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Patterns of reproductive behaviour in Millerichthys robustus

(Cyprinodontiformes: Cynolebiidae)

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aqua, International Journal of Ichthyology

Patterns of reproductive behaviour in Millerichthys robustus

(Cyprinodontiformes: Cynolebiidae)

Stefano Valdesalici1, Omar Domínguez-Castanedo2, and Miguel Ángel Mosqueda-Cabrera3

1) Via Cà Bertacchi N.5, 42030 Viano (RE), Italy. E-mail: valdesalici.stefano@gmail.com

2) Programa de Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana,
Unidad Xochimilco, México
3) Laboratorio de Sistemas Acuícolas, Departamento El Hombre y su Ambiente, Universidad Autónoma
Metropolitana-Xochimilco, Calzada del Hueso No. 1100, Col. Villa Quietud, Delegación Coyoacán,
04960, México, DF., México

Received: 30 July 2014 – Accepted: 12 October 2016

Keywords phase d’invitation de s’immerger/plonger n’était pas lancée

Almirante mexicano, killifish, annual fish, Veracruz, Mex-
ico.
Abstract
Millerichthys robustus is the only annual cynolebiid species
in Mexico. The purpose of the present study is to describe
patterns of reproductive behaviour in this species. Seven in-
dividual units of behaviour were identified in males and five
in females, some of which exhibit differences that can be in-
terpreted as specifically derived conditions. In Millerichthys
robustus, the phase of invitation to submerge/dive was not
initiated by the male, but by the female.
par le mâle, mais par la femelle.
Sommario
Millerichthys robustus è l'unica specie di cinolebide annuale
in Messico. Lo scopo di questo studio è di descrivere i mo-
delli di comportamento riproduttivo di questa specie. Nei
maschi sono state identificate sette regole individuali di
comportamento mentre solo cinque nelle femmine, alcune
delle quali mostrano differenze che possono essere interpre-
tate specificamente come condizioni derivate. In M. robustus
la fase di invito a sommergersi / immergersi non è avviata dal
maschio, ma dalla femmina.

Zusammenfassung
Millerichthys robustus ist die einzige jährliche Cynolebiid-
Spezies in Mexiko. Der Zweck der vorliegenden Studie ist
die Beschreibung von Mustern des Fortpflanzungsverhaltens
bei dieser Spezies. Sieben individuelle Einheiten des Verhal-
tens wurden bei Männern und fünf bei Frauen identifiziert,
von denen einige Unterschiede aufweisen, die als spezifisch
abgeleitete Bedingungen interpretiert werden können. In
Millerichthys robustus wurde die Phase der Einladung zum
Eintauchen / Tauchen nicht durch das Männchen, sondern
durch das Weibchen initiiert.
Résumé
Millerichthys robustus est la seule espèce de cynolebiidé du
Mexique. Le but de la présente étude est de décrire les com-
portements reproductifs de cette espèce. Sept patrons indi-
viduels de comportement ont été identifiés chez les mâles et
cinq chez les femelles, dont certains montrent des dif-
férences qui peuvent être interprétées comme des conditions
spécifiquement dérivées. Chez Millerichthys robustus, la
INTRODUCTION
The family Cynolebiidae is a diversified group of
killifishes, occurring between southern Florida and
northeastern Argentina (Costa 2008a). Several gen-
era have evolved a unique annual life cycle (Costa
1998). Annual fishes inhabit temporary ponds that
dry out seasonally and the adaptations to survive this
extreme condition include an elaborate courtship
behaviour (Vaz-Ferreira et al. 1964; Vaz-Ferreira &
Sierra 1972; Belote & Costa 2002, 2003, 2004;
García et al. 2008), which culminates in the deposi-
tion of drought-resistant eggs in the substrate, where
they undergo a diapause stages (Wourms 1972).
In Mexico the only annual cynolebiid species is
Millerichthys robustus (Huber 1992; Costa 1995;
Domínguez-Castanedo et al. 2013; 2016). The pur-
pose of the present study is to describe the reproduc-
tive behavior patterns of this species.

