Comment on 'Parasite defensive limb movements enhance acoustic signal attraction in male little torrent frogs'

  1. Nigel K Anderson
  2. Doris Preininger
  3. Matthew J Fuxjager  Is a corresponding author
  1. Department of Ecology, Evolution, and Organismal Biology, Brown University, United States
  2. Department of Evolutionary Biology, University of Vienna, Austria
  3. Vienna Zoo, Austria

Abstract

Zhao et al. recently reported results which, they claim, suggest that sexual selection produces the multimodal displays seen in little torrent frogs (Amolops torrentis) by co-opting limb movements that originally evolved to support parasite defense (Zhao et al., 2022). Here, we explain why we believe this conclusion to be premature.

Introduction

Many animals communicate by performing multimodal displays that showcase vocal and gestural signals (Partan and Marler, 1999; Bro-Jørgensen, 2010; Higham and Hebets, 2013; Starnberger et al., 2014b; Mitoyen et al., 2019). Recently, Zhao et al. attempted to study how these displays might evolve, at least with respect to the process by which discrete limb movements can be incorporated into more complex signaling routines. They did this by studying little torrent frogs (Amolops torrentis), which inhabit noisy streams throughout Hainan Island in Southern China (Zhao et al., 2022). They concluded that: (i) male frogs produce a set of discrete arm and leg maneuvers to help swat away blood-sucking parasites; (ii) these same limb movements enhance the attractiveness of male calls to females. Zhao et al. then argued that natural selection for parasite-induced movements creates an opportunity for sexual selection to generate a multimodal display by integrating these movements into the species’ signaling routine. However, we argue that these conclusions are premature because they are based on misinterpretations of the study’s main results.

Results and discussion

Only “un-preferred” movements are produced around parasites

For the main conclusions of Zhao et al., 2022 to be correct, the following must be true: (i) limb movements must function to protect frogs from parasitism; (ii) this defense tactic must have emerged before the species evolved either its social limb displays or its multimodal communication strategy (True and Carroll, 2002; Borgia and Keagy, 2015; Schwark et al., 2022). However, Zhao et al. do not to provide compelling evidence for either point. For example, they report male frogs sometimes produce certain gestures when parasites land on them or when parasites fly in the frog’s “vicinity” (although this term is not defined). Moreover, they do not statistically analyze their data to assess whether frogs are more likely to produce gestures when parasites are around. We therefore ran such an analysis, and we found that only two movements —limb shaking (LSA) and wiping (W)—were more likely to occur in the presence of parasites than one might expect by chance (Figure 1). Importantly, these specific movements were not the ones that females preferred in choice tests (Figure 5C and 5D in Zhao et al., 2022). At the same time, we found that both hind foot lifting (HFL) and arm wiping (AW) were not more likely to occur in the presences of parasites (Figure 1), even though these were the two limb movements that females seemed to prefer in choice tests (Figure 5A and 5B in Zhao et al., 2022). Our results therefore suggest that parasite presence is associated with only certain limb movements that Zhao et al. studied, but none that are positively linked to female preference (but see below for concerns about female preference tests).

Re-analysis of whether male little torrent frogs (Amolops torrentis) produce limb displays in the presence of parasites.

