 13.431). The best unrooted

maximum-likelihood tree out of 1931 examined had a In likelihood of 760.407.

Within the R. balcanica populations, there were two major clusters of populations

corresponding to the samples from Epeiros (sites 1-6) and to those from Macedonia (7-16).

Despite the occurrence of the unique LDH-2 allele b in a sample from the Gallikos River

(site 11), this sample was included in the Macedonia cluster in the two unrooted trees

(figs. 2-3), whereas the two rooted dendrograms (UPGMA, FITcH-MARGOLIASH with

contemporary tips) failed to recognize this association and placed this population as an

outgroup of all other R. balcanica populations. AII reconstructions coincided in assigning

the population from the Nestos River a position apart from the main clusters, but clearly

within the branch of R. balcanica. Nevertheless, Balkan frogs from the Nestos River

differed from those of all other sites by the introgression of R. ridibunda genome (e.g.

a-GPD, LDH-1, MProt, 6-PGD).

Eight out of ten R. ridibunda populations (sites 21-28) joined one major cluster similar

to those representing R. balcanica in Epeiros and Macedonia. However, again two

populations (sites 19-20: Kompsatos River and Thermes of Echinos) differed considerably

from all other conspecifics due to the presence of a rare allele of the LDH-I.

DISCUSSION

The allelic variation of proteins among 28 populations of water frogs in northern

Greece corroborates the previous bioacoustic finding that R. balcanica inhabits the region

west of the Nestos River and R. ridibunda the eastern region (SCHNEIDER & SINSCH, 1992).

The populations at sites 1-18 (Table I) correspond to R. balcanica, those at sites 19-28 to

R. ridibunda. The alternative fixation of the ADA alleles permitted an unequivocal

biochemical distinction of the two species under these electrophoretic conditions.

As we deliberately selected the polymorphic loci for our purpose of taxonomic

distinction, and did not follow the presumably monomorphic loci detected, we chose

CAVALLI-SFORZA’S chord distance instead of Ner's (1972) genetic distance as a measure of

genetic differentiation. The calculation of Ner's genetic distances from our data set would

have led to an overestimation of the real genetic differentiation due to restriction on

polymorphic loci. Therefore, our study presents relative values of genetic differentiation

among the populations studied which cannot be compared directly with Ners distances

published elsewhere. However, CAVALLI-SFORZA’S chord distance is a useful measure to

decide whether the genetic differentiation between R. balcanica and R. ridibunda supports

the proposed species status or not.

Source : MNHN, Paris

104 ALYTES 12 (3)

Rana balcanica Rana ridibunda

EPEIROS

MACEDONIA THRACE

Nestos River

Fig. 2. — Firc-MARGOLIASH tree (best one out of 7093 examined; sum of squares: 14.61, average

percent standard deviation: 13.43) based on CAVALLI-SFORZA’s chord distance matrix (Table III)

of all pairwise combinations of the samples. Due to the absence of an outgroup this tree

is arbitrarily rooted at the mean distance between the most similar populations of R. balcanica

and R. ridibunda. The localities corresponding to the numbers of the populations are listed in

Table I.

INTRASPECIFIC GENETIC DIFFERENTIATION

The chord distances separating conspecific populations within a given region

(Epeiros, Macedonia, Thrace) are low, but increase with geographical distance as should

be expected. HoTz & UZZELL (1982) also found low NErs distances (D = 0.00-0.02) in

water frogs of southwestern Greece (now classified as R. balcanica), and NEvO & YANG

(1982) in water frogs of Israel (now classified as R. levantina; D = 0.006-0.056). This

genetic homogeneity is probably due to the extensive exchange of individuals among

neighbouring populations. This is not surprising as R. balcanica is known to migrate over

large distances (SOFIANIDOU & SCHNEIDER, 1989), as do other European water frogs (15 km

within one activity period in adult R. lessonae and R. esculenta; TUNNER, 1992), and

juveniles usually disperse over even larger distances than adults (SINSCH, 1991, 1992).

Source : MNHN, Paris

SOFIANIDOU, SCHNEIDER & SINSCH 105

Rana balcanica Rana ridibunda

EPEIROS

MACEDONIA THRACE,,

Fig. 3. — Maximum-likelihood tree (best one out of 1931 examined with In likelihood: 760.407) based

on the allele frequencies (Table IT) of each sample. Due to the absence of an outgroup this tree

is arbitrarily rooted at the mean distance between the most similar populations of R. balcanica

and R. ridibunda. The localities corresponding to the numbers of the populations are listed in

Table I.

Nevertheless, we detected two notable exceptions to the general pattern, the R.

ridibunda populations at the Kompsatos River (site 19) and Thermes of Echinos (site 20),

and the R. balcanica populations at the Nestos River (site 17-18). These four populations

conspicuously deviated from the other conspecific ones, but still were clearly assignable to

their respective species because of the alternatively fixed alleles of the ADA-locus. The R.

ridibunda from the Kompsatos River and Thermes of Echinos differed from all others in

the frequency of the b allele at the LDH-I locus. Both localities are situated in the

hignlands, and altitude associated with warm water at the Thermes of Echinos may

represent a selective pressure in favour of this rare allele.

At the Nestos River, the situation is more complicated. We know from bioacoustic

and allozyme studies (SCHNEIDER et al., 1993; SINSCH & EBLENKAMP, 1994) that R.

balcanica and R. ridibunda occur syntopically in this region. SCHNEIDER et al. (1993)

detected a considerable character displacement in several mating call parameters which

maximize the differences of the mating call between the two species. SINSCH & EBLENKAMP

(1994), in turn, detected specific differences in the genotypes of several enzymes in R.

Source : MNHN, Paris

106 ALYTES 12 (3)

ridibunda of the Nestos region as compared to the genotypes from the brook at Valtos.

This study has revealed the introgression of R. ridibunda genome into the R. balcanica

genome at the Nestos River, e.g. allele b of the &-GPD and alleles b and c of the LDH-1.

The alternatively fixed alleles of the ADA locus show that the occurrence of typical R.

ridibunda aïleles at other loci is due to introgression and not to an erroneous classification

of some individuals of the Nestos samples. AIl these findings emphasize the importance of

a further thorough study of the water frog populations in this area of distributional

overlap between the two species.

INTERSPECIFIC GENETIC DIFFERENTIATION

Our study clearly demonstrates that the chord distances between conspecific

populations, even if they are separated by a large geographical distance, are always smaller

than those between any interspecific pair. This genetic divergence holds also in the contact

zone in the region around the Nestos River which apparently represents an ancient hybrid

zone.

There are few estimates of NErs distances between the three species of water frogs

which were formeriy referred to as R. ridibunda because they have only recently been

distinguished: (1) R. ridibunda — R. balcanica: D = 0.082 (SINSCH & EBLENKAMP, 1994);

(2) R. ridibunda — R. levantina: D = 0.178 (SINSCH & EBLENKAMP, 1994); (3) R. balcanica

— _R. levantina: D = 0.247 (NEVO & FirippuccI, 1988) and D = 0.196 (SinscH &

EBLENKAMP, 1994). AII estimates of the genetic distances between the three species clearly

fall into the normal range of genetic differentiation at species level in Amphibia (D = 0.1

— 3.0; AvisE & AQUADRO, 1982).

PROTEIN ANALYSES AND BIOACOUSTICS

This investigation of the enzymatic and non-enzymatic proteins of the water frogs in

northern Greece provides excellent corroboration of the results of previous bioacoustic

analyses (SCHNEIDER & SOFIANIDOU, 1985, 1986; SCHNEIDER & SINSCH, 1992; SCHNEIDER et

al., 1984, 1993). It confirms both the presence of the two species R. ridibunda and R.

balcanica and the local differences within R. balcanica (see figs. 2-3). The mating calls of

R. balcanica in Epeiros have on average fewer pulse groups per call, and longer intervals

between the pulses groups, than those of R. balcanica in Macedonia (SCHNEIDER &

SortANIDOU, 1986). But despite these local differences the mating calls of all populations

are clearly classifiable as R. balcanica calls (SCHNEIDER et al., 1993).

In view of these protein analysis results, it seems desirable to continue both the

bioacoustic and the electrophoretic studies in Greece, especially in the vicinity of the

Nestos River and adjacent regions in Thrace and Macedonia, in order to determine where

R. ridibunda and R. balcanica are sympatric and whether hybrids of the two species are

present there. Hybrids have been found in other regions where two water frog species are

sympatric: for example, hybrids of R. ridibunda and R. lessonae (BERGER, 1964, 1973), of

R. ridibunda and R. perezi (GRAF et al., 1977), and of R. balcanica and R. epeirotica

Source : MNHN, Paris

SOFIANIDOU, SCHNEIDER & SINSCH 107

(SOFIANIDOU & SCHNEIDER, 1987). Therefore, it seems rather likely that hybrids of À.

ridibunda and R. balcanica will eventually be found.

ACKNOWLEDGEMENTS

T. S. SOFIANIDOU and H. SCHNEIDER thank the Volkswagen Foundation for generous financial

support within the Partnership Project “Taxonomy and Faunistics of the Water Frogs in Greece”. AIl

authors thank Mrs. U. DUNG and Mrs. M. SCHLICH for much appreciated technical assistance.

Comments of E. BALLETTO, G. GOLLMANN and P. ROTH are greatly acknowledged.

LITERATURE CITED

AKEF, M. S. & SCHNEIDER, H., 1989. — The eastern form of Rana ridibunda (Anura: Ranidae) inhabits

the Nile delta. Zool. Anz., 223: 129-138.

Avis, J. C. & AQUADRO, C. F., 1982. — A comparative summary of genetic distances in the

vertebrates. Evol. Biol., 15: 151-185.

BERGER, L., 1964. — Is Rana esculenta lessonae Camerano a distinct species? Ann. Zool., 22: 245-261.

Se 1973. — Systematics and hybridization in European green frogs of Rana esculenta complex. J.

Herpet., 7: 1-10.

CavaLui-Srorza, L. L. & EbwarDs, A. W. F., 1967. — Phylogenetic analysis: models and estimation

procedures. Evolution, 32: 550-570.

FELSENSTEIN, J., 1985. — Confidence limits in phylogenies: an approach using the bootstrap.

Evolution, 39: 783-791.

GRAF, J. D., KarCH, F. & MOREILLON, M. C., 1977. — Biochemical variation in the Rana esculenta

complex: a new hybrid form related to Rana perezi and Rana ridibunda. Experientia, 33:

1582-1584.

GÜNTHER, R., 1990. — Die Wasserfrüsche Europas (Anura — Froschlurche). Wittenberg Lutherstadt,

A. Ziemsen Verlag: 1-288.

Horz, H. & UzzeLx, T., 1982. — Biochemically detected sympatry of two water frog species: two

different cases in the Adriatic Balkan (Amphibia: Ranidae). Proc. Acad. nat. Sci. Philad., 134:

50-79.

JOERMANN, G., BARAN, I. & SCHNEIDER, H., 1988. — The mating call of Rana ridibunda (Amphibia:

Anura) in western Turkey: bioacoustic analysis and taxonomic consequences. Zool. Anz., 220:

225-232.

MERTENS, R. & WERMUTH, H., 1960. — Die Amphibien und Reptilien Europas. Frankfurt am Main,

W. Kramer: i-xi + 1-264.

Net, M., 1972. — Genetic distance between populations. Am. Nat., 106: 283-292.

NEvo, E. & Firippucci, M. G., 1988. — Genetic differentiation between Israeli and Greek

populations of marsh frog, Rana ridibunda. Zool. Anz., 221: 418-424.

NEVO, E. & SCHNEIDER, H., 1983. — Structure and variation of Rana ridibunda mating call in Israel

(Amphibia: Anura). Jsr. J. Zool., 32: 45-60.

