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Citation: Nielsen, M.-E.; Johnson, J.B.
Quantifying Shape Variation in an
Antisymmetrical Trait in the Tropical
Fish Xenophallus umbratilis.Symmetry
2023,15, 489. https://doi.org/
10.3390/sym15020489
Academic Editors: Vilfredo De
Pascalis and Sergei D. Odintsov
Received: 14 December 2022
Revised: 9 January 2023
Accepted: 2 February 2023
Published: 13 February 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
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4.0/).
symmetry
S
S
Article
Quantifying Shape Variation in an Antisymmetrical Trait in the
Tropical Fish Xenophallus umbratilis
Mary-Elise Nielsen 1, * and Jerald B. Johnson 1,2
1
Evolutionary Ecology Laboratories, Department of Biology, Brigham Young University, Provo, UT 84602, USA
2Bean Life Science Museum, Brigham Young University, Provo, UT 84602, USA
*Correspondence: ellie.johnson.512@gmail.com; Tel.: +1-(385)-229-5791
Abstract:
Antisymmetry is a striking, yet puzzling form of biological asymmetry. The livebearing fish
Xenophallus umbratilis exhibits antisymmetry in the male intromittent organ and provides a system
that is well-suited for studying the nature of variation in antisymmetrical traits. Using geometric
morphometrics, we test the hypothesis that because the gonopodium is critical to fitness there will
not be significant differences in gonopodium shape between the two gonopodial morphs in this
species. Our results are consistent with this prediction, though we found that gonopodium shape
differed with gonopodium size.
Keywords: gonopodium; asymmetry; Poeciliidae; geometric morphometrics
1. Introduction
Understanding and explaining morphological variations has long been a topic of
interest in evolutionary biology. Some of the most widespread and easily discernable
forms of variation are breaks in symmetry, also known as asymmetry. Several types of
asymmetries exist. Fluctuating asymmetries are subtle, random deviations from symmetry
that are classically associated with parasites, environmental stress, and homozygosity [
1
–
5
].
This type of asymmetry typically results from aberrations in development and has been
shown to reliably signal genome quality as well [
6
–
8
]. Directional asymmetries are those
where all individuals share the same direction of asymmetry and the direction of asym-
metry usually has a genetic basis [
9
,
10
]. Finally, antisymmetry is a type of asymmetry
wherein “left-handed” and “right-handed” forms are both present within a population [
11
].
Antisymmetry is sometimes referred to as random asymmetry because the direction of
asymmetry in such traits appears to be random and is sometimes not heritable [12].
Some have suggested that traits that are non-heritable, e.g., many asymmetrical and
antisymmetrical traits, are evolutionarily unimportant [
13
,
14
]. However, such conclusions
are not universally accepted [
13
,
15
]. One of the challenges in understanding the evolution-
ary impact of antisymmetrical traits is that researchers often focus on traits that may not be
readily linked to fitness. What is needed is a study that examines antisymmetry in a trait
that is clearly and directly linked to fitness, such as those used for reproduction or rearing
offspring.
We have identified a species of livebearing freshwater fish, Xenophallus umbratilis [
16
],
that fits these criteria. This species exhibits antisymmetry in the male intromittent organ
called the gonopodium, a modified anal fin that is used to inseminate females [
17
,
18
].
Gonopodia in livebearing fish are often elaborated, featuring barbs, claws, and serrae
that presumably help males anchor more securely to the female urogenital pore during
copulation [
17
,
19
]. The gonopodium in X. umbratilis terminates with a hook-like structure
that curves to the left (sinistral) or to the right (dextral) (Figure 1).
Symmetry 2023,15, 489. https://doi.org/10.3390/sym15020489 https://www.mdpi.com/journal/symmetry
Symmetry 2023,15, 489 2 of 9
Symmetry 2023, 15, x FOR PEER REVIEW 2 of 9
Figure 1. Photographs of ventral view male specimens with sinistral (A) and dextral (B) gonopodia.
