Submitted to: Invertebrate Biology
Diverge nt demographic patterns and conservation of endemic species in extreme environments: the case study of the spring snail Melanopsis etrusca (Gastropoda: Melanopsidae)
Journal:For Invertebrate Peer Biology Review
Manuscript ID: InvBio-2015-Apr-023
Manuscript Type: Original Manuscript
Date Submitted by the Author: 21-Apr-2015
Complete List of Authors: Bartolini, Fabrizio Aquiloni, Laura Nisi, Barbara Nuccio, Caterina Vaselli, Orlando Cianfanelli, Simone
endemic snails, hot spring, growth , demographic patterns, Melanopsis Keywords: etrusca
An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 1 of 30 Submitted to: Invertebrate Biology
1 2 3 Divergent demographic patterns and conservation of endemic species in extreme 4 5 environments: the case study of the spring snail Melanopsis etrusca (Gastropoda: 6 Melanopsidae). 7 8 9 1* 2 3 2 4,5 1 10 Bartolini F. , Aquiloni L. , Nisi B. , Nuccio C. , Vaselli O. , Cianfanelli S. 11 12 13 1 Natural History Museum of the University of Florence, Zoological Section "La Specola" 14 15 Florence, Italy 16 2 University of Florence, Department of Biology, Florence, Italy 17 18 3 CNR IGG Institute Forof Geosciences Peer and Earth Review Resources, Pisa, Italy 19 4 20 University of Florence, Department of Earth Sciences, Florence, Italy 21 5 CNR IGG Institute of Geosciences and Earth Resources, Florence, Italy 22 23 *Corresponding author: [email protected] 24 25 26 27 Abstract 28 29 30 The conservation of endangered species poses challenges driven by contrasting targets 31 and needs. Populations’ isolation can indeed be a value or a limitation due to promoted 32 33 evolutionary change and risk of bottlenecks and/or other genetic detrimental processes. 34 35 In the present study we illustrate the case of Melanopsis etrusca (Brot, 1862), an endemic 36 snail commonly associated with some thermal springs from Central Italy. These habitats 37 38 represent a climatic refuge in response of dramatic paleo climatic events. 39 40 Populations are currently isolated within headstream stretches by the gradual changes in 41 physico chemical conditions downstream. Furthermore, strong differences in spring 42 43 thermal regimes have a role in determining population dynamics and life history, as 44 45 showed by differential patterns of individual growth and mortality. 46 Snails experiencing temperate conditions with relatively high temperature fluctuations (13 47 48 29 °C) showed seasonal patterns of growth with good level of age cohorts structuration. 49 50 Conversely, fast and continuous growth, with shorter life cycles, is typical of snails 51 constantly inhabiting warmer streams (31 34°C). Interestingly, population structures similar 52 53 to those observed in cooler conditions were recognized in a hot channel (36 38°C), where 54 55 snails spend long period outside the water, thus exploring an additional thermal niche. 56 Furthermore, a partial shift of snails’ diet reflects in terms of epilithic communities, which 57 58 change towards cyanobacteria dominated environment in warmer streams. 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 2 of 30
1 2 3 Hot adapted populations of M. etrusca appear to have larger potential for recolonization 4 5 after habitat perturbation or population collapse events, although they may be at risk of 6 bottlenecks. On the other hand, the isolation in climatic refuges seems to have promoted 7 8 behavioral strategies to cope with extreme thermal environments. 9 10 In a general perspective, thermal streams appear valuable model systems to investigate 11 patterns of populations’ adaptation and divergence, as those triggered by climate change. 12 13 14 15 16 1. Introduction 17 18 For Peer Review 19 20 Freshwater mollusks are among the most endangered metazoan groups, representing 21 almost 42% of the total recorded animal extinctions in the last 500 years (Lydeard et al. 22 23 2004). Such a trend appears determined by several factors among which scarce mobility 24 25 of these animals, frequent water bodies isolation (Strayer 2006; Olden et al. 2010) and 26 general limited resilience of freshwater habitats affected by anthropogenic pressure (e.g. 27 28 Lysne et al. 2008). 29 30 Such threatens appear magnified for endemic species limited to springs habitats, often 31 characterized by particular physicochemical conditions, which may impose the 32 33 development of specific physiological/behavioral adaptations to survive extreme 34 35 conditions. These habitats are considered biodiversity hotspots (Di Sabatino et al. 2003) 36 due to the very peculiar invertebrate and fish community herein hosted (e.g. Vinyard 1996; 37 38 Hershler et al. 2007). 