Alsodes norae, Cuevas, César C., 2008

Cuevas, César C., 2008, A new species of the genus Alsodes (Anura: Neobatrachia) from the Nothofagus forest, Coastal Range, Southern Chile, identified by its karyotype, Zootaxa 1771, pp. 43-53 : 45-51

Alsodes norae sp. nov.

(figs. 2, 3)

Holotype. IZUA 3541, an adult male with well developed sexual secondary attributes collected by César Cuevas, Javiera Cuevas, and Ignacio Cuevas, on 12 May 2006; Parque Oncol (39º41’S, 73º18’W; 720 m elevation; 29 km NW, 35 minutes by road from Valdivia city), Valdivia Province, Western slopes of the Coastal range (fig. 1).

Paratypes. IZUA 3534-3535: two males, IZUA 3536, 3540; two females, and IZUA 3537 – 3539; three juveniles, collected on 5 may 2003 by César Cuevas and Javiera Cuevas at the type locality. Same data as holotype.

Diagnosis. Alsodes norae sp. nov. can be distinguished from its congeners by the following combination of characters: 1) maximum snout-vent length in males (SVL) 61.4 mm, 2) spine of contrasting vivid, yellow and black colours, 3) head with yellowish triangle between anterior border of the eyes and the snout tip, 4) snout slightly truncated in dorsal and lateral profile, 5) iris brilliant bronze-yellowish, 6) postocular fold well developed extending beyond of the insertions of arms, 7) flanks very granular, 8) Inner palmar tubercle oval and enlarged, bigger than the outer, 9) carpal elements of hands cartilaginous, 10) dorsum of legs with thin dark transversal bars, 11) toes thin, long and strongly fringed, 12) 2n = 30 (22 biarmed and 8 uniarmed chromosomes), FN = 52, 13) NOR on pair 4.

Comparison: Alsodes norae can be distinguished from A. valdiviensis because the new species present the carpal elements cartilaginous (ossified in A. valdiviensis ). The new species differs of A. barrioi because this species possesses a strongly truncated snout, and little developed palmar, metatarsal and subarticular tubercles. It differs from A. vanzolinii , because this species has the loreal region flat (concave in A. norae ), and the postocular fold reaching only the insertion of arms (beyond in A. norae ). It differs from A. nodosus by the form of nuptial bilateral patches on the chest. In A. nodosus these are formed by eight to nine little rounded patches composed of tiny thorns (a big one in A. norae ). From the chromosome perspective, Alsodes norae can be easily distinguished from the other Alsodes species from the Coastal Range; A. valdiviensis , A. barrioi , A. vanzolinii , and A. nodosus , because these species have 2n = 26; NF = 52, 2n = 34; NF = 52, 2n = 26; NF = 50, and 2n = 22; NF = 44 respectively. Moreover, A. norae has four uniarmed pairs (fig. 4), contrasting with A. barrioi (eight), A. vanzolinii (one) and A. valdiviensis (fig. 6A, all biarmed pairs). When C-banding patterns of Alsodes norae are compared with those of other species, the five species located in the Coastal Range display autoapomorphic patterns of C- bands (fig. 5). Finally, A. norae is restricted to a very small area and has not been encountered in sympatry with any of the other congeneric species (see fig. 1).

Description of the Holotype. A male with body robust, arms and legs well developed. SVL 61.4 mm. Head depressed 1.1 times wider than long. Snout slightly truncated in dorsal and lateral profiles (fig. 2 A, B). Canthus rostralis well developed slightly curved. Loreal region slightly concave in cross section and skin smooth. Nostrils located anterolaterally, nearest to the tip of the snout than anterior border of the eyes. Eye diameter near 1:1 the eye-nostril distance. Interorbital distance 1.6 times internarial distance. Tympanum absent, postocular fold well developed extending beyond the insertion of arms. Tongue rounded, with a notch at the tip. Choanae rounded; dentigerous processes (4 - 5) of vomer between the choanae. Forelimbs of male not hypertrophied. Fingers long with white rounded tips, in order of increasing length: I = II – III – IV. Webbing on hand absent. Palmar tubercles well developed; the inner bigger than the outer which is divided in two (fig. 3A). Male with dorsal surface of first and second finger with thorny excrescences (fig. 3B). Hind limbs normally developed. Tibiotarsal joint reaches the anterior border of the eye. Toes long, thin, with white rounded tips and strongly fringed, in order of increasing lengths: I – II – III – V – IV (fig. 3C). Inner metatarsal tubercle oval and prominent. Tarsal fold well developed, covering ¾ of the tarsus. Flanks very granular; dorsal surface of body moderate in rough aspect. Arms, legs and ventral surface of body smooth. Skin around vent and posterior thighs grainy. Chest of male with two clearly delimited bilateral rounded patches (fig. 3D). Measurements of the type series are shown in the Table 1 .

