Aquatic Invasions (2018) Volume 13, Issue 2: 277–288 DOI: https://doi.org/10.3391/ai.2018.13.2.08 Open Access © 2018 The Author(s). Journal compilation © 2018 REABIC Research Article

Range expansion of an invasive goby in the Tiber basin (Central Italy): effects on the abundance and shelter occupancy of a congeneric native goby

Laura Pompei1,*, Daniela Giannetto2 and Massimo Lorenzoni1 1Dipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia, via Elce di Sotto, 06123 Perugia, Italy 2Department of Biology, Faculty of Sciences, Muğla Sitki Koçman University, 48000 Muğla, Author e-mails: [email protected] (LP), [email protected] (DG), [email protected] (ML) *Corresponding author

Received: 21 April 2017 / Accepted: 24 January 2018 / Published online: 7 March 2018 Handling editor: Jean Vitule

Abstract

Padogobius nigricans and are two gobies native to Italy and characterized by an allopatric distribution: P. nigricans is endemic to the Tyrrhenian catchment of central Italy, while P. bonelli is endemic to the Adriatic catchment of Northern Italy. In the past two decades P. bonelli has successfully established in central Italy outside of its original area of distribution, coming into contact with populations of P. nigricans. The superiority of P. bonelli in competition for breeding sites is well documented by laboratory studies; however, little evidence from the field has been provided. In this study, the occurrence and abundance of the two gobies were investigated in the River Tiber basin (central Italy) to assess the impacts of P. bonelli expansion on P. nigricans populations. Moreover, to investigate nest interference by P. bonelli towards P. nigricans, shelter occupancy of the two Italian gobies was investigated in the field with respect to individual density. Sampling was conducted by electrofishing in 77 sites throughout the upper River Tiber basin and nest occupation was analyzed in 8 river stretches. Padogobius bonelli has further expanded its distribution in recent years, following both natural and human-mediated dispersal patterns. It is numerically dominant over P. nigricans and fewer P. nigricans juveniles were found in the invaded than the uninvaded areas. Depending on adult density, P. bonelli can occupy up to 97% of nest sites and can seize a significant percentage of shelters even at low adult abundance. The expansion of P. bonelli into newly colonized areas in recent years has increased concern for the conservation of P. nigricans. In the invaded areas, the survival of P. nigricans populations is mostly through migration of individuals from the uninvaded areas located upstream of weirs, allowing isolated populations of this vulnerable and endangered to persist. Key words: Padogobius bonelli, Padogobius nigricans, non-native species, biological invasions, competition

probably introduced unintentionally during restocking Introduction activities with species of fisheries interest from northern Italy (Bianco and Ketmayer 2001). The co- Padogobius nigricans and Padogobius bonelli are occurrence of P. nigricans and P. bonelli has been two vicariant freshwater goby species native to Italy. reported from several locations in central Italy Padogobius nigricans is endemic to the Tyrrhenian (Zerunian 2002; Lorenzoni et al. 2006), including the drainages of central Italy (Bianco 1995), while River Tiber basin, the main watercourse of the Italian P. bonelli is endemic to northern Adriatic basins, peninsula. Here P. bonelli was first noticed in 1996 from the river Vomano (Italy) to the Krka drainages when it was found in small tributaries of the River (Croatia) (Kottelat and Freyof 2007) (Figure 1A). Tiber (Mearelli et al. 1996). From there, the species However, in the past two decades, P. bonelli has has colonized the upper and middle course of the widely extended its distribution range in the River Tiber and has spread into several adjoining Tyrrhenian basins of central Italy, where it was tributaries (Lorenzoni et al. 2007).

277 L. Pompei et al.

Figure 1. A) Original distribution of Padogobius nigricans (light grey) and P. bonelli (dark grey) in Italy and localization of the River Tiber basin (striped area); B) detail of the study area divided into sub-basins showing the 77 sampling sites investigated during 2011–2015 (1: Tiber, 43 sites; 2: Chiascio-Topino, 12 sites; 3: Paglia, 16 sites; 4: Nestore, 6 sites).

