A New Species Of Alsodes (Amphibia : Anura : Leptodactylidae) From Southern Chile

10 Pages·1998·3.6 MB·

by  Formas

PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON
lll(3):521-530. 1998.
A new species ofAlsodes (Amphibia: Anura: Leptodactylidae) from
southern Chile
J. Ramon Formas, Cesar Cuevas, and Jose Nunez
Instituto de Zoologia, Universidad Austral de Chile, Casilla 567, Valdivia, Chile
—
Abstract. A new species offrog, Alsodes kaweshkari, is described fromthe
temperate Nothofagus forests of South America in Southern Chile. From the
karyological point of view this species is included in the monticola group (2N
= 26) of the genus Alsodes.
The genus Alsodes Bell 1843, is distrib- caliper to the nearest 0.1 nmi according to
uted in central and southern Chile and along Cei (1962). The following measurements
the eastern slope ofthe Andes, in Argentina were taken: snout-vent length (SVL), head
(south of Mendoza city). Frost (1985) in- length, head width, nostril-snout distance,
cluded the following species in the genus: tibia length, and foot length. Internarial dis-
A. barrioi, A. gargola, A. Hiatus, A. laevis, tance was measured according to Cei
A. montanus, A. monticola, A. nodosus, A. (1980) and eye diameter according to
pehuenche, A. tumultuosus, A. vanzolinii, Duellman (1970).
and A. verrucosus. Formas (1989) studied The chromosomal characteristics were
the identity and synonymy of the Chilean obtained from the holotype (IZUA 1624)
frog Eupsophus vittatus and concluded that according to the following methods: the an-
this species must be included in the genus imal was treated with 0.1% colchicine for
Alsodes asA. vittatus. The taxonomic status two hours, then anaesthetized with diethyl
ofA. illotus is not clear (Cei 1980) and ac- ether, cut open ventrally under sterile con-
cording to Frost (1985) A. laevis has not ditions and its intestine carefully removed.
been collected since its description by Phi- Metaphase plates were obtained by squash-
lippi (1902). The most remarkable charac-
ing intestinal epithelium fragments that
teristic of the males of this genus is the were hypotonically treated with distilled
presence ofthorny structures on the fingers
water, then fixed in acetic-alcohol (1:3), and
and round spiny patches on the chest. finally placed in 45% acetic acid. Small tis-
During the course of collecting frogs in
sue fragments were squashed between a
southern Chile we found a new species of
glass slide and cover slip and dipped in liq-
Alsodes which is here described. Moreover,
we describe the karyotype and C-banded uid nitrogen; thereafter, the cover slip was
chromosomic pattern ofthe new taxon. The removed with a razor blade to allow chro-
bands of hemoglobin of the new species mosomes to air dry. After 3 days, the chro-
mosomes were stained for 15 minutes in
were characterized electrophoretically and
compared with those ofA. monticola (type Sorensen's phosphate buffer (pH 6.8), con-
species of the genus). taining 4% Giemsa solution (Formas 1991).
Centromeric positions were determined ac-
cording to Levan et al. (1964). Secondary
Methods and Materials
constrictions were included in the measure-
Specimens were depositedin the Instituto ments. Chromosomes were stainedto reveal
de Zoologia, Universidad Austral de Chile C-band pattern position (Sumner 1972).
(IZUA). Adults were measured with a dial Microscopic slides andthe specimen(IZUA
522 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON
M. fl<f.mrt ^7
cm
2
Fig. 1. Alsodes kaweshkari Holotype, IZUA 1624.