a According to Bogart 1970, b long arm / short arm, and c 1.0 – 1.7 = M = metacentric; 1.8 – 3.0 = SM = submetacentric; 3.1 – 7.0 = st = subtelocentric; 7.1 - ∞ = T = telocentric. * Secondary constriction position.

Pectoral girdle. The paratype IZUA 3540 has the pectoral girdle arciferal and the xiphisternum notoriously expanded and deeply notched with a little hole in the centre (fig. 3E).

Colour in life. Dorsal ground of head, forelimbs, hindlimbs and body of vivid yellow colour with moderately granular appearance. A dark bat figure with spread wings is observed along the dorsum of head ending at the middle of body. Hindlimbs dorsally with thin dark transversal bars. Ventral surface of thighs yellowish in colour in the groin region. Venter light cream with dark reticulations. Upper part of the iris golden yellowish with black reticulations (fig. 2A).

Colour in preservative (alcohol 70 %). The holotype (IZUA 3541) has the dorsal ground of head, forelimbs, hind limbs and body slight grey with a dark bat-like figure with spread wings in form, starting from the middle of eyes until the insertion of arms, ending towards the vent as many parallel longitudinal lines in appearance (fig. 2B). Head with a light gray triangle between eyes and the tip of the snout. A dark line extends over postocular fold. Belly, throat, ventral areas of arms and thighs whitish in the holotype, but with dark reticulations in the younger specimens.

Variation. One female (IZUA 3537), a preadult (IZUA 3534) and a juvenile (IZUA 3538) exhibit the same general characteristics of the holotype with some minor differences. The dorsum, flanks, and dorsal surfaces of the legs are covered with minute granules. The postocular fold reaches the middle of the body. Measurements of these specimens are shown in Table 1 .

Chromosomes. Chromosomal polymorphisms among individuals analyzed were not detected. So, the standard karyotype of Alsodes norae sp. nov. possesses a diploid number of 30 chromosomes (eleven biarmed and four uniarmed pairs) with a fundamental number (FN) 52. A moderate gap size it is discernible between pairs 5 and 6. According to their relative lengths, pairs 1 – 2 are large (> 100 units), 3 – 5 intermediate (80 – 100 units) and 6 – 15 are small (<80 units). By their centromeric ratio (r), pairs 1, 4, 10, 13 – 15 are metacentric (M); 7 and 9 submetacentric (SM); 2, 3 and 5 subtelocentric (ST), and 6, 8, 11 and 12 telocentric (T). A secondary constriction of type IV ( King 1980) was detected on the long arm of pair 4 (M). Karyotype data are shown in Table 2 .

The C-banded karyotype (fig. 4B) exhibits: centromeric bands on all except pairs 6, 7, 11, 12, and 13; telomeric bands on short arms of pairs 1, 3, and 5; paracentromeric bands on telocentric pairs 8, 10, 11 and 12, and interstitial bands on both short and long arms of pair 1, and in long arms of pairs 5, 7 and 14. Finally, heterochromatic blocks were detected on long arms of pairs 2, 3, and 6. The C-banded ideogram is represented in the figure 5B. Silver staining of chromosomes showed the NORs (with tandem duplication, fig. 4D) to be located within the secondary (nucleolar) constrictions on the short arm of pair 4 (fig. 4C).

Distribution and ecology. The new species is only known from the type locality, Parque Oncol, a small woody area of only 754 ha, recognized as a fragment of the original Valdivian rain forest ( Veblen et al. 1980).