Aggressive behaviour, broad tolerance to environ- common reproductive strategies (Zerunian et al. 1988; mental alterations (Zerunian 2002) and high energy Mecatti et al. 2010). In both species, females lay eggs allocation to reproduction (Pompei et al. 2016a) have on the ceiling of the shelter that is created under favoured the establishment of viable P. bonelli popu- stones, and males are responsible for parental care lations in the newly invaded ecosystems. Padogobius (Mecatti et al. 2010). Padogobius nigricans only bonelli was assessed as a major threat to the survival exhibits territoriality after laying eggs (Zerunian et of P. nigricans, which is listed as “vulnerable” al. 1988); in contrast, P. bonelli males defend a specific according to the IUCN Red List of Threatened territory, driving out intruders (Torricelli et al. 1986). Species (Crivelli 2006) and the Red List of Italian There are no differences in timing of the breeding Vertebrates (Rondinini et al. 2013). One of the most season (Pompei et al. 2016a, b) and where the two negative impacts ascribed to goby invasions is com- species cohabit, P. bonelli can outcompete P. nigricans petition with native which is more severe for for spawning sites preventing its reproduction through those species that share similar ecological requirements aggressive behaviour (Mecatti et al. 2010). Interspe- (Grabowska et al. 2016). Padogobius nigricans and cific antagonistic interactions may be the main cause P. bonelli share an analogous ecological niche, a of the decline of P. nigricans populations, along high degree of diet overlap (Pompei et al. 2014) and with pollution and habitat alterations (Crivelli 2006).

278 Distribution, abundance and nest occupancy of two congeneric gobies

Nevertheless, no studies on interference competition Methods in the field are available and the hypothesis of reproductive failure of P. nigricans caused by Distribution and abundance P. bonelli is based only on indirect evidence. In The investigated area is situated in the upper and some river courses, rapid decline of P. nigricans was middle basins of the River Tiber, the third-longest observed in coincidence with P. bonelli estab- river in Italy. The area was divided into four sub- lishment (Nocita and Zerunian 2007); in other cases, basins: three corresponding to those of the main a low number of young-of-the-year P. nigricans tributaries of the River Tiber (Chiascio-Topino, were found in the presence of P. bonelli (Pompei et Nestore and Paglia ), while the main course of al. 2016b). The main aims of the present study were the River Tiber and the minor tributaries were to update the information on the abundance and considered as a separate sub-basin, henceforth called distribution of the non-native P. bonelli in the upper Tiber (Figure 1B). River Tiber basin twenty years after its introduction. Sampling focused mainly in the Tiber, since During monitoring, particular attention was given to P. bonelli was not found in the other sub-basins sites not yet invaded by P. bonelli (i.e. with “intact” during previous monitoring (Lorenzoni et al. 2010). populations of P. nigricans). These sites were During 2014–2015 surveys, 43 stations located in usually located upstream of weirs (small dams up to the Tiber were sampled. These data have been inte- 3 m high that interrupt river continuity) (Pompei et grated with the data collected from 34 sampling al. 2016b). Thus the possible impact of P. bonelli on stations in the period 2011–2014 during the the native goby was assessed by comparing the monitoring for the 1st update of the Regional densities and population structure of P. nigricans in Map, a project which aimed to monitor the fish invaded and uninvaded systems. We hypothesize fauna of watercourses of Umbria Region (Lorenzoni that P. bonelli has further spread into new adjacent et al. 2010). Thus, 77 sampling stations in 36 river areas in the past few years, overcoming the native courses were considered in the analyses (Figure 1B). species and, in some cases, replacing it. We expected Fish sampling was carried out by electrofishing a reduction in young-of-the-year and an alteration of (electroshocker model: ELT62II-GI, direct current, the population structure of P. nigricans in the 300 V, 10–100 Hz) using the removal method (Moran presence of P. bonelli due to nesting interference. 1951; Zippin 1958) in the low flow period (June– We further hypothesize that uninvaded popula- September). Sampling was conducted by the same tions of P. nigricans, usually located upstream of staff to ensure consistent effort among sites. For small weirs, could support the conservation of the each river, the length of the stretch to be sampled invaded populations downstream by migration of was defined as 10 times the width of the wetted individuals, since downstream drifting of early stages channel. Length and width of the river sector examined is a common dispersal mechanism for some goby were measured to obtain the area necessary to 2 species (Hayden and Miner 2009; Brownscombe and calculate density (individuals/m ) of the two species. Fox 2012; Janáč et al. 2013). To test this, the popu- Specimens were lightly anaesthetized with 2-phen- lation structure of P. nigricans in the invaded sites oxyethanol to facilitate handling without harming was analyzed distinguishing between populations individuals. For those specimens whose distinction downstream of weirs (i.e. with an uninvaded on a morphological basis aroused doubts, a small population of P. nigricans upstream) and the other (< 5 mg) caudal fin piece was collected and stored in absolute alcohol at −20 °C for further molecular invaded populations located in streams without any identification through a specially designed PCR- weirs. Moreover, to gain insight the mechanism of RFLP protocol (see below). For each specimen, total competition for breeding territory in the field, the length (TL) and weight (W) were measured to the occupation of nest sites by the two species with nearest 0.1 cm and 0.1 g, respectively. A sample of respect to individual density was analyzed. Padogobius scales was removed from dorso-lateral or ventro- bonelli was expected to occupy the majority of the lateral rows of the caudal peduncle of each individual nests sites as well as bigger nests, because nest size (Pompei et al. 2014) and stored in 33% ethanol. is a predictor of male quality (Lindström 1988; After the field operations, all individuals were released Bisazza et al. 1989), and males of both species are at the site of capture. likely to compete for larger stones which may allow For age determination, fish scales were observed mating with more than one female. Lastly, nest size under a stereo microscope and the annuli were and the number of eggs in the nest were analyzed counted; two independent age determinations were and compared between the two species. made by two different operators. An additional age

