Tropical anurans mature early and die young: Evidence from eight Afromontane Hyperolius species and a meta-analysis

Age- and size-related life-history traits of anuran amphibians are thought to vary systematically with latitude and altitude. Because the available data base is strongly biased towards temperate-zone species, we provide new estimates on eight afrotropical Reed Frog species. A meta-analysis of the demographic traits in 44 tropical anuran species aims to test for the predicted clinal variation and to contrast results with variation detected in temperate-zone species. The small-sized reed frogs reach sexual maturity during the first or second year of life, but longevity does not exceed three to four years. Latitudinal effects on demographic life-history traits are not detectable in tropical anurans, and altitudinal effects are limited to a slight size reduction at higher elevations. Common features of anuran life-history in the tropics are early sexual maturation at small size and low longevity resulting in low lifetime fecundity. This pattern contrasts with that found in temperate-zone anurans which mature later at larger size and grow considerably older yielding greater lifetime fecundity than in the tropics. Latitudinal and altitudinal contraction of the yearly activity period shape the evolution of life-history traits in the temperate region, while trait variation in the tropics seems to be driven by distinct, not yet identified selective forces.


Introduction
Demographic life-history traits of amphibians are thought to vary systematically with latitude and altitude among species and also among conspecific populations [1]. Available evidence on interspecific variation suggests that in fact average age and longevity augments from equatorial regions towards the poles and also or exclusively with increasing altitude [2][3][4]. At the intraspecific level, populations of the European anurans Epidalea (Bufo) calamita and Rana temporaria showed similar trends in the latitudinal and altitudinal variation of age at maturity and longevity, whereas age-adjusted size was insensitive to altitudinal effects and weekly affected by latitude [4][5][6]. Unfortunately, current evidence available for the analysis of demographic trends is considerably biased towards temperate-zone species (>23.44˚N or S) rendering inferences on tropical amphibians inhabiting the equatorial belt from 23.44˚N to 23.44˚S tentative [7]. Detectable latitudinal variation of traits in tropical species is not probable because the location of the intertropical convergence zone varies over time in annual cycles and moves southwards during the past 600 years [8].
The major source of information on the age of amphibians without previous recapture history is the retrospective estimation by skeletochronology [4,9,10]. Lines of arrested growth (LAG) interrupting round bone growth are the result of a genetically based, circannual rhythm synchronised with seasonal cycles, usually hibernation in temperate-zone amphibians [11]. LAGs are also formed in tropical habitats in which seasonality is mainly based on precipitation regime [12]. Skeletochronological studies on the demography of tropical anurans focus currently on Asia [13][14][15], South America/Caribbean [7,16], and Madagascar [17,18], whereas knowledge on afrotropical species is limited to currently three species (S1 Table) [19].
We aim to scale down the apparent gap of knowledge on tropical amphibians and specifically on African frog species by providing demographic data on eight of the eleven currently known Hyperolius species (Anura, Hyperoliidae) inhabiting Rwanda [20]. The genus Hyperolius is among the most diverse sub-Saharian anuran genera (141 species) [21], but to our best knowledge this is the first report of estimates on demographic key-traits such as age at maturity, longevity and age-adjusted size. Moreover, the populations sampled at latitudes of 1.6-2.6˚S are closer to the equator than those of any other anuran species studied skeletochronologically so far, and cover an altitudinal range of 1,643-2,379m asl. Specific aims of our demographic analysis of Afromontane frog species are (1) to describe the post-metamorphic life history represented by age at maturity, median age and longevity, and (2) to identify sex-specific differences in traits and growth patterns in those species represented by larger samples. We complement the new evidence on eight afromontane species with a meta-analysis of published evidence on a total of 37 anuran species inhabiting the tropical belt all over the world (S1 Table). We selected those studies reporting age and size data derived from at least five individuals per gender to test for among-species variation of age and snout-vent length at maturity, and of longevity and maximum size, and their association with latitude and altitude as proxies for environmental variation in the tropics. The first comprehensive and quantitative analysis of demographic traits emphasizes that trait evolution in tropical anurans varies in several aspects from that in temperate-zone anurans.