(A) Proportion of different limb displays observed passively in a population of males (n=69) either in the presence of parasites (blue bars) or in the absence of parasites (orange bars). Note that these data are weighted by the number of limb movements each male produced, which were highly skewed in the original dataset. In other words, in the first analysis by Zhao et al., some males produced >90 displays, whereas other males produced zero (Zhao et al., 2022). See Methods for details about how we weighted values. For all subsequent analyses (G-tests for goodness of fit), the proportion of toe trembling produced in the presence and absence of parasites was used as the null hypothesis, setting our expectation of how often displays should be produced by chance in the presence or absence of parasites (see Methods for justification). (B–F) Density plots of the boot strapped chi-squared (χ2) statistics from the G-test of goodness of fit analysis. On the y-axis is the density of chi-squared (χ2) statistics after 1,000 iterations, and on the x-axis is the chi-squared (χ2) value. Solid green lines denote mean chi-squared statistics associated with each distribution of values, whereas solid red lines represent the cut-off for statistical significance (P<0.05) with 1 degree of freedom. If the green line falls on the right side of the red line, then the result is statistically significant (i.e., male frogs appear to perform the given display in the presence of parasites more than we might expect by chance, as determined by the null model set through toe trembling). By contrast, if the green line falls on the left side of the red line, then the result is not significant (i.e., male frogs do not perform the given display in the presence of parasites more than we might expect by chance). We found that (B) toe trembling (TT) was (as expected) not statistically significant (χ2=0.084, P=0.772), nor was (C) hind foot lifting (HFL; χ2=0.487, P=0.485) or (D) arm wiping (AW; χ2=2.772, P=0.096). Importantly, these were the behaviors the females supposedly preferred, though see the main text for a discussion of the limitations associated with this assay. We found that (E) limb shaking behavior (LSA) was statistically significant (χ2=5.0314, P=0.025, denoted with asterisk), as was (F) wiping (W) (χ2=4.212, P=0.040, denoted with asterisk). These latter two behaviors (LSA and W) were not preferred by females in the behavioral assay. Note that when comparing A to both E and F (LSA and W, respectively), the proportions in A would suggest that the effect reported in F would be more robust, compared to the effect in E. However, there were several males that did not wipe (0 values), which may have broadened the Chi Squared curve and decreased the statistical power in the analysis.

Parasitism and limb movements are correlational, and not causal

Zhao et al. also report a positive correlation between the number of parasite visits males receive and the number of limb movements males produce. They interpret these data as further support for the hypothesis that parasites are the cause of limb movements. However, correlation does not equal causation. Even if males who encountered more parasites were also more likely to have produced limb displays, this relationship does not necessarily mean that parasites directly “induced” or “evoked” this behavior, as Zhao et al. assert. Other explanations for the association include the possibility that higher quality males who display more vigorously also occupy spots along the breeding stream that contain more parasites. Micro- and macro-ecological factors that determine the abundance and distribution of blood-sucking parasites that target frogs are poorly understood (outlined recently by Virgo et al., 2022), but other work in midges implies a wide range of factors associated with the local landscape and ecology can influence their distribution and abundance (Kluiters et al., 2013; Rigot et al., 2013). Alternatively, parasites might be attracted to male calls (Bernal et al., 2006; Aihara et al., 2016; Toma et al., 2019), which males might produce more often when they are using their limbs to display during bouts of male-male competition (Grafe et al., 2012). Indeed, in both cases here, we would expect positive correlations between parasite levels and limb movements, without a causal link between the two.

Understandably, one might ask why exactly frogs would evolve limb movements like hind foot lifting (HFL) and arm wiping (AW), if they are not involved in parasite defense. This question seems even more logical given that Zhao et al. classify limbs movements produced in the absence of parasites as “spontaneous,” which implies that they are performed at random or without being triggered by an external stimulus. An alternative view, however, is that these so-called “spontaneous” limb movements are actually generated as social signals that help males compete with sexual rivals during agonistic interactions. Most frogs that use gestural signals do so for this purpose (see Table 1), and thus the behavior is assumed to evolve through intrasexual selection (Preininger et al., 2013b; Preininger et al., 2013c; Mangiamele and Fuxjager, 2018; Anderson et al., 2021a). Zhao et al. do not determine how many of the limb movements produced in the absence of parasites (e.g., “spontaneous”) were actually the result of male-male interactions, but they do indicate that little torrent frogs use these movements in such contexts.

Table 1
List of anuran species that perform limb displays or gestural signals.

For Dendrobatoidea, see Hödl and Amézquita, 2001. Note that in most cases the term courtship in the Behavioral Function column refers to instances in which females make choices about male mates, while males use gestural signals to simultaneously compete.