Nevo, E. & YANG, S. Y., 1982. — Genetic diversity and ecological relationships of marshfrog

populations in Israel. Theor. appl. Genet., 63: 317-330.

PASTEUR, N., PASTEUR, G., BONHOMME, F., CATALAN, J. & BRITTON-DAVIDIAN, J., 1988. — Practical

isozyme geneties. Chichester, Ellis Horwood Ltd.: 1-215.

SCHNEIDER, H. & EGIASARIAN, E. M., 1989. — Bioacoustic investigations of lake frogs (Ranidae: Rana

ridibunda) in Armenia as a contribution to the study of distribution of the eastern form. Biol.

J. Armenia, 42: 926-935.

Source : MNHN, Paris

108 ALYTES 12 (3)

es 1991. — The structure of the calls of lake frogs (Rana ridibunda: Amphibia) in the terra typica

restricta. Zool. Anz., 227: 121-135.

SCHNEIDER, H. & SisCH, U., 1992. — Mating call variation in lake frogs referred to as Rana ridibunda

Pallas, 1771: taxonomic implications. Z. zool. Syst. Evol.-forsch., 30: 297-315.

SCHNEIDER, H., SINSCH, U. & NEVO, E., 1992. — The lake frogs in Israel represent a new species. Zool.

Anz., 228: 97-106.

SCHNEIDER, H., SINSCH, U. & SOFIANIDOU, T., 1993. — The water frogs of Greece: bioacoustic

evidence for a new species. Z. zool. Syst. Evol.-forsch., 31: 36-47.

SCHNEIDER, H. & SOFIANIDOU, T. S., 1985. — The mating call of Rana ridibunda (Amphibia, Anura)

in northern Greece as compared with those of Yugoslavian and Israeli populations: proposal

of a new subspecies. Zool. Anz., 214: 309-319.

ce 1986. — Bioacoustic study of water frogs (Ranidae) in Greece. Jn: Z. ROGEK (ed.), Studies in

herpetology, Proc. 3rd ord. gen. Meet. Soc. Europ. Herpet.: 561-564.

SCHNEIDER, H., SOFIANIDOU, T. S. & KYRIAKOPOULOU-SKLAVOUNOU, P., 1984. — Bioacoustic and

morphometric studies in water frogs (genus Rana) of Lake Ioannina in Greece, and description

of a new species (Anura, Amphibia). Z. zool. Syst. Evol.-forsch., 22: 349-366.

SinscH, U., 1991. — Orientation behaviour in amphibians. Herpet. J., 1: 541-544.

pe 1992. — Amphibians. Jn: F. PAP1 (ed.), Animal homing, London, Chapman & Hall: 213-233.

SINscH, U. & EBLENKAMP, B., 1994. — Allozyme variation among Rana balcanica, R. levantina and

R. ridibunda (Amphibia: Anura): genetic differentiation corroborates the bioacoustically

detected species status. Z. zool. Syst. Evol.-forsch., 32: 35-43.

SoranDou, T. S. & SCHNEIDER, H., 1987. — Hybrid types of water frogs living sympatrically with

Rana epeirotica and Rana ridibunda in Greece. In: J. J. VAN GELDER, H. STRUBOSCH, & P. J. M.

BERGERS (eds.), Proc. 4rd ord. gen. Meet. Soc. Europ. Herpet.: 247-252.

as 1989. — Distribution range of the Epeirus frog Rana epeirotica (Amphibia: Anura) and the

composition of the water frog populations in western Greece. Zool. Anz., 223: 13-25.

Tuner, H. G., 1992. — Locomotory behaviour in water frogs from Neusiediersee (Austria,

Hungary). 15 km migration of Rana lessonae and its hybridogenetic associate Rana esculenta.

Proc. 6th ord. gen. Meet. Soc. Europ. Herpet.: 449-452.

Corresponding editor: Günter GOLLMANN.

Source : MNHN, Paris

Alytes, 1994, 12 (3): 109-121. 109

Ecological observations

on Rana pretiosa in western Utah

Orlando CUELLAR

Department of Biology, University of Utah, Salt Lake City, Utah 84112, U. S. A.

The ecology of the spotted frog Rana pretiosa was studied in a small

isolated marsh in westem Utah (U. S. A.) during the spring of 1992. À total of

354 egg clusters were counted, averaging 444 eggs per cluster. The majority

were laid in shallow water and were attached to other clusters, forming large

communal masses. Eighty spotted frogs were marked and released, comprising

28 females, 25 males and 27 juveniles. Snout-vent length and weight of the

females averaged 60.5 mm and 18.5 g respectively, of the males 51.6 mm and

11.0 g, and of the juveniles 33.3 mm and 2.2 g. Females were significantly

larger and heavier than males. Population density was approximately 100 frogs

per hectare. The rarity and highly isolated occurrence of spotted frogs in Utah,

and the general decline of ranid frog species in western North America, call for

protection of this population.

INTRODUCTION

Compared to other species of ranid frogs in North America, the natural history of the

spotted frog Rana pretiosa is still only partially documented (TURNER, 1958, 1960; MorRIs

& TANNER, 1969; LiCHT, 1969, 1974, 1975). The spotted frog occurs predominantly in

British Columbia, Washington, Oregon, northern Idaho, and western Montana, but also

sporadically in Wyoming, Nevada and Utah, where the populations tend to be small and

isolated. According to STEBBINS (1985), some populations of the spotted frog in Oregon

and Washington are nearly extinct because of competition with leopard frogs and

bullfrogs. Dumas (1966) previously reported that the leopard frog is competitively

dominant over the spotted frog and replaces it wherever they occur together. In Utah, the

spotted frog is presently known from two areas, the west desert and along the Wasatch

range (STEBBINS, 1985). In 1991, the United States Fish and Wildlife Service listed the

spotted frog as a candidate (category 2) under the Endangered Species Act (Federal

wide declines in amphibian populations. Similar declines have been recorded in several

western North American frogs (CORN & FOGLEMAN, 1984; HAYES & JENNINGS, 1986;

BRADFORD, 1991). Although TURNER (1958, 1960) conducted an extensive population

study of the spotted frog in Yellowstone Park, and Morris & TANNER (1969) reported on

the reproductive biology of a population from north central Utah (Provo), not a single life

Source : MNHN, Paris

110 ALYTES 12 (3)

history account has been published from the extreme western populations of Utah, in

particular from the Gandy Salt Marsh of Snake Valley. To date, the only evidence of

spotted frogs at this site is observational. The present study was conducted to document

the existence of the spotted frog in the Gandy Salt Marsh, and to assess its population

structure and breeding dynamics.

STUDY AREA

The Gandy Salt Marsh is located at the southern end of Snake Valley between the

ranching communities of Gandy and Trout Creek. The specific locality is at longitude 113°

55° 13” W and latitude 39° 28’ 48” N. The marsh consists of several spring-fed ponds

covering approximately 0.5 X 3.5 km, which drain eastward into an alkali lake bed

(fig. 1). The substrate of the larger ponds is covered with a thick layer of muck, ranging

in depth from about 30 cm to 1-2 m. The western edge of the marsh is flanked by an

extensive mud flat, which is sparsely vegetated with salt grass (Distichlis spicata), grease

wood (Sarcobatus vermiculatus), alkali rabbitbrush (Chrysothamnus albidus) and common

reed grass (Phragmites australus). During spring, the mud flat is saturated and slick. The

dominant vegetation surrounding the ponds (fig. 2) consists of bulrush (Scirpus acutus),

cattails (Typha) and sedges (Cyperus). The dominant bottom vegetation consists of the

stonewart alga (Chara sp.), common marestail (Hippuris vulgaris), filamentous green alga

(Spirogyra sp.) and rush (Juncus). Most of the small, deep springs are covered with

watercress (Nasturtium officionale). The study was conducted at the north end of the

marsh, encompassing an area approximately 300 x 50 m with several small to medium

sized ponds (Table I, fig. 1).

The virtual absence of native perennial grasses on the western boundary of the Gandy

Salt Marsh, such as Indian rice (Oryzopsis hymenoides), galleta (Hilaria jamesü), needle

(Stipa comata), and dropseed (Sporobolus sp.), indicates that this region has been heavily

overgrazed (personal observation). This is further supported by the stunted growth and

scarcity of the various native shrubs, such as greasewood (Sarcobatus vermiculatus), spiny

horse brush (Tetradymia spinosa), shadscale (Atriplex confertifolia), snake weed (Gutier-

rezia sorothrae), big sage (Artemesia tridentata), indian tea (Ephedra nevadensis) and hop

sage (Grayia spinosa). Most of the ground is exposed, trampled, and encrusted with

alkaline deposits. The only ground cover are sporadic clumps of salt grass (Distichlis

spicata), a halophytic species indicating high salinity. Because the western boundary slopes

towards the marsh, salts are undoubtedly leaching into the ponds. Numerous cow trails

lead directly into the marsh, enhancing the runoff and possibly the salinity. Although the

ponds are continuously flushed by the springs, enough salts could accumulate over time

to alter the freshwater invertebrate community, upon which the frogs depend for food.

Source : MNHN, Paris

North

Big North à }

Little North AAA

West Minnow ci Ds

North Minnow . 5

Minnow

Muskrat

Cow

9-11 Eutrophic Ponds

50 meters

Fig. 1. — Generalized map of the study site (north end of the Gandy Salt Marsh) showing the various

ponds described in the text, with inset of U.S.A. and State of Utah. The asterisk shows the

location of U. S. Bureau of Land Management (BLM) Cadastral Survey, south west corner

monument (1/4 S19 S30, T15S R18 W). Large numbers represent the ponds. Small numbers

represent BLM metal stakes marking the location of spring heads. Letters represent the

approximate site where each frog was collected. J: juvenile (earling); F: female; M: male. Adults

were collected predominantly along exposed banks.

Source : MNHN, Paris

mes Ra ee 0

Fig. 2. — Southwest view of Big North Pond. Photo taken during the spring of 1993. Note ice along

the edges and a flag marking the location of a frog captured the previous year. Mountains in

the background are in the State of Nevada.

Fig. 3. — Five recently laid egg clusters attached to each other and to the grass substrate. Note two

male spotted frogs adjacent to the egg clusters. This photograph was taken during the spring of

1993.

Source

MNHN, Paris

CUELLAR HE

Table I. - The number and location of Rana pretiosa egg clusters observed. Most

clusters were attached to substrate and to each other in masses. Distance and

depth in meters (depth = from water surface to bottom vegetation).

Date No. of Shore Cluster Type of

observed clusters distance depth substrate

3/16 25

3/16 4

3/17

3/20

3/21

Little North 3/23

Big North 4/3

0.15 Juncus

0.15 Chara

0.20 Juncus

0.10 Juncus

0.20 Typha

0.30 Typha

0.40 Juncus

0.15 Juncus

0.35 Juncus

0.15 Juncus

0.10 Nasturtium

0.15 Juncus

surface Juncus

0.30 Juncus

BSOSOOSSSOUROSYLN OCT RE ES

pouuuumuuoouooouuwbeouu

0.20

MATERIALS AND METHODS

Field work was conducted from March 16 to April 10, 1992. AIl ponds were searched

at least twice a day, mornings and afternoons. Captured frogs were individually marked

by clipping one or two different toes, and were then released at the site of capture after

recording sex, snout-vent length (SVL), weight, specific location and temperature. The site

of initial capture was marked with a road-construction flag bearing the number and sex

of the frog. SVL was measured from the ventral side. Weight was recorded with a spring

scale (Pesola), and frog (cloaca), water and air temperature were measured with a digital

thermometer. The location and number of egg clusters observed was also recorded and

marked with flags. All egg clusters in the marsh were counted. The number of eggs per

cluster was estimated from 22 clusters by counting the number of eggs in a small sample

of each cluster, weighing the sample, weighing the total cluster, multiplying the number of

eggs in the small sample by the total weight of the cluster, and dividing by the weight of

the small sample. Because spotted frogs lay their egg clusters together as a mass (fig. 3),

Source : MNHN, Paris

114 ALYTES 12 (3)

and the clusters are intimately attached to each other, counting the exact number in the

larger masses (> 45) was difficult. The southern end of the marsh system contains three

large, somewhat interconnected ponds (Middle, South, Fenced), from which only eggs

were enumerated (Table I).