Bone sampling and skeletochronological processing
Each individual was sexed and snout-vent length (SVL, distance between snout tip and cloaca) measured to the nearest 0.1mm using a calliper. With the exception of nine H. castaneus (kept in captivity) all specimens were sacrificed immediately after collection in accordance to the accepted standards of veterinary medicine by exposure to an overdose (buffered 1% solution for five minutes) of the anaesthetic MS-222 (deep anaesthesia within 10-25 seconds). Collection, sacrifice and export of the specimens were approved by the Rwandan Development Board (RDB, national agency of nature conservation). Specimens were stored individually in 70% ethanol at room temperature for future molecular and morphological examination. For skeletochronological age determination the 3 rd or 4 th digit of a forelimb was toe-clipped, and in some individuals a humerus or femur as well. Laboratory protocols followed the standard methods of skeletochronology [4,25]. The samples were embedded in Historesin™ (JUNG) and stained with 0.5% cresylviolet [26]. Diaphysis was cross-sectioned at 12μm using a JUNG RM2055 rotation microtome. Cross sections were examined light microscopically for the presence of growth marks at magnifications of 400x using an OLYMPUS BX 50. We distinguished strongly stained lines of arrested growth (LAGs) in the periosteal bone, separated by faintly stained broad growth zones, and the line of metamorphosis (LM), separating larval from postmetamorphic bone [4,27]. We selected diaphysis sections in which the size of the medullar cavity was at its minimum and that of periosteal bone at its maximum. The number of LAGs was assessed independently by the authors to estimate age.

Meta-analysis of demographic life-history traits of tropical anurans
The tropics are geographically limited to the region between the northern and southern latitude of 23.44˚. We considered all published skeletochronological studies on anuran species inhabiting this equatorial belt and reporting the age of at least five individuals per gender. Demography of tropical anurans Including our own data we obtained data on a total of 44 species, specifically on males of 43 species and on females of 29 species (S1 Table). If the studies did not explicitly associate age data with gender, we assumed that the majority of data were obtained from males due to the general capture bias towards males (see our Hyperolius data as an example). Plasticity of male and female life history was analysed in four traits: (1) minimum age at maturity (n LAGs) = age of the youngest adult of the sample; (2) minimum SVL (mm) of adults = size of the smallest adult irrespective of age; (3) longevity = maximum age detected within a sample (n LAGs); (4) maximum SVL (mm) of adults sampled irrespective of age. As a proxy for the climate at the sampling localities we used latitude and altitude above sea level. If these environmental variables were not explicitly given in the corresponding skeletochronological study, we estimated latitude and altitude by locating the sampling sites in electronic topographical maps. If anuran sampling occurred along an altitudinal transect, we calculated the average altitude to represent the transect climate.

Statistical analysis
All variables were first tested for normality. As size and age distributions of Hyperolius spp. were significantly skewed, descriptive statistics included median, minimum and maximum. Statistical comparison between gender and between taxa was based on the non-parametric Mann-Whitney-Wilcoxon W-test. Growth following metamorphosis was estimated using the von Bertalanffy equation [28]: where SVL t = average body length at age t; SVL max = asymptotic body length; SVL met = body length at metamorphosis; t = number of growing seasons experienced (n LAGs), and k = growth coefficient (i.e. shape of the growth curve). SVL met was assessed for each species from tadpoles of Gosner stages 40-43 collected in the field. The von Bertalanffy growth model was fitted to the average growth curve using the least square procedure (nonlinear regression). Estimates of SVL max and k are given with the corresponding 95% confidence interval. Sexual size dimorphism was tested for based on SVL max . The absence of overlap between confidence intervals was considered as a significant deviation at P<0.05.
The meta-analysis of size-and age-related life-history traits was performed separately on males and females because pronounced sexual dimorphism is present in many tropical anurans [7,18]. The association among life-history traits and latitude and altitude was tested for applying a factor analysis. Variables were standardized by dividing the difference between value and arithmetic mean by the standard deviation. Extraction criterion for principal components was an eigenvalue >1. Extracted principal components were submitted an orthogonal VARIMAX-rotation to yield factor loading of original variables close to 1 (strong association) or 0 (no association). Identified associations between a life-history trait and latitude and/or altitude were modelled using regression analyses (selection criterion: maximum R 2 ). Significance level was set at alpha = 0.05. All calculations were performed using the procedures of the program package STATGRAPHICS Centurion, version XVI (STATPOINT Inc.).