Species*Limb SignalsSexBehavioral
Function
EvidenceCountry
of Origin
EcologyActivity PatternReference
BrachychephalidaeBrachycephalus
ephippium
arm
waving
Maggressive,
defense
observationBrazilforest floordiurnalPombal et al., 1994,
Goutte et al., 2017
B. pitangaarm
waving
Maggressive,
defense
observationBrazilforest floor,
leaf litter
diurnalGoutte et al., 2017
BufonidaeAtelopus
limosus
arm
waving
Mcontext not
determined
observationPanamastreamdiurnalHödl and Amézquita, 2001
A. variusarm
waving
M, Faggressive,
to
defend sites
observationColombia,
Costa Rica,
Panama
streamdiurnalCrump, 1988
A. zetekiarm
waving
M, FM: agonistic,
territorial
vigilance, F:
intersexual
female-male,
courtship
experimental/
mirror image
PanamastreamdiurnalLindquist and Hetherington, 1996,
Lindquist and Hetherington, 1998
A. chiriquiensisarm
waving
Mcall response,
amplexus
attempt
observationCosta
Rica,
Panama
streamdiurnalLindquist and Hetherington, 1996,
Lindquist and Hetherington, 1998
leg-
kicking
Mduring egg
laying in
amplexus
observationLindquist and Swihart, 1997
LeptodactylidaeLeptodactylus
melanotus
foot
twitching &
back raise
MaggressiveobservationCentral
America,
Mexico
pondDiurnal,
nocturnal
Brattstrom, 1968,
Gregory, 1983
Crossodactylus
gaudichaudii
arm
waving
Mconspecific
in the vicinity
observationBrazilstreamdiurnalWeygoldt and Potsch de Carvalho e Silva, 1992
leg stretchMaggressiveobservationWeygoldt and Potsch de Carvalho e Silva, 1992
leg liftMaggressiveobservationWeygoldt and Potsch de Carvalho e Silva, 1992
C. schmidtiiboth legs
kicking
M, J*agonistic,
*context
not
determined
observationBrazilstreamdiurnalCaldart et al., 2014
leg kickingMagonisticobservationCaldart et al., 2014
toe flaggingM, FagonisticobservationCaldart et al., 2014
toe tremblingMagonisticobservationCaldart et al., 2014
limb lifting
(arm & leg)
M, F, Jagonistic,
M: courtship
observationCaldart et al., 2014
Hylodes asperfoot
flagging
Magonistic,
courtship
observationBrazilstreamdiurnalHaddad and Giaretta, 1999,
Hartmann et al., 2005
toe movement,
flagging
MagonisticobservationHaddad and Giaretta, 1999,
Hartmann et al., 2005
leg stretchingM, FM: agonistic;
F: mating
observation,
experimental
(mirror)
Haddad and Giaretta, 1999,
Hartmann et al., 2005
arm liftingMagonisticobservationHaddad and Giaretta, 1999
kickingMaggressiveobservationHaddad and Giaretta, 1999
leg liftingMagonisticobservationHartmann et al., 2005
H. cardosoileg stretching
(1 leg)
Madvertisement,
courtship
observationBrazilstreamdiurnalForti and Castanho, 2012
leg stretching
(2 legs)
Madvertisement,
courtship
observationForti and Castanho, 2012
limb liftingMadvertisement,
territorial
observationForti and Castanho, 2012
foot flaggingM, Fadvertisement,
courtship M:
territorial
observationForti and Castanho, 2012
foot flagging
+toe wave
Madvertisement,
courtship,
territorial
observationForti and Castanho, 2012
leg
kicking
Madvertisement,
courtship,
territorial
observationForti and Castanho, 2012
H. dayctylocinusfoot
flagging
Magonistic,
courtship
observationBrazilstreamdiurnalNarvaes and Rodrigues, 2005
toe
wiggling
MagonisticobservationNarvaes and Rodrigues, 2005
leg
stretching
MagonisticobservationNarvaes and Rodrigues, 2005
kickingMaggressiveobservationNarvaes and Rodrigues, 2005
arm
lifting
Mcontext not
determined
observationNarvaes and Rodrigues, 2005
H. japitoe
trembling
Magonistic,
advertisement,
courtship
observationBrazilstreamdiurnalde Sá et al., 2016
toe
flagging
Magonistic,
advertisement,
courtship
observationde Sá et al., 2016
toes
posture
Magonistic,
advertisement,
courtship
observationde Sá et al., 2016
foot
shaking
Magonistic,
advertisement,
courtship
observationde Sá et al., 2016
leg
stretching
Magonisticobservationde Sá et al., 2016
foot
flagging
Magonistic,
advertisement,
courtship
observationde Sá et al., 2016
hand
shaking
Magonistic,
advertisement,
courtship
observationde Sá et al., 2016
arm
lifting
M,Fagonistic,
courtship
observationde Sá et al., 2016
arm
waving
M,Fagonistic,
courtship
observationde Sá et al., 2016
H. meridionalistoe
flagging
MagonisticexperimentalBrazilstreamdiurnalde Sá et al., 2018,
Furtado et al., 2019
toe
trembling
Magonisticobservationde Sá et al., 2018,
Furtado et al., 2019
toe
posture
Magonisticobservation,
experimental