1624) were deposited in the amphibian col- new species and A. monticola. To proceed
lection of the Instituto de Zoologia, Univ- with the electrophoresis the samples were
ersidad Austral de Chile. denatured by heating (100°C, 5 minutes) in
Hemoglobin was obtained according to presence of 2% of betamercaptoethanol.
the following methodology: blood was col- Electrophoresis of the hemoglobins from
lected by cardiac puncture from the holo- Alsodes kaweshkari and A. monticola was
type and a specimen of Alsodes monticola carried out individually in vertical slab gels
(control species) using a sterile syringe with of polycrilamide (15%) in denaturant con-
mA
3.8% ofsodium citrate as anticoagulant; the ditions at 20 for 20 hours (Laemmeli
red cells were washed 3 times with 0.85% 1970). Gels were stained with Coomassie
NaCl and centrifuged at 700 g for 5 minutes Blue R-250 for 2 hours and destained with
at room temperature (20°C). The packed acetic acid (7%). The molecular weight of
cells were hypotonically treated with dis- the hemoglobin bands was estimated using
tilled water (10 volumes) for 30 minutes a protein standard for molecular weight
and centrifuged at 3500 g for 10 minutes. (Sigma Co).
The supernatant was stored at —70°C. The
hemolyzate was treated according to the Systematics
method described by Muir (1981) with
modifications. We used a minicolumn (5 X Alsodes kaweshkari, new species.
70 mm) of Sephadex G-50 equilibrated Fig. 1
mM —
with 1.0 of potassium phosphate (pH Holotype. IZUA 1624, an adult male
7.0). Fractions of500 |jl1 were collected and collected by J. Ramon Formas on 8 March
their absorbances measured at 410 nm, the 1995, at Puerto Eden (49°8'S, 74°25'W, 10
wave length at which the porphyrin ring of m), Wellington Island, Ultima Esperanza
the hemoglobin molecule absorbs specifi- Province, Magallanes and Chilean Antartic
cally. Those fractions with the highest ab- Region (XII—Region), Chile (Fig. 2).
sorbance value were used for the electro- Paratype. IZUA 1625, an adult male
phoretic analysis of the hemoglobin of the collected by Pablo Corti on 21 Oct 1995,
VOLUME NUMBER
111, 3 523
-46*
Peninsula de \ REPUBLICA
Taitao
J
ARGENTINA
-48*
Isia
Wellington
a
50'
100 km.
76*
i_
Fig. 2. Type locality ofAlsodes kaweshkari.
at Seno Huemules (48°43'5S, 74°25'W, 8 Body robust, arms and legs well developed.
m). UltimaEsperanzaProvince, Magallanes Head depressed, slightly wider than long,
and Chilean Antartic Region (XII Region), its length 29% of snout-vent length. Snout
Chile (Fig. 2)—. truncated from above, rounded in lateral
Diagnosis. Alsodes kaweshkari can be profile; canthus rostralis slightly rounded,
distinguished from its congeneric species loreal region concave in cross section; nos-
by the following combination ofcharacters: trils anterolateral, midway between tip of
SVL 56.5-62.2 mm; toes totally fringed; snout and anterior border of eye; eye di-
webbing of feet present between all toes, ameter greater than distance between eye
but reduced; dorso-lateral surfaces granular; and nostril; intemarial distance less than in-
skin around vent and posterior thighs gran- terorbital distance. Tympanum absent; post-
ular. — ocular fold evident, reaching insertion of
Description. Based on the type series. arm. Tongue rounded, without notch at tip.
524 PROCEEDINGS OFTHE BIOLOGICAL SOCIETY OF WASHINGTON
Fig. 3. Morphological details of the holotype. (A) Palmar view of the right hand. (B) Plantar view of the
right foot.
Choanae oval-shaped; dentigerous process- tips. Inner palmar tubercle ovoid, outerpal-
es ofvomer between choanae. Forelimbs of martubercle rounded; subarticulartubercles
males robust. Fingers in order ofincreasing rounded, (Fig. 3A). First finger with strong
length: II, I, IV, III. Webbing of hand ab- thorny excrescences; second finger with
sent. Fingers long with moderately globular narrow band of spines (Fig. 4B). Palmar
VOLUME NUMBER
111, 3 525
MjWfo
l**!
