Usova (2014) Age and growth rate of green frogs of the Nyzhnii Dobrytskyi pond

Usova E. E. Age and growth rate of green frogs (Pelophylax esculentus complex) of the Nyzhnii Dobrytskyi pond (Zmiiv district, Kharkiv region) // Visnyk Kharkivskoho natsionalnoho universytetu imeni V. N. Karazina. Seriia: biolohiia. – 2014 – Vyp.20, №1100. P. 204–212. UDC: (57.022+57.032):591.84:591.158.1:597.851 Age and growth rate of green frogs (Pelophylax esculentus complex) of the Nyzhnii Dobrytskyi pond (Zmiiv district, Kharkiv region) E. E. Usova V. N. Karazin Kharkiv National University (Kharkiv, Ukraine) e.e.usova@gmail.com Using skeletochronology, 104 specimens of Pelophylax esculentus complex (18 P. ridibundus, 66 diploid P. esculentus, and 20 triploid P. esculentus) from the hemiclonal population system of the Nyzhnii Dobrytskyi pond (N 49°33'22", E 36°18'39"; Zmiiv district, Kharkiv region, floodplain of the Homolsha River), located within the territory of the Homilshanski Lisy National Nature Park, were studied. The age of the examined individuals was determined from cross-sections of the 4th phalanx of the longest toe of the hind limb, and their annual increments were calculated. The maximum growth rate, for both males and females, is observed before the 4th hibernation, which corresponds to the attainment of sexual maturity at age 5. Differences in the growth rate of P. ridibundus, diploid, and triploid P. esculentus are statistically insignificant. Key words: Pelophylax esculentus complex, Pelophylax ridibundus, growth, skeletochronology. Age and growth rate of green frogs (Pelophylax esculentus complex) of the Lower Dobritsky pond (Zmiyiv district, Kharkiv region) E. E. Usova With the use of skeletochronology 104 specimens from the population of Pelophylax esculentus complex (18 P. ridibundus, 66 diploid P. esculentus, and 20 triploid P. esculentus) inhabited the Lower Dobritsky pond (N 49°33'22", E 36°18'39"; Zmiyiv district of the Kharkiv region, the floodplain of the Gomolsha River at the National Natural Park "Gomilshanski lisy") were studied. The age of the individuals was estimated histologically by the sections of the longest toe's 4th phalanx and determination of annual growth marks. The annual growth rates were calculated. The maximum growth rate was observed between the 4th and 5th hibernations for both males and females. It has been shown that differences in the growth rates between P. ridibundus, di- and triploid P. esculentus are statistically insignificant. Key words: Pelophylax esculentus complex, Pelophylax ridibundus, growth, skeletochronology. Introduction Skeletochronological study of age and growth rate is a well-known yet still underappreciated method for investigating poikilothermic vertebrates. This method yields empirical data that not only characterise the age and growth dynamics of the animals under study, but also allow the assessment of other parameters important from a population biology perspective, such as the relative survival of different sympatric forms. However, conducting such studies rigorously requires careful organisation and the fullest possible standardisation of primary data processing methods. In the present work we examine in detail the processing of skeletochronological data characterising green frogs (members of Pelophylax esculentus complex) from a population system of particular research interest. The hybridogenous complex of green frogs, Pelophylax esculentus complex (= Rana esculenta complex), comprises two parental species, Pelophylax lessonae (Camerano, 1882) and Pelophylax ridibundus (Pallas, 1771), together with their various di-, tri-, and tetraploid hybrids (Plötner, 2005), to which we apply (Shabanov et al., 2009) the species-equivalent name Pelophylax esculentus (Linnaeus, 1758). The coexistence of hybrids with parental species is maintained through hemiclonal inheritance (the clonal transmission of certain genomes by hybrids, without recombination), which is of considerable theoretical interest. Sympatric representatives of parental species and their various hybrids, united by shared reproduction and by the intergenerational transmission of both clonal and recombinant genomes, have been designated HPS — hemiclonal population systems (Shabanov et al., 2009; Shabanov, Litvinchuk, 2010). Among the most complex hemiclonal populations of green frogs known to science are those recorded in the upper Siverskyi Donets river basin, in a region termed the Siverskyi Donets diversity centre of green frogs (Shabanov et al., 2009; Shabanov, Litvinchuk, 2010). The greatest diversity of green frog forms has been recorded within the Homilshanski Lisy National Nature Park, situated in Zmiiv district, Kharkiv region (Korshunov, 2010). The most interesting of the studied HPS of Pelophylax esculentus complex is that inhabiting the Nyzhnii Dobrytskyi pond (49°33'22" N, 36°18'39" E), located in Dobrytskyi Ravine before it opens into the floodplain of the Homolsha River (a right tributary of the Siverskyi Donets). The triploid hybrid fraction within this HPS is substantial (Korshunov, 2010), and even isolated tetraploids have been recorded in the vicinity (Borkin et al., 2004). Materials and Methods The composition of our study sample is characterised in Table 1. The majority of examined individuals (75 specimens) constitute a single near-random sample collected on 10 July 2013. By the time this sample was collected, frog spawning had concluded. Frogs were caught by walking the perimeter of the pond during the first half of the night using bright hand-held torches and collecting all observed individuals found on the bank or in the water at a short distance (up to 1–2 m) from the bank. Frogs were blinded by torchlight and caught by hand. Using this capture method, approximately 90% of observed frogs could be collected. It may be assumed that the taxonomic (P. ridibundus or P. esculentus), sex (female or male), and size (large or small) composition of individuals that escaped the collectors roughly corresponded to that of the collected sample. Table 1. Study material Form Sample, year of collection Juveniles, specimens Females, specimens Males, specimens Total, specimens P. ridibundus 2004–2009 3 5 1 9 2013 - 8 1 9 P. esculentus, 2n 2004–2009 10 4 5 19 2013 - 10 37 47 P. esculentus, 3n 2004–2009 1 - - 1 2013 - 11 8 19 Total 14 38 52 104 Processing of collected individuals comprised the following steps: – determination of taxonomic identity (as P. ridibundus or P. esculentus) from external morphological characters (Korshunov, 2010), principally the size and shape of the inner metatarsal tubercle, the relative length of the shank and thigh, and the coloration pattern of the thighs and body as a whole; – sex determination from external characters (presence of nuptial pads and vocal sacs in males); – measurement of snout–vent length using a calliper; – photography of the dorsal surface of each individual; – removal of four phalanges of the longest toe of one hind limb (right); – expression of a drop of blood onto a glass slide and preparation of a standard smear by spreading the drop across the slide surface with the edge of another slide; – cauterisation of the wound surface to stop bleeding. Following these procedures, the examined frogs were released at the point of capture. As a result, a proportion of individuals in this HPS was marked, which may be used (in the event of subsequent recaptures) to estimate population size. Toe-clipping serves as a group mark; for individual identification of examined individuals upon future recapture, photographs of their dorsal colour patterns are planned to be used. Collection of the sample, processing of captured frogs, and examination of blood smears involved second-year undergraduate students of the Biology Faculty of V. N. Karazin Kharkiv National University who were completing their teaching field practice in vertebrate zoology at the university biological station in the vicinity of the village of Haidary, under the supervision of Associate Professor D. A. Shabanov. Blood smear processing according to the previously described method (Bondareva et al., 2012) was carried out under the supervision of E. V. Meleshko. Smears were air-dried, photographed under a microscope, and mean erythrocyte size was determined; on the basis of this measurement, the ploidy of examined individuals was established. Thus, for diploid individuals in the 2013 sample, their identification as P. ridibundus or P. esculentus was based on external morphological characters, while identification of triploid P. esculentus (externally difficult to distinguish from diploid P. esculentus, and sometimes from P. ridibundus) was based on erythrocyte size measurements. In addition to the 2013 sample, we examined preserved specimens collected at the Nyzhnii Dobrytskyi pond in 2004–2009. The genotype of these individuals was determined by S. N. Litvinchuk and Yu. M. Rozanov at the Institute of Cytology of the Russian Academy of Sciences (St Petersburg) using flow DNA cytometry (Borkin et al., 2004). In the course of the skeletochronological study, the 4th phalanx of the longest toe of the hind limb was cleaned of soft tissues, decalcified, and 20–22 μm transverse sections of the mid-diaphysis were obtained on a freezing microtome, stained with Ehrlich's haematoxylin, mounted in glycerine, coverslipped, photographed as temporary preparations, and measurements and counts of lines of arrested growth (LAGs) were subsequently performed from photographs using Adobe Photoshop CS5. Measurement results were converted to micrometres using a conversion coefficient determined from a photograph of a stage micrometer taken under the same conditions as the section photographs. Measurement results were entered into an electronic database created in Microsoft Office Excel 2010. The method for interpreting and processing the obtained results, enabling retrospective calculation of annual increments in the studied frogs at different life-history stages, is described in detail below. Data on registered frog increments were transferred to Statistica 8 (StatSoft Inc.) and analysed by analysis of variance (ANOVA), treating frog increment in a given year of life as the dependent variable and the age of the frogs, their genotype, sex, and the calendar year in which the observed increment was registered as grouping factors. Interpretation of phalangeal section photographs The skeletochronological method is based on the fact that as an individual grows, the transverse diameter of its long bones increases (Smirina, 1983; Roitberg, Smirina, 2006, and other works). During growth arrests corresponding to hibernation periods (and sometimes to summer cold spells, which considerably complicates interpretation of sections) lines of arrested growth (LAGs) form in the bone, clearly visible in stained sections (Fig. 1). [IMG_1] Fig. 1. Photomicrograph of a phalangeal cross-section. Labels: I — endosteal part of the bone; II — periosteal part of the bone; A, B, C — LAGs in the endosteal part of the bone; b — boundary between endosteum and periosteum; b.v. — blood vessel; 1–10 — LAGs in the periosteal part of the bone. A substantial complication in interpreting sections arises from the fact that, as the outer part of the bone (periosteum) grows, the endosteal cavity in the centre expands, destroying some of the periosteal LAGs. At a certain stage, expansion of the endosteal cavity ceases and it is filled by endosteal bone tissue growing toward the centre of the cavity. The result of age determination from skeletochronological data depends on the number of recorded LAGs and on the assumption made regarding the number of LAGs resorbed during endosteal expansion. Errors in LAG counting (e.g., when a trace from a brief growth arrest is taken for a hibernation mark) or in estimating the number of LAGs resorbed during endosteal expansion lead to errors in age determination. In the authors' view, the more challenging task is precisely the determination of the number of resorbed LAGs, as this requires estimating a quantity not directly visible in the section. Interpretation of periosteal LAGs is simpler in this respect, since these lines are visible both in photographs and in sections. When a well-founded decision is difficult to reach from a single photograph, obtaining additional photographs taken at different microscope settings (in particular, at a different focal plane of the section being analysed) or photographing other sections of the same bone can be helpful. When estimating the probable number of resorbed LAGs, the following should be taken into account: – the number of visible remnants of resorbed LAGs; – the size of the endosteal cavity; – the sizes of preserved LAGs and their correspondence to the sizes characteristic of LAGs formed in members of the given population at different ages. The last two parameters for our studied individuals are reflected in Fig. 2. [IMG_2] Fig. 2. Variability of parameters whose values informed assumptions about the number of resorbed LAGs in each individual. Body length of the frog during hibernation periods is calculated on the assumption that the increase in the transverse diameter of the bone is proportional to the increase in body length (Roitberg, Smirina, 2006; Usova, Shabanov, 2009). We therefore assume that lastlmed/L = ilmed/iL, where lastlmed is the mean transverse diameter of the bone at the time of examination, L is body length at the time of examination, ilmed is the transverse diameter of the bone during one of the hibernation periods as determined from LAG dimensions, and iL is the body length of the individual during the corresponding hibernation period. It follows that iL = L * ilmed / lastlmed. If the sizes of two successive LAGs in the bone are known (and the corresponding calculated body lengths of the frog as a whole), it is possible to calculate how much body size increased during the year between those hibernation periods. In addition, it is possible to determine frog size and growth dynamics for specific calendar years (in our case, 2012, 2011, 2010, etc.). Results and Discussion The overall result of the skeletochronological study is shown in Fig. 3. [IMG_3] Fig. 3. Relationship between frog size and age. The magnitude of the annual size increment can be compared with the frog's age in that year, total lifespan, calendar year, sex, genotype, and other parameters. The set of data on such annual increments can be processed using analysis of variance. For example, Fig. 4 shows the results of a two-way ANOVA in which annual increment magnitude was treated as the dependent variable and frog age and sex as the influencing factors. As can be seen, the growth dynamics of males and females (combined into a single group with immature individuals) are quite similar. Maximum growth rate is attained before the fourth hibernation. It should be inferred that this pattern indicates the attainment of sexual maturity between the fourth and fifth hibernations. In the analysis reflected in Fig. 4, both the dependence of increment on age and the dependence of increment on sex are significant; the interaction term (differences in how increments change with age among individuals of different