Histological features of round bone diaphysis sections in Hyperolius spp.
In all species examined, discernible growth marks were present in the stained diaphysis cross sections of humeri, femura, first and second phalanges (e.g. H. castaneus, Fig 2). The periosteal bone produced during the larval period stained darker than that produced during the terrestrial stage and was separated by a faint line of metamorphosis (LM Lines of arrested growth (LAGs) were easily distinguishable from the less-stained growth zones in the post-metamorphic periosteal bone (Fig 2). The number of LAGs was the same in the phalanx and femur or humerus of randomly chosen preserved individuals (two per species) demonstrating that non-lethal phalange sampling allows for precise LAG estimation.
The position of the last LAG and the corresponding collection date of the individual allowed for an estimation of the season in which bone growth was arrested. The periphery of bones collected between March and May showed narrow to broad growth zones without a terminal LAG, whereas the bones of individuals collected in September or October showed a peripheral LAG without or with a very small terminal growth zone. We conclude that growth was arrested during the dry season between June and September (Fig 1). This growth pattern was evident in all species examined. As double lines, i.e. multiple LAGs, were never observed, there was no indication for more than one period of arrested growth per year.
LAG formation was also observed in 7 out of 9 H. castaneus which were captured in October 2010 and kept in captivity in terraria until March 2012 (Fig 3A and 3B). The LAG formed in captivity was located at the periphery of the bone, with very little additional bone growth. Two individuals did not show any periosteal bone growth during captivity. Age distribution and longevity in Hyperolius spp The Hyperolius species studied were generally short-lived with a longevity ranging from one year (= survived dry season) in H. lateralis to four in H. rwandae (Table 1, Fig 4). In the samples including a significant number of females, median age did not differ significantly between sexes (H. castaneus: Mann-Whitney-Wilcoxon W-test, W = 379.5, P = 0.187; H. glandicolor: Mann-Whitney-Wilcoxon W-test, W = 74.0, P = 0.179; Table 1). Sexual maturation often occurred in the same year of metamorphosis, but at latest following the first dry season (Table 1). Males were considered mature reproductive, if their throats were coloured yellow, females were identified by having egg masses visible through the transparent parts of the abdominal ventral skin. Small-sized species had a greater life expectancy than the large ones ( Fig 5).

Growth pattern in Hyperolius spp
Using the von Bertalanffy growth model on the age-size data of amphibians requires knowledge of the snout-vent length at metamorphosis and of the duration of the growth period between metamorphosis and the first arrestment of growth. Size at metamorphosis was available for seven of the eight species and ranged from 8.3mm in H. cf. cinnamomeoventris to 14.0mm in H. viridiflavus (Table 1). Metamorphs and tadpoles of all stages were found at the beginning and end of the rainy period suggesting continuous reproductive activity and subsequently continuous recruitment of metamorphs in H. castaneus, H. discodactylus, H. kivuensis, H. lateralis and H. viridiflavus. Age class 0-LAGs individuals of these species had indeed SVL ranging from the size at metamorphosis to the size of 1 LAG old mature specimens (Fig 6). As the duration of growth period of the larger age class 0-LAGs individuals was an unknown fraction of a year, they were excluded from growth model estimation. Asymptotic maximum SVL max as estimated by the von Bertalanffy model was significantly female-biased in H. castaneus and in H. glandicolor (assumed size at metamorphosis 14mm as in the closely related H. viridiflavus), whereas the growth coefficient k did not differ significantly (Table 2; comparison of CI, P<0.05). There were also significant differences among species with respect to SVL max of males: H. lateralis < H. castaneus = H. glandicolor H. kivuensis = H. viridiflavus (Table 2; comparison of CI, P<0.05).
The analogous analysis of the data on females pertaining to 24 species yielded similar results as in the males. Two principal components (eigenvalue > 1) explained 76.3% of total variation (Table 3B). Factor loading by the original variables of the data set was as described for males. Multiple regression analysis demonstrated that only altitude explained a significant portion of variation the size variables. Again, there were significant correlations between SVL at maturity and maximum SVL, respectively, and altitude (R 2 = 0.255, F 1,24 = 9.23, P = 0.0059; R 2 = 0.284, F 1,24 = 10.5, P = 0.0036), described by multiplicative regression models (SVL maturity = e (4.550-0.156 Ã in(altitude)) ; SVL max = e (5.231-0.213 Ã in(altitude)) ; Fig 7).  Table 1 and text.    H. castaneus (A), H. kivuensis (B), H. lateralis (C) and  H. viridiflavus (D). All 1 LAG and few 0 LAG individuals were sexually mature (usually males, see Table 1). The bars represent 2mm size classes of the individuals.