RESULTS

The only signs of mating activity detected during this study were sporadic choruses

heard on March 16, 20, and 25. They were heard faintly from a distance, and the males

stopped calling when approached to within 15-20 meters of the pond. A total of 354 egg

clusters were counted during the study. Most were attached to other clusters, forming

masses (fig. 3). The number of clusters in a mass averaged 17, ranging from 2 to 85 (Table

1). Their average distance from shore was 1.4 m and their average depth from the surface

was 0.20 m. The majority were laid over grass, to which they were firmly attached.

Individual clusters were spherical, ranging in diameter from about 6 to 12 cm. The number

of eggs per cluster averaged 444, ranging from 325 to 710. Most masses contained clusters

in different stages of embryonic development. The average weight of clusters with embryos

was significantly greater than the weight of clusters with recently laid eggs, 157 g and

113 g respectively (t = 2.3, df = 20, P < 0.05), but both averaged the same number of

embryos, 441 and 421 respectively. Recently laid eggs were approximately 2 mm in

diameter. Each egg was surrounded by two clear gelatinous capsules, the outer one about

12 mm in diameter, and the inner one about 6 mm. Tail bud embryos were about 3 mm

in length. Hatching was first observed on March 25, with total length of the emerging

larvae about 8-10 mm. On April 6, 36 tadpoles measured at North pond averaged 13.4 mm

(range = 11-15 mm, SD 1.5). A sample of thirty-two (all 15 mm long) weighed 1.9 g,

averaging 0.06 g / tadpole. By this time, most had dispersed from the masses to the grassy

shoreline. Ten laboratory reared tadpoles weighed on May 19 averaged 1.98 g / tadpole

(range = 1.55-3.05 g). Their bodies averaged 24.5 mm (range = 20-25) and tails 31.4 mm

(range = 25-35). All had small developing rear legs.

Eighty spotted frogs were marked and released during the study, consisting of 28

females (35 %), 25 males (31 %) and 27 juveniles (34 %). The females averaged 60.5 mm

snout-vent length (range = 51-70, SD = 5.7), the males 51.6 mm (range = 45-59, SD =

3.0) and the juveniles 33.3 mm (range = 26-40, SD = 3.3) (fig. 6). The females averaged

18.5 g in weight (range = 12-28, SD = 4.2), the males 11.0 g (range = 7-15.3, SD = 1.6)

and the juveniles 2.2 g (range = 1.6-5.4, SD = 1.1). The females were significantly larger

and heavier than the males (SVL: t = 7.24, P < .001; WT: t = 10.3, P < .001). The

smallest size of a mature male was 45 mm. Mature males had distinctlÿ dark and swollen

nuptial pads on the thumbs. The dorsal coloration of both sexes was greenish brown with

faint dark spots (figs. 3-4) and the under surface was yellowish, especially in the axillary

and inguinal areas.

Lincoln-Peterson estimates conducted during the last five days of study averaged a

total population size of 146 frogs in the north marsh. This area covers approximately 1.5

hectares, so the total population density is about 100 frogs per hectare, 1/3 each females,

Source : MNHN, Paris

CUELLAR 115

Fig. 5. — A floating mass of eggx

s with the surface exposed to the air. Note bulging bubbles and

whitish, expanded capsules.

Source : MNHN, Paris

116 ALYTES 12 (3)

Rana pretiosa

2 H male

3 juveniles [] À female

$ [l

Ê I

ca]

re]

25 30 35 40 45 50 55 60 65 70

Snout-vent length (mm)

Fig. 6. — Snout-vent length distribution of the juveniles and adult male and female Rana pretiosa.

males and juveniles. Most of the adults were captured in the large ponds, whereas most

of the juveniles were captured in the small, grassy ponds and sloughs. The majority of

frogs were detected on shore, partly immersed in the water and concealed under the bank

vegetation. Of the 80 marked frogs, 24 (30 %) were recaptured once (Table II), 6 twice,

2 three times and 2 four times. Of the 24, 16 (67 %) were recaptured within 4 meters of

their original capture site, and of these, 6 were recaptured at the same site. Two females

(5 and 7) were recaptured three times at the same site. The average distance traveled away

from the point of first capture by females was 25 m, by males 14 m and by juveniles 0.9

m (Table IT). The longest distances traveled from the original capture site were 100 m, 150

m and 3 m, respectively. One male and one female migrated to other ponds. Mean body

temperatures of adult frogs were consistently higher than both water and air, about 18.4

compared to 15.8 and 14.9, respectively.

DISCUSSION

The virtual absence of calls after March 18-25 suggests that most of the breeding

occurred during early March. Because the majority of the egg clusters were found at the

beginning of the study, and some of the eggs had developed to the tail bud stage,

oviposition probably occurred several days prior to the study. The most recently laid

clusters were smaller (about 8 cm) than those with tail bud embryos (about 10-12 cm),

suggesting that the jelly capsules expand as development progresses. TURNER (1958) and

Morkis & TANNER (1969) report that the expansion is due to water absorption. Indeed,

clusters with embryos were significantly heavier than those with eggs, even though the

number of embryos and eggs was similar.

Source : MNHN, Paris

CUELLAR

117

Table II. - Number of recaptures and total distance Rana pretiosa traveled (meters)

from original point of capture. Al recaptures represent different days. 0 = same

place of capture.

No. of

recaptures

Distance and direction

from first capture

Total

distance

travele

Big North

Minnow

North

Minnow

West Minnow

Big North

Minnow

North

West Minnow

Minnow

West Minnow

Minnow

North

Minnow

Big North

Minnow

Little North

nm mm RS LLLS LS ST TT 0

25S

2W 2W

17S

Moved to N. Pond

Moved to Linkage

In addition to expanding in volume as development proceeds, the clusters begin rising

to the surface and the inner capsules develop a population of algae, giving the clusters a

greenish appearance (see SviHLA, 1935; Morris & TANNER, 1969). By hatching time, the

entire mass floats to the surface, where it spreads and assumes a frothy, yellowish-green

texture (fig. 5). The expanding bubbles force many larvae to the surface where they die

from dehydration. According to Morris & TANNER (1969), 10 to 20 percent of surface eggs

are destroyed this way. The bubbles probably develop from oxygen produced by the algae.

After hatching the free-swimming larvae remain below the egg mass, attached by their

mouths to the capsules, possibly feeding on the algae. The floating mass also apparently

serves as camouflage for the developing larvae, and may also enhance the speed of

development by increasing water temperature at the surface. The surface temperature of

one mass at Little North Pond was 24°C compared to 10°C at the bottom of the pond.

Source : MNHN, Paris

118 ALYTES 12 (3)

The fact that the majority of egg clusters were deposited together in masses suggests a

communal type of breeding in which several gravid females are attracted to the same place,

perhaps by several males calling together. Similar communal breeding was reported by

TURNER (1958), LicHT (1969), and Morris & TANNER (1969). Presumably, each egg cluster

represents a single clutch, but the exact number of clutches deposited by each female has

not been documented. The northern ponds contained 177 clusters (Table I), but the

number of females actually captured was 28. Even the Lincoln-Peterson estimate of 51

females corresponds to less than one third of the clusters. Therefore, either the population

is much larger than currently estimated, or some females lay more than one cluster.

The wide variation in number of eggs per cluster reported here, ranging from 325 to

710, suggests that some females may have split their clutches into two or more smaller

clusters. Other workers have also reported wide variation in egg numbers in the spotted

frog (TURNER, 1958: 206-802; LicHT, 1969: 249-935; Morris & TURNER, 1969: 148-1160).

Some of this variation could be related to differences in the size and age of females,

because female body size is positively correlated with clutch size in most frogs (PETTUS &

ANGLETON, 1967; SALTHE & DUELLMAN, 1973; BERVEN, 1982). Rana sylvatica is known to

deposit two or more egg masses in one breeding season (Davis & FOLKERTS, 1986). The

current estimate of 51 females in the north ponds suggests that each female on average

may lay 3.5 egg clusters (177/51).

Judging from the small number of frogs captured daily, only a fraction of the total

population appears to be active at one time. Moreover, spotted frogs at this site are highly

secretive and elusive. When approached, they usually slip into the water quietly, leaving

only a small swirl. Of the 80 captured frogs, only four were observed entirely out of the

water, basking on grass beneath the bank. Even so, the majority had a higher body

temperature than the water, suggesting they are only partly submerged in their perches.

Because most of the frogs were recaptured very close to the original site of capture, they

apparently use the same perches repeatedly, suggesting they are highly sedentary.

Although the overall sex ratio was nearly equal, some ponds had a predominance of one

sex (Table III). The only other anuran observed in this marsh was the northern leopard

frog, Rana pipiens. Six individuals were found at the extreme south end, one pair in

amplexus. Leopard frogs are apparently rare in this marsh, or emerge much later. They

also seem to prefer the larger ponds at the southern end. The northern end is inhabited

exclusively by spotted frogs. However, the abundance of spotted frog eggs in the southern

ponds indicates that they breed throughout the marsh system. At this time, leopard frogs

at this site do not seem to be replacing the spotted frog, as has been reported previously

by Dumas (1964). During the spring of 1993, however, I observed a single adult leopard

frog about 10 meters southeast of Muskrat pond.

The presence of cow dung in many of the ponds, especially the shallow ones, indicates

that cattle traverse them, trampling the aquatic vegetation, and possibly the frogs

themselves. Since the ponds provide the most luxuriant foliage in this region, and the only

source of fresh water, cattle tend to congregate around them, grazing the succulent

pasture. The highest concentration of cow dung and trails is along the banks, the critical

feeding, breeding and basking habitat of the frogs. Several ponds at the north end contain

a dark-reddish water, seemingly from dung eutrophication. One of these ponds (“Cow

Source : MNHN, Paris

CUELLAR 119

Table III. - The number of female, male and juvenile Rana pretiosa captured per pond.

Pond”) is littered with bones from a dead cow, which apparently died within it. Al such

ponds are devoid of aquatic vegetation, invertebrates and frogs. The remains of a single

dead cow in a fertile pond would undoubtedly destroy its entire frog population. A major

ranching problem in this region (Snake Valley) is the frequent drowning of cattle in the

deeper ponds, which are usually fenced to prevent miring (Terry HALE, pers. comm.).

Data from this study suggest that the population of spotted frogs at the Gandy Salt

Marsh is small, perhaps comprising less than three hundred individuals. The marsh itself

also is very small, consisting of a few isolated springs within a vast arid valley. According

to BEGON et al. (1990), when local populations are reduced to “several hundred”

individuals, they are prone to becoming extinct because of localized catastrophes, such as

drought. They also list the major causes of rarity according to the International Union for

the Conservation of Nature and Natural Resources. Most of these causes of rarity and

extinction apply to the ecology and distribution of the spotted frog in western Utah. Its

habitat is rare (isolated fresh water springs). Its resources are limited (confined to a few

spring-fed ponds). It cannot disperse to other similar habitats, and it is limited by cattle,

which degrade its aquatic habitat and trample its breeding and basking sites. In the interest

of protecting this vulnerable endemic species, livestock grazing should be discontinued

from the western boundary of the marsh, the principal watershed. This would allow

repopulation of the native grasses and shrubs, reducing erosion, evaporation, and

salinization.