279 L. Pompei et al. determination was carried out in cases of contrasting Once each nest was assigned to the correct results. The microscopic scalimetric method was species, the number of nests occupied by each species validated by means of length-frequency distribution was counted. Then, to check for species dominance, (Bagenal and Tesh 1985). a Chi-Squared test was performed for each station, The density of species (D, individuals/m2) was considering the number of nest of the two species as quantified for each sampling site and also expressed the observed values, while the expected values were as the relative percentage. A Wilcoxon Signed Rank estimated for each species as the number of nest test was then used to assess differences in densities expected on the basis of the relative percentage of between P. bonelli and P. nigricans in the sites the the adults found in the same station. two species cohabitate (invaded sites). For P. nigricans, The total surface areas of stones on which batches the densities found in the invaded and in uninvaded of eggs were found were measured (stone area) by sites were compared by Mann-Whitney U test to test examining the photos using ImageJ software ver. 1.51 if the presence of the non-native goby had affected (https://imagej.nih.gov/ij/). The number of unhatched the abundance of the native one. eggs in each nest was counted. Nests that contained The age composition of P. nigricans populations hatched eggs were excluded (Marconato et al. 1989). was analyzed separately for invaded and uninvaded To test the hypothesis that P. bonelli can both sites. Specimens were grouped into two categories: occupy the bigger nests and produce more eggs per young-of-the-year (age 0+ years) and adults (age > 1+ nest, a comparison between the two species in stone years). A Chi-squared test was used to assess diffe- area and number of eggs in the nest was carried out rences in the proportion of these two groups in the using Wilcoxon Signed Rank tests. These analyses invaded and uninvaded areas. An analogous compa- were conducted comparing the two species both rison was carried out for P. nigricans only in the within each of the six invaded stations and using the invaded areas, considering the populations inhabiting data of all the six stations pooled. the stations with weirs upstream and the populations Moreover, to investigate whether the presence of located in streams without any weir. the non-native species could have affected the shelter choice of the native one, the number of eggs in Nest occupation invaded and uninvaded areas were compared using a Mann-Whitney U test. These analyses were conducted Eight nesting stations were chosen. Six were located in using the pooled data of stone area and number of different tributaries of the upper River Tiber (Aggia1, eggs in the invaded and uninvaded areas. A linear Carpina1, Lanna, Resina, Soara and Vaschi rivers) regression was used, for each of the two species, to where both gobies are found, and two (Aggia2 and assess whether the number of eggs laid in a nest was Carpina2, upstream of the weirs) were located in correlated with the size of the nest stone. uninvaded areas (Figure 2). These sites were chosen because they were comparable in terms of environmen- Genetic analysis tal characteristics (Lorenzoni et al. 2007): they had pre- dominantly run morphology and the substrate mainly Padogobius nigricans and P. bonelli appear very consisted of cobbles and stones. The average area similar in shape, colour and size, so are difficult to sampled (± SD) was 210 ± 37 m2, water temperature distinguish in the field (Kottelat and Freyof 2007), was 18.4 ± 2.3 °C and mean depth was 0.38 ± 0.11 m. especially the smaller specimens (< 3 cm total length). Each station was sampled four times. The first Thus a PCR-RFLP technique using a 16S rRNA surveys were carried out soon after sampling for den- gene isolated from fin samples was performed. DNA sities of the two species; the other three nest surveys was extracted from fins using the Wizard Genomic were conducted weekly in June 2015. Each site was DNA Purification Kit (Promega) (Vercillo et al. 2004). searched for nest sites and every stone large enough Polymerase Chain Reaction (PCR) amplifications of to host a nest (approx > 50 cm2, Lugli et al. 1990) was the 16S rRNA segments were performed using turned over. Stones containing batches of eggs were primers Padof 5’-GTAGCGTAATCACTTGTCTT-3’ photographed. Because it is impossible to determine and Pador 5’-CAACATCGAGGTCGTAAAC-3’, the parent species from a nest, several samples of eggs obtaining a fragment of 426-bp. Amplifications used from each nest were collected, put in absolute ethanol a total volume of 25 μl, including 2 μl of total DNA, and stored at −20°C for molecular identification, follo- 10 μl of deionised sterile water (dsH2O), 0.2 μl of wing the same protocol as for the fin clips (see below). 2.5 mM of each primer and 12.5 μl of PCR Master After the field operations, stones were carefully placed Mix, following the Promega standard protocol. The in their original locations, taking care not to damage PCR thermal profile was as follows: 94 °C for 3’, 30 the eggs. cycles with 94 °C for 30’’, 54 °C for 30’’, 72 °C for 1’,