The tadpole of A. norae has yet not been collected. This woody area was characterised by Oberdorfer (1960) as dominated principally by Eucryphia cordifolia and Aextoxicum punctatum and less by Laureliopsis philippiana , a vegetational asociation named as Lapagerio-Aextoxiconetum and Laureliopso-Weimanniatumm Oberd, occurring in a zone of great humidity along the Pacific coast ( Veblen et al. 1980). The Valdivian rain forest region generally has a west coast maritime climate [Oceanic Region with Mediterranean influence (zone of oceanic tendency)] (di Castri 1968) characterized by an annual average temperature of 10.5 ºC (with a maximum average of 14.2 ºC and a minimum average of 6.9 ºC), relative humidity 84 % and rainfall 2000 - 2500 mm per year. Specimens of Alsodes norae sp. nov. were frequently found under fallen logs into the humid forest. Other frogs that occur in Parque Oncol are Batrachyla antartandica , Rhinoderma darwinii , Eupsophus roseus and E. vertebralis . However, at first sight animals of these species are easily distinguishable in their phenotypic appearance from those of Alsodes . Batrachyla antartandica , R. darwinii and E. roseus are smaller sized frogs (30 mm to 40 mm). Eupsophus vertebralis also is a large sized frog but it differs from Alsodes norae in possessing a white vertebral line and in lacking bilateral nuptial patches in the chest (Formas 1982).

Etymology. The specific name of the new taxa is the genitive Latin form of Nora, the author`s grandmother, an extraordinary Chilean woman.

Discussion. Although only three adult males (IZUA 3534, 3535, 3541), two females (IZUA 3536, 3537, 3540) and three juveniles (IZUA 3538 - 3539) of Alsodes norae were collected in the type locality, their generic assignments is justified by the absence of a tympanum in all specimens, added to the male morphologic attributes (thorny excrescences on the thumb and chest, arciferal pectoral girdle) (fig. 3), which are concordant with the generic diagnosis given by Cei (1980) and Formas et al. (1998). In addition, the skull of A. norae (here not described) presents a similar cranial pattern as those previously described for A. gargola , A. vanzolinii , and A. valdiviensis ( Lynch 1978, Formas and Vera 1982; Formas et al. 2002).

Formas et al. (2002) described A. valdiviensis from Cerro Mirador, Cordillera Pelada (40º08’S, 73º40’W; 1100 m altitude). This area of the Coastal Range is distant only 60 km (in a straight line) to the type locality of A. norae , however, both mountainous formations (Cerro Mirador and Parque Oncol) are separated by the Valdivia river estuary (fig. 1). The specimens of A. valdiviensis show morphological and chromosomal differences to those of A. norae . Alsodes valdiviensis has: opaque colours on the dorsum, unbarred legs, a postocular fold reaching the anterior border of arms, palmar tubercles more reduced in size, general aspect less granulose, prevomers with seven prevomerine teeth (4-5 in A. norae ). Its carpal elements are osseous while there are cartilaginous in A. norae , a condition only described before for Insuetophrynus acarpicus (Barrio 1970) , and Telmatobius chusmisensis ( Formas et al. 2006) . Finally, both species differ in their chromosomic complement (2n=26, A. valdiviensis ; 2n=30, A. norae ).

Most of Alsodes species have been described based on external adult morphology but some of them only have been recognized as different by displaying autoapomorphic chromosomic characteristic ( Veloso et al. 1979; 1981; Cuevas and Formas 2005b). In this framework, distributional criteria and chromosome evidence (2n = 30; FN = 52, Fig. 4 , 5 B) are the main arguments that justified the erection of the new taxon herein ( Cuevas and Formas 2005b). The genesis of 2n = 30 in A. norae probably involved Robertsonian rearrangements (fissions of biarmed chromosomes) ( Cuevas and Formas 2005b) which occurred in a hypothetical ancestor with 2n = 26, such as was proposed by Formas et al. (2002) for A. barrioi . This hypothesis can be illustrated by a didactical exercise, i.e., when all telocentric pairs of A. norae and those of A. barrioi are fused by the centromeric region conforming a karyotype with 2n = 26 from each other (fig. 6 B, C). The cases of A. nodosus (loss of four small pairs, NF = 44) and A. vanzolinii (presence of a telocentric pair, NF = 50), may be explained by translocations and pericentric inversions respectively ( Veloso et al. 1979; Cuevas and Formas 2003).

The discordance between chromosome and morphological evolution observed in Alsodes ( Cuevas and Formas 2001) , agrees with Lande`s (1979) hypothesis which claims that chromosome rearrangements do not involve drastic phenotypic changes. Such chromosome mechanisms would occurs in isolated populations (such as members of Alsodes underwent along the Coastal Range, fig. 1) involving demographic processes (small population size). This suggests a conserved morphological evolution in Alsodes such as has been observed in other amphibians ( Bogart 1991; Cherry et al. 1978; Stuart et al. 2006), as in the case of Eleutherodactylus .