Here, we evaluate the extent of shape variation in the degree of curvature in the gon-
opodium between sinistral and dextral males in X. umbratilis using geometric morpho-
metrics. Because the degree of curvature in the gonopodium likely impacts a male’s ability
to successfully transfer sperm (too curved or not curved enough may both inhibit copula-
tion), we predict that curvature will be maintained by common selective pressures regard-
less of chirality and will therefore be identical between the two morphs.
2. Materials and Methods
2.1. Study System and Sampling
Xenophallus umbratilis is a livebearing freshwater fish native to northern Costa Rica.
The species is typically found in small streams and is most abundant at the headwaters of
river drainages at high elevations [20]. As in all other poeciliid fishes, X. umbratilis em-
ploys internal fertilization and gives birth to live young. The gonopodium in X. umbratilis
is antisymmetrical, exhibiting a sinistral (left-handed) or dextral (right-handed) hook at
the terminus. Populations of X. umbratilis are usually composed of a mixture of sinistral
and dextral individuals, though several populations that are fixed for either morph have
been observed in the wild [21].
We studied X. umbratilis from ten different localities that contained both gonopo-
dium morphs collected from tributaries and streams in Costa Rica between 2005 and 2007
(Figure 2, Table 1). These specimens came from the Brigham Young University Bean Life
Science Museum collections. Fish were collected and humanely euthanized in the field
with an overdose of the anesthesia tricane methanesulfonate (MS-222) at a concentration
of 250 mg/L [22,23]. Fish were preserved in the field in ethyl alcohol and each specimen
was assigned a museum ID number.
Populations that were fixed for either the sinistral or dextral morph were excluded
from this study. We sorted each of the ten populations to remove females and juveniles,
and then sorted the remaining mature males by gonopodium morph. Using an Olympus
Figure 1.
Photographs of ventral view male specimens with sinistral (
A
) and dextral (
B
) gonopodia.
Here, we evaluate the extent of shape variation in the degree of curvature in the
gonopodium between sinistral and dextral males in X. umbratilis using geometric mor-
phometrics. Because the degree of curvature in the gonopodium likely impacts a male’s
ability to successfully transfer sperm (too curved or not curved enough may both inhibit
copulation), we predict that curvature will be maintained by common selective pressures
regardless of chirality and will therefore be identical between the two morphs.
2. Materials and Methods
2.1. Study System and Sampling
Xenophallus umbratilis is a livebearing freshwater fish native to northern Costa Rica.
The species is typically found in small streams and is most abundant at the headwaters of
river drainages at high elevations [
20
]. As in all other poeciliid fishes, X. umbratilis employs
internal fertilization and gives birth to live young. The gonopodium in X. umbratilis is
antisymmetrical, exhibiting a sinistral (left-handed) or dextral (right-handed) hook at the
terminus. Populations of X. umbratilis are usually composed of a mixture of sinistral and
dextral individuals, though several populations that are fixed for either morph have been
observed in the wild [21].
We studied X. umbratilis from ten different localities that contained both gonopodium
morphs collected from tributaries and streams in Costa Rica between 2005 and 2007
(Figure 2,
Table 1). These specimens came from the Brigham Young University Bean
Life Science Museum collections. Fish were collected and humanely euthanized in the field
with an overdose of the anesthesia tricane methanesulfonate (MS-222) at a concentration of
250 mg/L [
22
,
23
]. Fish were preserved in the field in ethyl alcohol and each specimen was
assigned a museum ID number.
Symmetry 2023,15, 489 3 of 9
Symmetry 2023, 15, x FOR PEER REVIEW 3 of 9
DP74 camera mounted on an Olympus MVX10 microscope (Tokyo, Japan), we took pho-
tographs of males with their ventral side facing up to the camera lens. We placed males
in a black, plastic trough for photographing to make their positioning under the lens easier
to control and more consistent across images. Following photographing, we returned
males to their original museum collection jars.
Figure 2. Map of the ten localities sampled in Costa Rica. Rivers and tributaries are shown in gray
and localities are denoted by black dots. Localities were sampled between 2005 and 2007. Image
generated with ArcGIS.