280 Distribution, abundance and nest occupancy of two congeneric gobies

Figure 2. A) Distribution of Padogobius bonelli in the River Tiber basin in the period 2006–2010 (black solid line, from Lorenzoni et al. 2010); B) Distribution and relative abundance, represented by pie charts, of P. bonelli (black) and P. nigricans (white) in the sampling stations of the Tiber (dark gray area) and other sub-basins (light gray areas) of the upper River Tiber basin during the investigated period (2011–2015). Asterisks indicate the first records of P. bonelli in the Rivers Aggia and Cerfone in 1996 (Mearelli et al. 1996). The numbers in the pie charts indicated the sites in which the nest investigation was conducted (1: Vaschi1; 2: Soara1; 3: Aggia1; 4: Aggia2; 5: Carpina1; 6: Carpina2; 7: Lanna; 8: Resina1); C) Frequency distribution of P. bonelli relative abundance in the Tiber sub-basin (N stations = 37) sampled in the period 2011–2015. For details see Supplementary material Table S1.

281 L. Pompei et al. and a final extension at 72 °C for 10’. Amplicons were species was totally replaced by the non-native one. purified with ExoSAP kit (ExonucleaseI and Shrimp- Only 10 uninvaded sites were found. Among them, Alkaline-Phosphatase, Amersham) and sequenced two were located in the southern part of the Tiber with an automated DNA sequencer (BMR Genomics) sub-basin and the other eight were located upstream (Genebank accession numbers: KM406308 for P. nigri- of weirs. In these 10 sites, the density of P. nigricans cans and KM406309 for P. bonelli). The sequences (median and inter-quartile range of density: 0.90, obtained were scanned with RestrictionMapper 0.31–1.77 individuals/m2) was significantly higher (http://www.restrictionmapper.org) and two endonu- than the density reached in the 23 sites where P. cleases were identified: SacII (CCGC_GG) produces bonelli is also present (Mann-Whitney U test: n = 43, two fragments of 239-bp and 187-bp only in Z = 3.685, p = 0.021). P. nigricans, while AflII (C_TTAAG) produces two Padogobius nigricans in the invaded areas was fragments of 147-bp and 279-bp only in P. bonelli. characterized by a lower proportion of juveniles Both restriction enzymes were assayed on amplicons. (9.4%) when compared to uninvaded areas (24.1%) The restriction mixtures (10 μl of amplicon, 3 μl of (χ2 = 18.176, df = 1, p < 0.01) (Figure 3A, B). bdH2O, 1.5 μl of enzyme-buffer and 0.5 μl of restric- When examining the age composition of P. nigri- tion enzyme) were incubated at 37 °C for 3 h, and cans in the invaded stations downstream of the weirs separated through 2% agarose gel electrophoresis. (i.e. with intact population of P. nigricans upstream) The digestion with SacII and AflII endonucleases (N = 8) and invaded stations without weirs (N = 15), produced restriction patterns characteristic for each some interesting considerations emerged. Stations species allowing the distinction. with intact populations of P. nigricans upstream had a more juveniles (N = 16, 