Diet composition, body condition and sexual size dimorphism of the common African toad (Amietophrynus regularis) in urban and agricultural landscape

Land use and land cover change (LULCC) are major drivers of global biodiversity loss. The conversion of natural habitats into human-modified landscapes poses novel and multifaceted environmental stressors to organisms, influencing their ecology, physiology, life history and fitness. Although the effects of LULCC have been studied extensively at the community level, there is scant information about its effect on population and individual characteristics. We assessed the diet composition, body condition, and sexual size dimorphism of the common African toad (Amietophrynus regularis) in urban and agricultural landscape. Diet composition was evaluated using gut content analysis, while body condition was measured using residual mass index. Overall, 935 prey items comprising six classes, at least 18 orders and 31 families were obtained from toads. This broad dietary niche suggested that Amietophrynus regularis is a generalist predator. The family Formicidae was the most consumed prey item, with a frequency of occurrence above 80% at both sites. We found no sex- or habitat-biased dietary partitioning in the toads. A statistically significant positive correlation existed between snout-vent-length (SVL) and diversity of prey items (Pearson’s correlation r = 0.999, p ≤ 0.0001) for toads from farmland, which also had better body conditions. The toads showed female-biased sexual size dimorphism, but males had longer tibio-fibula, radio-ulna, foot, and distal fore limbs. This study is probably the first to assess the diet composition, body condition and sexual size dimorphism of Amietophrynus regularis simultaneously. The ecological, evolutionary and conservation implications of our findings are discussed.


Introduction
Agricultural intensification and urbanization are occurring at an alarming rate globally, leading to land cover changes and simplification of natural environments (Mullu, 2016;Carraso et al., 2017). Land use and land cover changes are recognized as major drivers of global biodiversity loss (Concepcion et al., 2015;Lindenmayer, 2019;Liu et al., 2018). The conversion of forests and other natural vegetation into human settlements and agricultural landscapes poses multifaceted challenges and novel environmental stressors that threaten the survival of local species (Wilson et al., 2016;Rakotoniana et al., 2016;Salomao et al., 2018). Organisms inhabiting small habitat fragments may experience high levels of environmental stressors, such as climate change, increased competition, predation, parasitism, and decreased food availability (Mikolas, 2016). This may cause poor body condition, reduced fecundity, genetic diversity, reproductive success and fitness of organisms (Janin et al., 2011), which ultimately reduce the diversity and abundance of fauna and flora species and cause alterations in community composition and biotic interactions (Newbold et al., 2015;Segan et al., 2016;Fletcher, 2018;Liu et al., 2018;Jiménez-Peñuela et al., 2019).
The characterization of the ecological niche of a given species is based on its distinct life history, particularly its use of food resources as one of the most studied by ecologists (Moser et al., 2017). Food is a fundamental resource that influences organisms in a variety of ways (Marshal et al., 2009). The pursuit and consumption of food affects the behavior, ecology, and life history of organisms (Lee-Thorp and Sponheimer, 2006). The ability of an individual to safely harvest and process sufficient quality food to fulfill its requirements for growth, maintenance, and reproduction is a key determinant of its fitness (Marshal et al., 2009). Knowledge of the diet of an organism and its changes in space and time is According to sexual selection theory, sexual dimorphism arises from competition for mates and is attributed to sexual differences in reproductive roles (Seglie et al., 2010). Traits such