5mm 5mm
Fig. 4. Secondary sexual characters of the holotype. (A) Ventral view of the chest and throat. (B) Dorsal
view ofthe left hand.
surface with few thorns irregularly distrib- sal surfaces of head and areas below dor-
uted. Spines also present at lower border of solateral fold with granules.
mandible (Fig. 4A). Toes long, thick and The measurements of the holotype and
fringed; in order of increasing length: I-II- the paratype a—re given in Table 1.
III-V-IV; tips moderatelyrounded. Webbing Coloration. In alcohol, dorsal surfaces
present, thick, but reduced. Inner metatarsal of ground, arms and legs dark gray. Venter
tubercle oval, elongate; outer tubercle whitish, gular area dark gray. Arms and
smaller than the inner, oval. Tarsal fold legs whitish ventrally. The granular surface
present; reaching middle of tarsus. Flanks around posterior thighs dark gray. In life,
and dorsal surface granular; ventral skin dorsal surface, arms and legs brown. Belly
smooth with minute granules; skin around whitish yellow. Areas around nostrils, lips
vent and posterior thighs granular. Chest of and eyes light brown. The iris with a cop-
males with two bilateral rounded black pery reticulum.
—
patches ofkeratinous spines (Fig. 4A). Dor- Distribution and ecology. The species
is known from two Chilean localities (Puer-
— to Eden and Seno Huemules) (Fig. 2). The
Table 1. Measurements (mm) ofthe type series of type locality (Puerto Eden) is a small beach
Alsodes kaweshkari. of the Wellington Island, near a small fish-
ing town. There are small forests (Notho-
Holotype Paratype
IZUA 1624 IZUA 1625 fagus betuloides, Embothrium coccineum,
Character male male
Maytenus magellanicus, and Drymis win-
Snout-vent length 62.2 59.9 teri) surrounded by typical tundra with
Head length 17.7 16.4
plants of the genera Donatio, Astelia, and
Head width 24.2 22.8
Eye diameter 7.0 6.7 Azorella and rushes of the families Cyper-
Nostril-snout distance 4.0 3.6 aceae and Juncae. This area is situated in
Intemarial distance 5.7 5.4 the oceanic cold temperate region (di Castri
Tibia length 27.7 27.4 1968). The annual mean temperature ofthis
Foot length 33.8 30.9
region is 8.8°C; the relative humidity is
526 PROCEEDINGS OFTHE BIOLOGICAL SOCIETY OF WASHINGTON
u
u
If
\l \i
n U
tfi tl ilr
6 7 8 9 10
tX lA i«
11 12 13
10 jjm
B
n
Irir^s
I1I 2
3 4 5
II II It II 11
8 9 10
^ ^ ft VII
11 12 13
Fig. 5. Chromosomes ofthe holotype. (A) Karyotype. (B) C-banded karyotype.