39 40 Freshwater prosobranchs belonging to the genus Melanopsis are widespread all around 41 the Mediterranean basin where this group had a long evolutionary history. Melanopsis 42 43 fossils from brackish/marine deposits are known since the lower Cretaceous (e.g. Stewart 44 45 et al. 1991). However, the largest radiation of the genus occurred at the end of the 46 Miocene during the Messinian Mediterranean salinity crisis (about 5 Ma), when brackish 47 48 basins were abundantly present in several regions (e.g. Esu 1980; Esu & Girotti 1989; 49 50 Geary 1990; Guerra Marchán et al. 2010). 51 Within this genus, M. etrusca is a small (15 20 mm in shell length), poorly known endemic 52 53 species whose relict populations dwell few thermo mineral springs in Southern Tuscany 54 55 (Central Italy; Glaubrecht 1993). Climatic shifts through glaciation events are commonly 56 recognized as one of the main extinction factors of the closest ancestors, once abundant 57 58 and diversified in this geographic area, until the early Pleistocene (e.g. Esu et al. 1993; Di 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 3 of 30 Submitted to: Invertebrate Biology
1 2 3 Bella et al. 2002). However, in the last century, relict populations of this species underwent 4 5 further extinctions due to anthropogenic pressure (Cianfanelli et al. 1989). Exploitation of 6 thermal springs for spas as well as therapeutic, wellness and fitness activities, draining 7 8 and contamination of aquifers related to agricultural and/or mining purposes can be 9 10 regarded as the primary causes for the populations loss (Cianfanelli et al. 1989). Owing to 11 its critical status, M. etrusca has recently been classified as ‘Endangered’ by IUCN red list 12 13 of threatened species (Cianfanelli 2010). 14 15 The almost complete lack of knowledge about general aspects of the biology of this 16 species renders difficult to propose any effective actions for its protection (e.g. Watson & 17 18 Ormerod 2004). Furthermore,For thePeer investigation Review of life history traits in populations adapted 19 20 to diverging environmental conditions provides basic knowledge for both conservation and 21 understanding of organisms’ potentials for acclimatization and adaptation in evolutionary 22 23 terms. This appears particularly worthy considering the challenge posed to freshwater 24 25 habitats and species conservation by the rapid human induced climatic changes (Heino et 26 al. 2009; Woodward et al. 2010). 27 28 The present study was aimed to clarifying possible physico chemical constraints of M. 29 30 etrusca distribution, the importance of such parameters to its life history as well as aspects 31 of its trophic biology. Such issues were addressed by comparing M. etrusca grazing, 32 33 patterns of individual growth and population structure in different physico chemical 34 35 conditions, both along the same stream and between streams. 36 37 38 2. Material and Methods 39 40 41 2.1 Study area 42 M. etrusca occurs in six localities (Venturina, Aronna, Bruna, Podere Bagnolo, Poggetti 43 44 Vecchi and Roselle; Fig.1) of southern Tuscany characterized by thermo mineral waters 45 (Bencini et al 1977; Celati et al 1991), two of which (Poggetti Vecchi and Podere Bagnolo) 46 47 are severely endangered by anthropogenic brook desiccation (Fig. 1). 48 49 In the present study the following sites were considered: Venturina, Aronna, Bruna and 50 Roselle (Fig. 1). The thermal regime of the springs from which water courses originate is 51 52 quite different, since it varies from constantly high temperatures (>30°C), as the stream at 53 54 Venturina and the thermal channel at Roselle, to cooler (<30°C) sites such as those of 55 Bruna and Aronna. 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 4 of 30
1 2 3 The streams show narrow riverbeds (1.5 4 m) with shallow waters (5 40 cm). The current 4 5 regimes are from fast/moderate to very slow on hard (cobbles, gravels, rocky beds) and 6 soft (muddy) substrates, respectively. 7 8 M. etrusca is mainly distributed in the very first hundreds meters of the streams, while in 9 10 downstream areas this species is only occasionally observed, likely due to current 11 dragging of isolate specimens. 12 13 14 15 2.2 Physical and chemical parameters of the waters 16 Sampling stations were selected in order to identify the main physico chemical parameters 17 18 accounting for the typicalFor restricted Peer distribution Review of snails into headstream positions. For 19 20 each stream, two sampling stations were selected: 1) upstream (hereafter called 21 ‘upstream’), where dense assemblages of M. etrusca occurred and 2) downstream 22 23 (hereafter called ‘extinction’), where local extinction of the population was observed. 