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Article
Expect the unexpected: a new species of killifish from a
highly stochastic temporary wetland near Iguazú Falls
(Cyprinodontiformes: Rivulidae)
Felipe Alonso a,b, Guillermo Enrique Terán b,c, Pablo Calviño b, Wilson Sebastián Serra Alanís b,d,e, Martin
Miguel Montes b,f , Ignacio Daniel García b,g, Jorge Adrián Barneche b,f , Liliana Ciotek h, Pablo Giorgis h,and
Jorge Casciotta i
aInstituto de Bio y Geociencias del NOA (IBIGEO), National Scientific and Technical Research Council (CONICET)-UNSa. Rosario de
Lerma, Salta Province, Argentina; bKillifish Foundation, La Plata, Buenos Aires Province, Argentina; cUnidad Ejecutora Lillo
(UEL)-CONICET-Fundación Miguel Lillo. San Miguel de Tucumán, Tucumán, Tucuman Province, Argentina; dSección Ictiología, Dpto.
de Zoología, Museo Nacional de Historia Natural, Montevideo, Uruguay; eCentro Universitario Regional del Este (CURE) Sede Rocha,
Rocha, Uruguay; fCentro de Estudios Parasitológicos y de Vectores - [CCT-CONICET -La Plata] Centro Científico Tecnológico Conicet,
La Plata - [Conicet] Consejo Nacional De Investigaciones Científicas y Técnicas. La Plata, Buenos Aires province, Argentina; gInstituto
de Limnología “Dr. Raúl Ringuelet” (ILPLA)-UNLP– CONICET. La Plata, Buenos Aires province, Argentina; hAdministración de Parques
Nacionales, Parque Nacional Campos del Tuyú (), General Lavalle, Buenos Aires province, Argentina; iUniversidad Nacional de
La Plata, Facultad de Ciencias Naturales y Museo, División Zoología Vertebrados. La Plata, Buenos Aires province, Argentina
Corresponding author: Felipe Alonso (email: felipealonso@gmail.com)
Abstract
We describe Argolebias adrianae, a new species of killifish from a small temporary wetland in the Paraná Forest ecoregion
with no regular or predictable temporal pattern of water availability. This habitat is in the Lower Iguazú River Basin, known
for its high fish endemism, but until now, only two species of Rivulidae were reported from it, but from the Araucarian Forest
ecoregion. The genus Argolebias was previously only known from the lower portions of the Paraguay, Paraná, and Uruguay
basins and middle Paraná. The new species is distinguished from all congeners by its unique coloration, which includes a
conspicuously dark grey anterior third portion of the dorsal fin and the absence of iridescent spots on the basal half of the
pectoral fin in live adult males, as well as dark grey spots on the anterocentral portion of the flanks of females. Our phylogenetic
analysis shows A. adrianae to be closely related to Argolebias guarani from the adjacent Middle Paraná basin. We also provide data
on the ecology, ontogeny of coloration, and chorion ornamentation of this species. Our findings have important implications
for understanding the biogeography, ecology, and evolution of mechanisms that enable organisms to thrive in highly stochastic
environments like this one.
Key words: biogeography, endemisms, temporary wetlands, annual fishes, Aplocheiloidei, Argolebias adrianae
Introduction
In the face of the current global biodiversity crisis, one of
the major challenges is the taxonomic impediment——the dif-
ficulty in recognizing and describing species before they go
extinct (Engel et al. ). Neotropical freshwater fishes rep-
resent the most diverse vertebrate fauna on the planet, with
over valid species (Albert et al. ). Among them, the
Cyprinodontiform seasonal killifishes belonging to the Rivul-
idae family are particularly vulnerable due to their small
body size, narrow geographic ranges, and reliance on sea-
sonal aquatic habitats that are highly impacted by human ac-
tivities. (Costa ;Alonso et al. ). In fact, nearly % of
Cyprinodontiform killifish species in the Neotropical region
are categorized as threatened (Tagliacollo et al. ). The
Rivulidae genus Argolebias Costa was originally established
as a subgenus of Austrolebias (Costa ) to include several
small-bodied species previously identified as the “Austrolebias
alexandri species group” (Costa ). However, Loureiro et
al. () using a combined dataset of molecular and mor-
phological characters found that Argolebias nigripinnis (Regan,
), the subgenus’-type species, was not closely related
to the other “Austrolebias alexandri species group” members,
and thus restricted the subgenus to this species alone. Ar-
golebias paranaensis (Costa ), a species very similar to Ar-
golebias nigripinnis, was not included in their analysis. Later,
Alonso et al. () elevated Argolebias to the genus level
based on a new phylogenetic hypothesis of the “Austrolebias
genus group” (Austrolebias sensu lato) obtained from and ex-
tended dataset based on genes (six nuclear and four mito-
chondrial) and morphological characters, including %
298 Can. J. Zool. 102: 298–314 (2024) | dx.doi.org/10.1139/cjz-2023-0132
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Fig. 1. Argolebias adrianae n. sp. IBIGEO-I , male, holotype,
. mm standard length, in left lateral view. (A) Live spec-
imen, picture taken in aquarium; and (B) preserved in ◦
ethanol after fixation in formaldehyde.
of the total valid species of this genus. They also described
another species from this genus, Argolebias guarani Alonso,
Calviño, Terán, Serra, Montes, García, Barneche, Almirón,
Ciotek, Giorgis and Casciotta, , from the Middle Paraná
basin, and included Argolebias paranaensis in this genus, bring-
ing the total number of species in the genus to three. After
new collections of specimens from the Iguazú basin, here we
describe a fourth species of this genus and assess its phyloge-
netic position.
