Research Article |
Corresponding author: Max Mühlenhaupt ( max.muehlenhaupt@sunrisepc.de ) Academic editor: Sandro Bertolino
© 2021 Max Mühlenhaupt, James Baxter-Gilbert, Buyisile G. Makhubo, Julia L. Riley, John Measey.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Mühlenhaupt M, Baxter-Gilbert J, Makhubo BG, Riley JL, Measey J (2021) Growing up in a new world: trait divergence between rural, urban, and invasive populations of an amphibian urban invader. NeoBiota 69: 103-132. https://doi.org/10.3897/neobiota.69.67995
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Cities are focal points of introduction for invasive species. Urban evolution might facilitate the success of invasive species in recipient urban habitats. Here we test this hypothesis by rearing tadpoles of a successful amphibian urban coloniser and invader in a common garden environment. We compared growth rate, morphological traits, swimming performance, and developmental rate of guttural toad tadpoles (Sclerophrys gutturalis) from native rural, native urban, and non-native urban habitats. By measuring these traits across ontogeny, we were also able to compare divergence across different origins as the tadpoles develop. The tadpoles of non-native urban origin showed significantly slower developmental rate (e.g., the proportion of tadpoles reaching Gosner stage 31 or higher was lower at age 40 days) than tadpoles of native urban origin. Yet, tadpoles did not differ in growth rate or any morphological or performance trait examined, and none of these traits showed divergent ontogenetic changes between tadpoles of different origin. These findings suggest that prior adaptation to urban habitats in larval traits likely does not play an important role in facilitating the invasion success of guttural toads into other urban habitats. Instead, we suggest that evolutionary changes in larval traits after colonization (e.g., developmental rate), together with decoupling of other traits and phenotypic plasticity might explain how this species succeeded in colonising extra-limital urban habitats.
AIAI hypothesis, development, growth rate, invasion biology, morphology, performance, tadpole, urban evolution
Invasive species pose a major threat to global biodiversity, human wellbeing, and the economy (
Recently, evolutionary biologists have begun studying the adaptive divergence of traits in urban populations compared to populations from rural habitats (
To date, few studies have investigated whether prior adaptation to urban habitats facilitates invasion success in introduced habitats (
Amphibians provide an excellent model system for examining the relationship between urban adaptations and invasions. Currently, there are more than 120 amphibian species with recognised invasive populations globally (
Here, we will examine the trait divergence in tadpoles of the guttural toad (Sclerophrys gutturalis) of three different origins in South Africa: native rural (Durban Rural), native urban (Durban Urban), and non-native urban (Cape Town, an invasive population that originated from Durban;
The guttural toad is a large bufonid (maximum snout-vent length (SVL)) of 140mm;
Overview of the study system A a guttural toad (Sclerophrys gutturalis): this female was photographed in Cape Town B the species’ natural and non-native distribution in South Africa. The approximate locations of sampling sites are demarcated for Durban Rural (green), Durban Urban (yellow), and Cape Town (red). Further we show the general appearance of C the Durban Rural D durban Urban, and E Cape Town sampling sites.
The species has successfully established invasive populations in Mauritius, Réunion, and near Cape Town (Constantia, South Africa) (
Breeding-sized adults (Suppl. material
Shortly after collection, toads were transported to an experimental facility located at the University of KwaZulu-Natal (Westville Campus) situated at one of our sampling locations for Durban Urban. In the facility, toads were housed by sex and collection site in large plastic mesocosms (110 cm L × 130 cm W × 50 cm H) until they were used for breeding. Each mesocosm contained at least two water bowls (~ 15 cm L × 10 cm W × 5 cm H) on a 10 cm layer of soil mixed with leaf litter collected outside of the greenhouse. Crickets (Acheta domesticus) were fed to adults ad libitum every other day.
