Breeding, Growth and Population Monitoring of Endemic Anurans Under Changing Climate in Murree and Ayubia National Park, Pakistan

Abstract

The mean earth temperature has increased 0.6 °C in the last century. Temperature is a key component of climate directly influence amphibian biology due to their ectothermic nature. Effects of various temperatures on breeding and growth of amphibians have been studied in Palearctic, Nearctic, Neotropical and Australian regions. However, empirical data are lacking from Oriental region. The present study examined that how tadpoles of Hazara Torrent Frog (Allopaa hazarensis) and Murree Hills Frog (Nanorana vicina) responded to different temperatures through metamorphosis, body size, developmental complications or deformities, fitness and survival. A preliminary experiment was carried out for the selection of temperature gradient for the trials. The temperatures were 18, 20, 22, 24, 26, 28, 30 and 32 °C. Five adult males and females, and twenty larvae (Gosner Stage 25 and 30) were subjected of each species for 24 hours at each treatment. The final selected temperatures were 18, 22, 24, 26 and 28 °C for A. hazarensis and 18, 20, 22, 24 and 26 °C for N. vicina. The temperatures (30, 32 °C) were excluded for they caused 100% mortality. A total, 360 adults (n=180, ♂= 90, ♀=90 for each A. hazarensis and N. vicina were collected from the wild. Of these 160 adults were untreated for investigation of breeding season (seasonal sex hormones concentration) and 200 adults were exposed at the selected temperature gradient to investigate effects of temperature on sex hormones concentration. The untreated frogs were captured from field and blood was extracted without any exposure to temperature, the adults of treated group were exposed to selected temperature for 24 hours. The blood was extracted from cardiac puncture of A. hazarensis and N. vicina by using insulin syringe (30 gauge, 1mL/cc). The plasma was separated by blood centrifuge at 3000 rotations per minute (rpm) for 30 minute, and Enzyme linked immunosorbent assay was performed following kits protocols. Box plots were used to present hormonal concentration data; figures were generated using the ggplot and ggpubr package. The concentrations of sex hormones in the wild individuals were found to be highest during May to August indicating their breeding season for both species. The concentration (pg/ml) of estradiol and testosterone increased with the rise in temperature in treated adult individuals. The highest concentrations of estradiol and testosterone were recorded as (A. xiv hazarensis, 146 ± 126, 1610± 56 pg/ml at 28 °C); (N. vicina, 186 ± 7, 1615± 58 pg/ml at 26 °C), respectively. A total 1000 larvae (n=500) of each species were subjected to the trials to investigate impacts of temperature on growth and development. These were divided into five batches of (100 individuals per batch, 25 tadpoles per replication) for each species. The larvae were kept in glass aquaria (each with L×W×H 38× 30× 42 cm) containing 15 L tap water. Temperature regulators (range 16-34 °C ± 0.5) were used to regulate temperature of water with a filter (kept on for four hours/ day) and aerator with a stone for air circulation. About half of the water was changed twice a week. The tadpoles were fed twice in a day (10 am and 4 pm) with boiled spinach with supplement food for fish and frogs. The development stages (Gosner 25-46) were recorded once a week. The trials duration was 21 and 24 weeks for the tadpoles of A. hazarensis and N. vicina, respectively. To determine mortality, number of, dead tadpoles were counted on daily basis. The morphometric measurements of tadpoles were recorded once time a week, along with scoring for visible gross morphological deformities present as edema (swelling of abdomen) and tail kink (twist/curve in tail). Box plots were used to present tadpoles morphometric measurement data. Heat maps were plotted and Pearson’s correlation coefficient was calculated to analyze data on growth and development data; figures were generated using the ggplot and ggpubr package. Forest plots were used to compare thermal sensitivity on adverse effects between the two frog species. The results revealed the tadpoles of A. hazarensis and N. vicina showed the same response to elevated temperatures under laboratory conditions, including faster metamorphosis, reduction in the body size, more frequent developmental complications or deformities such as edema and tail kinks, lower fitness and higher mortality at elevated temperatures (>26 °C). The comparison of thermal sensitivity between the two species showed that N. vicina was more sensitive to higher temperatures. Protocol was developed and validated for eDNA based monitoring of A. hazarensis and N. vicina. Species sequences were obtained from tissue-derived DNA of targeted species and species-specific primers were designed and validated based on specificity and sensitivity. At each site, seven 1 L water samples were collected. Filtration of all water samples was carried out within 24 hours of collection, using hand-held vacuum pump and 0.45- xv μm pore size cellulose nitrate filter (47mm diameter) and stored in 95% ethanol in a 2ml vial at room temperature until DNA extraction. Total DNA was recovered from each filtered sample using the QIAGEN DNeasy Power Water kit. Real-time PCR (qPCR) were carried out for amplification DNA. Separate multispecies occupancy models was fit to the visual detection data and the eDNA data in order to account for imperfect detection. Multispecies occupancy models allow for simultaneous modeling of covariates influencing occupancy and detection probability of individual species as well as covariates that influence the interaction of the species. A ~550 bp region of the 16S rRNA gene sequence of A. hazarensis and N. vicina was obtained using Sanger sequencing. Based on the resulting alignment of sequences, five primer sets were designed (Allopaa-1 to Allopaa-5) targeting only A. hazarensis and fourteen (Nano 1 to Nano 14) primers were designed targeting only N. vicina DNA. After evaluation, Allopaa-4 targeting (~50 bp) and Nano-3 (~90) selected for A. hazarensis and N. vicina, respectively. The top-ranked model for occupancy included effects of elevation and year on occupancy of the two individual species, but did not include an interaction term. Elevation had a significant negative effect on the occupancy of A. hazarensis, and a significant positive effect on occupancy of N. vicina. Detection probability using eDNA method was greater for both species, but particularly for A. hazarensis. Estimated occupancy probability was also higher using the eDNA data relative to visual detection data, although the magnitude of the difference was smaller and 95% confidence intervals overlapped for both species. The findings contribute to filling the information gap regarding impacts of temperature rise on biodiversity of high altitude forested montane ecosystems and is an important contribution to future studies associating biodiversity and climate change. The large scale eDNA based monitoring is suggested for status and distribution for the species. The eDNA method can be replicate for other aquatic organisms for their surveillance and monitoring. Keywords: Climate change, growth, breeding, environmental DNA, anurans