16.5% of the total) than Results invaded stations without weirs (N = 7, 4.7% of the total) (χ2 = 11.339, df = 1, p < 0.01; Figure 3C, D). Distribution and abundance A total of 2315 gobies (696 P. bonelli, 1619 Nest occupation P. nigricans) were caught at the 77 sites. A total of 313 nest sites were examined in 8 locations. Padogobius bonelli was not found in the Nestore In rivers Aggia1 and Resina, the majority of the and Paglia sub-basins. In contrast, in the Chiascio- nests were occupied by P. bonelli. In both tributaries Topino basin three specimens of the non-native goby only 2 nests out of 77 and 33 nests, respectively, were found during the last survey in the northern- belonged to P. nigricans. In the River Resina the most station of the River Chiascio (Density P. bonelli 2 number of nests observed for the two species did not = 0.006 individuals /m ; Density P. nigricans = 0.24 2 2 differ from the expected values (χ = 0.185, df = 1, individuals /m ) (Figure 2B, Supplementary material p = 0.999), while in the River Aggia, P. bonelli Table S1). occupied a greater number of nests than the expected values based on species abundance (χ2 = 12.501, df = 1, Tiber sub-basin p < 0.01). In River Carpina1, P. bonelli also spawned To date, P. bonelli has colonized nearly the entire in most of the nests (Table 1). In Vaschi1 the number Tiber sub-basin, spreading into new water courses of nests observed did not differ from the number of (Figure 2A, B, Supplementary material Table S1). nest expected (χ2 = 0.008, df = 1, p = 0.99), and the Among the 37 sites investigated in 2014–2015 in proportion of nests occupied by the two species which gobies were sampled, the non-native goby exactly reflects the relative abundance of individuals was found in 27 sites (Table 1) and both gobies co- (Table 1). Similarly, in Lanna the percentage of the occurred in 23 sites. Padogobius bonelli was nests are close to those of the individuals. Padogobius numerically dominant to P. nigricans in 20 of the nigricans occupied 60% of the nests and accounted co-occurrence sites, representing more than 75% of for 67% of individuals. In Soara1, P. nigricans the gobies sampled in 12 sites (Figure 2C). occupied a slightly greater percentage of sites Padogobius bonelli achieved significantly higher compared to P. bonelli, despite the higher abundance densities than P. nigricans in the 23 co-occurring of adults (Table 1). sites (median and inter-quartile range: P. bonelli = Padogobius nigricans occupied larger nests and 1.61, 1.00–2.50 individuals/m2; P. nigricans = 0.50, produced more eggs per nest in the invaded areas 0.160–1.10 individuals/m2; Wilcoxon Signed Rank (Table 2), even if the differences between the two test: n = 23, Z = 3.376, p < 0.01). species in the mean number of eggs laid were not Four local extinctions of P. nigricans were significant (Table 2). For P. nigricans, no differences recorded (Figure 2B). In these sites, the native in nest stone areas nor the number of eggs per nest