To initiate breeding, we injected adults with a synthetic gonadotrophin, leuroprorelin acetate (Lucrin Depot, Abbott), diluted 1:20 with Ringer’s solution using 0.666 ml of that dilution for females and 0.333 ml for males (
Prior to the experiment, large plastic mesocosms (110 cm L × 130 cm W × 50 cm H; n = 33) located in the experimental facility were filled with 600 L of tap water and left to age for a week. Subsequently, the water was inoculated with water from a standing water tank to induce establishment of phyto- and zooplankton communities within the mesocosms. This water tank was located in the greenhouse (i.e., preventing access from toads and fish) and had live aquatic vegetation and algae growing within it. After another week, 50 g of rabbit chow (Rabbit Pellets, Westerman’s Premium; 9% protein, 1.25% fat, 0.75% calcium by weight) was added for additional nutrients (
At clutch age of three days, 20 tadpoles from one clutch were randomly selected for measurements (see below) and were returned to their respective mesocosms afterwards. At the age of ten days, and subsequently every ten days (i.e., age 20, 30, 40, 50 and 60 days), 20 tadpoles were randomly selected from each mesocosm, and measurement procedures were repeated (for sample sizes see Table
Sample sizes for guttural toad (Sclerophrys gutturalis) tadpoles across age: numbers below the specific traits correspond to the total number of tadpoles measured at the specific age. The total number of clutches used in this experiment was 10 (3/7), 14 (8/6), and 9 for Durban Rural, Durban Urban and Cape Town, respectively. The numbers in brackets correspond with the specific number of clutches derived from adults collected in either the first or seco nd sampling location for Durban Rural or Durban Urban, respectively (see Methods). For a more detailed report of the sample sizes in this experiment see Suppl. material
Traits Age (Days) |
Durban Rural | Durban Urban | Cape Town | ||||||
---|---|---|---|---|---|---|---|---|---|
SVL, body width, tail length | Body height, tail fin height, tail muscle height | Maximum velocity, maximum acceleration | SVL, body width, tail length | Body height, tail fin height, tail muscle height | Maximum velocity, maximum acceleration | SVL, body width, tail length | Body height, tail fin height, tail muscle height | Maximum velocity, maximum acceleration | |
2–4 | 199 | 199 | 199 | 272 | 271 | 272 | 180 | 180 | 179 |
10–12 | 200 | 199 | 200 | 240 | 235 | 240 | 160 | 160 | 160 |
20 | 200 | 200 | 200 | 180 | 179 | 180 | 160 | 160 | 160 |
30 | 200 | 199 | 200 | 180 | 180 | 180 | 80 | 80 | 80 |
40–41 | 160 | 160 | 160 | 180 | 180 | 180 | 80 | 80 | 80 |
50 | 60 | 60 | 60 | 180 | 180 | 180 | 60 | 60 | 60 |
60 | 60 | 60 | 60 | 100 | 100 | 100 | 40 | 40 | 40 |
After the measurements (see below), tadpoles were returned to their respective mesocosms. We acknowledge that our replicates cannot be assumed to be fully independent (e.g., a tadpole that was randomly picked for measurements at an age of three days might have been picked for another subsequent measurement at a higher age). However, since each mesocosm housed 1000 tadpoles at the start of the experiment, it is unlikely that a single tadpole was recurrently picked for measurements. As such our statistical analyses use the assumption that repeated measurements of an individual tadpole did not occur.
We measured standard morphological variables of tadpoles: SVL, tail length, body width, body height, tail muscle height, and tail fin height to 0.001 mm (
All tadpoles that underwent morphological measurements were also tested for swimming performance on the same day. Performance trials were carried out in a clear plexiglass tank (30 × 5 cm) filled with 3 cm of aged tap water (
High-speed videos were recorded from a dorsal and lateral perspective to the tadpole, using an angled mirror attached to the tank. As a swimming bout we defined the movement of a tadpole, initiated by rapid lashes of the tail fin in response to an approach or a touch by the metal wire, from the beginning to the end of the displacement. Only swimming bouts that were carried out on a horizontal plane with a displacement at least 2 cm away from its initial position were selected. For each tadpole, we analysed three swimming bouts that were judged to yield the highest values for velocity and acceleration. Using the image and video analysis software Tracker (
All statistical analyses were conducted using R version 4.0.1 (
To examine differences in growth rate and body volume, we fitted linear mixed effect models (LMM) using the R package “lme4” (
We also examined trait differences at a specific point in development. We chose age 40/41 days because previous literature has shown that at this age individual traits can diverge between populations (e.g., in European common frogs, Rana temporaria;
To examine differences in developmental rate between tadpoles from different origin populations, we fitted a generalised linear mixed effects model (GLMM) with logit link function using the R package “lme4” (
None of the observed morphological or performance traits showed significant differences between tadpoles of different origin (Table
Outcomes of linear mixed effect models testing for differences in morphological and performance traits between guttural toad tadpoles of native rural origin (Durban Rural), native urban origin (Durban Urban), and invasive urban origin (Cape Town) raised in a common garden environment. All variables were log-transformed prior to analysis. Presented are model coefficient estimates (β) with their corresponding standard errors (SE) for fixed effects and variance estimates (σ²) for random effects and residuals. Test statistics (t) are given, and all significant values (p < .05) are presented in bold. For categorical variables, reference levels are presented in brackets behind the variable name.