87%, and the rain fall ranges from 2000- and fingers (Fig. 4A, B). The following am-
mm
2500 per annum. The frog was col- phibians were also collected in this area:
lected under a log between the forest and Bufo variegatus, Batrachyla antartandica
the tundra. The male holotype shows its re- and Eupsophus calcaratus. Lynch (1975)
markable thorny excrescences on the chest pointed out the presence of Atelognathus
VOLUME 111, NUMBER 3 527
—
Table2. Relativelength,armratio(meanandstan- bands can be discerned at some telomeres,
dceanrtdridce;visamtio=n),suabnmdettaycpeentorficc;hrsotm=ossoumbeteslo(cmen=trimce)tao-f eAsptehciinalilnysteirntitchioalsebaonfdpawiarss o1,bs2e,rv6eadnidn t1h0.e
metaphase chromosomes ofAlsodes kaweshkari. Rel-
ativelengthwascalculatedaccordingtoBogart(1970). long arm of pair 5. Pair 4 presents a poly-
Arm ratio was calculated by dividing the short arm morphic situation: one chromosome shows
intothe long arm. *Chromosomeswith secondarycon- a remarkable band of pericentromeric po-
strictions. sition, but in the other the band is located
in the centromeric region. This situation
Pairn° Relativelength Armratio Type
1* 148.72 -h 18.32 1.97 + 0.20 sm wasHeombosgelrovbeidni.n—allFitghuereexa6mAinesdhoplwastes.the
2 135.58 ± 18.76 1.71 -¥ 0.63 sm
chromatographic profiles of the hemoly-
3 126.80 H- 10.50 4.69 -¥ 0.48 St
4* 116.74 -H 12.13 2.28 -+_ 0.74 sm sates of Alsodes kaweshkari and A. monti-
5 102.27 -y- 12.27 1.43 + 0.50 m cola obtained from the fractions collected
6* 92.39 -k- 7.21 2.05 -¥- 0.60 sm in the Sephadex G-50 minicolumn. The
7 74.03 + 8.03 2.10 + 0.32 sm fractions with the higher absorbance values
98 6661..1766 -+H 66..4060 11..4320 +-H 00..2274 mm at 410 nm (fraction 9 in Alsodes kaweskari
10 57.29 -H 6.43 1.20 -H 0.18 m and fraction 8 in A. monticola) were elec-
11 51.95 -H 5.54 1.90 ± 0.15 sm trophoretically analyzed under the experi-
+ m
12 48.73 -H 3.71 1.50 0.27 mental conditions used in this study. Figure
+ m
13 39.75 5.59 1.40 -t- 0.12 6B indicates the electrophoretical patterns
obtained for these species. Both taxa share
a similar two-banded pattern with an anodal
grandisonae and Diaz & Nunez (1988) re- band (Hb,) of 14.2 KDa an other catodal
ported Alsodes verrucosus (adult and tad- one (Hbj) of 16.8 KDa.
—
poles). The other locality where Alsodes Etymology. The new taxon is named
kaweshkari was collected is Seno Huemu- for the Kaweshkar Indians, a brave people,
les. This area has the same ecological char- hunters of sea-lions, who still live in Puerto
acteristics as Puerto Eden. The male para- Eden, the type locality of the new species.
type, collected at a border of a cold stream,
also has the nuptial asperities on fingers and Comparisons
chest.
—
Chromosomes. Examination of 10 When the adult snout-vent length ofAl-
metaphase plates from the holotype re- sodes kaweshkari (males, 59.9-62.2 mm) is
vealed a diploid number of 2N = 26. All compared with that of the other Alsodes
chromosomes are bi-armed and the funda- species, it is observed that this frog is as
mental number (NF) is 52. Pairs 5, 8-10, large as A. nodosus (58.5 mm), A. barrioi
12, 13 are metacentric, pairs 1, 2, 4, 6, 7, (59.9 mm) and A. tumultuosus (61.5 mm).
11 are submetacentric, and pair3 is subtelo- Other members of the genus are small [A.
centric. Pairs 1 and 4 have secondary con- vittatus (37.4 mm), A. gargola (40.8 mm),
strictions on the smaller arm, and pair 6 ex- andA. verrucosus (42.4 mm)] and medium-
hibits secondary constrictions on the longer sized [A. monticola (49.1 mm), A. montan-
arm. The karyotype ofAlsodes kaweshkari us (50.2 mm), A. vanzolinii (51.1 mm), and
is shown in Fig 5A. A summary of the rel- A. pehuenche (52.3 mm)]. Among the
ative length, arm ratio, and type of chro- large-sized frogs, the external morphology
mosomes is presented in Table 2. ofthe foot provides useful characteristicsto
The C-banded karyotype, based on five identify each taxon. One example of this is
plates (Fig 5B) shows constitutive hetero- A. nodosus, a species characterized by the
chromatin in the pericentromeric region of absence of lateral fringes (present on the
all chromosomes. Thin heterochromatic toes of A. barrioi, A. kaweshkari, and A.
528 PROCEEDINGS OFTHE BIOLOGICAL SOCIETY OF WASHINGTON
B
0.6 T
KDa 1 2
kaweshkari 29.0—
/\.