282 Distribution, abundance and nest occupancy of two congeneric gobies

Table 1. Abundance of Padogobius nigricans (Pn) and P. bonelli (Pb) in the 37 sites of the Tiber sub-basin sampled during 2014–2015 expressed as density (D ind m-2) and relative abundance (%), and number and percentage of nests of the two species collected in four sampling dates in the 8 sites investigated in 2015 for the analysis of nest occupation. The results of comparisons by Chi-squared test (χ2) between the number of the nests and the abundances of the two species are provided. D Relative abundance Nest percentage Nest number Chi-squared test Sites (individuals m-2) (%) (%) Pn Pb Pn Pb Pn Pb Pn Pb χ2 p df 1 Vaschi1 0.83 0.87 48.8 51.2 23 24 49.0 51.0 0.008 0.999 1 2 Soara1 1.04 0.18 85.2 14.8 18 14 56.2 43.8 10.596 <0.01 1 3 Aggia1 0.48 1.72 21.8 78.2 2 75 2.6 97.4 12.501 <0.01 1 4 Aggia2 0.61 0.00 100.0 0.0 22 – 100.0 – 5 Carpina1 0.63 0.88 41.7 58.3 24 44 35.3 64.7 0.408 0.650 1 6 Carpina2 1.77 0.00 100.0 0.0 14 – 100.0 – 7 Lanna 0.16 0.08 66.7 33.3 12 8 60.0 40.0 0.221 0.770 1 8 Resina1 0.10 1.03 8.8 91.2 2 31 6.1 93.9 0.185 0.999 1 9 Assino1 0.07 0.87 7.2 92.8 10 Assino2 0.56 1.98 22.2 77.8 11 Assino3 2.30 9.33 19.8 80.2 12 Assino4 0.45 1.63 21.6 78.4 13 Cerfone1 1.05 3.20 24.7 75.3 14 Cerfone2 0.57 5.29 9.7 90.3 15 Cerfone3 0.00 7.00 0.0 100.0 16 Canale Sovara 0.61 1.83 25.0 75.0 17 Mussino 0.00 0.11 0.0 100.0 18 Naia1 0.22 0.00 100.0 0.0 19 Naia2 0.40 0.00 100.0 0.0 20 Niccone1 0.14 1.62 8.1 91.9 21 Niccone2 0.08 0.00 100.0 0.0 22 Puglia1 5.67 1.00 85.0 15.0 23 Puglia2 3.90 0.00 100.0 0.0 24 Resina2 0.32 0.00 100.0 0.0 25 Rio di Bosco 2.10 0.00 100.0 0.0 26 Soara2 0.00 1.90 0.0 100.0 27 Sovara1 1.10 2.50 30.6 69.4 28 Sovara2 0.50 2.63 16.0 84.0 29 Sovara3 1.00 0.00 100.0 0.0 30 Tevere1 0.30 1.20 20.0 80.0 31 Tevere2 2.30 4.30 34.8 65.2 32 Tevere3 0.39 0.69 35.9 64.1 33 Tevere4 0.14 1.10 11.6 88.4 34 Nestore1 0.00 8.83 0.0 100.0 35 Nestore2 0.13 1.63 7.5 92.5 36 Vaschi2 1.73 0.00 100.0 0.0 37 Ventia 0.23 1.10 17.5 82.5 were found between invaded and uninvaded areas The establishment of P. bonelli in the upper River (Mann-Whitney U test: nest stone areas: n = 117, Tiber and the further expansion into newly colonized Z = 1.200, p = 0.230; no. eggs: n = 98, Z = 1.631, areas has increased concern for the conservation of p = 0.103) (Table 3). For both species the number of P. nigricans. Several alarming trends have emerged eggs in the nests were positively correlated with from the present research, which has implications for stone area (P. bonelli: r2 = 0.193, r = 0.439, p < 0.01; the survival of the native populations. P. nigricans: r2 = 0.392, r = 0.731, p < 0.01) (Figure 4). The presence of P. bonelli is more concentrated in the upper part of the Tiber basin: its distribution and Discussion abundance are closely tied to the distance from the points of first report i.e., to the time-lapse from the Large numbers of fish species have been introduced first introductions of the species. On average, into the River Tiber basin in recent decades P. bonelli reached significantly higher densities than (Lorenzoni et al. 2006). Some have had damaging P. nigricans in the invaded areas. In the sites with effects on native fish fauna (Giannetto et al. 2012). a longer invasion history, it was often the numerically