as bright coloration, hypertrophied morphological features or large body size that enhance access of one sex to the opposite sex, are favoured (Lovich and Gibbons, 1992). Larger males are favoured, and gain an advantage in intrasexual competition with other males (Martin et al., 2012;Zhang and Lu, 2013;Colleoni et al., 2014;Magalhaes et al., 2016;Altunisik, 2017). Sexual selection may also lead to dietary differences between sexes (Magalhaes et al, 2016). Unlike sexual selection, fecundity selection favors larger females, possibly promoting larger clutch sizes and enhanced reproductive capacity (Lowe and Hero, 2012;Martin et al., 2012;Zhang and Lu, 2013;Colleoni et al., 2014). For instance, females are known to be larger than males in 90% of anurans and 61% of caudates (Marzona et al., 2004;Seglie et al., 2010;Zhang and Lu, 2013;Altunisik, 2017).
Amphibians are among the most species-rich groups of terrestrial vertebrates, with hundreds of new species still being discovered annually (Musah et al., 2019). They are important components of ecosystem, influencing food webs and energy flow by their feeding behaviour (Le et al., 2018) and by serving as prey for larger vertebrates (Park et al., 2018). Despite their ecological importance, diversity and high vulnerability, Afro-tropical amphibians have received relatively less attention in the ecology, evolution and conservation literature. Most of the studies on amphibians are conducted in the Neotropics and concentrate on their distribution and speciation. There is scant information on amphibian population dynamics and the influence of land cover/land use changes on their ecology and life-history traits.
Most amphibians are generalist and opportunistic invertebrate feeders (Balint et al., 2008;Crnobrnja-Isailovic et al., 2012;Kittel and Sole, 2015;Do Couto et al., 2018;Santana et al., 2019). However, some species are specialist predators (Menin et al., 2015;Do Couto et al., 2018). The feeding habit and diet of amphibians may be influenced by seasonal availability and abundance of food (Crnobrnja-Isailovic et al., 2012), presence of competitors, andpredation risk (Lopez et al., 2009;Kittel and Sole, 2015;Santana et al., 2019). Therefore, the ability of an individual amphibian to safely harvest and process sufficient quality food may vary with environmental differences. This variation may be reflected in the diet composition and body condition of individuals. Also, individuals living in environments with high competition and risk of predation may develop adaptive traits that may differentiate them from individuals in less stressful environments.
The common African toad (Amietophrynus regularis) is common in Sub-Saharan Africa (Vancocelos et al., 2010;Iyaji et al., 2015). However, little is known about its ecology and life history as well as its trophic niche and how this is influenced by LULCC. Currently, there is lack of knowledge on the extent to which urban and agricultural landscapes influence their diet composition and body condition, as well as their adaptations for fitness enhancement under these stressful environments. Such information is of ecological and evolutionary significance and also important for predicting the impact of environmental changes on individuals and populations and for formulating effective conservation strategies.
The present study therefore assessed the diet composition, body condition and sexual dimorphism of the common African toad from agricultural and urban landscapes in Accra, Ghana. We hypothesized that agricultural and urban landscapes may have different effects on the diet composition, body condition and prominence of sexual size dimorphism of the common African toad. We expected toads from farmland to have more diverse diet and better body condition than those from the urbanized areas. Like most anurans, we expected females to have bigger body size than males. We also expected males from the urban area to have prominent traits that enhance sexual selection because of intense competition for mates.

