Zootaxa 1940: 1–15 (2008) ISSN 1175-5326 (print edition)

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ZOOTAXA
ISSN 1175-5334 (online edition)

Austrolebias reicherti Loureiro & García, a valid species of annual fish

(Cyprinodontiformes: Rivulidae) from Uruguay

MARCELO LOUREIRO1,2 & GRACIELA GARCÍA3

1
Sección Vertebrados, Departamento de Biología Animal, Facultad de Ciencias, Montevideo. E-mail: mapy@fcien.edu.uy
2
Departamento de Ictiología, Museo Nacional de Historia Natural y Antropología, Montevideo.
3
Sección Genética Evolutiva, Departamento de Biología Animal, Facultad de Ciencias. E-mail: ggarcia@fcien.edu.uy

Abstract

This article analyzes the available morphological (morphometric, meristic, and coloration patterns) and molecular evi-
dence that supports the taxonomic validity of Austrolebias reicherti. The species can be differentiated from A. charrua
Costa & Cheffe, recently proposed as a senior synonym (Costa 2006), by the following combination of characters (char-
acters of A. charrua in parenthesis): 1) origin of dorsal fin anterior to that of the anal fin (origin of dorsal fin posterior to
that of the anal fin), 2) 18 to 24 caudal fin rays (23–26), 3) black supraorbital band well developed (moderately devel-
oped), 4) a vertical band present on the posterior borders of dorsal and anal fins (band absent), 5) base of dorsal fin
banded and anal fin uniformly pigmented (both fins dotted at least on their proximal section), and 6) vertical dark bands
on body flank narrower or equal in width to the lighter space between bands (vertical bands equal or wider than space
between bands). According to morphological and molecular data, it is also concluded that A. salviai Costa, Litz & Lau-
rino is a junior synonym of A. reicherti.

Keywords: Rivulidae, Taxonomy, Patos-Merín drainage system

Introduction

Annual fishes of the Austrolebias adloffi species group (Family Rivulidae) are endemic to the lowlands of the
Patos-Merin drainage system (Costa 2006). Taxonomy of this group is problematic due to the overall morpho-
logical similarity among species and, at the same time, the high morphological variation within species. Since
the description of Austrolebias adloffi (Ahl 1922), the existence of high morphological (Vaz-Ferreira & Mel-
garejo 1984; Loureiro 1996; Reichert 1994), chromosomal (García et al. 1993, 1995, 2001), and molecular
(García et al. 2002) variability has been reported for this taxon throughout the southern Laguna Merin basin.
Subsequently, part of the variability was recognized as representing species level differences. Costa & Cheffe
(2001) described three new species from specimens previously assigned to A. adloffi: A. minuano Costa &
Cheffe, A. charrua, and A. nigrofasciatus Costa & Cheffe. More recently, Loureiro and García (2004)
described A. reicherti from an area between the distributions of A. charrua and A. nigrofasciatus, without an
explicit comparison with adjacent species. Costa (2006) considered A. reicherti a junior synonym of A. char-
rua based solely on the species distribution. Subsequently, Costa (2006) described two additional species in
this group: A. salviai, based on specimens from Río Tacuarí basin, Uruguay, and occupying the reported range
of A. reicherti (Loureiro & García 2004), and A. nachtigalli Costa & Cheffe, from specimens originally
described as A. nigrofasciatus from Río Yaguarón and Aº Grande basins (Brazil) (Fig. 1).
The goal of this study is to report on molecular and morphological evidence that supports Austrolebias
reicherti as a valid taxon of the A. adloffi species group, and diagnosable from A. charrua and A. nigrofascia-

Accepted by M.R. de Carvalho: 18 Sept. 2008; published: 24 Nov. 2008 1

tus. In addition, the taxonomic status of A. salviai and A. nachtigalli is considered given the nomenclatural
priority of A. reicherti.

FIGURE 1. Map of southern Patos-Merin system, showing distributions of A. charrua (black dots), A. reicherti (white
dots), A. salviai (white star), and A. nachtigalli (gray circle). YR = Yaguarón river; TR = Tacuarí River; PS = Parao
stream; OR = Olimar river; CR = Cebollatí river; SLS = San Luis stream; NL = Laguna Negra.