177 aqua vol. 22 no. 4 - 22 October 2016

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Patterns of reproductive behaviour in Millerichthys robustus (Cyprinodontiformes: Cynolebiidae)
MATERIALS AND METHODS
Fishes were obtained through field sampling from
a seasonal pond near Papaloapan River in Tlacotal-
pan, Veracruz, Mexico (18°37’39.3” N, 95°38’53.0”
W) using hand nets. The other fish species present
at the site were Cynodonichthys tenuis (Cynolebi-
idae), Gambusia sexrradiata, Xiphophorus maculatus,
Poecillia sphenops, Poeciliopsis gracilis, Belonesox be-
lizanus (Poeciliidae), Astyanax sp. (Characidae), Dor-
mitator maculatus (Eleotridae), Atherinella sp.
(Atherinopsidae), Mayaheros urophthalmus, and
Thorichthys sp. (Cichlidae).
Millerichthys robustus specimens were transported
to the laboratory in order to establish a captive
stock. Adult fishes were housed in 38 litres glass
aquaria, with a ratio of one male per two females.
No substrate was used in order to simplify cleaning
and discourage unwanted spawning, while physical
and chemical conditions simulated the collection
site. Mechanic, biological and chemical filtration
was also applied continuously with weekly renewal
of 1/5 of the water. Temperature was maintained at
26°C ± 1, with the photoperiod set to 14 light hours
and 10 dark hours. Subjects were fed three times a
day with Artemia salina nauplii.
Courtship behavior was observed when a 12 x 12 x
20 cm glass container covered with sterilized peat
moss 6 cm deep was placed in each aquarium, thus
resembling the substrate in the species’ natural habi-
tat. Each trial was recorded for a period varying
from 1 to 6 minutes, recorded with a in fact was a
Nikon coolpix s6100 video camera. Each individual
was used only once and the total number of trials
was 8. First, we defined the behavioural units of the
courtship pattern as per Garcia et al. (2008) using
0.25x display speed. Subsequently, timing for every
session was noted. After the experiments, some fish-
es were anaesthetised with clove oil (0.3%) and sac-
rificed with an overdose, fixed in formalin (10%) for
a period of 24 hrs, and stored in ethyl alcohol
(70%). Voucher specimens were deposited in the
Colección Nacional de Peces, Departamento de Zo-
ología, Instituto de Biología of the Universidad Na-
cional Autónoma de México (IBUNAM) (CNPE-
IBUNAM 19097, 2 males 33.0 -35.0 mm SL and 2
females, 35.2-35.4 mm SL).
RESULTS
Seven behavioural units in males and five in fe-
males were identified. Units appearing only occa-
sionally (one time) were removed from the analysis.
The reproductive behaviour begins with alternating
aqua vol. 22 no. 4 - 22 October 2016
lateral displays by the male (LD, Fig. 1); and the fe-
male responds by following the male (FO, Fig. 2)
near the substrate; this is followed by alternating lat-
eral displays with vibration by the male, folding and
twisting dorsal and anal fins, and sigmoid displays
above the substrate (VS, Fig. 3); the female responds
by diving (DI, Fig. 4), and when ¾ to almost com-
pletely inside the substrate the male follows (FF, Fig.
5) and mating (MA) occurs. After 30-35 sec the
male or female emerges (EM, Figs 6-7), followed af-
ter few second by the partner. If the male does not
follow the female (FF), then the female again dives
into the substrate (DI).
DISCUSSION
The ability to survive in temporary pools involves
specialisations such as eggs with a thickened chori-
on, a complex embryonic development with dia-
pause stage (Wourms 1972), and highly-derived
spawning behaviour which are convergent in annual
killifishes (Costa 1998). All known annual species
spawn into the substrate (Garcia et al. 2008; Pċ &
Reichard 2011), apart few exceptions, e.g. members
of the cynolebiid genus Papiliolebias (Nielsen &
Brousseau 2014) and members of the nothobranchi-
id subgenus Aphyobranchius (Wildekamp 2004),
whereas non annual killifishes, spawn near the sur-
face (Huber 1992; Wildekamp 1993), and this dis-
parity probably constitutes an adaptive condition
closely related to annualism.
The reproductive behaviour observed in Mil-
lerichthys robustus is similar to that of South Ameri-
can annual species (Belote & Costa 2002, 2003,
2004; Garcia et al. 2008; Volcan et al. 2011). Differ-
ences in some stages of the reproductive process can
be alternatively interpreted as specifically derived
conditions. Fins twisting was observed in cynopoe-
ciline genera such as Cynopoecilus, Leptolebias, and
Campellolebias (Vaz-Ferreira & Sierra 1972; Costa et
al. 1988; Costa 2008b), and fins folding in some
species of Simpsonichthys and Hypsolebias (Belote &
Costa 2002), for example.
The degree of conservancy in courtship displays
within South American annual fishes, indicates that
specific recognition, representing a barrier to hy-
bridization, may not occur, unless there are quanti-
tative differences in courtship displays or in the
aforementioned non-visual signals.
The major dissimilarity in Millerichthys compared
with with South American annual fishes is that the
phase of invitation to submerge/dive was not initiat-
ed by the male but by the female. This happen
178
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Stefano Valdesalici, Omar Domínguez- Castanedo and Miguel Ángel Mosqueda-Cabrera

1 2

3 4

5 6

Fig. 1. Alternating lateral displays by the male (LD).

Fig. 2. Female responds by following the male (FO).
Fig. 3. Alternating lateral displays with vibration by the
male, with folding of dorsal and anal fins and sigmoid
displays above the substrate (VS).
Fig. 4. Female responds by diving (DI).
Fig. 5. When ¾ to almost completely inside the substrate
the male follows (FF).
Fig. 6. Female emerging (EM).
Fig. 7. Male emerging a few seconds later.
Video by O. Domínguez-Castanedo

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Patterns of reproductive behaviour in Millerichthys robustus (Cyprinodontiformes: Cynolebiidae)
sometimes also in members of the genus Xenurole-
bias (Belote & Costa 2002).
This certainly indicates a specific recognition but it
is unclear if a hybridization barrier exists because the
only sympatric cynolebiid at the study site was Cyn-
odonichthys tenuis which is a not an annual fish (Hu-
ber 1992). Indeed this unique reproductive charac-
ter in Millerichthys robustus clearly separates this lin-
eage from all other family members. A comprehen-
sive molecular phylogenetic analysis may resolve the
precise relationship between Millerichthys robustus
and other annual cynolebiid, but does not lie within
the scope of the present study.
ACKNOWLEDGEMENTS
The authors wish to thank to Francesca Fontana
for the assistance with the first draft of the manu-
script, to Andrea Fontanesi for the assistance on
video cutting sequences, Matt Ford for correction of
spelling and grammar and Roberto Bolaños Villar-
real for its assistance in capture of videos. The collect
of the specimens in supported for the authorization:
SGPA/DGVS/02404/14 with the date of March 26,
2014, of the Subsecretaría de Gestión para la Protec-
ción Ambiental, Dirección General de Vida Silvestre
of the SEMARNAT.
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