de Sá et al., 2018,
Furtado et al., 2019
arm
lifting
M, FM-agonistic,
F-reproductive
observation,
experimental
de Sá et al., 2018,
Furtado et al., 2019
arm
waving
M, FM-agonistic &
reproductive,
F-reproductive
observation,
experimental
de Sá et al., 2018,
Furtado et al., 2019
leg liftingM, FM-agonistic &
reproductive,
F-reproductive
observation,
experimental
Furtado et al., 2019
foot flaggingMagonisticobservation,
experimental
Furtado et al., 2019
foot shakingMagonisticobservationde Sá et al., 2018
both
legs kicking
FagonisticobservationFurtado et al., 2019
H. nasustoe
wiggle
Magonistic
(threat signals)
observation,
experimental
BrazilstreamdiurnalWeber et al., 2004
arm
waving
Magonistic
(threat signals)
observation,
experimental
Weber et al., 2004
leg
stretch
Magonistic
(threat signals)
observation,
experimental
Weber et al., 2004
H. phyllodesfoot
flagging
Magonisticobservation,
experimental
BrazilstreamdiurnalHartmann et al., 2005,
Augusto-Alves and Toledo, 2021
leg
stretching
Magonistic,
courtship
observation,
experimental
Hartmann et al., 2005
arm
lifting
Magonistic,
advertisement
observation,
experimental
Hartmann et al., 2005,
Augusto-Alves and Toledo, 2021
arm
waving
Mcontext not
determined
observationAugusto-Alves and Toledo, 2021
leg
lifting
Magonistic,
advertisement
observation,
experimental
Hartmann et al., 2005,
Augusto-Alves and Toledo, 2021
two limbs
lifting
Mcontext not
determined
observationAugusto-Alves and Toledo, 2021
toe
flagging
Magonisticobservation,
experimental
Hartmann et al., 2005,
Augusto-Alves and Toledo, 2021
foot
shaking
Mcontext not
determined
observationAugusto-Alves and Toledo, 2021
two-leg
kicking
MagonisticobservationAugusto-Alves and Toledo, 2021
MyobatrachidaeTaudactylus
eungellensis
leg
stretching
Mcontext not
determined
-streamdiurnalHödl and Amézquita, 2001
foot
flagging
Mcontext not
determined
-Hödl and Amézquita, 2001
HylidaeBoana
albomarginata
Limb
lifting
Magonisticexperimental
(mirror)
Brazilpond
margins
vegetation
nocturnalHartmann et al., 2005,
Furtado and Nomura, 2014
(Hypsiboas
albomarginatus
)
face
wiping
Magonisticexperimental
(mirror)
Furtado and Nomura, 2014
(Hyla
albormarginata
)
toe
trembling
Magonisticexperimental
(mirror)
Hartmann et al., 2005,
Furtado and Nomura, 2014
leg
kicking
Magonisticexperimental
(mirror)
Hartmann et al., 2005,
Furtado and Nomura, 2014
B. ranicepslimb
lifting
Magonisticexperimental
(mirror)
Brazilponds or
wetlands
nocturnalFurtado et al., 2017
(Hypsiboas
raniceps
)
toe/finger
trembling
Magonisticexperimental
(mirror)
streamnocturnalFurtado et al., 2017
Litoria
cooloolensis
foot
flagging
MagonisticobservationAustraliatreenocturnalMeyer et al., 2012
L. genimaculatafoot
flagging
MagonisticobservationAustraliastreamnocturnalRichards and James, 1992
L. irisleg
flicking
Mcall responseobservationPapua New GuineastreamcrepuscularMeyer et al., 2012
L. nannotisfoot
flagging
MagonisticobservationAustraliastreamnocturnalRichards and James, 1992
arm
waving
MagonisticobservationnocturnalRichards and James, 1992
L. pearsonianahand
waving
MagonisticobservationAustraliastreamnocturnalMeyer et al., 2012
leg
flicking
MagonisticobservationMeyer et al., 2012
L.rheocolaleg
stretching
MagonisticobservationAustraliastreamnocturnalRichards and James, 1992
arm
waving
MagonisticobservationRichards and James, 1992
L. fallaxfoot
flagging
MagonisticobservationAustraliapondnocturnalMeyer et al., 2012
foot
flickering
MagonisticobservationMeyer et al., 2012
kickingMaggressiveobservationMeyer et al., 2012
Lysapsus
limellum
Limb liftingMagonisticexperimental
(mirror)
Brazillentic
water
bodies
nocturnalFurtado et al., 2017
Dendropsophus
nanus
Limb liftingMagonisticexperimental
(mirror)
BrazilpondsnocturnalFurtado et al., 2017
Dendropsophus
parviceps
foot flaggingMagonisticobservationVenezuelastreamside
ponds
nocturnalAmézquita and Hödl, 2004
Hyla
parviceps
arm wavingMagonisticobservationAmézquita and Hödl, 2004
Hyla sp.
(aff. ehrhardti
)
body
wiping (foot)
courtshipobservationBrazilforest,
bromeliads
nocturnalHartmann et al., 2005
face
wiping (arm)
M, FcourtshipobservationHartmann et al., 2005
foot flaggingMcourtship
(far from
females)
observationHartmann et al., 2005
limb lifting
(arm +leg)
McourtshipobservationHartmann et al., 2005
Phyllomedusa
boliviana