Materials and methods

Morphological data for Austrolebias reicherti were taken from Loureiro & García (2004). New measurements
and observations were made upon reexamination of type specimens and additional specimens (topotypes)
deposited at the Fish Collection, Facultad de Ciencias (Institutional Code: ZVC-P; Appendix I), Universidad
de la República (Uruguay). Data for A. salviai and A. nachtigalli is that of Costa (2006) whereas data for A.
charrua is from Costa & Cheffé (2001) and Costa (2006). Tissues for genetic analysis are deposited at the
Sección Genética Evolutiva (acronym: GP), Facultad de Ciencias, University of Uruguay; Gen Bank acces-
sion number are given in Appendix II.
Meristic counts and traditional measurements follow Costa (1989), except for: 1) peduncle length, that
was measured from a vertical through the last anal fin ray to the posterior margin of the hypural plate; and 2)
snout length which was measured from the anterior tip of the upper lip to a vertical through the anterior mar-
gin of the eye. Measurements were taken with a dial caliper and recorded to the nearest 0.1 mm and given as
percentages of the standard length (SL) or head length (HL). Cleared and double-stained, for bone and carti-
lage, specimens preparations were made following Dingerkus & Uhler (1977). Vertebral counts included the
compound caudal centrum as one element.
Shape variation was analyzed through morphometric data acquired as homologous landmark coordinates

2 · Zootaxa 1940 © 2008 Magnolia Press LOUREIRO & GARCÍA

on the lateral body of the specimens. Landmarks were obtained through photographs of preserved specimens.
Fourteen landmarks per specimen were digitized in lateral view according to D’Anatro & Loureiro (2005).
Landmark configurations for all specimens were aligned by the generalized Procustes superimposition proce-
dure (Bookstein 1991; Monteiro & dos Reis 2000). The thin plate spline and uniform component approxima-
tion were used to project the specimens into a linear tangent space in order to perform linear multivariate
analysis of shape variation and covariation (Bookstein 1991). A Discriminant Function Analysis (DFA) was
performed over the partial warp matrix and the uniform component scores generated by the software tpsRegr
ver. 1.24 (Rohlf 2003) to examine the extent to which landmarks reveal phenetic clusters of individuals.
Canonical axes were evidenced in shape space by multivariate regression of partial warps while uniform com-
ponent scores were assessed on canonical scores of the two roots obtained with tpsRegr.

Sampling and DNA extraction

A total of 67 individuals used in the phylogenetic analysis were collected from 30 natural ponds across the
geographic range of A. viarius Vaz-Ferreira, Sierra & Paulete , A. reicherti, A. arachan Loureiro, Azpelicueta
& García, and A. charrua in Uruguay, and A. adloffi from R. Grande do Sul, Brazil. In order to clarify intrage-
neric phylogenetic relationships samples of the following species were included in the present analyses: A.
bellottii Steindachner , A. cinereus Amato, A. melanoorus Amato, A. nioni Berkenkamp, Reichert & Prieto, A.
vazferreirai Berkenkamp, Etzel, Reichert & Salvia, A. alexandri Castello & López, A. cyaneus Amato, A.
juanlangi Costa, Cheffe, Salvia & Litz, A. affinis Amato, A. duraznensis García, Scvortzoff & Hernández, A.
gymnoventris Amato, and A. luteoflammulatus Vaz-Ferreira, Sierra & Paulete. Plesiolebias glaucopterus
Costa & Lacerda, was used as the outgroup in the analyses.
Genomic DNA was isolated from liver tissue of freshly sacrificed animals (fixed in ethanol 95%) using
extraction with sodium chloride protein precipitation, followed by ethanol precipitation (modified from
Medrano et al. 1990).

Mitochondrial cytochrome b (cyt-b) sequences

A 815-bp fragment of cyt-b was amplified using primers CB3-H and Gludg-L (Palumbi et al. 1991) fol-
lowing the PCR cycle profile: 94ºC for 1 min, 45ºC for 1 min, 72º for 1 min; 30 cycles). PCR products were
purified with CONCERT Kit rapid PCR purification System (Life Technology) and subsequently sequenced
in both directions using the amplification primers in a Perkin-Elmer ABI Prism 377 automated sequencer.
Sequences alignments were performed using CLUSTAL X (Thompson et al. 1997).