Table 1. Population identification and location information.
Population Museum ID Tributary/River Coordinates
1 009294c Rio Corinto 10° 12.674′ N 83° 53.114′ W
2 009301b Rio Esquivetto 10° 41.231′ N 85° 04.002′ W
3 009302 Trib. to Rio Bijagua 10° 43.887′ N 85° 03.318′ W
4 009310 Rio Tenerio 10° 41.285′ N 85° 04.561′ W
5 009320 Trib. to Rio Bijagua 10° 43.453′ N 85° 03.982′ W
6 009325 Quebrada Hormiguero 10° 41.454′ N 85° 05.019′ W
7 009338c Quebrada La Palma 10° 33.614′ N 84° 56.442′ W
8 009339 Quebrada Hormiguero 10° 41.445′ N 85° 05.036′ W
9 009340 Quebrada Isabel 10° 38.387′ N 84° 50.757′ W
10 009354 Quebrada Azul 10° 29.955′ N 84° 59.138′ W
2.2. Geometric Morphometric Analysis
We employed landmark-based geometric morphometrics to quantify gonopodium
shape in X. umbratilis [24]. Prior to landmarking, we used Olympus cellSens software [25]
to screen images and ensure that specimens were in focus and that the gonopodium was
level in the dorso-ventral and anteroposterior axes. We re-photographed specimens to
correct any rotation or focus errors and excluded males with damaged or underdeveloped
gonopodia from our analysis (n = 3). Additionally, for our analysis, we rotated or flipped
images so that all gonopodia were oriented such that they appeared to be dextral. This
reduced observer bias by making it impossible to visually distinguish sinistral and dextral
gonopodia. In total, 246 males (135 sinistral, 111 dextral) were included in this study.
Figure 2.
Map of the ten localities sampled in Costa Rica. Rivers and tributaries are shown in gray
and localities are denoted by black dots. Localities were sampled between 2005 and 2007. Image
generated with ArcGIS.
Table 1. Population identification and location information.
Population Museum ID Tributary/River Coordinates
1 009294c Rio Corinto 10◦12.6740N
83◦53.1140W
2 009301b Rio Esquivetto 10◦41.2310N
85◦04.0020W
3 009302 Trib. to Rio Bijagua 10◦43.8870N
85◦03.3180W
4 009310 Rio Tenerio 10◦41.2850N
85◦04.5610W
5 009320 Trib. to Rio Bijagua 10◦43.4530N
85◦03.9820W
6 009325 Quebrada
Hormiguero
10◦41.4540N
85◦05.0190W
7 009338c Quebrada La Palma 10◦33.6140N
84◦56.4420W
8 009339 Quebrada
Hormiguero
10◦41.4450N
85◦05.0360W
9 009340 Quebrada Isabel 10◦38.3870N
84◦50.7570W
10 009354 Quebrada Azul 10◦29.9550N
84◦59.1380W
Populations that were fixed for either the sinistral or dextral morph were excluded
from this study. We sorted each of the ten populations to remove females and juveniles, and
then sorted the remaining mature males by gonopodium morph. Using an Olympus DP74
camera mounted on an Olympus MVX10 microscope (Tokyo, Japan), we took photographs
of males with their ventral side facing up to the camera lens. We placed males in a black,
plastic trough for photographing to make their positioning under the lens easier to control
and more consistent across images. Following photographing, we returned males to their
original museum collection jars.
Symmetry 2023,15, 489 4 of 9
2.2. Geometric Morphometric Analysis
We employed landmark-based geometric morphometrics to quantify gonopodium
shape in X. umbratilis [
24
]. Prior to landmarking, we used Olympus cellSens software [
25
]
to screen images and ensure that specimens were in focus and that the gonopodium was
level in the dorso-ventral and anteroposterior axes. We re-photographed specimens to
correct any rotation or focus errors and excluded males with damaged or underdeveloped
gonopodia from our analysis (n= 3). Additionally, for our analysis, we rotated or flipped
images so that all gonopodia were oriented such that they appeared to be dextral. This
reduced observer bias by making it impossible to visually distinguish sinistral and dextral
gonopodia. In total, 246 males (135 sinistral, 111 dextral) were included in this study.