sexes) is statistically insignificant. Figure 5 shows the results of a two-way ANOVA in which age and genotype were treated as the main factors influencing frog increments. The effect of age, as in the previous case, remained significant, while the effect of genotype, surprisingly, proved insignificant. In Fig. 4 it can be seen that the curves corresponding to different genotypes run nearly parallel, and the difference between them may be regarded as statistical noise. Thus, skeletochronological data from all forms of green frogs distributed in the Siverskyi Donets diversity centre (namely P. ridibundus and diploid and triploid P. esculentus) can be analysed jointly. The approach used in the present study also allows assessment of which years were more or less favourable for frog growth. Such a comparison must, of course, be made using increments at the same age. Without this control, the age-related deceleration in frog growth rate (clearly visible in Fig. 4) would create the illusion that each successive calendar year is worse than the previous one. For this analysis we used increment data from the 4th, 5th, and 6th years of life, since these are the years for which we have the most numerous data. The results of this analysis are shown in Fig. 6. [IMG_4] Fig. 4. Dependence of annual increment magnitude on frog age and sex. [IMG_5] Fig. 5. Dependence of annual increment magnitude on frog age and genotype. [IMG_6] Fig. 6. Dependence of annual increment magnitude on calendar year. Comparison of annual increments across different calendar years revealed non-significant differences; however, a certain tendency is nonetheless apparent in Fig. 6. Of recent years, 2009 proved the most favourable for frogs, after which a certain declining trend in annual increments was recorded. A similar comparison conducted on a larger dataset would probably yield more reliable results. Acknowledgements The author of the article expresses deep gratitude to D. A. Shabanov for scientific supervision of this work and assistance in interpreting its results. The author also thanks A. V. Korshunov and O. V. Mykhailova for assistance in obtaining the 2013 sample. Its collection and processing involved second-year undergraduate students completing their teaching field practice in vertebrate zoology: M. S. Alkhovska, M. Yu. Kyt, A. O. Mykhailenko, A. A. Klysa, D. V. Kruhovyi, Yu. I. Kostiuk, and A. O. Mykhailychenko. An especially valuable contribution to the processing of this sample was made by E. V. Meleshko. The results of flow DNA cytometry kindly provided by S. N. Litvinchuk and Yu. M. Rozanov were of critical importance for interpreting the 2004–2009 collection data. In developing the methodology described in the present article, the author received invaluable assistance from E. M. Smirina, a pioneer of skeletochronological research in herpetology. References Bondareva A. A., Bibik Yu. S., Samylo S. M., Shabanov D. A. Cytogenetic characteristics of erythrocytes of green frogs from the Siverskyi Donets diversity centre of Pelophylax esculentus complex // Visnyk Kharkivskoho natsionalnoho universytetu imeni V. N. Karazina. Seriia "Biolohiia". – 2012. – Vyp.15 (№1008). – P. 116–123. Korshunov A. V. Ecological patterns of distribution of Pelophylax esculentus complex in the biotopes of the upper Siverskyi Donets river basin. Abstract of dissertation for the degree of Candidate of Biological Sciences / 03.00.16 — ecology. – Dnipropetrovsk, 2010. Smirina E. M. In vivo age determination and retrospective assessment of body size in the common toad (Bufo bufo) // Zoologicheskii zhurnal. – 1983. – Vol.63, No 3. – P. 437–444. Usova E. E., Shabanov D. A. On optimising the method of retrospective assessment of body size dynamics in members of Pelophylax esculentus complex (Amphibia, Ranidae) using skeletochronology // Zoocenosis-2009. Bioriznomanittia ta rol tvaryn v ekosystemakh. – Dnipropetrovsk, DNU, 2009. – P. 278–280. Shabanov D. A., Korshunov O. V., Kravchenko M. O. Which green frogs inhabit Kharkiv region? Terminological and nomenclatural aspects of the problem // Biolohiia ta valeolohiia. – Kharkiv: KhDPU, 2009. – Vyp.11. – P. 116–125. Shabanov D. A., Litvinchuk S. N. Green frogs: life without rules or a special mode of evolution? // Priroda. – 2010. – No 3. – P. 29–36. Borkin L. J., Korshunov A. V., Lada G. A. et al. Mass occurrence of polyploid green frogs (Rana esculenta complex) in Eastern Ukraine // Russian Journal of Herpetology. – 2004. – Vol.11, No 3. – P. 194–213. Plötner J. Die westpaläarktischen Wasserfroesche. – Bielefeld: Laurenti-Verlag, 2005. – 161 p. Roitberg E. S., Smirina E. M. Age, body size and growth of Lacerta agilis boemica and L. strigata: a comparative study of two closely related lizard species based on skeletochronology // Herpetological Journal. – 2006. – Vol.16. – P. 133–148.

Tablitsa 1. Doslidzhena materia

Forma

Vybirka,
rik zboru

Juvenilʹnykh,
ekz.

Samok,
ekz.

Samtsiv,
ekz.

Vseho,
ekz.

P. ridibundus

2004–2009

2013

P. esculentus,
2n

2004–2009

2013

P. esculentus,
3n

2004–2009

2013

Vseho

104