24 25 Four water samples were collected at each of the described locations, between August 26 2009 and May 2010. Water samples were collected and filtered (0.45 m) and transferred 27 28 into polyethylene bottles for the subsequent chemical analyses in laboratory. Temperature 29 30 (°C) and pH were determined in the field with an Hg thermometer and a portable Eutech 31 PC650 waterproof instrumentation, respectively. 32 33 The main anions and cations were analyzed by ion chromatography (761 Compact IC 34 35 Metrohm and 861 Advanced Compact IC Metrohm respectively). Analytical errors were 36 generally <3% for the main components. TDS (Total Dissolved Solids) was referred to the 37 38 sum (in mg/L) of the cationic and anionic species. 39 40 41 2.3 Population structure , individual growth and mortality rates 42 43 Population structures from all the four sites were assessed on two different moments of 44 45 the year, July 2009 and February 2010, in stream stretches with hard substrate and similar 46 conditions in terms of depth, current and distance from the spring. Snails density and size 47 48 were measured, as follows: a specific sampler was built by using a 20×20 cm plastic bottle 49 50 opened at both ends. The “cylinder” had a wall mounted with a thin conic aquarium mesh 51 (0.2 mm), whereas the opposite one was punched. The sampler was placed on the stream 52 53 bottom and the enclosed surface scraped with a brush. As a consequence, snails and 54 55 other material in suspension were naturally pushed into the net by the current flowing 56 through the punched wall. A soft rubber made rib fixed onto the lower edge allowed a 57 58 complete adherence of the sampler to the riverbed. 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 5 of 30 Submitted to: Invertebrate Biology
1 2 3 Eight quadrates, assigned to two transects, were sampled in each stream. This appeared 4 5 to be a reasonable amount of replicates considering both the very high abundance of 6 snails and the relative uniformity of the riverbed conditions. An additional sampling was 7 8 carried out at Roselle (hereafter Roselle Amph ), in a headstream stretch were an atypical, 9 10 massive amphibious behavior was observed, with inactive specimens forming a dense belt 11 along the stream bank, few centimeters above the water level. 12 13 The collected material was carefully cleaned. Snails were then placed on a plastic tray with 14 15 their shell opening turned up and a picture was taken for successive specimens counting 16 and size estimates. The snails were released at the end of this procedure. 17 18 Picture analyses For were carried Peer using theReview free software Image J (available at 19 20 http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institutes of Health, 21 Bethesda, MD). Such an indirect method of estimation was necessary due to the local very 22 23 high density of snails. 24 25 Growth rates were measured in semi natural conditions, by considering different thermal 26 environments. The Bruna stream and Venturina channel were thus selected as 27 28 experimental sites hosting cool and warm adapted populations of M. etrusca , respectively . 29 30 Four 26×10×16 cm plastic boxes (hereafter “mesocosms”) were placed in each stream in 31 the same localities of population structure sampling. Large windows of thin zinced mesh 32 33 were present on each side of the mesocosm, except the bottom, in order to allow a 34 35 constant water flow inside (Fig. 1). Mesocosms were set up with cobble collected from the 36 riverbed. A total of 30 snails were introduced in each mesocosm. Shell length ranged 37 38 between 3.1 and 16.8 mm for Bruna and between 3.1 and 10.8 mm for Venturina, 39 40 representing the natural differences in maximum size reached by the two populations. 41 Each animal was individually tagged (n=120) by coloring the top of the shell with several 42 43 chromatic codes by using permanent markers. 44 45 The experiment started at the beginning of September 2009 and ended in July 2010. 46 Increment in shell height and shell width was monthly measured until November, again 47 48 after two months, in January 2010, and subsequently every 3 months (accuracy 0.01 mm). 49 50 Every time, the number of dead specimens inside the mesocosms was recorded and 51 mortality rates were computed. 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 6 of 30