RESUMEN

La ecologia de la rana pinta Rana pretiosa fue estudiada en varios lagitos aislados en

la parte norteña de la cienega salada de Gandy en la frontera occidental de Utah durante

la primavera de 1992. Un total de 354 nidadas fueron contadas con un promedio de 444

huevos por nidada. La mayoria fueron puestas a orillas de lago pegadas con otras,

formando masas comunales. Un total de 80 ranas fueron marcadas y soltadas consistiendo

de 28 hembras, 25 machos y, 27 añeras. El promedio del tamaño (hocico-cloacal) y peso

Source : MNHN, Paris

120 ALYTES 12 (3)

de hembras fueron 60.5 mm y 18.5 g, respectivamente de machos 51.6 mm y 11.0 g, y de

añeras 33.3 mm y 2.2 g. Hembras son significativamente mâs grandes y mâs pesadas que

machos. Densidad poblacional es aproximadamente 100 ranas por hectarea. Debido a la

rareza y distribuciôn muy aislada de esta rana en Norte América deberia protejerse la

poblaciôn en Gandy de su posible extincion.

ACKNOWLEDGEMENTS

I am grateful to Ronald A. BOLANDER and Mark PIERCE of the U. S. Bureau of Land

Management (Utah Office) for encouraging me to conduct this study and for logistic support. I thank

Suzi and Terry HALE of Gandy, Utah for hospitality and field assistance, Eleanor M. ULIBARRI for

helping me to photograph the frogs during the spring of 1993, and Fausto R. MÉNDEZ for statistical

and technical assistance. I especially thank Günter GOLLMANN, Deputy Editor of Alytes, and

anonymous reviewers, for useful editorial suggestions, and Alain Dugois for inviting me to submit this

manuscript to Alytes.

LITERATURE CITED

BARINAGA, M., 1990. — Where have all the froggies gone? Science, 247: 1033-1034.

BEGON, M., HARPER, J. L. & TOWNSEND, C. R., 1990. — Ecology: individuals, populations and

communities. Boston, Blackwell Scientific Publications: 1-945.

BERVEN, K. A., 1982. — The genetic basis of altitudinal variation in the wood frog Rana sylvatica.

I. An experimental analysis of life history traits. Evolution. 36: 962-983.

BRADFORD, D. F., 1991. — Mass mortality and extinction in a high elevation population of Rana

muscosa. J. Herpet., 18: 147-152.

Con\, P. S. & FOGLEMAN, C., 1984. — Extinction of montane populations of the northern leopard

frog (Rana pipiens) in Colorado. Herpetologica, 25: 174-177.

Davis, M. S. & FoLKkERTS, G. W., 1986. — Life history of the wood frog, Rana sylvatica Le Conte

(Amphibia: Ranidae), in Alabama. Brimeleyana, 12: 29-50.

Dumas, P. C., 1966. — Studies of the Rana species complex in the Pacific Northwest. Copeia, 1966:

60-74.

HAYEs, M. P. & JENNINGS, M. R., 1986. - Decline of ranid frog species in western North America:

are bullfrogs responsible. J. Herpet., 20: 490-509.

LicHT, L. E., 1969. — Comparative breeding behavior of the red-legged frog (Rana aurora aurora)

and the western spotted frog (Rana pretiosa pretiosa) in southwestern British Columbia. Can.

J. Zool., 47: 1287-1299.

+ 1974. — Survival of embryos, tadpoles, and adults of the frogs Rana aurora aurora and Rana

pretiosa pretiosa sympatric in southwestern British Columbia. Can. J. Zool., 52: 613-627.

1975. — Comparative life history features of the western spotted frog Rana pretiosa, from low

and high elevation populations. Can. J. Zool., 53: 1254-1257.

Morkis, R. L. & TANNER, W. W., 1969. — The ecology of the western spotted frog, Rana pretiosa

Baird and Girard, a life history study. Great Bas. Nat., 29 (2): 45-81.

Perrus, D. & ANGLETON, G. M., 1967. — Comparative reproductive biology of montane piedmont

chorus frogs. Evolution, 21: 500-507.

Puiccirs, K., 1990. — Where have all the frogs and toads gone? BioScience, 40: 422-424.

SALTHE, S. N. & DUELLMAN, W. E., 1973. — Quantitative constraints associated with reproductive

mode in anurans. /n: J. L. VIAL (ed.), Evolutionary biology of the anurans, Columbia, University

Missouri Press: 229-249.

Source : MNHN, Paris

CUELLAR 121

STEBBINS, R. C. , 1985. — À field guide to western reptiles and amphibians. Chicago, Houghton Mifflin

Co: 1-336.

SvinLA, A. 1935. — Notes on the western spotted frog, Rana pretiosa pretiosa. Copeia, 1935: 119-122.

TURNER, F. B., 1958. — Life history of the western spotted frog in Yellowstone National Park.

Herpetologica, 14: 96-100.

—— 1960. — Population structure and dynamics of the western spotted frog Rana pretiosa in

Yellowstone Park, Wyoming. Ecol. Monogr., 30 (3): 251-271.

Corresponding editor: Günter GOLLMANN.

Source : MNHN, Paris

Alytes, 1994, 12 (3): 122. Announcement

Défense et illustration de l’histoire naturelle

avec Muséum 2000

Jamais époque n’a connu atteinte aussi grave à l'encontre de la nature: extinctions

sans cesse plus nombreuses d’espèces animales et végétales, pollution des eaux, altération

de l’atmosphère, dégradation de la pellicule pédologique, auxquels s’ajoutent appauvris-

sement des cultures et malaise généralisé de l’humanité. Ces drames sont le plus souvent

des retombées d’une évolution scientifique, technologique, démographique foisonnante et

mal maîtrisée. Face à eux, le citoyen pourrait penser qu'il s'impose de privilégier les études

sur cette nature - sur tous les aspects de cette nature - ainsi mise à mal. Or, et ce n’est pas

le moindre paradoxe de notre époque, en France tout spécialement, l’histoire naturelle est

la mal-aimée des pouvoirs publics et des technocrates qui les orientent. A tous les stades

de l’enseignement aussi bien que dans la recherche, les sciences naturelles régressent,

l'observation est méprisée, l'approche globale est ignorée, le tout au profit de disciplines

et de techniques plus “modernes”: or n'est-il pas évident que ces dernières - dont l'intérêt

est immense - ne sont plus rien si les objets de leur étude comme l'interprétation de leurs

résultats ne s’insèrent pas dans le cadre que seules peuvent leur apporter les disciplines

naturalistes de base.

Depuis des siècles, le Muséum national est en France le bastion de l’histoire naturelle.

Il est à ce titre depuis quelques années la cible de l’hostilité tenace et destructrice des

contempteurs de nos disciplines. L'association Muséum 2000 (association sous la loi de

1901, fondée le 29 novembre 1990) s’est fixé pour objectif de défendre cet établissement

prestigieux et irremplaçable. Cette vocation implique une volonté inébranlable de défendre

l'histoire naturelle dans notre pays et hors de celui-ci. Cette association est ouverte à tous.

Nous faisons appel à tous ceux que peut motiver notre idéal, chercheurs, enseignants,

professionnels ou amateurs, citoyens sensibles au drame de notre temps, pour qu’ils nous

apportent leur soutien, leur concours, leur dynamisme, leur adhésion.

Muséum 2000, 3 rue Rollin, 75005 Paris, France

Cotisation annuelle 1994: membre actif, 120 F; étudiant, 60 F; membre bienfaiteur,

à partir de 250 F.

Cahiers de Muséum 2000: N°1, “Manifeste 1992”, juin 1993, 25 F; N°2, décembre

1993, 25 F.

Conseil pour 1994: Président, Professeur Georges Busson (40.79.34.78); Vice-

Présidents, Marc SILBERSTEIN (43.55.54.43) et Professeur David SmirH (40.79.35.27);

Secrétaire Général, Jean-François VoisiN (40.79.30.68); Trésorier, Professeur Jean-Marie

DEMANGE (40.79.35.83); Trésorier Adjoint, Bernard MÉTIVIER (40.79.30.96); autres mem-

bres du Conseil: Claude BUREAUX, Professeur Alain COLLENOT, Professeur Alain DuBois,

François FRÔHLICH, Christiane TIREL.

Renseignements par lettre ou téléphone.

Source : MNHN, Paris

Alytes, 1994, 12 (3): 123-134. 123

The problem of declining

amphibian populations in the Commonwealth

of Independent States and adjacent territories

Sergius L.KUZMIN

Institute of Evolutionary Morphology and Ecology of Animals, Russian Academy of Sciences,

Leninsky prospect, 33, Moscow 117071, Russia

Published evidence of amphibian declines within the territory of C.LS. and

adjacent temitories (formerly U.S.S.R.) are reviewed. Populations of more than

half of all the species are subject to decline. Most of the recorded declines and

extinctions are local; the main causes are anthropogenic factors, especially

deforestation, destruction of water bodies, pollution and urbanization. Few

species are expanding under anthropogenic influences. The history of the

expansion of Rana ridibunda is briefly discussed.

INTRODUCTION

The study on amphibian ecology in the former U.S.S.R. has been conducted for

several decades. The recent dismemberment of the U.S.S.R. has led to the formation of the

Commonwealth of Independent States (C.I.S.) and few republics have not joined this.

Here I summarize the published and some unpublished data on the phenomenon of

declining amphibian populations within this territory. Analysing such information, the

researcher faces several problems. How to distinguish between stable declines and

temporary fluctuations in numbers? What are the extents of declines? Which conclusions

on the declines were made on the basis of subjective estimations, and which on the basis

of exact quantified data? Unfortunately, not all the papers reviewed contained the answers

to these questions. However, a comparative analysis permits some conclusions on the

population trends in different regions. In the analysis I have used puplications concerned

with almost all the republics of the former U.S.S.R. Papers with data only on population

number and lacking data on dynamics were not used.

Generally, the population changes may be analysed on two time-scales: long-term and

short-term. The first includes, conventionally, changes in geological and historical times:

covering millions to hundreds of years. The second includes changes from a century to one

year or less, and concerns fluctuations in population numbers.

Source : MNHN, Paris

124 ALYTES 12 (3)

LONG-TERM CHANGES

One can say with certainty that global changes of climate have influenced amphibian

numbers and distributions. À large proportion of contemporary Eurasian amphibian

species already existed in the late Pliocene. Glaciations and regressions of glaciers,

expansions and reductions of forests and steppes during the last 600-700 thousand years

had a great impact on the distribution and dynamics of amphibians (BORkIN, 1984). For

example, Quaternary remains of a toad related to Bufo raddei (now representative of the

Mongolian fauna) were found in European Russia. Quaternary bones resembling those of

Salamandrella keyserlingii have been found c. 800 km SW from the species’ contemporary

range (RATNIKOV, 1989). In addition, many bones of Quaternary tailless amphibians

attributed to extinct species have been found in Russia. Thus, we can ascertain the

extinctions of amphibian species within the territory of former U.S.S.R. during the last few

hundred thousand years.

It is evident that more recent changes have also occurred. For example, changes have

probably taken place during the last thousand years in the territory of Tatarstan

(GARANIN, 1989). During the cold periods the northern taiga species S. keyserlingii

occurred there and, in warm periods, Hyla arborea. Forest species, Triturus cristatus, Bufo

bufo, and Rana temporaria, receded northwards due to forest destruction. In the V‘*-XIII‘*

centuries and later, the increase of agriculture may have caused the spreading northwards

of the southern species Bombina bombina and Pelobates fuscus. Thus, long-term changes

in climate and landscape may have influenced species in different ways, according to their

specific ecological requirements.

SHORT-TERM CHANGES

POPULATION DECLINES

Taxa

From 13 species of Caudata inhabiting the former U.S.S.R., declines are documented

in 11 (Table 1). The remaining two, Triturus dobrogicus and Hynobius turkestanicus are

simply unexplored. From 27 anuran species, declines are documented in 15 (Table I). This

probably reflects the better state of anuran populations compared to caudates, rather than

the scarcity of information on some anuran species. Most reports concern the declines of

widespread and numerous species, such as common toad, brown frogs, etc. The declines

of endemic and relic species (©. fischeri, R. sibiricus, M. caucasica, T. vittatus, T.

montandoni, T. alpestris, T. karelini, P. caucasicus, P. syriacus) are probably local.

