DOI: 10.5061/dryad.59zw3r2h6
title: Characterization of the reproductive strategy of invasive round goby (Neogobius melanostomus) in the Upper Danube River

description: Originating from the Black and Caspian Seas, the round goby (Neogobius
 melanostomus) has become one of the most successful invaders of freshwater
 ecosystems. This dataset was created to provide a characterization of the
 reproductive strategy of an established round goby population in the Upper
 Danube River including sex ratio, fluctuations of gonadosomatic index
 (GSI), analysis of timing of spawning as well as of clutch and egg size.
 Data was collected for 1.5 years and time-series analysis was used to
 examine the influence of temperature, photoperiod and discharge on the
 reproductive strategy of round gobies. Further, clutch and egg sizes of
 round gobies as well as sex ratio of the population was analyzed.
description: <strong>Study area: </strong>The Danube is the second
 longest river with the second largest catchment area in Europe.The studied
 population of round gobies was identified based on previous populations
 screenings across the Upper Danube River and was located between Bad
 Abbach (48°56'22.9"N 12°02'18.3"E) and Saal an der
 Donau (48°54'35.9"N 11°55'27.6"E), Bavaria, Germany.
 The sampling site was located in the navigable part of the River, where
 river banks consisted mainly of artificial rip-rap structures without
 canopy cover. <strong>Field sampling:
 </strong>Round gobies were collected from April 2022 until November
 2023 twice a month by electrofishing or baited fish traps at three
 locations between Bad Abbach and Saal an der Donau, Bavaria, Germany. In
 total, eight electrofishing campaigns between June 2022 and June 2023 were
 conducted along the rip-rap structures at the shoreline of the River.
 Three 30m-replicate stretches were sampled at each location during
 electrofishing.  On all other sampling dates, baited fish traps were
 placed along the same stretches in <strong>~</strong>0.4-1.5m
 depth for 24h per sampling date (35 sampling dates in total). After
 collection, all gobies were euthanized using an overdose of the
 anaesthetic MS-222 (Tricaine), while all native fish were carefully
 returned to the River. To avoid degradation of tissue, gobies were
 transported to the laboratory (Aquatic Systems Biology, Technical
 University of Munich) on ice in a cooler and were frozen afterwards at
 -20°C for further analysis. Temperature data was obtained from a nearby
 measuring station in Kelheim (48° 55' 6.24 N 11° 53' 11.76 E
 )(Gewässerkundlicher Dienst Bayern 2024). Discharge data was obtained from
 the same measuring station (Hochwassernachrichten Dienst Bayern 2024).
   <strong>Gonado-somatic index (GSI):
 </strong>A total of 1041 females (size 84mm +/- 21) and 1026 males
 (size 84mm +/- 21) were dissected. Before dissection, gobies were patted
 dry with a paper towel, total length was measured to the nearest
 millimeter and fish were weighed to the nearest 0.001g using precision
 scales (Kern ADJ 100-4). After dissection, the wet weights of the gut
 content and gonads were recorded. The somatic Mass
 (M<sub>s</sub>) and GSI were calculated according to Brandner
 et al. 2018: M<sub>s</sub> =
 M<sub>t</sub> – (M<sub>indexed organ</sub> +
 M<sub>g</sub>) with M<sub>t</sub> =  total body
 mass and M<sub>g</sub> = gut content mass
 GSI = 100 x M<sub>gonads</sub>
 M<sub>s</sub> <sup>-1</sup>
 <strong>Clutch size: </strong>Round goby eggs were
 retrieved using spawning traps consisting of a roof tile with a paving
 stone attached on top with cables ties. This method allowed easy
 detachment of stones from the roof tiles and access to the clutches
 without damaging them. The spawning traps were placed at the same
 locations where round gobies were collected and additionally in the Marina
 in “Saal an der Donau” (48°54'36"N 11°55'31"E) close
 to the uppermost stretch of goby collection. The traps were installed in a
 depth of <strong>~</strong>0.4-1.5m in the Danube and ~3m in
 the Marina, respectively and were checked for clutches every week. If
 clutches were present, a translucent foil with an imprinted grid was
 placed over the clutch and multiple pictures were taken. The photos were
 used to determine the clutch area in cm² and the average number of eggs
 per cm². Subsequently, the number of eggs per clutch was calculated
 (clutch area in cm² x number of eggs per cm²). Eggs were taken from each
 clutch using tweezers and preserved in 96% (v/v) ethanol. Height and width
 of eggs were measured using a stereo microscope (Binocular Olympus SZX10,
 Olympus Germany GmbH) with a 20-fold-magnitude and the software cellSens
 (Olympus corporation). <strong>Statistical
 Analysis </strong> <strong>Sex ratio:
 </strong>For the analysis of sex ratio, data was pooled and analyzed
 per individual month. Additionally, an overall sex ratio was calculated.