1 2 3 2.4 Food items on hard substrates/crop contents 4 5 An explorative study was carried out through a semi quantitative method in order to 6 investigate the main microphytobenthic groups naturally present on streams cobbles and 7 8 to perform a comparison with the food items ingested by M. etrusca 9 2 10 An overall surface of 130 cm was sampled from each site in unshaded homogeneous 11 riffle stretches (depth 5 10 cm) densely populated by M. etrusca , following the general 12 13 indications given in the European standard EN 13946:2003 for epilithic diatoms. Samples 14 2 15 were obtained by randomly scraping single 13 cm units from cobbles and stones surfaces 16 by means of a steel blade. Collected samples were preserved into 4% formalin solution 17 18 and carried to laboratoryFor for taxonomicPeer identificat Reviewion (Bouchard & Anderson 2001). For 19 20 each site, six aliquots of 50 µL were observed into 3 mL sedimentation chambers, using an 21 inverted microscope (Zeiss IM) with 400x magnification. Sampling was carried out in 22 23 August 2009 and February 2010. 24 25 M. etrusca gut contents were obtained by dissecting (Bilgin 1973) 20 snails collected 26 during August 2009, in the same area of cobbles collection of two sites characterized by 27 28 different thermal conditions: Bruna and Venturina. For each site, five 50 µL subsamples of 29 30 the pooled extract were observed similarly to what described for phytobenthic analysis. 31 32 33 34 2.5 Data analysis and statistics 35 Principal Component Analysis (PCA) was used to visualize patterns of variability among 36 37 samples and to determine the contribution of each single physicochemical parameter to 38 39 the overall observed variability. Permutational analysis of variance (PERMANOVA; 40 Anderson 2001) was used to test the null hypothesis of no differences in normalized 41 42 physicochemical water parameters, among the four sites (factor “Site”: four levels, random 43 44 and orthogonal) and position along streams (factor “Position”: two levels, fixed and 45 orthogonal). Similarly, the hypothesis of no difference between shell length increments 46 47 (normalized on the initial size) among sites with different thermal regimes was tested, by 48 49 considering the first month of experiments. The following factors were considered: “Site” 50 (two levels: Bruna and Venturina; fixed and orthogonal); “Position” (two levels: upstream 51 52 and extinction; fixed and orthogonal); “Mesocosm” (four levels; random and nested in 53 54 “Position”). 55 Permutations of residuals under a reduced model (more than 9900) were based on 56 57 similarity matrices computed on Euclidean distances of normalized data and Type III sum 58 59 of squares. 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 7 of 30 Submitted to: Invertebrate Biology
1 2 3 The SIMPER procedure (Clarke 1993) was used to find out the percentage contribution of 4 5 the overall variability observed among samples. Univariate statistical analysis was applied 6 to the most relevant variables and carried out by using ANOVA. The same models as in 7 8 the multivariate analysis were considered. Cochran’s C test was performed to test the 9 10 normality of distributions prior of performing ANOVAs and square root transformations 11 were used when opportune. SNK test was used to test for differences among levels of 12 13 factors found to be significant by ANOVA. 14 15 Average size and density of snails from summer samplings were analyzed by ANOVA, 16 taking into account two factors: “Site” (four levels; fixed) and “Transect” (two levels; 17 18 random and nested Forin “Site”). ThePeer stretch including Review amphibious specimens in Roselle was 19 20 not included in the analysis due to the different patterns of animal occurrence that is 21 mainly outside the ‘riverbed'. For both PERMANOVA and ANOVA, square root 22 23 transformation of data was carried out when appropriate. 24 25 Chi square test of independence was used to compare the absolute abundance of the 26 main microphytobenthic groups between two sites characterized by different thermal 27 28 regimes (Bruna and Venturina). A similar analysis was carried out to compare microalgal 29 30 occurrence in snail crops and natural substrates. Pooling of data was carried out when 31 appropriate (minimum expected frequencies less than 5). 32 33 Multivariate analyses of variance were performed using PRIMER v. 6.1 (Clarke & Gorley 34 35 2006) and PERMANOVA+ for PRIMER routines (Anderson et al. 2008). ANOVAs were 36 performed using the software GMAV 5 for Windows (Underwood et al. 2002). 37 38 Age cohorts identification was carried out following the method of Battacharya (1967), 39 40 using FiSAT (FAO ICLARM Stock Assessment Tools, www.fao.org/fishery/) free software 41 (Gayanilo et al. 1996). 42 43 44 45 46 3. Results 47 48 49 50 51 3.1 Physico-chemical water parameters 52 Streams strongly differed in terms of thermal regime. During the study period water 53 54 temperature of Bruna stream underwent seasonal variations of around 12 °C (13.7 25.7 55 °C) at upstream position and together with Aronna (19 22.7 °C), represented the two 56 57 cooler streams. Conversely, Venturina and Roselle were characterized by temperatures 58 59 constantly above 30 °C. The highest temperature were reached at Roselle Amph (≥38°C, 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 8 of 30