Seasonal killifish live in temporary wetlands, which period-
ically dry up, rendering the habitat unsuitable for adult sur-
vival. Nevertheless, these fish lay desiccation-resistant eggs
that undergo metabolic and developmental arrests, known
as diapauses, which are regulated by environmental cues and
hatch when the ponds are filled with rainwater (Podrabsky
and Hand ;Furness ). Seasonal killifish experience
rapid growth and reach maturity within a short lifespan,
which enables them to live in these harsh conditions (Costa
;Berois et al. ). Interestingly, as far as we know all
studied seasonal killifish species that have been studied in-
habit temporary wetlands where the dry and flooding sea-
sons have relatively fixed timing and duration throughout
the year, with some interannual variation linked to weather
variability (e.g., Alonso et al. ;Reichard et al. ;
García et al. ). Their life cycle is closely tied to rela-
tively predictable patterns of filling and desiccation of their
habitat (Podrabsky and Hand ;Polaˇ
cik and Podrabsky
;Furness ) and synchronize their hatching with
the onset of the aquatic phase of the pool (Reichard et al.
). Seasonal killifish must balance energetic investments
in growth, reproduction, and survival, which shape their life
history strategies under dierent environmental conditions
(Podrabsky and Hand ;Furness ).
Previously, the known distribution of the Argolebias genus
encompassed the floodplains associated with the middle and
lower Uruguay River (sensu Bessonart et al. ), lower
Paraguay, middle and lower Paraná (sensu ˇ
Ríˇ
can et al. ),
and Río de la Plata, spanning Argentina, Uruguay, Brazil, and
Paraguay (Alonso et al. ). In this work, we describe a
new species within this genus that we discovered in
in a small seasonal wetland near the Iguazú Falls located at
Parque Nacional Iguazú in the Paraná Forest ecoregion. This
region is situated within the highly endemic Iguazú basin
(Baumgartner et al. ). The discovery of this new species
holds great significance from a biogeographical perspective,
as it represents the first known occurrence of the genus in the
Iguazú basin and the fifth species of seasonal killifish from
the Paraná plateau (Kröhling et al. ). The new species
can be distinguished from its congeners by a unique color
pattern. We also include this species in a phylogenetic anal-
ysis and discuss its phylogenetic relationship with the other
species in the genus. This new species inhabits a highly un-
predictable environment with no clear patterns of drying–
filling cycles and high interannual variability, posing a chal-
lenging scenario for this species from an evolutionary per-
spective highlighting the need for further research on the
evolutionary mechanisms that enable organisms to thrive in
unpredictable environments like this one.
Materials and methods
Specimens were euthanized by immersion in an anesthetic
solution (.% -phenoxyethanol), and then fixed in a %
formaldehyde for week, washed in water for day, and
transferred to a % ethanol solution for preservation. Col-
lection permit was granted by Administración de Parques Na-
cionales Argentina (DRNEA , RNV -). Color pattern de-
scriptions are based on photographs of both sides of living in-
dividuals. Measurements and counts follow Costa ().Mea-
surements are presented as percentages of standard length
(SL), except for those related to head morphology, which are
expressed as percentages of head length. Fin ray counts in-
clude all elements. Number of vertebrae and gill rakers were
recorded only from the cleared and stained specimens; the
compound caudal centrum was counted as a single element.
The osteological preparation was made according to Taylor
and Van Dyke (). Terminology for cephalic neuromast se-
ries follows Costa (). Abbreviations C and S mean speci-
mens cleared and stained for bone and cartilage. Appropriate
actions were taken to minimize pain or discomfort of fish,
and this study was conducted in accordance with interna-
tional standards on animal welfare, as well as being compli-
ant with national regulations and the “Comité Nacional de
Ética en la Ciencia y la Tecnología” of Argentina. Type ma-
terial is deposited in the following ichthyological collections
of Argentina: Instituto de Bio y Geociencias del Noroeste Ar-
gentino (IBIGEO-I), Rosario de Lerma, Salta and Fundación
Miguel Lillo (CI-FML), and Tucumán. Comparative material
is listed in Alonso et al. (,). Comparisons with
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300 Can. J. Zool. 102: 298–314 (2024) | dx.doi.org/10.1139/cjz-2023-0132
Fig. 2. Details of main diagnostic characters of Argolebias adri-
anae n. sp. (A) Anterior portion of the dorsal fin, about its
anterior third, in adult males, conspicuously dark grey to
black (vs. absent or reduced to first dorsal fin rays); (B) iri-
descent spots on basal half of pectoral fin in adult males ab-
sent (vs. present in Argolebias nigripinnis and Argolebias guarani,
unknown in Argolebias paranaensis); and (C) anterocentral por-
tion of flanks in females with dark grey spots (vs. without in
A. nigripinnis and A. paranaensis, shared with A. guarani).
Argolebias paranaensis are based on Costa () original de-
scription of that species.
DNA extraction and sequencing
Three individuals of Argolebias adrianae n. sp. were sam-
pled and stored separately in % ethanol. DNA was extracted
using WizardGenomic DNA Purification Kit (Promega) ac-
cording to the manufacturer’s protocol. There were obtained
– bp fragments of cytochrome b(Cyt-b), – bp
of s ribosomal unit (s), – bp of recombination ac-
tivating protein (Rag) and – bp of glycosyltrans-
ferase (Gly) amplified using primers CytB_CB-H/Gludg-L and
ARL/BrH (Palumbi et al. ) for the first two; Pachyp-
F (Phol et al. ) and H_RABex (Near et al. )
for Rag; and GLYT_/GLYTR_ (Li et al. ) for Gly;
PCR cycles were used following Loureiro et al. (). Bidi-
rectional sequencing was performed by Macrogen. Sequences
were aligned and double checked by eye using the plat-
form GENEIOUS .. (http://www.geneious.com;Kearse et
al. ). New sequences were deposited in GenBank un-
der accession numbers: OR, OR, OR for
Cyt-b; OR, OR, OR for s; OR,
OR, OR for Rag; and OR, OR,
OR for Gly.