Model | Variable Names | Model Output | |||
---|---|---|---|---|---|
Snout-Vent Length | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 0.412 | 0.042 | 9.740 | .011 | |
Origin (Durban Rural) | 0.064 | 0.052 | 1.239 | .341 | |
Origin (Durban Urban) | 0.025 | 0.052 | 0.492 | .672 | |
Age | 0.006 | < 0.001 | 11.970 | < .001 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | 0.002 | ||||
Residuals | 0.008 | ||||
Body Volume | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 1.208 | 0.151 | 7.983 | .016 | |
Origin (Durban Rural) | 0.220 | 0.186 | 1.187 | .358 | |
Origin (Durban Urban) | 0.114 | 0.185 | 0.614 | .602 | |
Age | 0.020 | 0.002 | 12.606 | < .001 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | 0.026 | ||||
Residuals | 0.085 | ||||
Tail Muscle Height | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | -0.544 | 0.035 | -5.690 | .003 | |
Snout-Vent Length | 0.542 | 0.017 | 31.681 | < .001 | |
Origin (Durban Rural) | -0.013 | 0.042 | -0.306 | .789 | |
Origin (Durban Urban) | 0.002 | 0.042 | 0.041 | .971 | |
Age | -0.003 | < 0.001 | -5.578 | < .001 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | 0.001 | ||||
Residuals | 0.008 | ||||
Tail Fin Height | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | -0.009 | 0.064 | -0.139 | .902 | |
Snout-Vent Length | 0.393 | 0.012 | 31.917 | < .001 | |
Origin (Durban Rural) | -0.049 | 0.078 | -0.577 | .623 | |
Origin (Durban Urban) | -0.040 | 0.078 | -0.517 | .657 | |
Age | -0.004 | 0.009 | -0.434 | .670 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | 0.004 | ||||
Residuals | 0.004 | ||||
Tail Length | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 0.265 | 0.009 | 29.937 | < .001 | |
Snout-Vent Length | 0.729 | 0.011 | 68.726 | < .001 | |
Origin (Durban Rural) | 0.009 | 0.010 | 0.992 | .437 | |
Origin (Durban Urban) | 0.014 | 0.009 | 1.522 | .290 | |
Age | 0.001 | < 0.001 | 6.995 | < .001 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | < 0.001 | ||||
Residuals | 0.003 | ||||
Maximum Swimming Velocity | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 1.529 | 0.090 | 17.066 | .003 | |
Snout-Vent Length | 0.667 | 0.026 | 25.656 | < .001 | |
Origin (Durban Rural) | 0.030 | 0.109 | 0.277 | .808 | |
Origin (Durban Urban) | 0.046 | 0.109 | 0.423 | .714 | |
Age | -0.004 | 0.001 | -3.348 | .004 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | 0.008 | ||||
Residuals | 0.018 | ||||
Maximum Swimming Acceleration | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 2.819 | 0.042 | 66.838 | < .001 | |
Snout-Vent Length | 0.592 | 0.032 | 18.303 | < .001 | |
Origin (Durban Rural) | 0.056 | 0.049 | 1.142 | .374 | |
Origin (Durban Urban) | 0.051 | 0.049 | 1.036 | .414 | |
Age | -0.001 | < 0.001 | -2.505 | .017 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | 0.002 | ||||
Residuals | 0.028 |
Trait changes across 60 days post-hatching in tadpoles: none of the observed traits were significantly different between origins (native rural – Durban Rural, native urban – Durban Urban, non-native urban – Cape Town). Presented are A snout-vent length (growth rate) B body volume C tail length D tail muscle height E tail fin height F maximum swimming velocity, and G maximum swimming acceleration. All morphological and performance variables were log-transformed prior to analysis and predicted data was back-transformed before plotting. Circles represent predictions from linear mixed effect models and the lines represent predicted linear regressions with 95% confidence intervals.