 To test for differences between the observed sex ratio and an expected 1:1
 equilibrium, a chi²-test was computed.
 <strong>Clutch Size and Gonadosomatic Index (GSI):
 </strong>Shapiro-Wilk and Levene Test were performed to test for
 normal distribution and homogeneity of variances of data. In a first step,
 a regression of GSI with fish size was computed for both female and male
 round gobies to explore the relationship between the two parameters.
 Further, differences in GSI between fish caught by electrofishing and
 traps were examined to check whether both can be included in the following
 analysis. For the analysis of GSI, data were then pooled and differences
 between individual months were analyzed. Since assumptions for parametric
 tests were not met for GSI, non-parametric Kruskal Wallis test with
 Bonferroni correction for multiple testing was performed. Assumptions of
 normal distribution and homogeneity of variances were met for clutch and
 egg size. Therefore, ANOVA with post-hoc Tukey test was performed to
 analyze differences of clutch and egg sizes between sampling
 dates. <strong>Time-series analysi:
 </strong>To analyze the influence of water temperature, photoperiod
 and discharge on GSI, the correlation between these time-series was
 examined using cross-correlation. As data are required to be stationary
 and prewhitened for cross-correlation, Kwiatkowski-Philipps-Schmidt-Shin
 (KPSS) test was used to check for stationarity. All data showed to be
 stationary, so no further differencing (elimination of a linear trend by
 computing differences between consecutive observations to make a time
 series stationary; (Shumway and Stoffer 2016)) of data was conducted. To
 account for the influence of long-term trends in the input (temperature,
 photoperiod or discharge) or the output time series (GSI), that may hide
 or suggest a significant correlation between the two time-series,
 prewhitening of data was performed. For prewhitening of data an ARIMA
 Model (Box and Jenkins 2013) was fitted to the input time-series
 (temperature, photoperiod or discharge), which was then applied to the
 output time-series (GSI) according to Probst et al. 2012 and the
 cross-correlation between the two prewhitened time-series was performed.
 As round gobies were sampled on 35 occasions within 625 days one lag in
 the time series analysis (time gap between two time series) was calculated
 as follows: <strong>625 days/35 = 17.8
 (</strong><strong>≈ 18) days</strong>
 All analysis were computed using the statistical software R (Ver.
 4.1.1, R Development Core Team, 2009) and significance was accepted for
 <em>p</em>&lt;0.05.