We used the program tpsDig [
26
] to digitize landmarks on each specimen and to
measure variation in sinistral and dextral gonopodia. We used seven landmarks to outline
the shape of the gonopodium (Figure 3). Landmarks were placed by a single researcher on
each specimen and specimens were processes in random order.
Symmetry 2023, 15, x FOR PEER REVIEW 4 of 9
We used the program tpsDig [26] to digitize landmarks on each specimen and to
measure variation in sinistral and dextral gonopodia. We used seven landmarks to outline
the shape of the gonopodium (Figure 3). Landmarks were placed by a single researcher
on each specimen and specimens were processes in random order.
Figure 3. Landmark placement used to analyze gonopodium shape in X. umbratilis. Landmarks were
placed as follows: (1) origin of the gonopodium; (2) terminus of the gonopodium; (3) point of cur-
vature at the tip of the shaft; (4) midpoint between landmarks one and two; (5) midpoint between
landmarks two and three on the terminus; (6) midpoint between landmarks three and five; and (7)
midpoint between landmarks five and two. Landmarks one and two are homologous; landmark
three is a pseudo-landmark; and landmarks four, five, six, and seven are sliding landmarks.
We generated shape variables from our landmark data in tpsRelw [26]. After produc-
ing shape variables using a general Procrustes analysis [27], tpsRelw runs a principal com-
ponents analysis and calculates relative warps and centroid size. For this analysis,
tpsRelw calculated ten relative warps. We used the first six of these relative warps, which
accounted for 99.89% of total shape variation, for additional analysis, and excluded four
relative warps that accounted for less than 0.5% of variation. Excluding these relative
warps allowed us to avoid inflating the degrees of freedom in our shape analysis [28,29].
2.3. Statistical Analysis
We used a multivariate linear mixed model to evaluate the effects of morph (sinistral
or dextral), centroid size (a measure of size commonly used in geometric morphometrics)
[30], and locality on shape variation [28,29]. In each analysis, we used relative warps
(shape variables) as our response variable. Because relative warps come from a matrix of
shape variables, we had to convert the shape variable matrix into columns of vectors to be
used in our multivariate linear mixed model. This conversion subsequently required the
creation of an index variable that retained individual information for each of the relative
warps in our analyses. We included the index variable in our analysis as a predictor vari-
able and it is necessary to meaningfully measure the differences in shape variation be-
tween groups. Hence, it is the two-way interactions between morph and the index varia-
ble, centroid size and the index variable, locality and the index variable, and the three-
Figure 3.
Landmark placement used to analyze gonopodium shape in X. umbratilis. Landmarks
were placed as follows: (1) origin of the gonopodium; (2) terminus of the gonopodium; (3) point of
curvature at the tip of the shaft; (4) midpoint between landmarks one and two; (5) midpoint between
landmarks two and three on the terminus; (6) midpoint between landmarks three and five; and
(7) midpoint between landmarks five and two. Landmarks one and two are homologous; landmark
three is a pseudo-landmark; and landmarks four, five, six, and seven are sliding landmarks.
We generated shape variables from our landmark data in tpsRelw [
26
]. After pro-
ducing shape variables using a general Procrustes analysis [
27
], tpsRelw runs a principal
components analysis and calculates relative warps and centroid size. For this analysis,
tpsRelw calculated ten relative warps. We used the first six of these relative warps, which
accounted for 99.89% of total shape variation, for additional analysis, and excluded four
relative warps that accounted for less than 0.5% of variation. Excluding these relative warps
allowed us to avoid inflating the degrees of freedom in our shape analysis [28,29].