Study Area
This study was conducted on Legon campus (05°39'03"N, 00°11'13"W) of the University of Ghana. The campus has a total area of 1,300 hectares (13 km²) and is located about 13 km north-east of Accra, the capital city of Ghana (Gbogbo et al., 2017). The climate of the area is characterized by a pronounced gradient of mean annual rainfall ranging from 733 mm to 1,118 mm distributed over a major (May-July) and minor (September-October) rainy season and a daily mean temperature of about 30°C (Holbech et al., 2015;Gbogbo et al., 2017). The vegetation is generally coastal grassland, thicket and dry forest (Garshong et al., 2013), with most of the natural vegetation converted into developed areas (buildings, markets, roads and other man-made infrastructure) and farmland. The only remnant of the original vegetation is within the Legon Botanical Garden which is located north of the campus and covers an area of about 2.0 km 2 . The developed (urban) area, which covers about 7.3 km 2 , includes areas around students' halls, hostels, chalets, staff bungalows, academic facilities, central administration blocks, library block, bookshops, banking square, restaurants and canteens. The farmland is located adjacent to the botanical garden and covers an area of about 1 km 2 . Crops grown in the farm include mango, cashew, maize and vegetables.

Study Species
The common African toad, Amietophrynus regularis [Bufo regularis] (Amphibia: Bufonidae) (Iyaji et al., 2015), is a large, strong toad with warty skin and dark olive-brown dorsal surface. The underside of both sexes is white to beige, with males having a black sub-gular or throat pigmentation (Deef, 2019).
The common African toad also known as the square-marked toad, is listed as 'Least Concern' by the IUCN in view of its wide distribution in a broad range of habitats and presumed large population size (IUCN, 2016). This toad occurs in both moist and dry savanna, forest margins, montane grassland, and agricultural and other habitats associated with rivers (Deef, 2019). It is widely distributed in Sub-Saharan Africa, with its range extending from oases in Algeria and Libya to northern Nilotic Egypt (Ibrahim, 2008;Saber et al., 2016). The African common toad feeds on a wide variety of vertebrates and invertebrates, particularly insects (Ibrahim, 2008), serving as insect biocontrol in most agricultural fields (Saber et al., 2016). It is a good source of protein in many parts of sub-Saharan Africa, notably Nigeria and Burkina Faso (Akinsanya et al., 2020).

Data Collection
Using visual encounter surveys, samples were collected from farmland and developed areas on the University of Ghana, Legon campus. Prior to sampling, the study area was surveyed and breeding sites determined using points of male calls. Samples were collected from July to September between the hours of 22:00GMT and 08:30GMT. During the day, toads were searched for under leaf litter, fallen logs, rocks, and other microhabitats where toads are known to seek refuge. During chorusing nights, calling males or pairs in amplexus (Plate 1) were captured by hand following Quiroga et al., (2015) and Oropeza-Sanchez et al. (2018). The sexes of individuals were determined using the presence or absence of sub-gular pigmentation (Plate 2) following Vera-Candioti et al. (2019).
Morphometric parameters including snout vent length (SVL), weight (W), mouth width (MW), head width (HW), head height (HH), head length (HL), foot length (FT), radio-ulna length (RUL), humerus length (HuL), femur length (FL) and tibia-fibula length (TbFL) were measured for each captured toad (Table 1). The diet composition of toads was assessed using gut content analysis. The toads were dissected and stomach contents were emptied into a petri dish containing 70% ethanol. Identification of stomach content was done to the highest taxonomic level possible (order or family) using a dissecting microscope.

Plate 1: Toad pairs in amplexus encountered at night in the Developed Area
Plate 2: Differentiation of males from females through sub-gular pigmentation, which is darker in males.

Data Analysis
We calculated the Numerical Percentage (N%) as (Ni x 100)/Nt, where Ni is the number of prey category i, and Nt is the total number of all prey categories. The Frequency of Occurrence (F%) was estimated as the number of toad stomachs in which category i prey were found. Diversity of Diet Composition was estimated using the where 'pi' represents the proportion of total abundance represented by the i th species. The Sorenson's qualitative index and Bray-Curtis quantitative index were used to assess the similarity of diet composition between sexes and sites. The Pearson's correlation coefficient (r) was used to assess the correlation between SVL and diversity and abundance of prey categories consumed by the toads. The residual mass index was calculated using log 10 transformed data to insure the linearity of the relationship between weight (W) and snout-vent length (SVL). The theoretical body weight of each frog was obtained by introducing the SVL into the equation of the regression line. This was then subtracted from the measured body weights to obtain the residual value (Bacila et al., 2010;Vera-Candioti et al., 2019). Individuals with positive residual indices were considered to be in good condition, while those with negative residual indices were said to be in poor condition. The body condition scores were compared between sites and sexes, and the degree of significance evaluated using the t-test. The t-test was also used to assess the significance of the differences between morphometric parameters of males and females from farmland and developed areas. A trait was said to be female-biased if the male to female ratio was less than 1, and male-biased if the ratio is greater than 1. No sexual size dimorphism existed where the male to female ratio equalled 1. All analyses were in Excel and SPPSS version 22 and test of significance was at alpha of 0.05.