Is skeletochronology reliable for aging tropical anurans?
Afromontane anurans showed regular alternations between periods of periosteal growth and arrestment of growth analogous to those observed in temperate-zone amphibians. Bone growth was observed in all individuals collected during the rainy season (e.g. Fig 2). Our data suggest that reinforcement of the genetically-based circannual growth rhythm is mediated by the seasonal variation of precipitation rather than that of temperature. Seven out of nine H. castaneus specimens held in captivity (equivalent to a prolonged dry period) had formed an additional LAG as predicted (77.8%), whereas the bones of the deviant two individuals did not grow during the whole period of captivity (presumably quality and quantity of food were suboptimal). Similarly, 11 out 21 captive-held L. fallax showed the predicted number of additional  Table 3.

Factorial analyses of four demographic life-history traits of tropical anurans and corresponding latitude and altitude of collection sites.
Matrix of factorial loads following VARIMAX-rotation of principal components in (A) Males (n = 41 species) and (B) Females (n = 28 species). Details on species involved are listed in S1 LAGs (52%), the other one supernumerary LAG or one or two less than predicted [7] supporting the circannual periodicity of LAG formation even in the zoo environment [32]. In the natural habitat LAG formation during the dry period was also observed in the Asian frog Sylvirana nigrovittata [12] emphasising that low water availability combined with optimal temperatures may act as external zeitgeber for the circannual clock in the same way as hot temperatures and dryness in arid regions and cold temperatures in temperate climate zones (4).
There is no indication that skeletochronological age estimation may be generally unreliable in tropical amphibians because LAG formation is less pronounced as in the temperate zone ( [7]; but see [2] for a failure of LAG detection in perennial Litoria lesueuri). The rate of correct skeleotochronological age estimation is about 86% in temperate-zone amphibians younger than eight years [4] and available evidence for tropical anurans suggests that the rate is similar. We conclude that aging tropical frogs by counting LAGs yields a conservative estimate of longevity because in case of proven deviation from the actual lifespan longevity tends to be underestimated ( [2,7] this study). Unlike adults of temperate-zone anurans, many reproductive adults of tropical species do not show visible LAGs because sexual maturity is often reached before finishing the first year of life ( [13,14,33] this study).