foot flaggingMaggressiveobservationBoliviapondnocturnalJansen and Kohler, 2008
leg liftingMaggressiveobservationJansen and Kohler, 2008
leg stretchingMaggressiveobservationJansen and Kohler, 2008
P. burmeisterileg stretchingMagonisticobservationBrazilpondnocturnalAbrunhosa and Wogel, 2004
kickingMaggressiveobservationAbrunhosa and Wogel, 2004
P. sauvagiifoot flaggingMterritorialobservationArgentina,
Bolivia,
Paraguay,
Brazil
pondnocturnalHalloy and Espinoza, 2000
Scinax
eurydice
leg kickingM2 males
far from
each other
observationBrazilpond
(rainy season)
nocturnalHartmann et al., 2005
limb lifting
(arm +leg)
M2 males
far from
each other
observationHartmann et al., 2005
CentrolenidaeVitreorana
uranoscopa
limb lifting
(arm +leg)
Magonistic,
spontaneous
(no other
individual
present)
observationBrazilnocturnalHartmann et al., 2005
(Hyalinobatrachium
uranoscopum
)
RanidaePulchrana
(Rana) baramica
toe wavingMattract preyobservationSingaporeforestGrafe, 2008
Staurois
latopalmatus
arm wavingMagonisticobservationBorneostreamdiurnalPreininger et al., 2009
foot flaggingMagonisticobservationPreininger et al., 2009
S. guttatusfoot flaggingM, FagonisticF-experimental,
M-observation
streamGrafe and Wanger, 2007,
Preininger et al., 2016
leg drummingMcontext not
determined
observationGrafe and Wanger, 2007
foot raisingMcourtshipobservationGrafe and Wanger, 2007
arm wavingMagonisticobservationGrafe and Wanger, 2007
S. parvusfoot flaggingM, Jagonisticobservation,
experimental
BorneostreamdiurnalGrafe et al., 2012,
Preininger et al., 2012,
Preininger et al., 2013b
foot lifting (tap)Magonisticobservation,
experimental
Grafe et al., 2012,
Preininger et al., 2013b
MicrixalidaeMicrixalus
candidus
foot liftingMagonisticobservationIndiastreamdiurnalPreininger and
Fuxjager, pers.
observation
foot stretchingMagonisticobservationPreininger and
Fuxjager, pers.
observation
foot flaggingMagonisticobservationPreininger and
Fuxjager, pers. observation
M. elegansfoot liftingMagonisticobservationIndiastreamdiurnalPreininger and
Fuxjager, pers.
observation
foot stretchingMagonisticobservationPreininger and
Fuxjager, pers.
observation
foot flaggingMagonisticobservationPreininger and
Fuxjager, pers.
observation
M. kottigeharensisfoot liftingMagonisticobservationIndiastreamdiurnalPreininger et al., 2013c,
Anderson et al., 2021b,
Anderson et al., 2021d
foot stretchingMagonisticobservationPreininger et al., 2013b,
Preininger et al., 2013c
foot flaggingMagonisticobservationPreininger and
Fuxjager, pers.
observation
toe wigglingMagonisticobservationPreininger and
Fuxjager, pers.
observation
kickingMaggressiveobservationPreininger et al., 2013c
M. niluvaseifoot liftingMagonisticobservationIndiastreamAnderson et al., 2021d,
Preininger and
Fuxjager, pers.
observation
foot stretchingMagonisticobservationPreininger and
Fuxjager, pers.
observation
foot flaggingMagonisticobservationPreininger and
Fuxjager, pers.
observation
kickingMaggressiveobservationPreininger and
Fuxjager, pers.
observation
M. saxicolafoot liftingMagonisticobservationIndiastreamAnderson et al., 2021d,
Preininger and
Fuxjager, pers.
observation
foot stretchingMagonisticobservationPreininger and
Fuxjager, pers.
observation
foot flaggingMagonisticobservationPreininger and
Fuxjager, pers.
observation
toe wigglingMagonisticobservationPreininger and
Fuxjager, pers.
observation
kickingMaggressiveobservationPreininger and
Fuxjager, pers.
observation
M. speccafoot liftingMagonisticobservationIndiastreamPreininger and
Fuxjager, pers.
observation
foot flaggingMagonisticobservationPreininger and
Fuxjager, pers.
observation
M. uttaraghatifoot liftingMagonisticobservationIndiastreamPreininger and
Fuxjager, pers.
observation
foot stretchingMagonisticobservationPreininger and
Fuxjager, pers.
observation
foot flaggingMagonisticobservationPreininger and
Fuxjager, pers.
observation
toe wigglingMagonisticobservationPreininger and
Fuxjager, pers.
observation
RhacophoridaeBuergeria
japonica
leg-stretchesMagonistic
male-male
interaction
observationJapanaquatic and terrestrialAnderson et al., 2021c
B. otaifoot-flaggingMagonistic
male-male
interaction
observationTaiwanstreamYang, 2022
Theloderma
bambusicolum
foot-flaggingMterritorial
behavior
observationVietnamdense bushesOrlov et al., 2012
  1. *