Phylogenetic analysis
We expanded the dataset of Alonso et al. () by incorpo-
rating the morphological and genetic data of the new species
(Supplementary material S). Gblocks website (Castresana
;Talavera and Castresana ) was used to detect am-
biguously aligned hypervariable regions in the non-coding
genes dataset, according to a secondary structure model,
those were excluded from the analyses. Phylogenetic anal-
yses of the molecular and the combined dataset were per-
formed under maximum parsimony using implied weight-
ing (Golobo ,) with K=. Clade support was esti-
mated using symmetric resampling ( replicates, with
addition sequences, saving up to trees each), expressed as
GC values (groups present/contradicted) (Golobo et al. ).
A multiple sequence alignment was performed by using the
ClustalW tool implemented in MEGA (Kumar et al. ).
All searches have been performed with TNT (Golobo et al.
).
Ecological data
Over a period of about years, park rangers conducted vi-
sual observations and photography of the pond whenever
possible, resulting in observations. To track the depth of
the puddle, a stick graduated with centimeters was inserted
into its deepest part. In June , specimens were measured
and weighed, and Fulton’s condition factor (K) was calculated
using Htun-Han’s () method. The length–weight relation-
ship of the experimental fish was determined by linear re-
gression analysis, and scatter diagrams were created to plot
length and weight and follow the cube law proposed by Le
Cren ().
Nomenclatural acts
The electronic edition of this work follows the require-
ments of the International Code of Zoological Nomencla-
ture, and hence the new names contained herein are avail-
able under that Code from the electronic edition of this
article. This published work and the nomenclatural acts
it contains have been registered in ZooBank, the online
registration system for the ICZN. The ZooBank life sci-
ence identifiers (LSIDs) can be resolved, and the associated
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Table 1. Morphometric data for Argolebias adrianae n. sp. holotype and paratypes (n=).
Males (n=) Females (n=)
Holotype Range Mean SD Range Mean SD
Total length (mm) . .–. . . .–. . .
Standard length (mm) . .–. . . .–. . .
Percentages of standard length
Body depth . .–. . . .–. . .
Caudal peduncle depth . .–. . . .–. . .
Pre-dorsal length . .–. . . .–. . .
Pre-pelvic length . .–. . . .–. . .
Pre-anal length . .–. . . .–. . .
Length of dorsal fin base . .–. . . .–. . .
Length of anal fin base . .–. . . .–. . .
Caudal fin length . .–. . . .–. . .
Pectoral fin length . .–. . . .–. . .
Pelvic fin length . .–. . . .–. . .
Head length . .–. . . .–. . .
Percentages of head length
Head depth . .–. . . .–. . .
Head width . .–. . . .–. . .
Snout length . .–. . . .–. . .
Lower jaw length . .–. . . .–. . .
Interorbital width . .–. . . .–. . .
Eye diameter . .–. . . .–. . .
Meristic data——c o u n t s
Dorsal fin rays – . . – . .
Anal fin rays – . . – . .
Caudal fin rays – . . – . .
Pectoral fin rays – . . . . .
Pelvic fin rays . . – . .
Longitudinal series of scales – . . – . .
Transverse series scales – . . – . .
Scales rows around caudal peduncle – . . – . .
Note: SD: standard deviation. Range, mean, and SD include holotype.
information viewed through any standard web browser by ap-
pending the LSID to the prefix “http://zoobank.org/”. The LSID
for this publication is urn:lsid:zoobank.org:pub:BFC-
BE-AC-BC-DFF.
Results
Argolebias adrianae, new species
(Figs. –,Tables –)
: urn:lsid:zoobank.org:act:BF-FAF-BF-C-
AFDCF.
: IBIGEO-I , male, . mm SL, Argentina, Mi-
siones province, Parque Nacional Iguazú, seasonal ponds of
the waterfall Arrechea stream, tributary of Lower Iguazú
River, near Km of Macuco trail (aprox. −.,
−., m absl); Coll.: Felipe Alonso and Martín. M.
Montes, October (Fig. ).
: IBIGEO-I , males, .–. mm SL; fe-
males, .–. mm SL; CI-FML males .–. mm
SL ( C and S), females .–. mm SL ( C and S), all col-
lected with the holotype.
: Argolebias adrianae n. sp. can be distinguished
from other Argolebias species by its unique coloration, char-
acterized by () conspicuously dark grey to black anterior
third portion (about to the th or th rays) of the dor-
sal fin in adult males (vs. black portion reduced to first to
third anterior dorsal fin rays); () iridescent spots on basal
half of pectoral fin in adult males absent (vs. present in
Argolebias nigripinnis and Argolebias guarani); and () antero-
central portion of flanks in females with dark grey spots
(vs. without in Argolebias nigripinnis and Argolebias paranaensis)
(Fig. ).
argolebias : Argolebias adri-
anae n. sp. can be also dierentiated from Argolebias guarani by
presenting () – pelvic fin rays (vs. ); () flanks with some
scales with light blue anterocentral portion arranged in ir-
regular vertical lines in mature males (vs. anterocentral por-
tion of all scales on flanks iridescent turquoise); () anterior
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Fig. 3. Live color pattern variability and ontogenetic changes in Argolebias adrianae n. sp. males. (A) Juveniles around cm
standard length (SL); (B) young adult male about cm SL; (C–G) adult males; (H) mature older male; (D, F, G, and H) dominant
territorial males.
nostrils dorsolateral directed (vs. ventrolateral); () absence of
scattered small dark brown to grey irregular blotches on the
laterodorsal surface of head and anterior portion of trunk (vs.
present); and () brownish grey background coloration on the
base of unpaired fins in juveniles and young adults (vs. orange
yellowish).