description: # Characterization of the reproductive strategy of invasive round goby
 (*Neogobius melanostomus*) in the Upper Danube River
 [https://doi.org/10.5061/dryad.59zw3r2h6](https://doi.org/10.5061/dryad.59zw3r2h6) Originating from the Black and Caspian Seas, the round goby (*Neogobius melanostomus*) has become one of the most successful invaders of freshwater ecosystems. This dataset was created to provide a characterization of the reproductive strategy of an established round goby population in the Upper Danube River including sex ratio, fluctuations of gonadosomatic index (GSI), analysis of timing of spawning as well as of clutch and egg size. In the Danube, the round goby population was found to be female dominated, however fluctuations in magnitude of female bias were observed between months. Monitoring of the population across 1.5 years revealed that GSI was highest from April to June, while lowest values were observed in August and September. Using time-series analysis, a delayed effect of temperature on GSI was found, while a quicker response of GSI levels to photoperiod and discharge was observed. GSI increased with body size and eggs were found to be significantly larger in May, however clutch sizes did not differ between months. ## Description of the data and file structure **Rawdata_GSI.csv:** Rawdata collected during dissection of gobies; column names: date, season, month, size: total length in cm; total weight: weight in g, weight gut content: weight of gut content in g, weight tissue: weight of fish without gut content in g; weight gonads: weight of gonads of fish in g, weight liver: weight of liver in g, sex, GSI: gonadosomatic index (see description of methods for calculation of GSI), method: method used to collect the fish; zero values for “weight gut content” mean that no gut content was found; zero values for “weight gonads” and “weight liver” mean that the organs were not found or were too small to be weighed; zero values for “weight gonads” subsequently led to zero values for “GSI”, as GSI could not be calculated **Rawdata_Clutches.csv:** Rawdata of clutches; column names: date: date of collection; location: location of collection; Month: month of collection; number: number of clutches: serial number of clutches per sampling date; Eggnumbers: number of eggs in the clutch; egg/cm: number of eggs per cm, clutchsize: Size of the clutch in mm², height: mean height of eggs in m, width: mean width of eggs in m **Rawdata_Discharge.csv:** Rawdata of discharge for the entire sampling period; column names: month, date, mean: mean discharge of the day, maximum: maximum discharge of the day, minimum: minimum discharge of the day **Rawdata_Photoperiod.csv:** Rawdata of photoperiod for the entire sampling period; column names: date, month, Time_long: long format of photoperiod hours:minutes:seconds, time: short format of photoperiod **Rawdata_Temperature.csv:** Rawdata of temperature for the entire sampling period; column names: Date, Temperature: mean temperature of the day, month **Rawdata_sexratio.csv:** Rawdata of sex ratio for electrofishing campaigns (only data of electrofishing campaigns was used as this method is the least selective); column names: month, sex, number: number of each sex per sampling date; zero values in the column “number” mean that no individuals of one sex were caught on this date **Time-series_Temperature.csv:** Data used to compute the time-series analysis for gonadosomatic index and discharge; column names: date, monthly mean: mean of temperature per month, temperature, GSI: gonadosomatic index **Time-series_Photoperiod.csv:** Data used to compute the time-series analysis for gonadosomatic index and discharge; column names: date, photoperiod: photoperiod in h, GSI: gonadosomatic index **Time-series_Discharge.csv:** Data used to compute the time-series analysis for gonadosomatic index and discharge; column names: date, discharge: discharge in m/s, GSI: gonadosomatic index **01 Visualization.R:** Code for visualization of GSI, temperature, photoperiod and discharge data using the files Rawdata_GSI.csv, Rawdata_Temperature.csv, Rawdata_Photoperiod.csv and Rawdata_Discharge.csv **02 GSI, Clutch and Egg Size, Sex Ratio.R:** Code for assumption tests (shapiro test, levene test), analysis of GSI data, clutch and egg sizes and visualization of egg sizes using the files RawdataGSI.csv, Rawdata_Clutches.csv, Rawdata_sexratio.csv, **03 Linear Regression.R:** Code for computing the linear regression of body size with gonadosomatic index (GSI) and visualization of Regression using the file Rawdata_GSI.csv **04 Time series analysis.R:** Code for test for stationarity of data, prewhitening of data and time-series analysis using the files Time-seriesTemperature.csv, Time-series_Photoperiod.csv, Time-series_Discharge.csv ## Sharing/Access information Temperature and Discharge data was derived from the following sources: * Temperature: [https://www.gkd.bayern.de/de/fluesse/wassertemperatur/kelheim/kelheim-10053009/messwerte/tabelle](https://www.gkd.bayern.de/de/fluesse/wassertemperatur/kelheim/kelheim-10053009/messwerte/tabelle) * Discharge: [https://www.hnd.bayern.de/pegel/donau_bis_kelheim/kelheim-10053009/tabelle?methode=abfluss&](https://www.hnd.bayern.de/pegel/donau_bis_kelheim/kelheim-10053009/tabelle?methode=abfluss&) ## Code/Software Data analysis was computed using the statistical software R (Ver. 4.1.1, R Development Core Team, 2009). The used R codes for data visualization and analysis are provided (see description of data and file structure).