    Species names in parentheses represent former names used in original publication.

  2. M=male; F=female; J=juvenile.

Limitations to the female preference tests

Zhao et al. also conduct experiments that examine whether females prefer to associate with males that produce supposed “parasite-induced” limb movements while calling. In theory, results from this study should provide the rationale for the hypothesis that sexual selection by female choice co-opts leg movements into reproductive displays. Yet, as we indicate above, this idea runs counter to many studies that suggest that gestural displays in frogs mediate agonistic encounters among males (Table 1). To our knowledge, there are currently no studies that clearly and definitively show that male frogs use the same limb movements described by Zhao et al. to attract female mates. There is certainly some observational evidence for visual displays employed during courtship, but such data are relatively rare and functionally ambiguous (examples: de Sá et al., 2018 has n=3 courtship interactions; Furtado et al., 2019 has n=1 courtship interaction). To this end, Zhao et al. only report four male-female interactions across two breeding seasons, and during these interactions males don’t produce any of the limb displays that are purported to be linked with parasite defense. Furthermore, when working in the field with torrent frogs, one must recognize that it is nearly impossible to distinguish male gestural displays directed to other males from those directed at females (see Table 1 and most “courtship” interactions listed therein). This is because males in the area will trigger these behaviors from each other, even as females approach (Preininger and Fuxjager, personal observations; Zhao et al., 2022).