Argolebias adrianae can be further dierentiated from Ar-
golebias nigripinnis by presenting () dorsal portion between
preopercle and opercle iridescent turquoise (vs. brownish
to black); () infraorbital bar thin and triangular (vs. wider,
approximately as eye width, and diuse); () abundant and
relatively big and subsquare irregular iridescent turquoise
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Fig. 4. Live color pattern variability and ontogenetic changes
in Argolebias adrianae n. sp. females. (A) Young female around
. cm standard length (SL); (B and C) adult females, >cm
SL.
to light blue blotches on unpaired fins, evenly distributed,
with those in the distal portion elongated and merging (vs.
rounded and smaller iridescent dots on unpaired fins, only on
the base and middle portion of the dorsal fin, which presents
a bright iridescent subdistal stripe with a wide black border);
() thin exterior border of scales below dorsal fin in dorsal
portion of trunk light blue in mature males (vs. grey); ()
deeper body of males (.%–.% vs. .%–.% SL); () a
shorter caudal fin length (.%–.% vs. .%–.% SL);
() a shorter pelvic fin length in females (.%–.% vs. .%–
.% SL); () smaller eye diameter in males (.%–.% vs.
.%–.% SL); () a shorter snout length in females (.%–
.% vs. .%–.% SL); and () more pectoral fin rays (–
vs. –).
It can be further dierentiated from Argolebias paranaensis
by () presenting dorsal fin origin anterior to anal fin ori-
gin (vs.posterior); () deeper body (.%–.% vs. .%–
.% SL in males and .%–.% vs. .%–.% SL in fe-
males); () deeper caudal peduncle in males (.%–.% vs.
.%–.% SL); () a shorter caudal fin length (.%–.%
vs. .%–.% SL); () shorter pectoral fin length (.%–
.% vs. .%–.% SL in males; and .%–.% vs. .%–
.% SL in females); () a wider head of males (.%–.%
vs. .%–.% SL); () smaller eye diameter (.%–.%
vs. .%–.% SL); and () more pectoral fin rays (– vs.
–). However, these measurement comparisons should be
taken with caution, as the Argolebias adrianae specimens in
this study are slightly larger (n=, .–. mm) than
those of Argolebias paranaensis from Costa () (n=, .–
. mm, SL).
: Morphometric data resumed in Table . Mor-
phometric and meristic data in Supplementary material (SI).
Males larger than females, largest examined male . mm
SL, largest female . mm SL. Elongate body, moderately
deep, laterally compressed. Highest body depth at dorsal
fin origin. Dorsal profile on lateral view slightly concave
from snout to vertical through anterior margin of preop-
ercle and convex from this point to posterior end of dor-
sal fin base, slightly concave on caudal peduncle. Ventral
profile on lateral view convex from lower jaw to anal fin
origin, nearly straight on anal fin base, and slightly con-
cave on caudal peduncle. Snout blunt and jaws short. Dor-
sal fin anterior portion shorter than posterior portion. Un-
paired fins rounded, with serrated borders in males. Dor-
sal fin distal posterior border slightly pointed in females.
Anal fin subrectangular. Anteromedian rays of anal fin of
females not lengthened. Caudal fin relatively short, sub-
triangular, and rounded. Pelvic fin bases close and not
united.
Anal fin origin posterior to dorsal fin origin, at vertical
through base of second to fourth dorsal fin rays. Base of dor-
sal fin origin at vertical between th and th pleural ribs
in males and between th and th pleural ribs in females.
Base of anal fin origin at vertical between th and th pleu-
ral ribs in males and between th and th pleural ribs in
females. Urogenital papilla not attached to anal fin, some-
time reaching base of st anal fin ray. Caudal fin rounded.
Pectoral fins elliptical, posterior margin on vertical reaching
pelvic fin base in male, not reaching pelvic fin base in fe-
males. Tip of each pelvic fin reaching base of st or nd anal
fin rays in males, and urogenital papilla in females. Pelvic
fin bases in close proximity, medial membrane not united.
Dorsal fin rays – in males and – in females. Anal fin
rays – in males and – in females. Caudal fin rays –
, usually . Pectoral fin rays –, rarely . Pelvic fin
rays –.
Scales large and cycloid. Trunk and head scaled, except ven-
tral surface of head. No scales on dorsal and anal fin bases,
and two rows of scales on caudal fin base. Frontal squamation
F-patterned, sometime H. Longitudinal series of scales –,
regularly arranged; transversal series of scales –; scale
rows around caudal peduncle . Contact organs present in
all analyzed males, – contact organs on each scale of flanks
and opercular region in males. Row of minute contact organs
on one or two uppermost pectoral fin rays in males; no con-
tact organs on unpaired fins.
Cephalic neuromasts: supraorbital –, parietal , an-
terior rostral , posterior rostral , infraorbital –,
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304 Can. J. Zool. 102: 298–314 (2024) | dx.doi.org/10.1139/cjz-2023-0132
Fig. 5. Coloration changes during ontogeny in Argolebias species. (A–E) Argolebias guarani,(F–I)Argolebias adrianae, and (J–O)
Argolebias nigripinnis. (J–O) photos by André Mestre.
preorbital , otic –, post-otic , supratemporal , median
opercular –, ventral opercular –, preopercular –,
mandibular –, lateral mandibular –.
Basihyal triangular, width about %–% of length; basi-
hyal cartilage short, about % of total basihyal length, with-
out lateral projections. Dermosphenotic ossification absent.
Ventral process of posttemporal short. Six branchiostegal
rays. Two teeth on second pharyngobranchial. Gill rakers on
first branchial arch +. Vertebrae: total: , caudal: ,
abdominal, intermediate.