4 8 12
Fractions
Fig. 6. Hemoglobins ofAbodes kaweshkari and A. monticola. (A) Chromatographic elution profiles ofA.
monticolaandA. kaweshkariin SephadexG-50minicolumn(B)ElectrophoreticalpatternsofAlsodeskaweshkari
andA. monticola. (1) Standard ofmolecular weight, (2) A. monticola, (3) Alsodes kaweshkari.
tumultuosus). In the case of this latter spe- nodosus). The presence of26 chromosomes
cies, its toes are long and thin, whilst in in Alsodes kaweshkari allows its inclusion
Alsodes kaweshkari and A. tumultuosus in the monticola group.
they are thick and strongly fringed. These Muir (1981) studied the electrophoretical
frogs also differ in characteristics of the patterns of the hemoglobin molecule of 14
outer metatarsal tubercle (large in Alsodes taxa ofthe frogs ofthe genus Xenopus. The
kaweshkari and small in A. tumultuosus). species and subspecies were characterized
On the other hand, both species differ in and grouped according the numberofbands
webbing; reduced in Alsodes kaweshkari obtained in the electrophoretical profiles.
and moderately developed in A. tumultuo- Xenopus borealis, X.fraseri, X. muelleri, X.
sus (Fig. 7A, B). ruwenzoriensis, X. tropicalis, X. vestitus
Formas & Vera (1983) studied the kary- andX. witteii show an electrophoreticalpat-
ological relationships among the memberof tern of two bands (Hb,, Hb2). Alsodes kaw-
the genus Alsodes. They recognized three eshkari and A. monticola show a similar
groups within the genus: the barrioi group two-banded electrophoretical pattern like
(2N = 34) (A. barrioi), the monticola group that found in some Xenopus species.
(2N = 26) {A. gargola, A. monticola, A. Though our biochemical study was unable
tumultuosus, A. vanzolinii, and A. verruco- to differenciate Alsodes kaweshkari and A.
sus), and the nodosus group (2N = 22) (A. monticola, this is the first attempt to char-
VOLUME 111, NUMBER 3 529
5mm
Fig. 7. Schematic plantar views ofthe left foot ofAbodes kaweshkari (A) and A. tumultuosus (B).
acterize the molecule of hemoglobin of de Chile (DBCG), Chile; Listituto de Biolo-
frogs of the genus Alsodes. gia Animal, Universidad Nacional de Cuyo
(IBA), Argentina; Instituto de Zoologia,
Specimens Examined Universidad Austral de Chile (IZUA), Chile;
—
Abbreviations. Carmen Ubeda (personal Museo Nacional de Historia Natural
collection) (CU), Argentina; Departamento (MNHN), Chile; Museo de Zoologia, Univ-
de Biologia Celular y Genetica, Universidad ersidad de Concepcion (MZUC), Chile.
—
530 PROCEEDINGS OF THE BIOLOGICAL SOCIETY OF WASHINGTON
—
Alsodes barrioi: IZUA 1629-1630; Cordil- dicatedbykaryologicalanalysis. Cytogenetics
lera Pelada, Provincia de Valdivia, 1020 9:369-383.
m, Chile. Cei, J. M. 1962. Batracios de Chile. Ediciones de la
Alsodes gargola: CU 6; Macizo Loncoluan, U.ni1v9e8r0s.idAamdphdiebCihainlse,oSfanAtrigaegnot,inCah.il—e,Mo1n2i8tpopr.e
Provincia de Neuquen, 1900 m, Argen- Zoologico Italiano (N.S.) Monografia 2:1-609.
tina. Diaz, N., & H. Nunez. 1988. Nuevo hallazgo de Al-
Alsodes montanus: IZUA 824; Estero Cob- sodes verrucosus (Philip—pi 1902) en Chile y
arrubias, Provincia de Santiago, 2400 m, descripcion de su larva. Boletin Museo Na-
cional Historia Natural, Chile 41:87-94.