Statistical analyses of mitochondrial cytochrome b sequences

Nucleotide composition and substitution patterns were calculated using the software MEGA (Kumar et al.
2001) and DNASP4 (Rozas et al. 2003). The corrected pair wise sequences divergence were obtained using
the Kimura two-parameter algorithm (K2P, Kimura 1980) as implemented in MEGA.

Phylogenetic Analyses
Phylogenetic reconstruction was done using three models implemented in PAUP*4.0b8 (Swofford 1998):
maximum-parsimony (MP), neighbour-joining (NJ), and maximum-likelihood (ML). An equally weighted
MP analysis was performed through heuristic search (MULPARS option, stepwise addition, tree-bisection-
reconnection [TBR] branch swapping, 100 replications). A strict consensus tree was obtained from the result-
ing 12 equally parsimonious trees. Distance trees were generated using corrected K2P distances (Kimura
1980), implemented in MEGA, and subjected to the neighbour-joining method of tree reconstruction (Saitou
and Nei 1987). The degree of confidence assigned to nodes in MP and NJ trees was assessed by bootstrapping
with 500 replicates.

AUSTROLEBIAS REICHERTI, A VALID SPECIES OF FISH Zootaxa 1940 © 2008 Magnolia Press · 3
 The best-fitting evolutionary model for ML analyses was the HKY85 (Hasenawa, et al. 1985) substitution
model with among-site variation and gamma distribution (HKY85 + I + G). The model was selected among
56 models using the Akaike information criterion (Akaike 1974) as implemented by the Modeltest 3.06
(Posada and Crandall 1998). A heuristic maximum-likelihood search (with 100 replications of stepwise addi-
tion and TBR branch swapping) was implemented in the PAUP*4.0b8.

Bayesian Inference
Bayesian method of phylogenetic inference (Rannala and Yang 1996) was implemented using the pro-
gram MrBayes 2.0 (Huelsenbeck 2000). Searches were run with four simultaneous Markov Chain Monte
Carlo chains (MCMC) for 1 million generations, sampling trees every 100 generations and applying tempera-
tures of 0.2. Chains reach stationary at about generation 442.000 th, the first 441.900 trees were discarded.
The remaining 558.000 were used to compute a 50% majority-rule consensus tree. The percentage of times
that a clade occurred among the sampled trees has been interpreted as the probability of the existence of that
clade (Huelsenbeck et al. 2002).

Analysis of molecular variance and Minimum Spanning Network

Genetic structuring among A. reicherti populations was assessed by the variance components among hier-
archical partitions in the data set through the Analysis of Molecular Variance (AMOVA) Excoffier et al.
(1992). The Euclidean metric of Excoffier et al. (1992) was used to construct the matrix of pair wise dis-
tances. Each population was assigned to two different groups and sub regions: north-eastern Parao stream
basin (southern hereafter) and Tacuarí river basin plus south-western Yaguarón river basin (northern hereaf-
ter). The genetic variation was partitioned into three components: among groups, among populations within
groups, and among individuals; disregarding either their original populations or groups.
The relationship among the cyt-b haplotypes was estimated through a minimum spanning tree based on
the mean number of pair wise differences among haplotypes using ARLEQUIN software package (Schneider
et al. 2000).
Population subdivision was measured assuming an infinite mutation model (Kimura and Crow 1964) and
calculating the FST (Slatkin 1991) for the whole population. Pair wise estimates of FST were calculated using
ARLEQUIN (Schneider et al. 2000) to generate pair wise estimates of levels of gene flow as follows:
Nfm ≈ ½ [(1/ FST) – 1](Wright 1951).

Results

Morphology

Austrolebias reicherti vs A. charrua

From the comparative data in Table 1, several morphological differences can be noted between these species:
1) Austrolebias reicherti males have the origin of the dorsal fin anterior to the origin of the anal fin, whereas
the dorsal fin of A. charrua originates posterior to the anal fin, 2) the number of caudal fin rays in A. reicherti
range from 18 to 24 and in A. charrua they range from 23 to 26, 3) males of A. reicherti have a well developed
supraorbital band while in A. charrua this band is moderately developed, 4) a characteristic vertical band is
present on the posterior border of the dorsal and anal fins of A. reicherti males, but it is absent in A. charrua,
5) males of A. reicherti have bars on the base of the dorsal fin and the annals fin is uniformly pigmented, in
males of A. charrua both fins are proximally or entirely dotted, and 6) males of A. reicherti have vertical black
bands on body flanks that are narrower or equal in width to the space between those bars, the vertical bands
are as wide or wider than the space between the bands in males of A. charrua.