Nevertheless, such declines may have severe consequences due to low total number and

narrow ranges.

Source : MNHN, Paris

KUZMIN 125

Time and regions

Most data on declines are from the period 1970-1990. Basically, this reflects increasing

interest in nature conservation at this time. At the same time, there was an increase in

anthropogenic influences on amphibian populations. Earlier declines in the 1920s and

1940s were connected mainly with the destruction of habitats due to urbanization,

industrialization, establishment of collective farms, and with the Second World War

(BANNIKOV & ISAKOV, 1967; BESKROVNY & BURMENSKAYA, 1970).

The most numerous declines have been registered in the European region (Table I).

This reflects not only the better exploration of this region, but probably the real situation.

Reports from Western Siberia and the Far East are much less numerous, though these

regions have been studied to a reasonable extent.

Natural factors

Natural factors (see legend of Table I) are rarely reported as causes of amphibian

declines. In these instances the main factor is probably natural community succession

leading to eutrophication and overgrowth of the breeding ponds. These factors are

indicated for four amphibian species, and may have led to extinctions of isolated

populations especially in urban areas.

Anthropogenic factors

Anthropogenic factors probably play the main role in the recent declines (see Table

D). Destruction of forests and other arboreal vegetation appears to be most widespread. This

factor is indicated in 23 % of papers containing information on the causes of declines. This

leads not only to destruction of terrestrial habitats, but also to the drying of breeding

ponds and brooks. Deforestation was indicated as the cause of declines of 40 % of species

studied. This is most harmful for the forest species such as newts and common toads. Pond

drainage has been reported as a second main factor of amphibian declines (20 % of papers;

indicated for 44% of species). Intensive use of artificial fertilizers and pesticides was

reported as a factor of declines in 14 % of reports and for 40 % of species. This influences

amphibians mainly by spoiling of water, frequenly caused by water pollution by cattle and

industrial wastes (each reported in 11 % of papers). Nevertheless, the second kind of

pollution appears to be more harmful than the first: this was reported for 40 and 16 % of

species studied, respectively. Other anthropogenic factors (see footnote to Table I) have

been reported relatively rarely (in 3-6 % of papers) and concerned, as a rule, only 4 to

13 % of species studied.

Only urbanization, as a complex factor, being not frequently indicated, concerns 36 %

of species. This has caused sharp declines of amphibians. In the large cities, such as

Moscow, Nizhny Novgorod and Ekaterinburg, amphibian numbers decreased from the

periphery of city to the centre (BANNIKOV & IsAKkOV, 1967; LEBEDINSKY, 1981; VERSHININ,

1987; KUZMIN, 1989; USHAKOV & BELOBORODOVA, 1989). B. viridis, R. temporaria, R.

arvalis and R. ridibunda are better adapted to life in urban areas than other amphibians,

but under excessive urbanization they also have disappeared. The process of extinction

Source : MNHN, Paris

Table I. - Amphibian declines in the territory of former U.S.S.R.

Years. —: unknown.

Lrai

Causes of declines. - A: anthropogenic factors (1: destruction of forests and other arboreal vegetation; 2: destruction of hiding places, 3:

streambank damage by cattle; 4: intensive cattle pasture; 5: intensive use of artificial fertilizers and pesticides in agriculture; 6: use of pesticides;

7: use of fertilizers, 8: removal of the rice fields, 9: earthwork;, 10: pond drainage, 11: pond destruction; 12: pond clearing and building of

embankments; 13: water pollution by cattle; 14: industrial pollution of water; 15: pollution of water by domestic wastes; 16: transportation of

logs by heavy traffic; 17: overflooding; 18: pollution and drying of streams, 19: trailing of the cut trees along the streams; 20: river bank clearing

and building of embankments; 21: introduction of the fish Percottus glehni; 22: introduction of Rana ridibunda, 23: urbanization; 24: collecting

for commercial aims; 25: collecting for educational aims; 26: collecting for scientific aims; 27: fishing; 28: intensive recreation), N: natural

factors (1: mudding of ponds; 2: partial drying of ponds; 3: increase of water eutrophication; 4: overgrowth and shallowing of ponds, 5

probable increase of temperature and decrease of humidity), —: unknown.

Sources of information. - 1-63: numbers of references in “Literature cited”, SK: personal unpublished data; RK: personal communication of R.

A. KuBykn.

Species

Regions

Years

Causes of declines

Sources of

information

Salamandrella keyserlingii

Onychodactylus fischeri

Ranodon sibiricus

Salamandra salamandra

Mertensiella caucasica

Triturus vulgaris

Triturus vittatus

Triturus montandoni

Triturus alpestris

Triturus karelini

Triturus cristatus

Siberia: Upper Angara River

Far East

Kazakhstan: Upper Cherkassai River

Kazakhstan: Dzhungarian Alatau

Ukrainian Carpathians

Georgia

Moscow

Moscow Province: Glubokoe Lake

Volga-Kama region

Lower Volga

Ukrainian Carpathians

North Caucasus

Georgia

Ukrainian Carpathians

Skobelevskie Beskidy, Lvov

Ukrainian Carpathians and Skobelevskie Beskidy, Lvov

Chechnya

Azerbaijan

Moscow

Moscow Province: Glubokoe Lake

Volga-nama District

Delta of Don

Ukrainian Carpathians

19705

1960-1970s

19805.

1920-1980s

1973-1990

1973-1988

1960-1980s

10-15 years

1948-1984

10-15 years

19805

1973-1990

1920-1945

A2, A10

A26

A3, A13, A27

Al, A24, A25

AI8

A19

AI, A12, A14, A23

A21

A6, A10, A14, A15

A8, A10

AT, A10, A13, NI, N2, N3

A0, A13, A4

A24, A25

A10

A10, A11, A14, A23, N3, N4

A21

A6, A10, A14, A15

43, 52

SK, 10, 36

(€) TI SHLATV

Source : MNHN, Paris

Table I. - Continuation.

Species Regions Years Causes of declines Sources of

information

Bombina bombina Moscow 1920-1980s A10, AI1, A23 10, 36

Volga-Kama region _ Al 19

Severny Donets 1980-1986 A13 21

Lower Volga 1973-1988 AS 33

Ukrainian Polesje _ AI0 63

Pelodytes caucasicus North Caucasus _ Al, A9, AI8 22

Chechnya _ AI8 3

Georgia _ AI8 26

Pelobates fuscus Moscow 1922-1966 All, A23 10, 36

Moscow Province _— A28 23

Lower Volga 1973-1988 AS 33

Severny Donets 1980-1986 A4 21

Steppe on Dnepr = AI4 39

Pelobates syriacus Georgia Æ A10, A13, AI4 26

Armenia _ _ 16

Bufo bufo Moscow 1922-1966 All, A23 10, 36

Voronezh 1950-1970 Al 19

Volga-Kama region LS Al 19

Novosibirsk 1939-1969 _ 54 7%

Altai Mountains 1980s A22 62 c

Baikal region 1960-1970 Al, A4 49 N

Steppe on Dnepr and Dnestr Rivers — AL, AS 30, 51 &

Bufo viridis Moscow 1922-1980 A10, A11, A14, A23 SK, 10, 36, 40 2

Severny Donets 1980-1986 A4 21

Lower Volga 1973-1988 AS 33

Tbilisi _ A2, A6 28

Hyla arborea Steppe on Dnepr = AS 30

Ukrainian Carpathians _ Al 43

‘Hyla japonica Siberia: Zea River = A17 25

Rana temporaria Moscow 1922-1980 All, Al4, A23, N4 SK, 10, 36

Moscow Province _ A6, AT 40

Rana arvalis Moscow 1922-1980s AIll, A14, A23, N4 SK, 10, 36

Moscow Province _ A6, AT 40

Serveny Donets 1980-1986 A4 21

Volga-Kama region 1953-1963 NS 18

Lower Volga 1973-1988 AS 33

Novosibirsk 1939-1969 _ 54

Altai Mountains 19805 A22 62

Rana macrocnemis Turkmenistan: Kopetdag Mountains _ Al 6

Rana asiatica Kazakhstan: Kapchagai District 1960s-1970s |A17 RK

Rana lessonae | esculenta Moscow 1920-1980 AI, A14, A23, N3, N4 SK, 10, 36

Ukrainian Polesje _ A0 63

Rana ridibunda Moscow 1920-1980 All, A14, A20 SK, 10, 36

Lower Volga 1973-1988 AS 33 Se

Ukrainian Polesje _ A10 63 N

Kyzgyzstan: Chu River _ A25, A26

Source : MNHN, Paris

128 ALYTES 12 (3)

starts from the fragmentation of a population by residential districts and main roads.

Then, as a result of isolation, susceptibility to anthropogenic and natural influences

(inbreeding, droughts, frosts, pollution, etc.) is increased. After the last breeding centres

have been destroyed, the animals may breed for a some time in shallow puddles. Finally,

the population becomes extinct after the last adults die. Population declines may occur

even if some ponds remain, if they have been cleared and concreted. The latter make the

shores unsuitable for most amphibians.

The influence of reservoirs is complex. Commonly these did not lead to sharp changes

in amphibian species composition. Some species (mainly terrestrial) may decrease in

number, but some time later (up to 10 years) they can recover (KALETSKAYA, 1953;

ILYASHENKO, 1989; SMIRNOVA & EGOROV, 1985; DERKACH et al, 1989; UsHAKOv &

PISARENKO, 1989). In the Carpathian Mountains, the construction of reservoirs permitted

some lowland species to enter the mountains, such as T. vulgaris, T. cristatus, R. ridibunda,

R. lessonaelesculenta (POLUSHINA, 1977).

Extent

The extent of amphibian declines varies among the species and is determined by

different causes. Local extinction, as a rule, is caused by a complex of anthropogenic

factors. These situations were indicated for all the species listed in Table I, with a few

exceptions: S. keyserlingii, T. karelini, H. japonica, and R. macrocnemis. Local extinctions

are most frequent in European region, reflecting the greater extent to which it has been

studied. Deforestation is the most frequent key factor in local extinctions (POLUSHINA,

1977; GARANIN, 1983). However, in a historical perspective, this may have more “global”

consequences for species (change of geographical ranges, etc.). Urbanization leads to local

declines. However, the rate of these declines is species-specific. In general, forest species

tended to decline and become extinct more rapidly than open-area species.

Some factors are reported to cause declines but not extinction. For example,

introduction of the fish Percottus glehni into some ponds in the Moscow Province led to

local declines of T. vulgaris and T. cristatus to less than 10 % of their previous abundance

(MANTEIFEL et al., 1991). Intensive cattle pasture led to marked declines in B. bombina and

R. arvalis numbers in different habitats of Severny Donets River basin (European Russia)

(GoGoLEvA, 1987).

Some factors have had complex negative influences. For example, felling of trees and

their trailing along the moutain streams led to almost total extinction of some Georgian

micropopulations of M. caucasica (personal observations). On the other hand, on the

Carpathian Mountains timber transportation by heavy traffic leads to the formation of

deep ruts in the roads, creating suitable conditions for newt breeding. The newts are

attracted by newly established water bodies and breed there. This led to the killing of T.

montandoni and T. alpestris (mainly eggs and larvae) by traffic and drying. As a result, the

population declined markedly (TARASZCZUK, 1985). Although overall amphibian mortality

on the roads may be high, its influence on population declines is unclear (GANEEV et al,

1985; RYZHEVICH, 1989).

Source : MNHN, Paris

KUZMIN 129

FLUCTUATIONS IN NUMBERS

Changes in population sizes have been documented in several regions of the former

U.S.S.R. Fluctuations caused by anthropogenic factors were briefly noted above.