, : Adult dominant males (Figs. ,
A,B,and) present a grey colored general background
with light blue iridescent markings. Scales on trunk, tail,
and dorsal portion of head with anterocentral portion grey
and darker borders, and in flanks some scales present its an-
terocentral portion iridescent light blue forming irregular
vertical lines (–), three of them anterior to anal fin ori-
gin, sometimes a fourth incomplete line is observed above
pectoral fin base. In very mature old males, these vertical
lines tend to faint as the grey central portion of many scales
in the flanks tend to be lighter and more light blue, al-
though the scales in vertical lines are still more iridescent
and bluish. The portion of the head posterior to the infraor-
bital band and ventral to the dorsal portion of the gill open-
ing is light blue to turquoise. Dark brown blotch around neu-
romast supraorbital series, posterior rostral series, and or-
bital area. Short infraorbital band approximately as wide as
pupil. Black pupil horizontally elongated. Eye with a verti-
cal black stripe about the wide of pupil. Iris mostly orange,
with lightly grey posterior border. Ventral portion of head
light blue. Ventral portion of abdominal region greyish. Area
over maxilla, premaxilla, dentary, and between dentaries,
grey.
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Fig. 6. Females after fixation, IBIGEO-I , preserved in %
ethanol.
Fins with a well-defined thin black border. Unpaired fins
base brownish grey in young and dark grey in mature males,
with abundant, evenly distributed, relatively big, and sub-
square irregular iridescent turquoise to light blue blotches,
with those in the distal portion elongated and merging. First
third of dorsal fin (about to the th or th rays) with a black
background color. Dorsal fin rays distal portion hyaline. Pec-
toral fin light grey, translucent, with medial distal portion
light blue to turquoise with diuse light blue blotches. Pelvic
fins dark grey with irregular light blue to turquoise blotches.
: Females (Fig. ) present a yellowish-brown body
background coloration with faint and diuse brown marble
pattern on trunk and tail composed of deep melanophores.
Vertically elongated, dark grey blotches on central portion
of trunk, anterior to dorsal fin origin, with some specimens
presenting these markings on the posterior portion of the
trunk also, composed of more superficial melanophores. The
marbled brownish patterns in the background are created by
deeper melanophores, while the greyer blotches consist of
more superficial melanophores. Small faint grey infraorbital
bar, thinner than iris, which is light orange with a grey dorsal
and posterior border. Black pupil. Ventral portion of head and
abdominal region whitish light yellow. Opercle light yellow
and slightly iridescent. Unpaired fins, with light brown rays,
except distal portion, with mainly hyaline interradial mem-
brane. Anal and dorsal fins present light grey to light brown
blotches, mainly concentrated on the basal portion and gen-
erally over the rays. Pair fins hyaline. Caudal fin hyaline.
, : Juveniles
around cm SL (Fig. A). Light grey background body color
with faint and diuse slightly dark vertical bands on the
trunk and tail, wider above the anal fin, interspersed by thin
clear bands. Caudal and paired fins hyaline. Dorsal, anal, and
pelvic fins with black borders. Dorsal and anal fins grey. Dor-
sal fin with dark and light grey diuse blotches on the base,
elongated in the basal–distal axis, and intercalated in each in-
terradial membrane space. Opercular region faint light blue.
Young adult males (Fig. B), about cm SL. Greyish light
brown body color. Scales of flanks with a wide grey border.
Vertical irregular lines appear in the flanks formed of light
blue iridescent blotches, each in the anterocentral portion
of each scale. First third anterior portion of dorsal fin back-
ground color dark black. Iridescent light blue spots appear
on unpaired and pelvic fins, while pectoral fins are hyaline.
All fins have a black border.
Adult males (Figs. C–G) have more intense colors. Medial
distal portion of pectoral fins light blue to turquoise. Pos-
terior border of scales below dorsal fin with thin light blue
border.
Mature older males (Fig. H). Anterocentral portion of
scales in the flanks lighter, making the vertical lines of iri-
descent light blue spots less evident, although they are still
more intense than adjacent scales.
Dominant territorial males (Figs. D,F,G,andH). Back-
ground coloration gets darker, especially above the anal fin
and caudal peduncle, while stressed or subordinate males
(like juveniles) or males in a white background environment
have a lighter and brownish background coloration. Changes
in color intensity occur in a short period, in a few minutes.
: Young females (Fig. A), about .cm SL present a
yellowish-brown background coloration of flanks and basal
portion of rays in unpaired fins. Anal and dorsal fins present
light brown diuse blotches on the basal portion. Pair fins
hyaline. Small, vertically elongated dark grey blotches on cen-
tral portion of body. At about cm SL (Figs. B and C), the
background coloration turns more brownish, and blotches
tend to get darker. No appreciable changes were detected in
female’s coloration with respect to their social context or en-
vironmental conditions.
: We present here the
coloration changes during ontogeny for all species of Ar-
golebias (Fig. ), except for Argolebias paranaensis, whose alive
coloration is unknown. Based on this, there are some com-
mon developmental patterns in the coloration that can be
highlighted:
) In the first developmental stages, about -month-old spec-
imens show vertical dark grey bands intercalated with
lighter vertical bands on the flanks, which continue in the
base of the dorsal and anal fins as short vertical light iri-
descent bands in their basal portions (Figs. A,B,F,and
L).
) As development continues, the vertical bands in the base
of the dorsal and anal fins become more iridescent and
start to divide, forming dots. Iridescent dots also appear
on the caudal fin, arranged in more or less circular lines.
On the flanks, iridescent spots appear in the anterocentral
portion of scales where the light bands were forming ver-
tically aligned lines of iridescent spots. The distal border
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306 Can. J. Zool. 102: 298–314 (2024) | dx.doi.org/10.1139/cjz-2023-0132
Fig. 7. Map showing the known distribution of species of the Argolebias genus. Stars represent type localities and circles other
records. Each symbol may represent more than one record. The following colors of symbols correspond Argolebias guarani
(red); Argolebias nigripinnis (yellow); Argolebias paranaensis (orange), and Argolebias adrianae n. sp. (violet). Distribution data taken
from Costa (),Calviño (),andAlonso et al. (). Map downloaded from DIVA-GIS version . (Hijmans et al. )
(projection: NAD, coordinate system: UTM).
of unpaired fins becomes dark black, the opercular region
becomes light blue iridescent, and the suborbital band
appears as a faint light grey diuse marking. (Figs. C,G,
and M).