Still, Zhao et al. attempt to test female preference for male limb movements by presenting females with video stimuli of males that were calling and either producing limb movements or not. However, these video stimuli are not ecologically relevant to female frogs. This is because each stimulus was manually altered to include a standardized audio channel, such that the male in the video would be perceived to have called without inflating its vocal sac. Free-living females do not naturally encounter such stimuli, particularly when they assess males by looking at them head-on (as females do in this experiment). Zhao et al. indicate that they designed the stimuli this way because they were afraid the effect of vocal sac inflation would mask any effect of limb movement on female preference. Vocal sac inflation has a powerful effect on sexual attractiveness and mate choice in frogs (reviewed by Starnberger et al., 2014a), including in little torrent frogs (Zhao et al., 2021). Importantly, if vocal sac inflation does mask effects of limb movements on female preference, then selection should not strongly favor the co-option of these movements into the display. We suspect that females showed a preference for males that produced HFL and AW movements because they were the closest resemblance of “fixed” vocal sac inflations, particularly when the alternative stimulus included calls without vocal sac inflations (Rosenthal et al., 2004; Narins et al., 2005; Taylor et al., 2008; Gomez et al., 2011; Preininger et al., 2013a). Visual and acoustic components might differ in context and dominance, but nevertheless strongly modulate mate choice (Taylor et al., 2011). One might argue against our point by saying that females can in fact observe males producing limb movements and calls without seeing vocal sac inflation, such as when females see males from behind. However, such visual perspectives of the male were not incorporated into the experimental design, and thus the current study cannot reveal how females would respond to seeing males perform limb movements from such alternate angles.

Conclusions

Here, we highlight concerns about a study by Zhao et al. that tried to explain the origins of multimodal display behavior in little torrent frogs (Zhao et al., 2022). By reanalyzing data from this study, we show that only certain limb movements are potentially performed more in the presence of parasites, and these are not the movements that females seem to prefer. The study by Zhao et al. also over-interprets correlational evidence to propose that limb movements evolved to avoid parasite attacks. Finally, Zhao et al. cannot determine whether limb movements are functionally significant during male-female interactions because female preference experiments were limited with respect to their ethological relevance.

We also have other concerns about this study. For example, data videos and drawings of limb movements are ambiguous and unclear (e.g., parasites are unclear in Video 1; gesture illustrations in Figure 1E and C show mirror images of the same movements), and there are no data showing how frequently frogs use limb movements to physically wipe away parasites, or whether frogs ever experience parasites in their “vicinity” without producing limb movements. It is also unclear why preference tests were carried out at night, which creates a temporal mismatch with day-recorded video stimuli. Nonetheless, as biologists who study gestural signals in frogs, we remain open to the possibility that visual displays might arise through the co-option of adaptive movements that are unrelated to communication. Similarly, we recognize that the role of female choice in the evolution of frog limb displays is poorly understood and merits further investigation. However, studies exploring these topics should be carried out using approaches that are clear and replicable, so that we can draw lasting conclusions.

Materials and methods

We used data from the original study (Table S1 in Zhao et al., 2022) to statistically test whether male frogs were more likely to produce the various limb movements when parasites were around than one would otherwise expect by chance. We reasoned that this analysis would help us understand whether behaviors that were more closely aligned with parasite presence were also associated with female preference tests. (Please see above for a discussion of the limitations associated with preference tests).