283 L. Pompei et al.

Figure 3. Age frequency distribution of 1) Padogobius nigricans considering all the stations of the Tiber sub-basin divided into A) invaded and B) uninvaded areas; and 2) P. nigricans considering only the stations of the Tiber sub-basin invaded by P. bonelli divided into C) stations with weirs upstream and D) stations without weirs upstream. Number of specimens and number of stations considered in each graph are indicated. dominant species. Padogobius bonelli has also invaded Hayden and Miner 2009; Janáč et al. 2013) and do new river stretches downstream, moving its range not exhibit strong swimming abilities (Bergstrom et edge farther south. The southernmost station in al. 2008). However several studies have pointed out which P. bonelli was found is located approximately exceptional dispersal abilities of gobies during range 140 km of river length from the site of the first expansion, especially in lakes and in downstream report in 1996. At this station the non-native species movements in the rivers (Steingraeber and Thiel was rarest, as observed in invasion patterns of several 2000; Irons et al. 2006; Roche et al. 2013), while other alien gobies (Raby et al. 2010; Gutowsky et al. rates of expansion upstream were usually much 2011; Brownscombe and Fox 2012). Colonization to slower (Bergstrom et al. 2008; Brownscombe and the south (downstream) seems to be a result of a Fox 2012). Padogobius bonelli was also found in a natural expanding migration over a continuous area newly isolated site of the Chiascio-Topino sub-basin, and highlights the ability of P. bonelli to disperse located more than 70 km from the confluence with over a considerable distances in a relatively short the River Tiber. Therefore, the most probable origin time. Given their small body sizes (maximum length of these gobies is an unintentional introduction during of males in the River Tiber basin: P. nigricans 9.2 cm; illegal stoking with uncontrolled material. Conside- P. bonelli 7.7 cm) and benthic habits, gobies are usually ring the invasion pathways of P. bonelli in the upper considered species with reduced vagility (Miller 1990; Tiber, it is very likely that this location will act as

284 Distribution, abundance and nest occupancy of two congeneric gobies

Table 2. Descriptive statistics for nest stones area (cm2) and number of eggs in the nest (N eggs) for Padogobius bonelli (Pb) and P. nigricans (Pn) in the 6 invaded sites investigated. The results of the comparison (Wilcoxon Signed Rank test) between the two species for each site and for the total sample (Total) is reported.

Wilcoxon Signed Rank test Station Species N Mean Min Max SE W p Pb 75 211.3 55.8 724.5 14.1 Aggia1 43 0.550 Pn 2 223.3 181.7 264.8 5.3 Pb 31 248.3 95.4 515.8 20.2 Resina1 37 0.689 Pn 2 206.1 174.6 237.6 31.5 Pb 44 173.2 57.1 357.5 11 Carpina1 243 <0.01 Pn 24 310.2 109 939.8 44.4 Pb 24 434.9 62.3 1548.8 90.3 Nest stone area (cm2) Vaschi1 99 0.010 Pn 23 250.2 68.4 2398.2 98.4 Pb 8 272.4 147.3 456.1 65.7 Lanna 24 0.069 Pn 12 476.6 164.8 820.8 116.1 Pb 14 287.8 121.6 944.2 65.6 Soara1 68 0.051 Pn 18 315.5 145.9 533 24.8 Pb 196 247.1 55.8 1548.8 16.1 Total 5822 0.018 Pn 81 301.1 68.4 2398.2 35.2 Pb 53 353.5 94 846 27.7 Aggia1 86 0.144 Pn 2 234.5 154 315 15.7 Pb 24 352.4 63 1219 58.5 Resina1 35 0.312 Pn 2 165 96 234 69 Pb 35 300.5 988 988 34.4 Carpina1 334.5 0.978 Pn 19 340.4 933 933 59.6 Pb 23 372.9 131 771 65.5 N eggs Vaschi1 217 0.215 Pn 21 484.6 72 972 69.1 Pb 6 235.3 91 315 50.9 Lanna 28 0.305 Pn 7 331.8 53 614 108.1 Pb 14 429 105 942 53.3 Soara1 60.5 0.228 Pn 18 469 101 2149 100.7 Pb 157 351.1 32 1219 18.5 Total 5083 0.748 Pn 69 401.8 53 2149 43

Table 3. Descriptive statistics of nest stone areas (cm2) and number of eggs in the nest (N eggs) for Padogobius nigricans in the 2 uninvaded sampling stations. The results of comparison (Mann-Whitney U test) between pooled data for the invaded (Table 2) and uninvaded areas are shown.