) In a further developmental stage, about months old,
the background color becomes darker, and the iridescent
turquoise markings intensify (Figs. D,H,andN).
: Like live
specimens but without iridescent colorations and with an
amber pale general ground with light grey markings (Figs.
B and ). Briefly, males with light grey fins with hyaline
dots and markings arrange in the same pattern as the light
blue to turquoise iridescent marking in live specimens de-
scribed above. Most scales present a wide grey border and a
thin most external clear border, most marked and contrast-
ing on the dorsal portion of the body. Faint suborbital band
below the eye. Females present hyaline fins with faint light
brown rays. Light brown diuse blotches on the basal portion
of anal fin, above rays, elongated in a basal–distal direction.
Dorsal fin with smaller and more scattered brown blotches.
Flanks with light brown diuse blotches forming a marbled
pattern. Dark grey blotches, more superficial on the skin,
concentrated on the trunk portion immediately anterior to
the dorsal fin origin at the height of the eye. Sometimes with
fewer and smaller dark grey blotches on the ventral portion
of caudal peduncle. Dorsal portion of head and trunk, ante-
rior to the end of dorsal fin, dark brown with scales present-
ing a thin light brown anterior border. Faint suborbital band
below the eye.
: Argolebias adrianae is only known from its type
locality (Fig. ), a seasonal pond near the Arrechea stream, an
Iguazú River auent (aprox. −., −., altitude m
above sea level) in Parque Nacional Iguazú (a national re-
serve). The Arrechea stream is a left bank tributary of the
Iguazú River, which has an independent fall (Arrechea Fall)
very close to the other Iguazú Falls nearby. It presents the typ-
ical fish species from the Lower Iguazú river upstream from
the Iguazú Falls (Casciotta et al. ).
: The pond has a surface area of approximately
× m and a maximum depth of cm. It is surrounded by
emerging basalt stones and completely covered by aerial veg-
etation from the surrounding jungle (Fig. ). During the six
dierent occasions, we observed the pond when it was dry,
the soil was covered by a layer of leaf litter, trunks, branches,
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Fig. 8. Type locality of Argolebias adrianae n. sp. (A) November ; (B) December ; (C) July ; (D) January
; (E) surface of the bottom of the pond when empty; and (F) clay with organic matter below the surface of the pond when
its empty.
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Fig. 9. Historical climatic data from Iguazú (aprox. −., −.). Data provided by the National Meteorological Service
(Servicio Meteorológico Nacional ).
Fig. 10. Depth of the pond and meteorological data from Iguazú airport nearby (provided by the National Meteorological
Service).
and some terrestrial vascular plants (Fig. ). Beneath this
layer, the soil appeared to be composed of clay mixed with
decomposing plant debris (Fig. ). The soil was consistently
moist, and we did not observe any dry portions. During the
seven dierent occasions, we observed the pond when it con-
tained water, it was generally clear to tea-colored, sometimes
slightly muddy in the deeper portions (Fig. ). There were no
aquatic plants present in the pond, but surrounding vegeta-
tion was partially submerged during periods of maximum
water level (Fig. ). The pond apparently overflows to sur-
rounding areas when precipitation fills it. Although the tem-
porary wetland can intermittently extend to nearby areas of
the jungle during these circumstances, these areas are not
very deep and appear to be more ephemeral. We were only
able to collect fish in the deepest part of this environment.
Historical climatic data show that the period of maximum
humidity occurs from May to July when temperatures are
relatively low, while the driest period is from August to De-
cember, with an intermediate period from January to April
(Fig. ). However, interannual variability in precipitation and
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Table 2. Length and weight of alive juvenile individuals in
June . Abbreviations: SD: Standard Deviation, SE: Stan-
dard Error, TL: Total Length, SL: Standard Length.
nMin Max Media SD SE
SL
Female . . . . .
Male . . . . .
Weight
Female . . . . .
Male . . . . .
TL–SL
Female . . . . .
Male . . . . .
Note: SL, standard length.
temperatures during the studied period was high (Fig. ),
and we observed at least four drying/filling cycles in this pond
over a -year period. It is possible that there were more cycles,
as not all months were sampled. We observed two long wa-
ter periods of approximately months each, from March to
June and from June to September , with no data
between those points. We also observed one long dry period
of months in (from August to December) and three
short dry periods of around month each in December ,
January , and March . According to Pablo Burchard
(pers. comm.), specimens of about cm were present in mid-
February , and on June Montes collected speci-
mens about cm SL from a second cycle cohort of that year.
Later, on August, adult fish were collected, and the pond
was full of water but about a week later ( August), the pond
was almost dry, but in October it still had water and adult
fish (Agustín Solari pers. comm.). Based on these results, it
appears that this pond experiences several desiccation events
per year due to the high air temperatures in the area, and its
filling timing is unpredictable due to the variable precipita-
tion pattern.
In June , specimens were collected, measured, and
weighed (Table ). A total of individuals were collected,
of which .% were females. Males were, on average, larger
(. cm, range: .–. cm) and heavier ( g, range: -
g) than females (. cm, range: .–. cm and . g,
range: – g, respectively) and showed a higher Ful-
ton’s condition factor (K) (., range: –.) compared
to females (., range: –.). The length–weight (log-
transformed) relationship determined by linear regression
(Le Cren ) yielded the following parameters: b=.,
a=.; and correlation coecient =..
:
The incubation period of the eggs at room temperature be-
fore hatching is around .– months. The character states
proposed by Thompson et al. () for this species have been
coded herein (Table ,Fig. ).