We did not find significant effects of tadpole origin on any morphological or performance trait at the age of 40/41 days (Table
Model output of linear mixed effect models examining differences in guttural toad tadpoles (Sclerophrys gutturalis) at the age of 40/41 days between guttural toad tadpoles of native rural origin (Durban Rural), native urban origin (Durban Urban), and invasive urban origin (Cape Town) raised in a common garden environment. All morphological and performance variables were log-transformed prior to analysis. Given are model coefficient estimates (β) with their corresponding standard errors (SE) for fixed effects and variance estimates (σ²) for random effects and residuals. Test statistics (t) are presented, and all significant values (p < .05) are presented in bold. For categorical variables, reference levels are presented in brackets behind the variable name.
Model | Variable Names | Model Output | |||
---|---|---|---|---|---|
Snout-Vent Length | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 0.659 | 0.026 | 25.291 | < .001 | |
Origin (Durban Rural) | 0.025 | 0.032 | 0.796 | .437 | |
Origin (Durban Urban) | 0.043 | 0.031 | 1.362 | .190 | |
Random Effects | σ² | ||||
Mesocosm ID | 0.002 | ||||
Parentage Site | 0.000 | ||||
Residuals | 0.006 | ||||
Body Volume | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 1.993 | 0.075 | 26.487 | < .001 | |
Origin (Durban Rural) | 0.070 | 0.092 | 0.757 | .459 | |
Origin (Durban Urban) | 0.141 | 0.090 | 1.564 | .135 | |
Random Effects | σ² | ||||
Mesocosm ID | 0.020 | ||||
Parentage Site | 0.000 | ||||
Residuals | 0.051 | ||||
Tail Muscle Height | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | -0.684 | 0.080 | -8.572 | .004 | |
Snout-Vent Length | 0.659 | 0.050 | 13.212 | < .001 | |
Origin (Durban Rural) | -0.010 | 0.089 | -0.113 | .921 | |
Origin (Durban Urban) | 0.029 | 0.089 | 0.327 | .775 | |
Mesocosm ID | 0.001 | ||||
Parentage Site | 0.005 | ||||
Residuals | 0.007 | ||||
Tail Fin Height | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | -0.038 | 0.025 | -1.525 | .147 | |
Snout-Vent Length | 0.456 | 0.031 | 14.914 | < .001 | |
Origin (Durban Rural) | -0.024 | 0.018 | -1.363 | .315 | |
Origin (Durban Urban) | -0.018 | 0.018 | -1.032 | .424 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | < 0.001 | ||||
Residuals | 0.002 | ||||
Tail Length | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 0.312 | 0.023 | 13.772 | < .001 | |
Snout-Vent Length | 0.734 | 0.031 | 23.409 | < .001 | |
Origin (Durban Rural) | 0.014 | 0.011 | 1.220 | .238 | |
Origin (Durban Urban) | 0.017 | 0.011 | 1.534 | .142 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | 0.000 | ||||
Residuals | 0.003 | ||||
Maximum Swimming Velocity | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 1.329 | 0.089 | 14.999 | < .001 | |
Snout-Vent Length | 0.754 | 0.075 | 10.012 | < .001 | |
Origin (Durban Rural) | -0.007 | 0.090 | -0.083 | .942 | |
Origin (Durban Urban) | 0.078 | 0.090 | 0.865 | .479 | |
Random Effects | σ² | ||||
Mesocosm ID | < 0.001 | ||||
Parentage Site | 0.005 | ||||
Residuals | 0.018 | ||||
Maximum Swimming Acceleration | Fixed Effects | β | SE | t | p |
Intercept (Cape Town) | 2.675 | 0.110 | 24.272 | < .001 | |
Snout-Vent Length | 0.737 | 0.099 | 7.421 | < .001 | |
Origin (Durban Rural) | 0.008 | 0.109 | 0.077 | .945 | |
Origin (Durban Urban) | 0.112 | 0.109 | 1.028 | .413 | |
Random Effects | σ² | ||||
Mesocosm ID | 0.001 | ||||
Parentage Site | 0.007 | ||||
Residuals | 0.029 |
The proportion of tadpoles having developed to or past Gosner stage 31 (
(A) Model output of a generalised linear mixed effects model used to examine differences in guttural toad (Sclerophrys gutturalis) tadpole developmental rates across origins. We present model coefficient estimates (β) and the corresponding standard errors (SE) for fixed effects, as well as variance estimates (σ²) for random effects. Test statistics (z) are presented and all significant values (p < .05) are presented in bold. For the categorical variables, reference levels are presented in brackets behind the variable name. (B) Results of post-hoc multiple comparisons testing for differences in developmental rate among guttural toad (Sclerophrys gutturalis) tadpole origins. Presented are conditional odds ratios with their corresponding standard errors (SE). Test statistics (z) and p-values (pcorr) corrected using a “tukey” adjustment (
Output from the generalised linear mixed effect model | ||||
---|---|---|---|---|
Variable Names | ||||
Fixed Effects | β | SE | z | p |