2.3. Statistical Analysis
We used a multivariate linear mixed model to evaluate the effects of morph (sinistral or
dextral), centroid size (a measure of size commonly used in geometric morphometrics) [
30
],
and locality on shape variation [
28
,
29
]. In each analysis, we used relative warps (shape
Symmetry 2023,15, 489 5 of 9
variables) as our response variable. Because relative warps come from a matrix of shape
variables, we had to convert the shape variable matrix into columns of vectors to be used in
our multivariate linear mixed model. This conversion subsequently required the creation
of an index variable that retained individual information for each of the relative warps in
our analyses. We included the index variable in our analysis as a predictor variable and it
is necessary to meaningfully measure the differences in shape variation between groups.
Hence, it is the two-way interactions between morph and the index variable, centroid size
and the index variable, locality and the index variable, and the three-way interactions
between morph, centroid size and the index variable, and locality, centroid size and the
index variable, that allowed us to comprehensively determine what factors significantly
influence gonopodium shape variation [31].
To test our hypothesis, we ran three models. We first needed to determine if centroid
size and locality were significant predictors of shape variation to inform how we constructed
subsequent models that tested for the effect of gonopodial morph on shape. Our first model
tested for the impact of centroid size on shape. The second model included centroid size
and locality. Our third model tested for the effects of morph and centroid size and included
locality as a random effect. Across all three models, relative warps (shape variables) were
used as the response variable (see Table 2for details on each model’s components). We used
the Akaike Information Criterion (AIC) to determine which of the three models provided
the best fit [32].
Table 2. Variables used in each model of the multivariate linear model analysis.
Response Variable Random Effect Predictor Variable
Model 1 Relative Warps –
Centroid Size
Index
Centroid Size ×Index
Model 2 Relative Warps –
Locality
Centroid Size
Centroid Size ×Locality
Index
Locality ×Index
Centroid Size ×Index
Locality ×Centroid Size ×Index
Model 3 Relative Warps Locality
Morph
Centroid Size
Centroid Size ×Morph
Index
Morph ×Index
Centroid Size ×Index
Morph ×Centroid Size ×Index
We estimated the degrees of freedom in our analyses using the Kenward-Roger
method [
33
] and ran our multivariate linear mixed models in SAS software, using the
Proc MIXED protocol (SAS version 9.4, SAS Institute Inc., Cary, NC, USA).
3. Results
Neither morph nor locality were significant predictors of gonopodium shape. That
is, we could not reject the hypothesis that the shape of dextral and sinistral gonopodial
morphs are the same (Table 3). Gonopodium shape in X. umbratilis did differ significantly
by centroid size in both models 1 and 3 (see the two-way interaction between centroid size
and the index variable from models 1 and 3 in Table 2). Of the three models, model 2 had
the lowest AIC score (
−
10,768.2) and best fit the data. The terminus of the gonopodium
becomes slightly more open moving from the smallest centroid size to the largest (Figure 4).
Although a significant predictor, the extent of shape variation that centroid size explains
appears to be relatively limited compared to the overall variation in shape captured across
Symmetry 2023,15, 489 6 of 9
Relative Warps 1 and 2 (Figure 5). In other words, the primary difference in gonopodium
as a function of centroid size was a slight change at the tip of the gonopodium (Figure 4).
Table 3.
Results from the multivariate linear mixed model analysis. We ran three different models
with different combinations of predictor variables. Predictor variable terms that include an interaction
with the index variable are those that evaluate if/how gonopodium shape changes.