4 · Zootaxa 1940 © 2008 Magnolia Press LOUREIRO & GARCÍA

AUSTROLEBIAS REICHERTI, A VALID SPECIES OF FISH Zootaxa 1940 © 2008 Magnolia Press · 5
 Geometric morphometry analysis indicates that both species also differ in males body shape (Fig. 2). Dif-
ferences are mostly in body depth (A. charrua deeper) and head size (A. charrua smaller head).

FIGURE 2. A. Discriminant analysis of shape change in males of A. charrua (white triangles) and A. reicherti (black
dots). B. shape changes associated to negative values of Root 1. C. shape changes associated to positive values of Root 1.
Ellipse represents 95% confidence interval.

Austrolebias reicherti vs A. nigrofasciatus

Comparative data is given in Table 1. Males of A. reicherti can be distinguished from those of A. nigrofascias-
tus in their anal fins, being uniformly ligth blue in A. reicherti whereas they are dark bluish gray with basal
light blue bars in A. nigrofasciatus.

Austrolebias reicherti vs A. natchigalli

Data presented in Tables 1 and 2 show that these two taxa differ in: 1) the position of the origin of the dorsal
fin in relation to the origin of the anal fin in males (anterior in A. reicherti and posterior in A. nachtigalli) and
2) the body depth of females (29.5–36.2 % of SL in A. reicherti vs 37.0–42.6% in A. nachtigalli).

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AUSTROLEBIAS REICHERTI, A VALID SPECIES OF FISH Zootaxa 1940 © 2008 Magnolia Press · 7
Austrolebias reicherti vs A. salviai

Table 2 shows that both taxa are differentiated in the body depth of the females (29.5–36.2 % of SL in A.
reicherti vs 36.3–38.7% of SL in A. salviai).

Mitochondrial cyt-b variation in the Austrolebias.

The present study includes 67 mitochondrial cytochrome b sequences of 18 taxa from different Aus-
trolebias species complexes. Among 815 bp of cyt b sequenced, 547 were variables sites and 372 were phylo-
genetically informative. Table 3 includes the mean value of the corrected K2P sequence divergence within
each taxon of the A. adloffi species complex; remarkably, A. charrua (3.4%, SE = 0.009) and A. reicherti
(3.2%, SE = 0.011) showed the highest values. Table 4 shows pair wise corrected genetic distances among
groups under the Kimura 2-P model of nucleotide substitution.

TABLE 3. Within group average genetic distances (d) under Kimura 2-parameter model and Standard Error (S.E): esti-
mated by bootstrap method in the A. adloffi species complex.

d S.E.
A. adloffi 0,000 0,000
A. charrua 0,027 0,007
A. arachan 0,018 0,013
A. reicherti 0,019 0,008

TABLE 4. Corrected genetic distances among groups under Kimura 2-P model (below diagonal), Standard Error (S.E)
estimated by bootstrap method (above diagonal): 1- Outgroup taxon; 2. A. luteoflammulatus and A. gymnoventris taxa; 3.
A. alexandri species complex; 4. A. bellottii species complex; 5. A. adloffi; 6. A. charrua; 7. A. arachan; 8. A. reicherti.

Phylogenetic analyses

All phylogenetic analyses recovered the same tree topology (Fig. 3). ML analysis produced a well-supported
phylogeny (-ln likelihood score = 9677,022 ). Bayesian values over the maximum likelihood branches showed
a high degree of confidence for the posterior probability of monophyly for the major and the minor clades in
the genus Austrolebias. A major clade includes the species of the A. bellotti and A. adloffi species complex
and a minor and basal one consists of haplotypes of the A. alexandrii species complex. Within the A. adloffi
species complex, a sister taxa relationship is well supported for A. charrua and A. reicherti, while A. arachan
and A. adloffi collapsed basal to this clade. Furthermore, all samples of A. charrua collapsed in a well sup-
ported basal star polytomy, whereas samples of A. reicherti clustered into two lineages, without bootstrap sup-
port.