Diet Composition
A total of 44 (21 males and 23 females) and 48 (27 males  and nine orders of prey items were consumed by females and males respectively from the Farmland, with all the orders of prey items found in males also being present in females (Fig. 2). The similarity (Sorenson's index) of prey items in terms of order was therefore very high (75%). A very strong statistically significant positive correlation (Pearson's correlation, r ≥ 0.9996, p < 0.00001) existed between SVL and diversity of prey items consumed for both males and females (Fig. 4). There was also a weak positive correlation (Pearson's correlation r ≤ 0.3693, p ≥ 0.0561) between SVL and the abundance of prey items consumed (Fig. 4).
A total of 507 prey items, comprising six invertebrate classes (Insecta, Arachnida, Chilopoda, Diplopoda, Gastropoda, and Polychaeta), from at least 15 orders and 28 families were obtained from toads in the Developed Area. The class Insecta formed the majority (91.7%) of the prey items, with the Hymenoptera (74%) and Coleoptera (10.1%) being the most abundant prey categories (Fig. 3). The Formicidae was the most abundant family (74%), and the most consumed prey category with 91.1% of individuals with gut content consuming it. Males consumed more quantities of prey items in terms of numerical proportion, but females consumed a higher diversity of prey items (H' = 1.364) than males (H' = 0.894). There was a weak positive correlation between SVL and the diversity of prey items consumed for both males and females (Pearson correlation coefficient r = 0.344, p = 0.192, male; r = 0.1199, p = 0.536, female) (Fig.7). There was also a weak positive correlation between SVL and the abundance of prey items consumed by females (r = 0.053, p = 0.846) (Fig. 8), but males showed a weak negative correlation between SVL and the abundance of prey items consumed (r = -0.168, p = 0.384) ( Fig. 9).

Body Condition
Generally, toads in the Farmland and Developed Areas had poor body condition (i.e., negative residual mass index), except females from the farmland. However, both male and female individuals from the Farmland had better body condition than those from the Developed Areas. Also, male toads from the Developed Areas appeared to possess better body conditions than their female counterparts, even though the difference was not statistically significant (t-test, alpha = 0.05, p = 1).

Diet Composition
Our results suggest that the common African toad has a wide taxonomic range of diet composition, and However, females consumed a greater diversity of prey items, which could be due to their higher energy requirements, particularly during the breeding season (Quiroga et al., 2015), when their higher metabolic rates support reproductive functions (Balint et al., 2008). Also, fecundity and egg size in amphibians depend on the nutritional quality of food (Van Ngo and Ngo, 2014). Females meet high nutritional quality demands by consuming a higher diversity of prey items. Also, the diet of individual toads from the Farmland and Developed Area remained largely constant (Sorenson's index = 83.9%; Bray-Curtis index = 91.6%), indicating the similarity in distribution of prey items between the two sites.
This was expected, given the proximity of the two sites and the fact that urbanization and agricultural practices have similar effects on invertebrate diversity and species composition (Jones and Leather, 2012). Indeed, a recent study of the ground dwelling insects in the study area showed similar species composition for the farmland and developed area, Hymenoptera (Formicidae) and Coleoptera (Carabidae) being the most abundant species (Asante, 2020). Our findings support the results obtained for Pelophylax ridibundus (Balint et al., 2008) and common toads (Bufo bufo) (Isailovic et al., 2012).
Prey items from the taxa Formicidae, Stratiomyidaea and Polychaeta were most consumed, indicating that these prey categories were the most abundant and accessible. Usually, it is the readily available and most abundant and accessible food sources that are used (Moser et al., 2017). The occurrence of Formicidae in the diet of most toads supports the findings of other studies on anurans (Moser et al., 2017;Oropeza-Sanchez et al., 2018). This may be due to the social behaviour and high environmental availability of these prey items (Moser et al., 2017). Additionally, such prey items may be relatively easy to capture and process, making their consumption energetically rewarding (Van Ngo and Ngo, 2014). The occurrence of high frequencies of Carabidae (Coleoptera) corroborates the findings for Pelophylax ridibundus (Balint et al., 2008). Common African toads are 'sit-and-wait' predators whose diets reflect the availability and accessibility of prey items. Their broad dietary niche therefore suggested that they are opportunistic generalist feeders.
The SVL of the toads correlated positively with prey diversity and abundance and this is consistent with the findings of Almeida et al. (2019). Larger individual toads have greater demand for energy uptake and so consume a higher variety and number of prey items. They also have larger stomach capacity that can accommodate large volumes of prey items compared to smaller individuals (Le et al., 2020).
Additionally, prey consumption is influenced by activity patterns during foraging. Larger individuals are more active and tend to encounter more diverse prey items and consequently to consume more prey categories (Mageski et al., 2018).