Fluctuations of the numbers of R. temporaria and R. arvalis are caused by periodic

droughts and frosts (SERGEEV & VETSHEVA, 1942; BANNIKOV, 1948; KALETSKAYA, 1953).

After droughts marked reductions in the numbers of brown frogs occur, caused by

breeding ponds drying up. Afterwards frog numbers recover due to successful breeding in

renewed ponds. Fall in population size is also caused by high mortality in hibernacula

during frosty winters with little snow. Different species show different reactions to both

factors. R. temporaria is less tolerant of drought than the more southerly R. arvalis. The

latter, however, is less tolerant of frosts due to its exclusively terrestrial hibernation. Green

frogs living in permanent waters express higher tolerance to both factors. Monitoring of

amphibian populations in Volga-Kama region has shown wide annual fluctuations of both

specific and total amphibian densities (sometimes by 20-30 times) against the background

of the total decline (fig. 1). In other cases, the fluctuations are not accompanied by this

trend (fig. 2).

POPULATION INCREASE AND DISPERSAL

Extensions of geographical ranges and population increases may occur not only in

geological time, but also during the short term. For example, in Belorussia the increase of

P. fuscus, B. calamita and H. arborea probably took place in the XX‘ century

(SAPOZHENKOV, 1961). Its causes are still unknown. In Moscow Province the increase in

number and the dispersal of southern species (B. bombina, P. fuscus, B. viridis, R. arvalis)

occurred in the 1920-1940s, whereas the northern species either reduced their numbers

(8. bufo), or remained almost unchanged (R. temporaria) (BANNIKOV,1955). As in B. bufo,

B. viridis and R. arvalis, this tendency was observed at least up to the 1980s (per-

sonal observations). This is probably related to the general warming of the regional

climate.

However, population increases and dispersals are basically related to anthropogenic

factors. In Moscow Province ploughing up the water meadows led to a local increase of

P. fuscus (GORBUNOV, 1989). Tree-felling and country-road building in forests resulted in

the formation of small artificial pond systems and promoted the increase and local

dispersal of S. keyserlingü, P. fuscus, B. bufo, B. viridis, R. temporaria and R. arvalis

(PoLusHINA, 1977; PikuLiK, 1985; KUTENKOV, 1990; ISHCHENKO, personal communica-

tion). These were documented in Karelia, Ural, Belorussia and Carpathians. The creation

of artificial water bodies (fishponds, channels, sedimentation reservoirs, etc.) has promoted

population increases in S. keyserlingii, T. vulgaris, T. vittatus, B. bombina, P. fuscus, B.

bufo, H. arborea, R. arvalis, R. temporaria, R. ridibunda, and R. lessonaelesculenta

(ToporkovA, 1977; KUBANTSEV & ZHUKOVA, 1981; KiREEV, 1983; TUNIYEV et al., 1986;

GoGoLEva, 1987; TARASZCZUK, 1987; KUTENKOV, 1990; personal observations). These

events were observed in Central and Southern Russia, Kalmykia, Karelia, Middle Ural,

Northern Caucasus, forest and steppe zones of the Ukraine.

Source : MNHN, Paris

130 ALYTES 12 (3)

1948 1950 1952 1954 1956 1958 1960 1962 1964 1966 1968 1970 1972 1974 1976 1978

years

Fig. — Annual fluctuations in amphibian numbers in a plot of Volga-Kama region, Russia (from

GARANIN, 1983).

El; 1

100| 2

EX 3

90] EE 4

— à

70]

60]

50]

40| | |

10]

L Li .

1968 196 1970 197 1972 1973 19M 1975

19%6

years

Fig.2. — Annual fluctuations of different amphibian species on lakeshores of the Marii El, Russia

(ASTRADAMOV & ALYSHEVA, 1979). 1: Rana arvalis; 2: Rana esculenta/lessonae; 3: Rana ridibunda;

4: Bombina bombina; 5: total number.

Source : MNHN, Paris

KUZMIN 131

There are some examples of deliberate introductions of Anura by man. Breeding B.

viridis were found in 1984 in Novosibirsk City — at 650 km from the range margin of this

species (ZOLOTARENKO, 1985). R. nigromaculata tadpoles were imported to Turkmenistan

from China together with herbivorous fry fishes about 30 years ago. The frog was

successfully acclimatized in the Kara-Kum Channel (ATAEV & ATAEVA, 1981).

The most marked expansions are shown by R. ridibunda. Probably, between 1903 and

1939 this species naturally penetrated the Lake Balkhash basin (Kazakhstan) from the

River Chu valley (Kyrgyzstan) (KORELOV, 1953). Between 1964 and 1970 the species

extended eastwards: into the surroundings of Uch-Aral Village (Alakul Hollow, Kazakh-

Stan) (GRACHEV, 1971). Subsequent dispersal of the species was conditioned by anthro-

pogenic factors: in the European steppe and desert regions, by land-reclamation; in

Siberia, by thermal pollution of the environment (pouring out of warm waters into the

permanent reservoirs). In the 1960s, the frog was introduced with fry fishes to Issyk-Kul

Lake from River Chu valley, Kyrgyzstan, and started to displace local Rana asiatica. In

1969, tadpoles were imported with fry fishes from Krasnodar (Southern Russia) into the

city of Verkhny Tagil (Ural); since 1976, the population has expanded into neighbouring

rivers (ToPoRKOVA, 1977, 1978). Introductions to other Uralian cities (Ekaterinburg in

1977 and Chelyabinsk in 1981) led to new populations being established (VERSHININ, 1990).

In 1970, the species was first recorded in the city of Gorno-Altaisk (Altai Mountains,

Eastern Siberia), where it was introduced from the Osh Province (Kyrgyzstan) with fishes.

Since that time, a stable population of R. ridibunda has been established, which has

displaced local B. bufo and R. arvalis at some sites (YAKOVLEV, 1990). At the same time

R. ridibunda was introduced into ponds in Yakutsk City (BELIMOV & SEDALISHCHEV, 1980).

Introductions in Kazakhstan (cities of Karaganda, Pavlodar and Ust-Kamenogorsk) were

probably related to the release of frogs from local universities and institutes (PRUS &

SMOLYANINOVA, 1989).

PERSPECTIVES

Quantitative data on declining amphibian populations in the former U.S.S.R. are not

numerous. Nevertheless, existing records show declines to be widespread. Anthropogenic

factors play the main role in this. Inexplicable declines, or extensive short-term declines

under natural factors, have not been registered here. The increase and dispersal of several

species under anthropogenic influences have taken place against a general background of

impoverishment of regional amphibian assemblages. “Perestroika” and the dismember-

ment of the U.S.S.R. have led to economic and political chaos. The latter may result in

both the retardation of environmental destruction due to the closing of factories, and in

non-controlled destruction of nature in other sites due to violation of laws. The latter may

already be occurring, e.g. in an unprecedented trade in amphibians, including protected

species.

In 1992, a C.I.S. Regional Group was established within the Declining Amphibian

Populations Task Force of I.U.C.N. This Group consists of amphibian biologists working

in 10 Sub-Regional Groups. We look forward to consolidating our studies on amphibian

declines within the territory of the former U.S.S.R.

Source : MNHN, Paris

132 ALYTES 12 (3)

ACKNOWLEDGEMENTS

Lexpress my gratitude to Dr. Vladimir G. ISHCHENKO and Mr. Rudolf A. KUByYkIN for the

information on amphibian local dispersals and declines, and to Annemarie OHLER and Roger BOUR

for redrawing the figures and preparing the table for publication.

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amphibians. /n: Problems of herpetology, Kiev, Naukova Dumka: 262-263.

(61) VERSHININ, V. L., 1987. — Adaptive peculiarities of Rana arvalis groups in the conditions of a

large city. Ekologiya, À (1): 46-50.

(62) YAKOVLEV, V. A., 1990. — On the ecology of Rana ridibunda on Altai. Ekologiya, 1 (1): 67-71.

(63) ZaBrODA, S. N., 1988. — Influence of the drying measures on the batrachofauna of Ukrainian

Polesje. /n: Ratsionalnoe ispolzovanie, okhrana, vosproizvodsivo biologischeskikh resursov

ekologiya vospitaniya, Zaporozhie: 215.

(64) ZoLoTARENKo, G. $., 1985. — On the record of Bufo viridis in the Novosibirsk surroundings. /n:

Problems of herpetology, Leningrad, Nauka: 80-81.

Corresponding editor: Tim HALLIDAY.

Source : MNHN, Paris

Alytes, 1994, 12 (3): 135-144. 135

lonic net fluxes through the in situ epithelia

of larval Caudiverbera caudiverbera

(Anura, Leptodactylidae)

Berta ZAMORANO* & Alfredo SALIBIAN**!

* Departamento de Fisiologia y Biofisica, Facultad de Medicina,

Universidad de Chile, Casilla 70005 - Correo 7, Santiago, Chile

** Câtedra de Fisiologia Animal, Facultad de Ciencias Naturales y Museo,

Universidad Nacional de La Plata, Paseo del Bosque, 1900 La Plata, Argentina.

The nature of the in vivo epithelial exogenous Cl and Na‘ uptake

mechanisms at gilled and gill-less stages of tadpoles of the Chilean frog

Caudiverbera caudiverbera was studied.

lon net fluxes (Jn) in tadpoles acclimated in tap-water (controls),

deionized water and in choline.Cl and Na,SO, solutions were measured.

Larvae took up both CI and Na* from diluted NaCI solutions. Tadpoles

kept in deionized water showed the highest fluxes; larvae from choline.Cl

solutions showed elevated JnNa* while those from Na, SO, had very high

dnCT.. The differences found in the Jn suggest that the ion uptake mechanisms

might be partially independent.

Acclimation solutions slightly affected the plasma ion concentration

values with respect to controls. Concentrations of CI" and Na* of animals from

deionized water showed a decreasing tendency; the level of those ions was

lower in plasma of tadpoles maintained in Na,SO, and choline.Cl, respecti-

vely.

It was postulated that the in vivo ion uptake must be active.

INTRODUCTION

The mechanisms of water and ion equilibrium in amphibian larvae are not as

extensively understood as in adults (see DUELLMAN & TRUEB, 1985). Several authors have

summarized our knowledge on different aspects of the osmoregulation in larvae of these

vertebrates (ALVARADO, 1979; WARBURG & ROSENBERG, 1990).

We attempted to improve our comprehension of the nature of the epithelial ionic

uptake mechanisms during the larval ontogenetic cycle of Caudiverbera caudiverbera. I is

a large aquatic leptodactylid endemic to Chile (Cr1, 1962; Diaz, 1983; SALIBIAN, 1974,

1. To whom reprint requests should be addressed.

Source : MNHN, Paris

136 ALYTES 12 (3)

1980), characterized by the fact that after metamorphosis juveniles remain in contact with

water throughout their lives. Only few anurans are known to share this unusual type of

life cycle with C. caudiverbera.

The sequence of morphological events during metamorphosis of this species is similar

to those described in the larval development of terrestrial anurans. ZAMORANO et al. (1988)

studied the pattern of urinary nitrogen excretion along their larval-juvenile transition; they

found a physiological peculiarity: in spite of their permanent aquatic condition, a shift

from ammoniotelism to ureotelism occurs as in the water-to-land model of metamorpho-

SIS.

Our main purpose was to study the characteristics of exogenous CI and Na* uptake

mechanisms through the intact epithelia of prometamorphic and climactic larval stages of

C. caudiverbera after acclimations in solutions of different chemical composition.

MATERIALS AND METHODS

PREPARATION OF THE ANIMALS

Larvae were collected in natural ponds at Melipilla, Chile. The animals were held in

the laboratory at 18°C in aquaria containing tap-water renewed daily and fed ad libitum

with cooked spinach. The photoperiod was LD 12/12. It has been demonstrated that the

ion transport mechanisms in amphibian larvae are affected by environmental factors such

as temperature (PARSONS, 1975) and season (Cox & ALVARADO, 1979). Since the

experiments reported here were conducted at constant temperature and photoperiod and

carried out during a short period of time, it is reasonable to conclude that our results were

not irregularly affected by those factors.