8 · Zootaxa 1940 © 2008 Magnolia Press LOUREIRO & GARCÍA

FIGURE 3. Maximum Likelihood under the HKY85 + I + G substitution model (Hasenawa et al. 1985) based on cyt- b
haplotypes of the A. adloffi species complex, A. viarius and other species complex from the Cynolebiatinae subfamily.
Plesiolebias glaucopterus was included as the outgroup taxon. Numbers above the branches represent values from Baye-
sian inference obtained with the MrBayes software, they represent the posterior probability that the clade is true.

AUSTROLEBIAS REICHERTI, A VALID SPECIES OF FISH Zootaxa 1940 © 2008 Magnolia Press · 9
DNA polymorphism and populations structuring in A. reicherti populations
High diversity 0.9 (SE = 0,059) among 12 haplotypes was found in A. reicherti. Except haplotype 3
(shared by three individuals) and haplotype 1 (shared by six individuals) the remaining haplotypes showed a
frequency of 1/n. The nucleotide diversity (π) in this taxon was 0.013 (SE =0.0030).
The variance of molecular suggests that most of the genetic variation among cyt-b haplotypes is distrib-
uted within-populations (94.32 %). The fixation index (FST : 0.05677) also corroborates the low level of
genetic structuring among populations of A. reicherti from to Northern and Southern regions. Moreover, the
minimum spanning network based on cyt-b gene confirmed the low level of genetic structuring among haplo-
types of A. reicherti populations (Fig. 4). The most frequent and central haplotypes, Hap3 and Hap1, separated
only by one step mutation, conform two linked star like topologies. All remaining haplotypes in A. reicherti
belong to five different ponds (42, 43, 55, 58, and 59) were connected with these two central haplotypes. Hap-
lotype 3 from ponds 58 and 59 (Fig. 4) connected with haplotype 6, which was separated through nine steps
from the haplotype 11 that belongs to the sister taxon, A. charrua. The indirect estimates of gene flow from
the mitochondrial haplotypes within A. reicherti showed high values of gene flow among ponds, ranging from
Nmf = 2 to 26 migrant per generation.

Discussion

Our analyses support that A. reicherti is a valid taxon within the A. adloffi species group. This is supported by
at least six morphological characters that differentiates this species from A. charrua and by molecular analysis
that recover the monophyletic A. reicherti. Furthermore, there is no gene flow between these two species.
Besides, A. reicherti is easy distinguishable from A. nigrofasciatus by the coloration pattern of their anal fins
(Loureiro & García, 2004; Costa, 2006). Furthermore, all phylogenetic analyses (Fig. 3) yielded robust sup-
port to the existence of a highly structured and monophyletic A. reicherti, and as the sister taxon of A. char-
rua, within the A. adloffi species complex.
Austrolebias salviai was described from specimens from a single locality in the Río Tacuarí basin (Costa
et al. 2006; Costa, 2006), a locality within the distribution originally reported in the description of A. reicherti.
We found one morphometric character (females body depth), that could distinguish these two taxa. However,
the molecular analyses showed that populations from south-western basin of Yaguarón river to north-eastern
basin of Parao stream have high levels of gene flow, suggesting the occurrence of single taxon in both South-
ern and Northern Tacuarí River. Consequently, we argue that A. salviai Costa, 2006, is a junior synonym of A.
reicherti.
The application of Bayesian analyses to discriminate simultaneous speciation from weak phylogenetic
signal seems to be a useful approach. In this analyses, A. reicherti and A. charrua have well supported basal
polytomies (Fig. 3) (“hard” polytomies, Hoelzer and Melnick 1994). Considering that A. reicherti and A.
charrua appear as the most recent derivatives within the A. adloffi species complex, the putative event of spe-
ciation must have occurred during the Late Pleistocene (100.000 years ago) in agreement with a molecular
clock estimate based on cyt-b sequences and in accordance with the complex geological events during the
Pleistocene and Post-Pleistocene marine transgressions in this area (Sprechman 1978; Montaña and Bossi
1995).
The high values of DNA polymorphism, AMOVA analysis, minimum spanning network, and the high
level of migration per generation among ponds corroborate the low level of genetic structure within popula-
tions of A. reicherti between the Southern and Northern basins. The high within-population estimates of gene
diversity from cyt-b data could be explained by historical events of wide spread floodings during the rainy
season that could have mixed populations across large distances generating high intrapopulation variation in
A. reicherti.