We ran all statistical analyses in R Studio (https://www.rstudio.com), an integrated environment for R 4.13 (https://www.r-project.org). For data preprocessing, we noted that Zhao et al. did not account for the drastic differences in number of behaviors produced by each frog. This oversight can lead to certain individuals in the population having an outsized effect on statistical outcomes. For example, a frog that produced ≈90 limb movements in the absence of parasites and 10 limb movements in the presence of parasites was compared to another frog that produced 10 limb movements in absence of parasites and 1 limb movement in the presence of parasites. The proportion of behaviors that these individuals produced in each context is the same, but the absolute total number of these behaviors is quite different; as a result, if raw values of behavior are compared between the groups (absence of parasites vs. presence of parasites), then the first frog will have a more robust impact than the second frog. Weighting values can be an important way to avoid such effects, and so we adopted this approach. We weighted following Garamszegi, 2014, where each display count, X, was multiplied by the inverse of the sum count of X for the given individual.

Next, to test how limb movements might correspond to the presence of parasites, we used a G-test (for goodness of fit) to statistically compare the proportion of limb movements produced in the absence of parasites (i.e., called “spontaneous” limb movements, see main text) and the proportion of limb movements produced in the presence of parasites. This test assumes independence between the proportions. To meet this assumption, we randomly sampled 35 individuals from the data set, and noted the total number of “spontaneous” limb movements these individuals produced. We then took the remaining 34 individuals from the data set and recorded only the total number of limb movements produced in the presence of parasites. We repeated this process 1,000 times, always resampling the dataset with replacement. In each case, we employed the g.test function from the AMR package to calculate a Chi Squared (χ2) test statistic, which produced a distribution of statistic values. We used the mean χ2 statistic associated with each limb movement to compute a corresponding p value. Importantly, these models were calculated using a null distribution that was determined by the level of toe trembling behavior in the absence (89%) and presence of parasites (11%). Past studies, including some that Zhao et al. cite (such as Hödl and Amézquita, 2001), show that toe trembling is not a parasite defense behavior; rather, it is commonly used either as a social signal (Lindquist and Hetherington, 1996; Rojas and Pašukonis, 2019) or as a feeding/hunger signal (Grafe, 2008; Hagman and Shine, 2008; Sloggett and Zeilstra, 2008; McFadden et al., 2010; Claessens et al., 2020). Either way, toe trembling provides a nice statistical heuristic to anchor our a priori expectations of how many of these limb displays should be produced when parasites are not around vs. when they are around. Accordingly, if the proportion of limb displays differed significantly from this expectation, then we could conclude that the given behavior was produced more often in the presence of parasites than expected by chance. By contrast, if the proportion of limb displays did not differ significantly from our null expectation based on toe trembling, then we cannot reject the null hypothesis.

Data availability

Figure 1 source data are included with original manuscript (Supplementary file 1) on which we are commenting.

The following previously published data sets were used
    1. Zhao L
    (2022) Dryad Digital Repository
    The data of parasite-induced and spontaneous displays in each limb movement for calling males, silent males and males that have females nearby.
    https://doi.org/10.5061/dryad.f1vhhmgzg

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Article and author information

Author details

  1. Nigel K Anderson

    Department of Ecology, Evolution, and Organismal Biology, Brown University, Providence, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2619-3405
  2. Doris Preininger

    1. Department of Evolutionary Biology, University of Vienna, Vienna, Austria
    2. Vienna Zoo, Vienna, Austria
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Methodology, Writing – original draft, Project administration
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6842-1133
  3. Matthew J Fuxjager

    Department of Ecology, Evolution, and Organismal Biology, Brown University, Providence, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration
    For correspondence
    matthew_fuxjager@brown.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0591-6854

Funding

National Science Foundation (OISE-1952542)

  • Matthew J Fuxjager

Vienna Zoo

  • Doris Preininger

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Nick Antonson, Nicole Moody, and Sofia Piggott for helpful discussions about this paper.

Version history

  1. Received: May 10, 2023
  2. Accepted: September 12, 2023
  3. Version of Record published: October 9, 2023 (version 1)

Copyright

© 2023, Anderson et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Nigel K Anderson
  2. Doris Preininger
  3. Matthew J Fuxjager
(2023)
Comment on 'Parasite defensive limb movements enhance acoustic signal attraction in male little torrent frogs'
eLife 12:e89134.
https://doi.org/10.7554/eLife.89134

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https://doi.org/10.7554/eLife.89134

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