Chile.
Alsodes monticola: IZUA 1550,1749; Cor- di Castri, F. 1968. Esquisse ecologique du Chili. Biol-
ogic de FAmerique australe. CI. Debouteville
dillera Pelada, Provincia de Valdivia, andE. Rappaport,eds.—EditionsduCentreNa-
1020 m, Chile. tional de la Recherche Scientifique, Paris IV:7-
Alsodes nodosus: IZUA 756,700; Aguas 52.
Claras, Provincia de Petorca, 150 m, Duellman, W. E.—1970. The hylid frogs ofthe Middle
Chile. America. Museum of Natural History, Uni-
A versity ofKansas Monographs 1:1-753.
Alsodes pehuenche: IB 1643; Valle del
Pehuenche, Provincia de Mendoza, 2500 Formas, J. R. 1989. Sinonimia e identidad de la rana
austral Chilena Eupsophus vittatus (Philippi
m, Argentina. 1902) (Anura: Leptodactylidae).—Boletin So-
Alsodes tumultuosus: DBCG 161-162; La ciedad de Biologia, Concepcion 60:123-127.
Parva, Provincia de Santiago, 2600 m, . 1991. The karyotypes of the Chilean frogs
Chile. Eupsophus emiliopugini and E. verte—bralis
Alsodes vanzolinii: MZUC 12063-12070; (Amphibia: Anura: Leptodactylidae). Pro-
ceedings ofthe Biological Society ofWashing-
Ramadillas, Provincia de Arauco, 100 m,
ton 104:7-11.
Chile. MNHN , & M. I. Vera. 1983. Karyological relation-
Alsodes verrucosus: 1506; Puerto ships among frogs of the genus Alsodes, with
Eden, Provincia de Ultima Esperanza, 10 description of the karyotypes of A. vanzolinii
—
m, Chile. andA. verrucosus. Copeia 1983:1104-1107.
Alsodes vittatus: MZUC (untagged); Cor- Frost, D. R. 1985. (ed). Amphibian species of the
dillera de Pemehue, Provincia de Malle- world. A taxonomic and geographic reference.
Allen Press and the Association ofSystematics
co, 1152 m, Chile.
Collection, Lawrence, Kansas, 732 pp.
Laemmli, U. K. 1970. Cleavage of structural proteins
Acknowledgments
during the assambly of the head of bacterio-
The authors would like to give special phage T4.—Nature 227:680-685.
thanks to Pablo Corti who collected the Levan A., Fredga A., & A. Sandberg. 1964. Nomen-
paratype in Seno Huemules. Alberto Velo- clature—for centromeric positions on chromo-
somes. Hereditas 52:201-220.
so, Patricia Iturra, Jose Navarro (Universi- Lynch, J. D. 1975. A new Chilean frog of the extra-
dad de Chile), Enrique Pereira (Universidad
andean assamblage of Telmatobius (Amphibia:
Nacional de Cuyo, Argentina) and Carmen Leptodactylidae).—Bulletin of Southern Cali-
Ubeda (Universidad del Comahue, Argen- fornia Academy ofSciences 74:160-161.
tina) kindly provided specimens for com- Muir, A. V. 1981. Comparison of hemoglobins from
parisons. Marcos Navarro expertly prepared the genus Xenopus (Amphibia: Salientia).
the drawings. This work was supported by Journal ofExperimental Zoology 218:327-333.
Direccidn de Investigacidn Universidad Philippi, R. A. 1902. Suplemento de los Batraquios
Chilenos descritos en la Historia Fisica y Poli-
Austral de Chile, Proyecto S 95-25.
tica de Chile de don Claudio Gay. Santiago, 61
pp.
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—
Bogart, J. P. 1970. Systematic problems in the am- strating centromeric heterochromatin. Experi-
phibian family Leptodactylidae (Anura) as in- mental Cell Research 75:304-306.