10 · Zootaxa 1940 © 2008 Magnolia Press LOUREIRO & GARCÍA

FIGURE 4. Unrooted haplotype network based on the cyt b gene including twelve A. reicherti haplotypes (A) and the
most related haplotype 11 from A. charrua (B). Circles areas are proportional to the haplotype frequencies. Each single
line indicates one mutation event between haplotypes (small bars dividing single lines represent missing haplotypes).

Costa (2006) considered A. reicherti as a junior synonym of A. charrua and indicated that specimens of A.
reicherti he collected from the type locality had the diagnostic characters of A. charrua. Unfortunately, these
specimens were not listed among the specimens analyzed in the re-description of A. charrua (Costa 2006) and
were not available for our examination. Our re-examination of the type specimens of A. reicherti plus addi-
tional lots of topotypes (n= 30) possess the diagnostic characters of A. reicherti reported in the original
description of the species and these characters clearly differentiate the two species. Costa (2006) also argued
that the Río Cebollatí basin is inhabited exclusively by A. charrua. Austrolebias reicherti type locality (plus at
least three additional localities) is located 500 meters away from Parao stream, a current tributary of the Río
Cebollatí. This entire region has undergone, at least over the last 100.000 year, a series of marine transgres-
sions and regressions (Sprechman 1978; Montaña and Bossi 1995). These fluctuations could easily account

AUSTROLEBIAS REICHERTI, A VALID SPECIES OF FISH Zootaxa 1940 © 2008 Magnolia Press · 11
for gene flow among populations that would regularly counteract populations differentiation by temporal iso-
lations.
Finally, more evidence is needed to confirm the taxonomic status of A. nachtigalli. This species differs
from A. reicherti in only two morphological characters. Furthermore, molecular evidence (García et al. in
revision) suggests that both species are not directly related. More specimens need to be analyzed to present
more conclusive results on this matter. For this reason, we keep A. nachtigalli as a valid taxon of the A. adloffi
species group.

Acknowledgments

We thank to R. O. de Sá for language and content correction of the manuscript. We are thankful to grants
(2006-2007) for molecular research to G. Garcia provided by Killi Data Organization Museum National d'
Histoire Naturelle, France. The authors are also grateful to the Government of Japan for the donation of equip-
ment.

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Appendix I. List of A. reicherti examined material; Colección de Vertebrados, Facultad de Ciencias,
Montevideo (ZVC-P)

Austrolebias reicherti
Uruguay
Holotype. ZVC-P 4362, male, 33.5 mm SL, Departamento de Treinta y Tres, temporary pond 300 m east from Route 18,
1 km north to Vergara City, approximately 32º55′ S, 53º54′ W, 6 Sep 2000, M. Loureiro, F. Teixeira, A. D’Anatro, E.
Charbonier.
Paratypes. ZVC-P 4363, 18 spms., 8 males, 29.2-34.9 mm SL, 10 females, 27.2-29.1 mm SL (9 C & S), collected with
the holotype. ZVC-P 4365, 5 spms., 3 males, 39.1-42.0 mm SL, 2 females, 35.8 mm SL (2 C & S), Departamento de
Treinta y Tres, temporary pond next to a small road that originates in Route 18 and runs parallel south to Tacuarí
River, approximately 32º46′ S, 53º38′ W, 15 Sep 2000. ZVC-P 4366, 5 spms., 2 males, 30.7-39.3 mm SL, 3 females,
28.8-31.4 mm SL, Departamento de Cerro Largo, temporary pond next to the road between Rio Branco City and
Balneario Laguna Merín, approximately 32º41′ S, 53º18′ W. ZVC-P 4367, 5 females 28.7-32.1 mm SL, Departa-
mento de Cerro Largo, temporary pond next to Route 26, 5 km W from Rio Branco City, approximately 32º35´ S
53º23´ W. ZVC-P 4370, 10 spms., 5 males, 24.6-35.5 mm SL, 5 females, 24.4-30.3 mm SL, Departamento de Tre-
inta y Tres, temporary pond next to Route 91, 29 km N of General Martínez Town, approximately 32º59′ S, 53º52′
W.
Topotypes. ZVC-P 4144, 5 spms., 3 males 34.6-38.1 mm SL, 2 females 29.3-36.0 mm SL, Departamento de Treinta y
Tres, temporary pond next to a small road that originates in Route 18 and runs parallel south to Tacuarí River,
approximately 32º46′ S, 53º38′ W; ZVC-P 4169, 1 male 27.7 mm. SL, Departamento de Treinta y Tres, temporary
pond next to a small road that originates in Route 18 and runs parallel south to Tacuarí River; ZVC-P 4177, 8 spms.
4 males 32.5-38.5 mm SL, Departamento de Treinta y Tres, temporary pond next to Route 91, 29 km N of General
Martínez Town, approximately 32º59′ S, 53º52′ W; ZVC-P 4337, 4 females 28.7-31.1 mm SL, Departamento de
Cerro Largo, temporary pond next to Route 26, 5 km W from Rio Branco City, approximately 32º35´ S 53º23´ W;
ZVC-P 4364 1 female 28.2 mm SL, Departamento de Treinta y Tres, temporary pond 300 m east from Route 18, 1
km north to Vergara City, approximately 32º55′ S, 53º54′ W, 6 Sep 2000, M. Loureiro, F. Teixeira, A. D’Anatro, E.
Charbonier; ZVC-P 6518, 2 spms. 1 male 34.4 mm SL, 1 female 36.1 mm SL, Departamento de Treinta y Tres, tem-
porary pond next to a small road that originates in Route 18 and runs parallel south to Tacuarí River.