Mann-Whitney U test Station Species N Mean Min Max SE Z p Aggia2 Pn 22 583.8 170.4 1692.1 121.3 Nest stone area (cm2) 1.200 0.230 Carpina2 Pn 14 183.8 40.5 346.1 21.4 Aggia2 Pn 18 831.2 166 1790 162.5 N eggs 1.631 0.103 Carpina2 Pn 11 194.6 31 733 53.9 a new spread point which suggests that the non-native notion of reduced P. nigricans reproduction. Nesting goby could spread further downstream, following a interference is common amongst gobies, and several “stepping stone and diffusion” dispersal mode. We studies have reported that native fishes can be exclu- hypothesize rapid density increases in recently inva- ded from reproductive territory through aggression ded areas due to availability of resources at initial from alien gobies (Janssen and Jude 2001; Van Kessel low density and to the high reproductive potential of et al. 2011; Kakareko et al. 2013; Jermacz et al. 2014; P. bonelli (Pompei et al. 2016a). Grabowska et al. 2016). However, the influence of This dispersal pattern, both natural and human- P. bonelli on shelter occupancy of P. nigricans was mediated, raises further concern for the conservation as strong as we expected only in some of the of the endemic goby. Padogobius bonelli seems to examined sites. In other cases, the proportion of nests affect P. nigricans population structure, as well as its was more aligned with the densities of the two species, abundance. In the invaded areas, the presence of and the prevalence of P. bonelli is less marked. This fewer juveniles than uninvaded areas supports the situation may depend on resource availability. Optimal

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Figure 4. Relationship between nest stone area (cm2) and the number of eggs in the nests (N eggs) for A) Padogobius nigricans (N = 98) and B) P. bonelli (N = 157) in stations of the upper River Tiber. shelter could potentially be a limiting resource, making invaded and uninvaded areas. Thus P. bonelli seems any competition between the two gobies much stronger to interfere not with the “quality” of P. nigricans (Błońska et al. 2016). If shelters are limited, it’s likely nests, but rather with the “quantity”. Moreover, that the more aggressive P. bonelli is a stronger shelter is not only an essential environmental resource competitor, ultimately excluding P. nigricans from during the spawning period for both species, it also the reproductive habitat. In laboratory conditions, plays significant roles in predator protection and P. bonelli males directly compete with conspecifics refuge from stream flows (Allouche 2002). for possession of larger nest sites, because females Occupying the best sites is apparently not enough use nest size as a predictor of male quality (Lindström for the conservation of P. nigricans. In the invaded 1988; Bisazza et al. 1989). The nest is a substrate areas, the survival of P. nigricans populations is mostly which may physically limit the size of eggs mass in through migration of individuals from uninvaded these species that lay eggs in a single layer, and thus, areas located upstream of weirs that prevented the the ability of a male to clutches (Lindström spread of the non-native species. Passive movement 1988; Marconato et al. 1989). For both species, as in of early ontogenetic stages may represent an important most gobiids (Lindström 1988; Marconato et al. 1989; dispersal mechanism in gobies (Janáč et al. 2013). In Takahashi et al. 2001), the number of the eggs in the this sense, small weirs have had a dual task: they faci- clutches depends on the lower surface area of the litate the survival of intact populations of P. nigricans, nest, thus it is likely that males compete for larger and allow recolonization of invaded areas, preventing stones which allow them to mate with more than one long-term inter-specific exclusion (Belkessam et al. female, thereby maximizing reproductive success 1997). River damming is one of the most damaging (Marconato et al. 1989). Moreover, in fish species anthropogenic alterations for freshwater environments with exclusive paternal care, females usually favour (Baxter 1977; Dynesius and Nilsson 1994; Franchi large nests (Takahashi and Khoda 2002). They prefer et al. 2014). Damming impedes free movement of to spawn with males whose nests already contain fish, emphasizing the consequences of isolation eggs (Forsgren et al. 1996) and large brood sizes may (Lorenzoni et al. 2006). Nevertheless, the presence induce more paternal care (Coleman et al. 1985). of such barriers substantially contributes to the The preliminary results of this study indicate that preservation of isolated populations of vulnerable P. nigricans occupied on average the larger nests and endangered species; for this reason actions to and produced more eggs per nest. Moreover, no restore connectivity between rivers must take into differences were found in nest characteristics (size account the risk of spread of invasive species (Van and number of eggs) of P. nigricans between the Kessel et al. 2016).

286 Distribution, abundance and nest occupancy of two congeneric gobies

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Supplementary material The following supplementary material is available for this article: Table S1. Occurrence of non-native P. bonelli and native P. nigricans (presence in the upper River Tiber observed in the period 2011–2015. This material is available as part of online article from: http://www.aquaticinvasions.net/2018/Supplements/AI_2018_Pompei_etal_Table_S1.xlsx

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