When gross chromosome morphology (2n, FN) is analyzed in relation to geographical range, Alsodes can be grouped into species with variable karyotypes i.e. 2n = 30, FN = 52 ( A. norae sp. nov.); 2n = 22, FN = 44 ( A. nodosus ); 2n = 26, FN = 50 ( A. vanzolinii ); 2n = 34, FN = 52 ( A. barrioi ); 2n = 26, FN = 52 ( A. valdiviensis ) (fig. 5) ( Veloso et al. 1981; Formas and Vera 1983; Cuevas and Formas 2003; 2005b) distributed along the Coastal Range (32º to 40ºS); and species that share a karyotype with 2n = 26, FN = 52 ( A. australis , A. gargola , A. hugoi , A. igneus , A. kaweshkari , A. montanus , A. pehuenche , A. tumultuosus , A. verrucosus ) ( Kuramoto 1990; Cuevas and Formas 2003; 2005b) distributed along the Andes Range (32º to 48ºS). An exception to this rule is A. nodosus with a range including both the Coastal Range and the Andes ( Bogart 1970) (fig. 1).

In the last 10 years, eight new Chilean anurans species (14.3% of total current members) have been described [six in Alsodes ( Formas et al. 1997; 1998; 2002; Cuevas and Formas 2001; 2005a), and two in Eupsophus ( Ibarra-Vidal et al. 2004; Veloso et al. 2005)]. These are examples of multiple cases of phenotypically resembling species which imply that amphibian diversity remains significantly underestimated along the Chilean territory. Thus, the outstanding chromosome heterogeneity (fig. 5), and restricted distribution at high altitudes encourage to carry on future analyses of specimens from new places of the Coastal Range (Chiloé Island 42.5ºS; Osorno Province 40.8ºS; Cautín Province 39.5ºS; and Cauquenes Province 36ºS). Such studies would likely result in the discovery of new chromosome numbers, and detect new species in the genus Alsodes . Finally, the recognition of the Coastal Range as an important center of diversification of the Chilean anuran fauna (unknown in all its extension), could constitute a cornerstone to the conservation of this unique geomorphologic and floristic formation.

Figures

FIGURE 4. Chromosomes of Alsodes norae (A) Standard karyotypes, (B) C-banded karyotype, (C) secondary constriction and (D) nucleolus organizer region position.

FIGURE 5. C-bands ideograms of the five Alsodes species from the Coastal Range of Chile (A) A. valdiviensis, (B) A. norae, (C) A. vanzolinii, (D) A. barrioi, (E) A. nodosus.

Tables

TABLE 1. Measurements (mm) of the type series, adults and juveniles of Alsodes norae sp. nov. Character abreviations are: SVL, snout vent length; HL, head length; HW, head width; ED, eye distance; IDi, internarial distance; ThL, thigh length; TL, tibia length; FL, Foot length. * Cleared and stained.

Characters Holotype	Paratype	Paratype	Paratype	Paratype	Paratype	Paratype Paratype
IZUA 3541	IZUA 3534	IZUA 3535	IZUA 3536	IZUA 3537	IZUA 3538	IZUA 3539 IZUA 3540
male	male	male	female	female	juvenile	juvenile female *
SVL 61.4	48.1	36.2	35.8	30.6	29.9	28.9 46.0
HL 23.2	18.0	13.2	13.0	12.8	10.2	10.5 17.7
HW 24.0	20.3	14.4	13.5	12.7	11.8	11.9 17.3
ED 8.9	5.2	4.4	4.9	3.8	3.5	3.9 5.3
IDi 5.6	4.6	3.7	3.4	3.5	3.3	3.4 5.0
ThL 32.1	24.5	17.2	15.8	15.2	14.1	14.0 22. 8
TL 31.9	26.6	16.4	15.9	14.8	13.4	12.4 22.6
FL 51.3	43.7	24.9	24.4	22.0	20.9	20.8 34.2

TABLE 2. Relative length, arm ratio (mean and standard deviation), and types of chromosomes of Alsodes norae sp. nov.

Chromosomes
1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
RL a 111.94	101.13	92.83	88.67	80.12	71.82	69.93	62.11	59.43	54.38	48.77	46.34	39.76	39.28	32.52
AR b 1.15	3.31	3.74	1.62	3.12	∞	1.88	∞	1.97	1.30	∞	∞	1.38	1.39	1.29
CC c M	ST	ST	M*	ST	T	SM	T	SM	M	T	T	M	M	M