Does variation of demographic life-history traits differ among tropical and temperate anurans?
Age at maturity. Tropical anurans mature on average one year earlier than temperatezone species (estimate based on compiled published data of 124 species). Temperature-and precipitation regime in the tropics usually allow for activity during most of the year so that the majority of species mature within their first or second year of life (e.g. Table 1), while the Demography of tropical anurans delayed maturity of temperate-zone anurans can be attributed to the shorter annual growth period. Morrison and Hero [1] predict that age at maturity generally increases along latitudinal and altitudinal clines. Case studies on species inhabiting wide geographical ranges provide support for this prediction in temperate-zone anurans [5,6]. In contrast, we did not find any evidence that age at maturity is affected by latitudinal or altitudinal variation in tropical species. Yet, the age variation detectable by skeletochronology has a resolution of one year, whereas a resolution of months would have been required possibly to identify potential latitudinal and altitudinal effects. We cannot completely rule out clinal geographical influence on the age at maturity in tropical anurans, but we expect adaptive delay of maturation to be rare because of the generally favourable environmental conditions.
Minimum size at maturity. The tendency of females being larger than males at attaining maturity was not statistically significant in the complete data set on tropical species, but well established in two Hyperolius species (see Table 2). Comparing median SVL at maturity of tropical anuran species (26.9mm) with that of temperate ones (41.8mm; estimate based on compiled published data of 18 species) the size threshold of maturity seems to be considerably lower in the tropics. Since female size is positively related to clutch size in most anuran species [34,35] and longevity of tropical species is low ( [17,30], this study), lifetime fecundity appears to be much smaller than in temperate-zone species, paralleling trait evolution in birds [36]. At the same time, the diversity of reproductive modes including parental care is far greater in Amazonian amphibians [37] than in those of temperate regions [38] suggesting a potential evolutionary advantage of k-strategists with small clutches in the Neotropics. Yet, the eight Hyperolius spp. analysed in this study and all other Rwandan anuran species are short-lived and unspecialized pond or stream breeders [20,23] demonstrating that the coupling between low lifetime fecundity and parental care in the Neotropics does not prevail in the Afrotropics. It is intriguing that size of recently matured tropical anurans decreases with increasing altitude, a factor explaining about a third of observed variance. Since the surface/volume-ratio is unfavourable for small individuals with respect to evaporative water loss and thermal relations [39], this tendency may indicate a trade-off between early maturation and size.
Longevity. The maximum lifespan of tropical anurans is about 2-3 years lower than that of temperate anurans (estimate based on compiled published data of 140 species). In longlived species the discrepancy is even greater, 13 LAGs in the tropical E. hexadactylus [31] compared with 17 LAGs in the temperate E. calamita [40] and 18 LAGs in R. temporaria [41]. This pattern seems to indicate that the risk of dying during the inactivity period of winter is probably lower than that of being predated during the season of activity. Again, longevity is predicted to increase along latitudinal and altitudinal clines [1]. There is ample support for this prediction in temperate-zone amphibians [3,5,6,42,43]. In contrast, longevity of tropical species did not co-vary significantly with latitude or altitude suggesting local constraints such as predator impact and parasite load being more important than macroclimate gradients.
Maximum size. Short lifespan and small maximum size are associated in most tropical species (e.g. Fig 5 for Hyperolius spp.). Maximum SVL (median: 33.2mm) is only about half of that of temperate-zone species (median: 65.0mm; estimate based on compiled published data of 80 species), but there are remarkable exceptions especially in aquatic tropical species (e.g. SVL up to 320mm in Conraua goliath [44] and 170.3mm in Telmatobius macrostomus [45]). Maximum SVL of European and North American anuran species increases with latitude [46], whereas the intraspecific pattern is more complex and SVL decreases with altitude [5,47]. The later tendency is present in tropical frogs as well, whereas latitudinal effects seem to be absent. Female size variation in temperate-zone species has been suggested to be the evolutionary byproduct of the optimization of lifetime fecundity [5,47], while that in tropical species remains enigmatic requiring further investigations.

Conclusions
Variations of demographic life-history traits in afromontane Hyperolius spp. are in line with those in anuran species inhabiting the tropical regions in South America, Madagascar and Asia. Common features are early sexual maturation at small size and low longevity resulting in low lifetime fecundity. This pattern contrasts with that found in temperate-zone anurans which mature later at larger size and grow considerably older, experiencing greater lifetime fecundity. Macroclimatic constraints mediated by latitude and altitude account for a large portion of age-and size-related life-history traits in temperate-zone species, whereas only sizerelated traits co-vary slightly with altitude and not at all with latitude in tropical species. We conclude that the contraction of activity period at increasing latitudes and altitudes shapes demographic life-history traits of anurans in the temperate region. In the tropical belt, however, climate does not constrain activity in the lowland at any latitude and only to a minor extent in the highlands indicating that timing of sexual maturation, short lifespan and size limitation respond to different evolutionary forces than those in the temperate zone.
Supporting information S1