: The specific epithet adrianae is created to honor
Adriana Almirón, in recognition of her valuable contribu-
tions to Neotropical Ichthyology. It serves to highlight the im-
portant role of women in the development of science and
advocate for a just and equal scientific system that does
not discriminate based on gender identity. We urge the sci-
entific community to take all necessary measures to elimi-
nate gender bias in scientific careers. The term adrianae is a
matronymic in the genitive case.
: The final phylogenetic hypothesis
resulted in Argolebias adrianae n. sp. being the sister species of
Argolebias guarani, forming a monophyletic clade along with
Argolebias nigripinnis and Argolebias paranaensis, which corre-
sponds to the genus Argolebias (Fig. ).
Discussion
Seasonal killifish balance of energetic investments in
growth, reproduction, and maintenance, shape their life his-
tory strategies under dierent environmental conditions.
This requires a delicate balance of allocating energy to dif-
ferent functions, depending on the availability of resources
and the conditions in the habitat. Therefore, their life cycle
is closely tied to relatively predictable patterns of filling and
desiccation of their habitat (Berois et al. ;Alonso et al.
;Reichard et al. ;García et al. ). However, the
cycle of filling and drying of the pond of the new species is
highly variable, with several desiccation events occurring per
year due to the high air temperatures in the area.
The pond where Argolebias adrianae inhabits is a very infre-
quent habitat for most Cynolebiasini seasonal killifish, being
a small pond inside the jungle and covered by trees. Cynopoe-
cilus notabilis Ferrer, Wingert & Malabarba, and probably
other Cynopoecilines occupy a similar habitat, associated to
dense fragments of the Altantic Forest. It fills up when precip-
itation occurs and can overflow to surrounding areas. Histor-
ical climatic data show that the period of maximum humid-
ity occurs from May to July when temperatures are relatively
low, while the driest period is from August to December, with
an intermediate period from January to April. However, the
filling timing of the pond is unpredictable due to high in-
terannual variability in precipitation and temperatures dur-
ing the studied period. Over the -year period of observation,
we recorded at least four drying/filling cycles in this pond,
with two long water periods of approximately months each
and one long dry period of months. Three short dry peri-
ods of around month each were also observed. Additionally,
up until August , two filling and drying cycles were ob-
served, and it was likely that a third cycle would occur later
in the year. Therefore, three hatching events are expected
for this year. This suggests that in this region, it is reason-
able to anticipate two to three cycles per year. In contrast,
as far as we know, all studied species of seasonal killifish
have a life cycle closely tied to the relatively predictable fill-
ing and desiccation patterns of their habitat, temporary wet-
lands that experience relatively fixed timing and duration of
dry and flooding seasons throughout the year, and have a syn-
chronized hatching with the onset of the aquatic phase of
the pool (Polaˇ
cik and Podrabsky ;Reichard et al. ;
García et al. ). Those killifish have a bet hedging strategy
that allows them to deal with a variable environment. They
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Table 3. Characters proposed by Thompson et al. () for the zona pellucida coded for Argolebias adrianae n. sp.
Characternumber
Characterstate ––––––
Fig. 11. Images of egg surface of Argolebias adrianae n. sp. (A) General view of egg and micropyle; (B–D) detail of chorion surface.
(A, C, and D) Scanning electron microscopy; (B) light microscopy.
distribute the risk by having embryos at dierent develop-
mental stages so that not all eggs will hatch in each filling
of the pond. This approach reduces the risk of total loss in
the event of a short filling cycle and allows them to avoid in-
vesting all their descendants in a single filling cycle. In highly
variable environments, a spread strategy is expected, result-
ing in a higher number of eggs at dierent stages. Conversely,
in predictable environments, fewer eggs at dierent stages
are expected (e.g., Domínguez-Castanedo et al. ;Furness
et al. ;Pinceel et al. ;Polaˇ
cik et al. ). There-
fore, the study of this new species from an extremely unpre-
dictable environment can provide valuable insights into the
evolution of life history strategies in unpredictable environ-
ments being this species a paradigmatic case of this.
Regarding the taxonomy and phylogenetic relationships of
Argolebias adrianae new species, we recovered it as the sis-
ter species of Argolebias guarani on our phylogenetic analy-
sis, forming a monophyletic group along with Argolebias ni-
gripinnis and Argolebias paranaensis. This clade is recognized
as the genus Argolebias, which is the sister clade to the re-
maining “Austrolebias genus group”, as found by Alonso et al.
(). Also, Argolebias adrianae possesses a unique combina-
tion of character states that are diagnostic of the genus Ar-
golebias (Alonso et al. ): () a gap between the preopercu-
lar and mandibular series of neuromasts, () a wide dark grey
to black border on most scales on the ventral half portion of
the flank, above the anal fin, in mature dominant males (ex-
cluding bands), () fused iridescent marks on the distal por-
tion of the dorsal fin, () a black blotch on the most anterior
region of the dorsal fin, () the absence of dark grey to black
spots on the flanks in males, and () the dorsal radial of the
pectoral fin being absent (see Fig. ).
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Fig. 12. Phylogenetic relationships of Argolebias including Argolebias adrianae n. sp. and other related Rivulidae genera, based
on morphological characters and molecular data from Alonso et al. () and this paper. Analysis under implied weighting
(K=). Numbers on nodes represent symmetric resampling. Terminal taxa colors represent data analyzed for the taxon as
follows: black, morphology and two or more molecular markers; green only on morphology; blue, two or more molecular
markers; orange, only one molecular marker.
It is noteworthy that the eggs of Argolebias adrianae and Ar-
golebias guarani lack filaments on the chorion surface (see also
Alonso et al. ). However, those filaments are present in
the other analyzed species of the “Austrolebias genus group”
(Thompson et al. ;Alonso et al. )butnotinother
seasonal rivulids like Campellolebias Vaz-Ferreira & Sierra de
Soriano, ; Leptolebias Myers, ; and Ophthalmolebias
Costa, (Thompson et al. ). It is worth noting that the
number of analyzed species of aplocheiloid killifishes is rela-
tively low. Therefore, studying the evolutionary significance
and diversification patterns of the zona pellucida in this
group of fishes remains a promising and interesting research
avenue.