Intercept (Cape Town) | -3.08 | 0.599 | -5.142 | < .001 |
Origin (Durban Rural) | 1.37 | 0.663 | 2.066 | .039 |
Origin (Durban Urban) | 1.534 | 0.654 | 2.347 | .019 |
Random Effects | σ² | |||
Mesocosm ID | 0.324 | |||
Parentage Site | < 0.001 | |||
Multiple Comparisons between Origins | ||||
Origin Comparison | Conditional Odds Ratio | SE | z | pcorr |
Cape Town – Durban Rural | 0.254 | 0.169 | -2.066 | 0.097 |
Cape Town – Durban Urban | 0.216 | 0.141 | -2.347 | 0.049 |
Durban Rural – Durban Urban | 0.849 | 0.344 | -0.404 | 0.914 |
Probability of reaching Gosner stage 31 or higher (
Here we show, in a common garden experiment, that the invasive urban Cape Town population of the guttural toad has a slower larval development (i.e., the proportion of tadpoles having developed to or past Gosner stage 31 at the age of 40/41 days) compared to the native urban population from Durban. Contrary to our predictions, tadpoles of urban/rural or native/invasive origin do not differ in growth rate, or any of the other morphological or performance traits we examined. From this, we suggest that prior adaptation to urban habitats (AIAI hypothesis sensu
One of the most prominent differences between the areas of Durban and Cape Town is the Mediterranean climate in Cape Town compared to subtropical Durban. The invasive toad population in Cape Town is confronted with a colder, drier, and more seasonal climate compared to the climate of the source habitat (
Given the substantial literature reporting differences in tadpole growth rates due to ecological or evolutionary factors, we were surprised that we did not find any differences in growth rate among the three location types. Several studies on body size differences among rural and urban, as well as among native and invasive populations, across a wide range of taxa report higher growth rates and larger adult body sizes for urban and/or invasive populations (
Urban bodies of water have been reported to frequently show high levels of modification (especially of the riparian zone) and, partly as a consequence, show reduced native biodiversity and high abundance of invasive species (reviewed in
In this common garden experiment, we raised the F1 progeny from toads collected in the wild. Thus, we cannot rule out maternal/paternal effects on differences or similarities between tadpoles (e.g.,
We show here that prior adaptation in larval growth rate as well as morphological and performance traits is unlikely to have facilitated the invasion success of guttural toads in Cape Town. Furthermore, the reduction in developmental rate likely arose after the introduction to Cape Town. Thus, bridgehead effects and decoupled evolution of traits are more likely to drive successful colonisation of new habitats in this species. Our findings suggest several promising avenues of future research. For example, we suggest investigations examine divergent selection for aquatic and terrestrial life stages in amphibian invaders, and how this might lead to coupling or decoupling of traits across life stages. We also know little about how certain habitat characteristics in urban environments, such as altered species composition or anthropogenic structures, might facilitate colonisation of invasive populations or how possible plastic traits can enhance invasion success across different habitat types - which are both important knowledge gaps to address.
We would like to thank NCC Environmental Services (Western Cape) for providing us with toads from Cape Town, and the managements of Durban Botanic Gardens, and Shongweni Dam and Nature Reserve for providing us with access to their facilities for toad collection. MM, JM & JB-G would like to acknowledge the DSI-NRF Centre of Excellence for Invasion Biology. We would also like to thank N.S. Dludla for invaluable assistance during the experiment and P. Kowalski for invaluable assistance during fieldwork. Furthermore, J. Jeschke provided helpful comments on the first draft of this manuscript and we would like to thank the lab members of the Measey lab at Stellenbosch University for productive discussions that helped shape this manuscript. We would also like to show our gratitude towards S. Bertolino, F.G. Ficetola, and an anonymous reviewer, who helped improve the quality of this manuscript. We acknowledge support by the OpenAccess Publication Fund of Freie Universität Berlin for paying the APC.
As the authors, we confirm that we have no conflict of interest, regarding the content of this study or its outcomes.
Supporting information
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