Predictor Variable Degrees of Freedom F-Value p-Value
Model 1
Centroid Size 1, 702 6.44 0.0113
Index 5, 657 4.9 0.0002
Centroid Size ×Index 5, 657 4.82 0.0002
Model 2
Locality 9, 794 1.02 0.4226
Centroid Size 1, 794 1.25 0.2365
Centroid Size ×Locality 9, 794 1.04 0.4092
Index 5, 659 2.04 0.0718
Locality ×Index 45, 1183 0.98 0.5024
Centroid Size ×Index 5, 659 2.07 0.0671
Locality ×Centroid Size ×
Index 45, 1183 0.98 0.5188
Model 3
Morph 1, 704 0.01 0.9238
Centroid Size 1, 619 5.09 0.0244
Centroid Size ×Morph 1, 703 0.04 0.8391
Index 5, 653 3.26 0.0065
Morph ×Index 5, 653 1.39 0.2273
Centroid Size ×Index 5, 653 3.07 0.0095
Morph ×Centroid Size ×
Index 5, 653 1.23 0.0095
Symmetry 2023, 15, x FOR PEER REVIEW 6 of 9
Table 3. Results from the multivariate linear mixed model analysis. We ran three different models
with different combinations of predictor variables. Predictor variable terms that include an interac-
tion with the index variable are those that evaluate if/how gonopodium shape changes.
Predictor Variable Degrees of Freedom F-Value p-Value
Model 1
Centroid Size 1, 702 6.44 0.0113
Index 5, 657 4.9 0.0002
Centroid Size × Index 5, 657 4.82 0.0002
Model 2
Locality 9, 794 1.02 0.4226
Centroid Size 1, 794 1.25 0.2365
Centroid Size × Locality 9, 794 1.04 0.4092
Index 5, 659 2.04 0.0718
Locality × Index 45, 1183 0.98 0.5024
Centroid Size × Index 5, 659 2.07 0.0671
Locality × Centroid Size ×
Index 45, 1183 0.98 0.5188
Model 3
Morph 1, 704 0.01 0.9238
Centroid Size 1, 619 5.09 0.0244
Centroid Size × Morph 1, 703 0.04 0.8391
Index 5, 653 3.26 0.0065
Morph × Index 5, 653 1.39 0.2273
Centroid Size × Index 5, 653 3.07 0.0095
Morph × Centroid Size ×
Index 5, 653 1.23 0.0095
Figure 4. Variation in gonopodium shape explained by centroid size. Shape deformation is repre-
sented by thin plate splines. The top thin plate spline visualizes shape at the largest centroid in this
dataset and the bottom thin plate spline visualizes the shape at the smallest centroid size.
Figure 4.
Variation in gonopodium shape explained by centroid size. Shape deformation is repre-
sented by thin plate splines. The top thin plate spline visualizes shape at the largest centroid in this
dataset and the bottom thin plate spline visualizes the shape at the smallest centroid size.
Symmetry 2023,15, 489 7 of 9
Symmetry 2023, 15, x FOR PEER REVIEW 7 of 9
Figure 5. Visualization of gonopodium shape variation along Relative Warp 1 (RW1) and Relative
Warp 2 (RW2). Thin plate splines along the x-axis represent shape deformation across Relative Warp
1. Moving from left to right, these splines show the terminus of the gonopodium becoming more
tightly curved. Thin plate splines along the y-axis represent shape deformation across Relative Warp
2. These splines show some bending in the midpoint of the gonopodium shaft and some changes in
the curvature of the gonopodium tip.
4. Discussion
This study provides insight on variation in antisymmetrical traits. We predicted that
the shape of gonopodial curvature would not differ between sinistral and dextral individ-
uals in X. umbratilis and our results support this prediction. We also found that centroid
size is a significant predictor of gonopodium shape. Males of many poeciliid species ex-
hibit determinate growth, so this finding suggests that the size at which males mature
may affect gonopodium shape [34,35]. However, it seems unlikely that centroid size im-
pacts gonopodium shape in a biologically meaningful way, as the variation due to cen-
troid size is minimal when considered with the overall shape variation in the gonopodium
we observed.