14 · Zootaxa 1940 © 2008 Magnolia Press LOUREIRO & GARCÍA

Appendix II. Detailed list of localities, geographic coordinates, Gen Bank access numbers of specimens
of A. charrua, A. reicherti, and A. nachtigalli, used in this article
Species Locality Lat/Long Gen Bank Access Number
Austrolebias charrua Dept. Rocha (Uruguay)
Road 19 km 6.5-7 (ch33) 33°41'19''S/ AY724390, AY724391,AY724379-
53°31'36''W AY724380
Road 9 km 272 (ch6) 34°12'54''S/ AY724377
53°46'18''W
Road 13 and Road 16 (ch8) 34°03'34''S/ AY724390-AY724409
53°51'17''W
Road 14 km 489.5 (ch28) 33°54'09''S/ AY724400
53°40'38''W
Road 9 km 302 34°01'39''S/
53°35'35''W
Road 9 km 336.5 (ch32) 33°45'19''S/ AY724399, AY724378-AY724410,
53°26'09''W AY724404
Road 19 km close to San Luis stream (ch35) 33°36'03''S/
53°43'29''W
Road 15 km 151.5 (ch37) 33°35'04''S/ AY724381, AY724406, AY724405
54°04'10''W
Road 16 km 34.5 (ch38) 33°59'48''S/
53°49'03''W
Road 15 km 173 (ch44) 33°28'13''S/
53°52''11''W
Road 9 km 272 (ch50) 34°12'18''S/
53°46'28''W
Dept. Treinta y Tres (Uruguay)
Road 91, close to Corrales del Parao stream 33°00'06''S/ AY724390, AY724390-AY724389
(ch54) 53°52'22''W
Road 8, close to Treinta y Tres city (ch66) 33°13'32''S/
54°23'45''W
Road 91 10 km to the North of Charqueada town 33°09'04''S/
(ch53) 53°52'27''W
Austrolebias reicherti Dept. Treinta y Tres (Uruguay)
Road 18 km 369.5 (ch42) 32º46'51''S/ AY724392, AY724401, AY724402,
53º38'40''W AY724403
Road 18 close to Vergara town (ch43) 32º55'21''S/ AY724398, AY724396
53º54'49''W
Road 91, 39 km North of Charqueada town 33º01'34''S/ AY724397, AY724395, AY724393,
(ch55) 53º52'46''W AY724394
Road 18 km 369.5 (ch56) 32º46'51''S/
53º38'40''W
Dept. Cerro Largo (Uruguay)
Road to Lago Merín town (ch58) 32º42'04''S/
53º18'32''W
Road 26 5km from Rio Branco city (ch59) 32º34'34'S/
53º26'20''W
Austrolebias natchigalli Rio Grande do Sul, Brazil
Road BR 116 km 15 AY724407

AUSTROLEBIAS REICHERTI, A VALID SPECIES OF FISH Zootaxa 1940 © 2008 Magnolia Press · 15