Body Condition
Body condition is a measure of energetic status, physiological state and individual quality or fitness (Cox and Calbeek, 2015;Warner et al., 2016). Generally, individuals in better body condition are predicted to have greater energetic status and better chance of survival compared to individuals with poorer body conditions (Labocha et al., 2014). In this study, body condition of individual toads was generally poor at both sites and for both males and females. This may be due to the fact that individuals experienced similar levels of food availability, accessibility, quantity and quality, and threats such as predation, competition, and parasite loads (Jarvis, 2015;Falk et al., 2017). Access to high quality food influences body condition and fitness, and thus plays a significant role in the success of animals (Scholz et al., 2020). The poor body condition of toads from the two study sites, Farmland and Developed Area partly suggested that the food sources are not of adequate quality. This phenomenon may be attributed to the impact of land-use/land-cover change. Even though females consumed a wider variety of prey items, their body condition was not significantly better than males. This could be due to the fact that body reserves for females might have been depleted during the breeding season (Bancila et al., 2010).

Sexual Dimorphism
Our results revealed significant sexual size dimorphism in African common toads. Mating system, parental investment, diet, habitat selection, reproductive success and population density are factors that may contribute to morphological differences between male and female toads (Mori et al., 2017). Sexual size dimorphism may be due to different selective pressures experienced by different sexes (Lowe and Hero, 2012). For both sites, females were larger in size (SVL and weight) than males, which is consistent with the general trend in anurans and other amphibians (Lee, 2001;Monnet and Cherry, 2002;Di Cerbo and Biancardi, 2012;Lowe and Hero, 2012;Labus et al., 2013;Seglie et al., 2013;Quiroga et al., 2015;Mori et al., 2017). The disparity in weight between males and females may be due to fecundity selection (Altunisik, 2017). According to the fecundity selection hypothesis, increased fecundity of larger females is associated with increased clutch size, and consequently high reproductive success (Labus et al., 2013). Also, with females carrying males during amplexus, it is energetically prudent for females to be larger than males. Small size comes with agility, which makes it easier for males to reach females (Mori et al., 2017). It has also been reported that male amphibians usually do not live long enough to attain large sizes (Shine, 1979).
Males from the Developed Area had longer radio-ulna, distal forelimb, tibio-fibula and feet than females. The difference in tibio-fibula length is contrary to results obtained by Quiroga et al. (2015) on Odontophrynus cf. barrioi. The increase in length of such body morphologies for males may be in response to requirements for amplexus (Magalhaes et al., 2016). During the process of amplexus, males tend to cling onto females very tightly. This requires more muscle strength to avoid displacement by competing males. Therefore, a significant variation may be positively correlated to reproductive success in males. However, the length of humerus was similar to studies carried out by Quiroga et al., (2012) using Rhinella arenarum. According to Quiroga et al., (2012), this difference helps females to support their own weight and that of males on their back during amplexus. Individuals from developed areas exhibited greater levels of sexual dimorphism than those from farmland. This may have possibly been influenced by different selective regimes associated with different environmental conditions, such as intraspecific competition and predation intensity acting separately on both males and females.

Ecological, Evolutionary and Conservation Implications
This study is probably the first to simultaneously assess the diet composition, body condition and sexual size dimorphism of Amietophrynus regularis, and provides vital information on the food niche and effects of human modified landscape on the body condition and sexual size dimorphism of this species.
Our findings have important ecological, evolutionary and conservation implications. Individual variation in resource use have consequences for intra-and inter-specific interactions and population dynamics.
Dietary patterns of individuals and populations is an essential component in trophic interactions and food web structure and provide insight into selective pressures on prey. Access to high quality food plays a significant role in the success of animals, as it influences their body condition and fitness (Scholz et al., 2020). Limited food availability can reduce reproductive success and survival rate, ultimately altering population densities, transient dynamics and persistence (Bolnick et al., 2011). The utilization of a wide range of food resources in common African toads can be advantageous in dynamic habitats with constantly changing food availability. Our results on body condition and sexual size dimorphism also provide information on how phenotypic plasticity is used by toads as an adaptation measure on an ecological time scale. In all, our data is important for understanding the feeding behaviour, population dynamics and adaptive potential of toads in changing environments. This is crucial for formulating effective conservation strategy in human-modified landscapes.