Before the experiments, animals were divided into four groups and left without food

for 15-20 days in one of the following solutions: (1) tap-water (control group}; (2)

deionized water (prepared from distilled water passed through a Kotterman 7002 resin

column); (3) 3.4 mEq.l'' choline.Cl; (4) 3.4 mEq.l'! Na,SO,. Animals tolerated acclimation

conditions without signs of perturbation. In previous studies carried out on adults of C.

caudiverbera (MORENO et al., 1978; SALIBIAN, 1970, 1973), we have demonstrated that

animals kept in tap-water could be considered as controls since the evaluated morpholo-

gical and chemical parameters were identical to those of frogs maintained in NaCI dilute

solutions.

The CI and Na* concentrations in the tap-water were periodically checked during the

acclimation period; they were (means + SEM, in mEgq.l!) 3.2 + 0.5 (n = 12) and 2.9 +

0.4 (n = 12), respectively.

Animals were staged adapting the notation of GosnER (1960). The considered stages

were the following: (1) gilled (or ETkIN's prometamorphic) stages: 36-37 (toes partially or

totally separated) and 38-39 (metatarsal tubercle and pigment free patches on the inner

surface of toes appear); and (2) gill-less (or climactic) stages: 42-43 (incomplete regression

of the tail) and 44-46 (complete resorption of the tail).

Source : MNHN, Paris

ZAMORANO & SALIBIAN 137

The body weight range (in grams) of tadpoles at stages 36-37 was 18.6-24.0, and

34.2-40.4 at stages 38-39; in the gill-less larvae the ranges were 28.9-34.0 (stages 42-43) and

19.5-21.3 (stages 44-46).

MEASUREMENT OF IONIC NET FLUXES

Animals were weighed in water with a digital Sartorius 2250 balance at the beginning

and at the end of the experiments; no statistically significant changes were observed

between these two data sets of values. A polyethylene PE 20 cannula was inserted into the

cloaca of the larvae and tightly fastened by a concentric subepithelial ligature; by this

means renal and intestinal contamination of the external bath was avoided.

Each cannulated tadpole was individually placed in a container with 200 ml of its

acclimation solution and maintained in that condition for about 16 h; thus physiological

alterations due to the manipulation stress were avoided, as it was found in our laboratory

(unpublished). Then the external solution was carefully replaced by siphoning with

150-200 ml of 1.7 mEgq.l' NaCI solutions. Aliquots of the external bath for subsequent

analyses of CI and Na* concentrations were taken at the beginning of the experiments

and every 30-45 mn during the following 6-7 h.

The CI and Na* concentrations in those samples were plotted against time and the

net fluxes calculated from the slopes of the regression lines. For this calculation a mean

volume of the external bath was considered. The experiments were performed in spring;

each tadpole was used in only one experiment. Animals that showed abnormally high

negative net fluxes during the first hour of the experiment or with signs of skin damage

were discarded.

Since our flux data corresponded to animals fasted for 2-3 weeks prior to the

measurements, food must be discarded as an additional source for balancing the Cl and

Na* losses. ALVARADO & MooDY (1970) have shown that even in the fasting condition,

the gut cannot be a physiologically relevant site for ion accumulation from the external

bath because the volume drunk by the tadpoles can be considered negligible.

BLOOD SAMPLING

Blood samples were drawn from animals anesthetized in 0.3% tricaine (Sigma)

solutions through a little cut in the extreme of the ventricle. Blood was collected in glass

tubes with heparin (Biochimie) at 5 IU.ml'! of blood. Plasma was separated by

centrifugation at 4°C in a Sorvall RC-2B centrifuge at 600 g for 15-20 min, and diluted

1/100 before the analyses.

ANALYTICAL TECHNIQUES

Chloride concentration was evaluated potentiometrically (SANDERSON, 1952) with a

Radiometer titration unit (pHmeter 26, TTT auto titrator and autoburette type ABU-1C)

or with a Buchler digital H-2500 chloridometer. Sodium concentration was determined by

emission photometry with an Eppendorf flame photometer.

Source : MNHN, Paris

138 ALYTES 12 (3)

EXPRESSION OF THE RESULTS AND STATISTICAL ANALYSES

AII data are given as means + SEM (standard error of the mean). Net ion fluxes are

expressed as pEq.h°'.100 g'' animal weight. Plasma ion concentrations are in mEq.l"'.

Student’s t test was used to test differences between means of ion net fluxes. Results

of plasma CI and Na* concentrations of tap-water, deionized water and choline.Cl

animals were tested for normality (Kolmogorov-Smirnov); then a two-way analysis of

variance followed by Scheffé-Dunnett tests for comparison of means were made. Plasma

ion concentrations of Na,SO, larvae only were tested for significant differences between

36-37 and 44-46 stages data of animals by means of Student’s t test.

RESULTS

NET IONIC FLUXES IN CONTROL TAP-WATER ACCLIMATED LARVAE (TABLE I)

The magnitude of CI and Na‘ epithelial net fluxes (Jn) were similar at stages 36-37

and 42-43; in the latter the fluxes were reduced by 50 %. At the end of metamorphosis

(stages 44-46), there was a further reduction and a partial dissociation of the fluxes, with

JnCT being significantly higher than JnNa*; this dissociation was not observed at the two

earlier stages.

NET IONIC FLUXES IN DEIONIZED WATER ACCLIMATED LARVAE (TABLE II)

When compared with those of controls from tap-water, the absolute JnCT and JnNa*

values in these larvae were always very high. At early stages (36-37 and 38-39), JnNa* was

higher than JnCl while the opposite occurred at the last gill-less stages of the

metamorphosis.

NET IONIC FLUXES IN CHOLINE.CL SOLUTIONS ACCLIMATED LARVAE (TABLE III)

In all studied stages the incubation of animals in Na-free solutions resulted in higher

JnNa* than JnCf. The differences between net fluxes remained unchanged during stages

with gills, but increased abruptly later as metamorphosis advanced. It is interesting to note

that in these animals the JnCÏ were comparable to those of control tap-water tadpoles of

the same stage.

NET IONIC FLUXES IN NAS04 SOLUTIONS ACCLIMATED LARVAE (TABLE IV)

Results show that at stage 37 both JnCf and JnNa* were low and similar. When

animals reached stages 44-46 fluxes were dissociated, with JnCT being seven times higher

than JnNa* which, in turn, was almost identical to that found in control animals of the

same age.

Source : MNHN, Paris

ZAMORANO & SALIBIAN

139

Table I. - CF and Na* net fluxes (Jn) through the in vivo epithelia of larval Caudiverbera

caudiverbera acclimated in tap-water. Fluxes were measured from 1.7 mEq.l' NaCI

solutions; data in uEq.h*.100 g' body mass (means + SEM); N: number of

experiments.

Larval stages MC JnNat

HhCI-MNat

36-37 +17.6 + 1.3 +18.0 + 1.9

42-43 +9.9 + 0.9 +10.9 + 0.8

44-46 +3.6 + 0.5 +3.0 + 0.3

—0.4+0.1

—10+0.1

+0.6+ 0.1

1. Mean differences of paired data + SEM.

2. Statistical significance of the differences.

Table IL. - CF and Na* net fluxes (Jn) through the in vivo epithelia of larval Caudiverbera

caudiverbera acclimated in deionized water. Fluxes were measured from 1.7 mEq.l'

NaCI solutions; data in HEgq.h*.100 g' body mass (means + SEM); N: number of

experiments.

Larval stages CF JnNat

JnCF-InNa*t !

+42.7+42 +71.443.2

233.L25 +32.9+2.3

+23.1 + 1.9 +218+2.1

+57.3 + 1.6 +29.5+ 1.1

1. Mean differences of paired data + SEM.

2. Statistical significance of the differences.

—28.7 + 0.9

—9.6+0.5

+1.3 + 1.0

+27.8 + 0.9

< 0.001

Table IN. - CF and Na* net fluxes (Jn) through the in vivo epithelia of larval

Caudiverbera caudiverbera acclimated in 3.4 mEq.h choline.Cl solutions. Fluxes

were measured from 1.7 mEq.l NaCI solutions; data in HEq.h*.100 g' body mass

(means + SEM); N: number of experiments.

Larval stages MC JaNat

HCF-MNa*t

36-37 +20.2 + 2.0 +28.9 + 1.5

38-39 +21.7 + 0.7 +29.4 + 2.8

42-43 +2,34 0.4 +14.9 + 1.6

44-46 +6.1 # 1.4 427.04 23

—8.7+2.3

—7.7+2.5

—12.6 + 0.9

—20.9 & 1.0

1. Mean differences of paired data + SEM.

2. Statistical significance of the differences.

Source : MNHN, Paris

140 ALYTES 12 (3)

Table IV. - CF and Na* net fluxes (Jn) through the in vivo epithelia of larval

Caudiverbera caudiverbera acclimated in 3.4 mEq.l' Na:SO, solutions. Fluxes were

measured from 1.7 mEq.l NaCl solutions; data in WEq.h*.100 g' body mass (means

+ SEM); N: number of experiments.

Larval stages IMCF JnNat InCF-MnNa* !

37 +2.9+0.2 +2.8 + 0.3 +0.1+ 0.1

44-46 +19.7 + 0.7 +2.7# 0.4 +17.0 + 0.1

1. Mean differences of paired data + SEM.

2. Statistical significance of the differences.

Table V. - Plasma chloride and sodium concentration of Caudiverbera caudiverbera

larvae acclimated in different solutions. Data in mEq.l (means + SEM), in

parenthesis, number of animals; N.M.: not measured.

Acclimation solutions

Choline.Cl Na2SO4

Tap-water Deionized water (G4mEq.f) (3.4 mEg+)

68.541.6(10) 564432(10) 71L6+1.7(10) 63.04 1.6 (7)

6934+1.8(10) 6384+20(10) 75.642.4(10) NM.

71.140.4(13) 742#1.7(10) 70.94 2.0 (8) NM.

754411(10) 693+03(11) 75442.1(10) 66.34+2.2(7)

79.64#30(10) 726#5.6(10) 76.2+1.6(8) 87.3+3.5(7)

8224+2.6(10) 67.5+1.7(10) 78.1 +2.4 (10) NM.

96.8+2.7(13) 873+1.2(10) 86.5+2.5 (8)! NM.

106.3+2.4(10)! 97.9+2.5(11) 95.2+1.8(10) 105.6 + 2.6 (7)

1. Within each acclimation solution, significantly different from the 36-37 stage larvae (p <

0.05).

2. Within each larval stage range, significantly different from the tap-water larvae (p <

0.05).

3. Significantly different from the 36-37 stage larvae (p < 0.001).

Source : MNHN, Paris

ZAMORANO & SALIBIAN 141

PLASMA CL' AND NA* CONCENTRATIONS (TABLE V)

In comparison to the Cl concentrations in plasma of control larvae, there was a trend

towards its reduction in animals from deionized water and Cl-free solutions. The CI

concentration in plasma of larvae kept in choline.Cl solutions remained unaltered with

respect to tap-water controls.

When compared with control tap-water acclimated tadpoles, the Na* plasma

concentration of animals from deionized water and Na-free solutions showed a tendency

to decrease; the plasma Na* concentration of Na,SO, acclimated animals was not

different from those of controls.

In all groups of animals the plasma Na * concentrations showed a significant increase

in the gill-less stages whereas the Cl concentrations did exhibit a similar but less

pronounced trend.