Asymmetry is typically considered in a morphological context, though asymmetry
can also be demonstrated behaviorally [36]. Previous research in X. umbratilis from John-
son et al. [21] found that gonopodial morphology reliably predicted detour behaviors and
eye-bias for potential mates and predators. In that study, dextral (right morph) males de-
toured to the right to view potential mate and predator stimuli. Sinistral (left morph)
males’ behaviors were completely opposite, with males detouring to the left for the same
set of stimuli. Our work aligns with these observed patterns. We found that sinistral and
dextral gonopodial morphs were essentially mirror images of each other, which is con-
sistent with a specific morph type predicting detour behavior in this species. Another
study found that individuals from a fixed sinistral population of X. umbratilis preferen-
tially positioned themselves wherein males were primarily on the left side of a female
during mating interactions [37]. However, individuals from fixed dextral populations did
not display side-biased positioning behavior. Our results provide some context for the
strong side-bias in the sinistral population and the lack of side-bias in the dextral popula-
tion by ruling out the possibility that unequal degrees of curvature in the gonopodium
influenced mating positioning behaviors.
Figure 5.
Visualization of gonopodium shape variation along Relative Warp 1 (RW1) and Relative
Warp 2 (RW2). Thin plate splines along the x-axis represent shape deformation across Relative Warp
1. Moving from left to right, these splines show the terminus of the gonopodium becoming more
tightly curved. Thin plate splines along the y-axis represent shape deformation across Relative Warp
2. These splines show some bending in the midpoint of the gonopodium shaft and some changes in
the curvature of the gonopodium tip.
4. Discussion
This study provides insight on variation in antisymmetrical traits. We predicted that
the shape of gonopodial curvature would not differ between sinistral and dextral individu-
als in X. umbratilis and our results support this prediction. We also found that centroid size
is a significant predictor of gonopodium shape. Males of many poeciliid species exhibit
determinate growth, so this finding suggests that the size at which males mature may
affect gonopodium shape [
34
,
35
]. However, it seems unlikely that centroid size impacts
gonopodium shape in a biologically meaningful way, as the variation due to centroid
size is minimal when considered with the overall shape variation in the gonopodium we
observed.
Asymmetry is typically considered in a morphological context, though asymmetry
can also be demonstrated behaviorally [
36
]. Previous research in X. umbratilis from Johnson
et al. [
21
] found that gonopodial morphology reliably predicted detour behaviors and
eye-bias for potential mates and predators. In that study, dextral (right morph) males
detoured to the right to view potential mate and predator stimuli. Sinistral (left morph)
males’ behaviors were completely opposite, with males detouring to the left for the same
set of stimuli. Our work aligns with these observed patterns. We found that sinistral
and dextral gonopodial morphs were essentially mirror images of each other, which is
consistent with a specific morph type predicting detour behavior in this species. Another
study found that individuals from a fixed sinistral population of X. umbratilis preferentially
positioned themselves wherein males were primarily on the left side of a female during
mating interactions [
37
]. However, individuals from fixed dextral populations did not
display side-biased positioning behavior. Our results provide some context for the strong
side-bias in the sinistral population and the lack of side-bias in the dextral population by
ruling out the possibility that unequal degrees of curvature in the gonopodium influenced
mating positioning behaviors.
A critical component of studying morphology and its evolutionary implications is
tying morphology to function and fitness. Though the gonopodium certainly is important to
Symmetry 2023,15, 489 8 of 9
fitness in X. umbratilis, very little is understood about the actual mechanisms that facilitate
insemination in this species and livebearing fish in general. Given that dextral and sinistral
gonopodia are essentially mirror images of each other, we might expect that functional
behavior associated with fertilization in this species will also reflect antisymmetry in the
gonopodium. Future work should focus on understanding the mechanisms involved in
copulation and how this compares between gonopodial morphs.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/sym15020489/s1.
Author Contributions:
Conceptualization, M.-E.N., J.B.J.; investigation, M.-E.N.; data curation, M.-
E.N.; formal analysis, M.-E.N.; visualization, M.-E.N.; writing—original draft preparation, M.-E.N.;
writing—review and editing, M.-E.N. and J.B.J. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement:
The data presented in this study are available in a supplementary
material file.
Acknowledgments:
We thank Riley Nelson and Tanner Van Orden for providing microscope and
imaging software, and for assistance photographing specimens, respectively. We also thank Mark
Belk for his help with image screening and data analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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