In this study, we also investigated the ontogenetic changes
in color patterns during the development of all Argolebias
species, except Argolebias paranaensis. Our findings highlight
the importance of including such data in future species de-
scriptions of aplocheiloid killifish and other species. This is
particularly relevant given that many important diagnostic
characters rely on the male’s color patterns, which are likely
to be influenced by sexual selection. However, in juveniles,
sexual selection may only be acting on characters that are
correlated with adult characters (i.e., Abbott and Svensson
), making color patterns more conserved, as observed
here. Thus, including these characters may provide insights
into the phylogenetic relationships among species, as well as
shed light on the evolution of these groups and color patterns
themselves.
Regarding the biogeography of seasonal killifish in the
Paraná plateau, it is noteworthy that only six species of Rivul-
idae have been recorded so far. These species include this new
species and Garcialebias botocudo (Lanés, Volcan & Maltchik,
), Garcialebias nubium (Lanés, Volcan & Maltchik, ),
and Amatolebias varzeae (Costa, Reis & Behr, ) in the upper
Uruguay basin, and Acrolebias carvalhoi (Myers, ) and Gar-
cialebias araucarianus (Costa, ) in the Iguazú basin (Serra
et al. ). The Iguazu River basin ecoregion spans from the
headwaters of the Iguaçu River to Iguaçu Falls and shows a
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high degree of endemism, with about species of fish be-
ing endemic (Reis et al. ). This is partly attributed to the
Iguazu Falls and other cascades, which have acted as eective
barriers to dispersal between the Paraná and Iguazu basins,
driving speciation. Overall, the region demonstrates about
% of the total species endemism (Hales and Petry ;
Baumgartner et al. ). The genus Argolebias was previously
restricted to the middle and lower Paraná basin and the lower
Uruguay basin (Alonso et al. ). This new species repre-
sents the first record of this genus in the Iguazú basin and the
northernmost and easternmost record, with the highest alti-
tude ( m absl) documented as well. The cladogenetic event
between Argolebias guarani from the Middle Parana nearby
and Argolebias adrianae from the Iguazu basin may be linked
to an isolation event due to the formation of the falls that
occurred around –. million years ago (Stevaux and La-
trubesse ).
In recent years, several new species of rivulid killifishes
have been described (e.g., Alonso et al. ;Serra and
Loureiro ;Volcan and Severo-Neto ,;Lanés et
al. ), highlighting the importance of continued investi-
gation into seasonal killifishes in South America, as many
species are likely still undescribed. This is particularly con-
cerning given the threat of habitat loss, primarily due to agri-
cultural expansion (e.g., Alonso et al. ;Tagliacollo et al.
). Fortunately, the discovery of the new species within a
Natural Protected area is encouraging, and it is possible that
this species also occurs in other habitats within the region.
However, despite several samplings over the past years, we
were unable to collect this species in other habitats beyond
the type locality, and it is currently the only seasonal rivulid
recorded in this area.
Acknowledgements
We would like to thank to Guillermo Gil and Facundo Luque
and all the personal from Iguazú National Park for their as-
sistance during our collecting trips in the park. To Killifish
Foundation members and to Marcos Mirande, Fernando Lobo,
Gastón Aguilera, Baltazar Bugeau, and Virginia Martínez for
their permanent support. We also want to express our grati-
tude to André Mestre for kindly providing us with pictures of
Argolebias nigripinnis ontogeny. This manuscript benefited by
the contribution of two anonymous reviewers and the editors
of the journal. TNT was provided free by the Willi Hennig So-
ciety.
Article information
History dates
Received: July
Accepted: October
Version of record online: March
Copyright
© The Author(s). Permission for reuse (free in most
cases) can be obtained from copyright.com.
Data availability
Data generated or analyzed during this study are provided in
full within the published article and its Supplementary ma-
terial.
Author information
Author ORCIDs
Felipe Alonso https://orcid.org/---
Guillermo Enrique Terán https://orcid.org/
---
Pablo Calviño https://orcid.org/---X
Wilson Sebastián Serra Alanís https://orcid.org/
---
Martin Miguel Montes https://orcid.org/---X
Ignacio Daniel García https://orcid.org/---
Jorge Adrián Barneche https://orcid.org/---
Liliana Ciotek https://orcid.org/---
Pablo Giorgis https://orcid.org/---
Jorge Casciotta https://orcid.org/---
Author notes
Felipe Alonso, Guillermo Enrique Terán, and Pablo Calviño
made an equal contribution to this work and should be con-
sidered joint first author.
Author contributions
Conceptualization: FA, GET, PC, WSSA
Data curation: FA, GET, PC, WSSA, MMM, PG, JC
Formal analysis: FA, GET, PC, WSSA, MMM
Funding acquisition: FA, GET, JC
Investigation: FA, GET, PC, WSSA, MMM, IDG, JAB, LC, PG
Methodology: FA, GET, PC, WSSA, MMM, IDG, JAB
Project administration: FA, GET, JC
Resources: FA, GET, PC, MMM, IDG, LC, JC
Software: FA, GET
Supervision: FA, GET, PC, JC
Validation: FA, GET, PC, WSSA
Visualization: FA, GET, PC
Writing – original draft: FA, PC
Writing – review & editing: FA, GET, PC, WSSA, MMM, IDG,
JAB, PG, JC
Competing interests
The authors declare there are no competing interests.
Funding information
PICT-- granted to FA and PICT- to
GET by Agencia Nacional de Promoción de la Investigación,
el Desarrollo Tecnológico y la Innovación.
Supplementary material
Supplementary data are available with the article at https:
//doi.org/./cjz--.
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