DISCUSSION

Both gilled and gill-less tadpoles of Caudiverbera caudiverbera showed the capacity to

take up CF and Na* from dilute external NaCI solutions. This ability was found to exist

independently of the age of the animals and of the acclimation conditions.

Our preparation does not allow to discriminate precisely the sites where transport

occurs at each stage. However, evidence reported by several authors (see ALVARADO, 1979)

indicates that during anuran metamorphosis there are gill-to-skin shifts as site for ion

translocations. In tadpoles of C. caudiverbera indirect evidence in favor of the postulated

shift along the larval-juvenile transition from the gill as the dominant organ towards the

skin as the main site for ion transport was reported by GONZALEZ et al. (1979). They

measured the specific activity of ouabain sensitive (Na* + K*)-ATPase in isolated gills

and skin of larvae at different stages of animals stored in tap-water. At stages 30-35 they

showed the existence of enzymatic activity only in gills; at the intermediate stages 36-39 the

activity was found both in gills and skin in the proportion of 1/3; later, close to the end

of metamorphosis (stages 40-43), most of the enzyme activity was detected on the skin.

Relative to the NaCI solutions used in flux measurements, plasma of C. caudiverbera

tadpoles were always markedly hyperionic with respect to CI and Na*; the ratios of their

concentrations in plasma to external bathing media varied between 40 and 62.

We conclude that our data allow us to postulate that the ion accumulation processes

of larval C. caudiverbera are active and must be attributed to epithelial mechanisms

located, according to the age of the animals, in gills and/or skin.

The acclimation condition affects the magnitude of ionic net fluxes when animals are

transferred to NaCI solutions. In control tap-water animals (Table I), the JnCT and the

JnNa* were practically similar; this could be interpreted as an indication of a linked

uptake of CI and Na*. However, at climactic stages a clear-cut difference among the net

fluxes appeared, suggesting the existence of a differentiation in their ion uptake

Source : MNHN, Paris

142 ALYTES 12 (3)

mechanisms, the animals being able to take up a fraction of the exogenous Cl by a

Na*-independent ionic exchange system.

After keeping tadpoles in deionized water (Table II), the ion net fluxes were

augmented at least three times with respect to controls; the increases were constant and not

dependent on the developmental stages. Similar response to ion-free incubation of the

animals was shown in intact R. catesbeiana tadpoles (ALVARADO & Moopy, 1970); in the

same species, BROONKOOM & ALVARADO (1971) demonstrated an increase in the (Na* +

K *)-ATPase activity in gill homogenate of “salt depleted” animals.

Tadpoles coming from Na-free solutions (Table III) took up Na* at higher rate than

CT, independently of the considered age. Conversely, the JnCl in animals from Cl-free

solutions (Table IV) were higher than the JnNa *; the ion net fluxes were among the lowest

registered in the early stages. In both groups the differences between fluxes were greatly

enhanced in climactic stages.

It must be emphasized that the observed differences in epithelial net ion flux values

between the controls and the remaining three groups cannot be attributed to changes in

the plasma CT and Na* levels (see Table V). It might be necessary to search for that

correlation in the intracellular condition of the epithelial cells involved in ion transport

processes rather than in that of the extracellular fluids (Cox & ALVARADO, 1983; LARSEN,

1988). The relative constancy found in the CI and Na* concentrations of the extracellular

compartment may explain the excellent tolerance of these animals to incubation for 2-3

weeks in artificial environmental conditions.

Tadpoles in ion-free media are exposed to a severe limitation in their capacity to

compensate for the normal ion and water losses. We postulate that the particular condition

of these animals may trigger an endocrine compensatory reponse, increasing the circulatory

levels of hormones involved in the hydromineral balance regulation through epithelia,

especially neurohypophysial peptides and aldosterone. The integrated action of these

hormones upon their effectors may act by promoting an increased reabsorption of water

and ions, principally Na*, thus reducing the losses and, consequently, maintaining the

plasma levels stable over the ion deprived incubation period of time. The existence of such

compensatory responses was shown in adults of several anuran species (BENTLEY, 1969;

CRABBE, 1963; Rojas et al., 1987) and in embryos of Bufo arenarum (CASTARÉ et al., 1987).

Finally, the magnitude of the in vivo ion net fluxes of control tadpoles tend to decrease

as metamorphosis goes on and become close to those measured in adults (GARCIA ROMEU

et al., 1969). This fact might be the consequence of the reduction in the ion exchange areas

due to resorption of the gills and tail and/or ontogenetic selective modifications in the

magnitude of the unidirectional fluxes.

RESUMEN

Se estudi la naturaleza de los mecanismos epiteliales de captaciôn in vivo de CT y

Na* exôgenos en estadios larvales branquiados y no branquiados de larvas de la rana

chilena Caudiverbera caudiverbera.

Source : MNHN, Paris

ZAMORANO & SALIBIAN 143

Se midieron los flujos iénicos netos (Jn) en larvas aclimatadas en agua potable

(controles), agua deionizada y en soluciones de Cl.colina y SO,Na,.

Los animales captaron CI y Na* desde soluciones diluidas de CINa. Los Jn mäs altos

se observaron en larvas de agua deionizada; las de Cl.colina tuvieron los JnNa* elevados

mientras que los JnCT fueron mayores en las de SO,Na.. Las diferencias halladas en los

Jn sugieren que los mecanismos de captaciôn i6nica podrian ser parcialmente indepen-

dientes.

Las soluciones de preadaptaciôn afectaron ligeramente las concentraciones plasmä-

ticas de CI y de Na* con respecto a las de los controles. Las de los animales de agua

deionizada mostraron una tendencia a la reducciôn; los niveles de esos iones resultaron

disminuidos en larvas mantenidas respectivamente en SO,Na, y Cl.colina.

Se postulé que el transporte de iones in vivo debe ser de caräcter activo.

ACKNOWLEDGEMENTS

This study was supported by a grant from the Universidad de Chile (DTI: PEA-003-791).

Professor A. SALIBIAN is a member of the Carrera del Investigador Cientifico Tecnolôgico, Comisin

de Investigaciones Cientificas de la Provincia de Buenos Aires, and Director of the Applied

Ecophysiology Program, Divisiôn Biologia, Departamento de Ciencias Bäsicas, Universidad Nacional

de Lujän, Argentina.

The authors are indebted to Mr. Matias O. SALIBIAN-BARRERA for carefully preparing the

typescript.

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Corresponding editor: Günter GOLLMANN.

Source : MNHN, Paris

AIMTES

International Journal of Batrachology

published by ISSCA.

EDITORIAL BOARD FOR 1994

Chief Editor: Alain Duois (Laboratoire des Reptiles et Amphibiens, Muséum national d'Histoire

naturelle, 25 rue Cuvier, 75005 Paris, France).

Deputy Editor: Günter GOLLMANN (Institut für Zoologie, Universität Wien, AlthanstraBe 14, 1090

Wien, Austria).

Editorial Board: Ronald G. AzriG (Mississippi State University, U.S.A.); Emilio BALLETTO (Torino,

Italy); Alain COLLENOT (Paris, France); Tim HALLIDAY (Milton Keynes, United Kingdom);

W. Ronald HEYER (Washington, U.S.A.); Walter HôDL (Wien, Austria); Pierre JOLY (Lyon,

France); Masafumi Marsu1 (Kyoto, Japan); Jaime E. PÉFAUR (Mérida, Venezuela); J. Dale

ROBERTS (Perth, Australia); Ulrich SinscH (Koblenz, Germany); Marvalee H. Wake (Berkeley,

US.A.).

Technical Editorial Team (Paris, France): Alain Dugois (texts); Roger Bour (tables); Annemarie

OKLER (figures).

Index Editors: Annemarie OHLER (Paris, France), Stephen J. RICHARDS (Townsville, Australia).

GUIDE FOR AUTHORS

Alytes publishes original papers in English, French or Spanish, in any discipline dealing with

amphibians. Beside articles and notes reporting results of original research, consideration is given for

publication to synthetic review articles, book reviews, comments and replies, and to papers based

upon original high quality illustrations (such as color or black and white photographs), showing

beautiful or rare species, interesting behaviors, etc.

The title should be followed by the name(s) and addresses) of the author(s). The text should

be typewritten or printed double-spaced on one side of the paper. The manuscript should be organized

as follows: English abstract, introduction, material and methods, results, discussion, conclusion,

French or Spanish abstract, acknowledgements, literature cited, appendix.

Figures and tables should be mentioned in the text as follows: fig. 4 or Table IV. Figures should

not exceed 16 X 24 cm. The size of the lettering should ensure its legibility after reduction. The

legends of figures and tables should be assembled on a separate sheet. Each figure should be

numbered using a pencil.

References in the text are to be written in capital letters (SOMEONE, 1948; So & So, 1987;

EveryBopY et al., 1882). References in the literature cited section should be presented as follows:

BourReT, R., 1942. - Les batraciens de l'Indochine. Hanoi, Institut Océanographique de l’Indochine:

i-x + 1-547, pl. I-IV.

GRAF, J.-D. & PoLLs PELAZ, M., 1989. - Evolutionary genetics of the Rana esculenta complex. In:

R. M. DAWLEY & J. P. BOGART (eds.), Evolution and ecology of unisexual vertebrates, Albany, The

New York State Museum: 289-302.

INGER, R. F., Vois, H. K. & VorIis, H. H., 1974. - Genetic variation and population ecology of some

Southeast Asian frogs of the genera Bufo and Rana. Biochem. Genet., 12: 121-145.

Manuscripts should be submitted in triplicate either to Alain Dumois (address above) if dealing

with amphibian morphology, systematics, biogeography, evolution, genetics or developmental

biology, or to Günter GOLLMANN (address above) if dealing with amphibian population genetics,

ecology, ethology or life history.

Acceptance for publication will be decided by the editors following review by at least two

referees. If possible, after acceptance, a copy of the final manuscript on a floppy disk (3 % or 5 4)

should be sent to the Chief Editor. We welcome the following formats of text processing: (1)

preferably, MS Word (1.1 to 6.0, DOS or Windows), WordPerfect (4.1 to 5.1, DOS or Windows) or

WordStar (3.3 to 7.0); (2) less preferably, formated DOS (ASCII) or DOS-formated MS Word for the

Macintosh (on a 3 % high density 1.44 Mo floppy disk only).

No page charges are requested from the author(s), but the publication of color photographs is

charged. For each published paper, 25 free reprints are offered by Alytes to the author(s). Additional

reprints may be purchased.

Published with the support of AALRAM

(Association des Amis du Laboratoire des Reptiles et Amphibiens

du Muséum National d'Histoire Naturelle, Paris, France).

Directeur de la Publication: Alain DuBois.

Numéro de Commission Paritaire: 64851.

Alytes, 1994, 12 (3): 93-144.

Contents

Theodora S. SOFIANIDOU, Hans SCHNEIDER & Ulrich SINSCH

Comparative electrophoretic investigation on

Rana balcanica and Rana ridibunda from northern Greece .......... 93-108

Orlando CUELLAR

Ecological observations on Rana pretiosa

TN El UE AS, SET ARE RSR EEE ER 109-121

Sergius L. KUZMIN

The problem of declining amphibian populations

in the Commonwealth of Independent States

ANONATIACONILENTITONMES RS een rennes eee tre 123-134

Berta ZAMORANO & Alfredo SALIBIAN

Ionic net fluxes through the in situ epithelia

of larval Caudiverbera caudiverbera (Anura, Leptodactylidae) ...... 135-144

Announcement

Défense et illustration de l’histoire naturelle avec Muséum 2000 122

Alytes is printed on acid-free paper.

Alytes is indexed in Biosis, Cambridge Scientific Abstracts, Current Awareness in Biological

Sciences, Pascal, Referativny Zhurnal and The Zoological Record.

Imprimerie F. Paillart, Abbeville, France.

Dépôt légal: 4°" trimestre 1994.

Source : MNHN, Paris