1 2 3 mean value = 37 °C). The observed ∆T between upstream and extinction positions during 4 5 the sampling period varied among different sites and according to the period of the year 6 (Tab. 1). 7 8 The values of the main physico chemical water parameters are listed in Table 1. The 9 10 sampled waters at both the upstream and extinction positions were characterized by 11 relatively high values of TDS from 1,381 mg/L (Aronna Ext) up to 2,678 mg/L (Roselle 12 13 Ups). The main cations were Ca 2+ and Mg 2+ whose concentrations were up to 592 mg/L 14 15 (Roselle upstream) and 125 mg/L (Roselle upstream), respectively. Among the anionic 16 2 species SO 4 and HCO 3 showed the highest contents with concentrations up to 1,664 17 18 mg/L (Roselle upstream)For and 280Peer mg/L (Bruna Review upstream), respectively. According to the 19 20 ‘Venice System’ (1959) such a kind of water can be classified as oligohaline (e.g. Por 21 1972; Hartog 1974). The bulk of variability among samples was explained by a geographic 22 23 factor, determining a clear segregation of samples in the multivariate space (Fig. 2). A 24 25 general decreasing trend of water temperature (Tab. 1)(riportare i valori) and main 26 dissolved anions and cations species was observed moving away from upstream positions 27 28 (Tab.1), whereas pH levels showed a rising pattern towards exctinction positions. 29 30 A significant interaction was found by PERMANOVA between distance and position (Tab. 31 2): in particular, pairwise comparisons performed for the different sites found significant 32 33 differences between upstream and extinction position in each stream, except for Bruna 34 35 (PERMANOVA, post hoc tests). 36 + SIMPER found pH, HCO 3 , NO 3 , K and temperature explaining almost 76% of the 37 38 differences observed between upstream and extinction positions averaged over different 39 40 sites. However, the univariate analysis showed pH being the only parameter significantly 41 changing at the extinction positions across streams (ANOVA, SNK test; Tab.3). On the 42 43 other hand, NO 3 was found to be significantly higher only at the extinction positions of 44 45 Venturina and Roselle (ANOVA, SNK test; Tab. 3). 46 47 48 49 3.2 Population density and size class distribution 50 Populations of M. etrusca were characterized by very dense assemblages, with peaks of 51 52 over 10,000 snails per square meter, as estimated from some quadrates in Roselle (Fig. 53 54 3A). Population from the warmest sites achieved the highest average densities, although 55 significant differences were found only between Roselle and the two cooler streams, Bruna 56 57 and Aronna (ANOVA, SNK test; Tab. 4). 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 9 of 30 Submitted to: Invertebrate Biology
1 2 3 The analysis of age cohorts performed with Bhattacharya method (Fig. 4A; Tab. 5), 4 5 showed significant differences among the various populations. During summer, the 6 population from Bruna appeared well structured, presenting at least four age cohorts. In 7 8 winter they were reduced to three, with a pick of small specimens. Population from Aronna 9 10 showed to be less structured with two age cohorts both in summer and winter (Fig. 4C; 11 Tab. 5). An almost non structured population was recorded at Venturina, with a first well 12 13 defined cohort and a second one scarcely represented in summer, while in winter only one 14 15 cohort was recognized (Fig. 4D; Tab. 5). A similar pattern was observed at Roselle, 16 although a second cohort was also found in winter (Fig. 4E; Tab. 5). For both these sites, 17 18 in winter, the first For cohort appeared Peer shifted Review towards smaller average sizes (Tab. 5). 19 20 Interestingly the population structure for the amphibious population at Roselle appeared to 21 be quite well structured, with three age cohorts during both seasons and with a well 22 23 represented occurrence of large specimens (Tab. 5). 24 25 26 27 3.3 Population structure, individual growth and mortality rates 28 29 After the first month of measurements, significant differences in growth rates were found 30 across positions of different sites and between upstream and extinction positions at 31 32 Venturina, whereas these did not significantly differ at Bruna (Fig. 5A; PERMANOVA, post 33 34 hoc tests; Tab. 6). 35 M. etrusca individual growth from Bruna showed, in both positions, a clear seasonal 36 37 pattern (Fig. 5A). Actually, the starting average shell length increment measured at the end 38 39 of summer dropped to almost zero in the period October January. Between January and 40 April 2010, upstream individual growth recovered to a level similar to the one measured at 41 42 the end of summer, although slightly lower, probably due to the ageing of specimens 43 44 jointed to the progressive reduction of the experimental replicates. It must be noted that 45 the value reported in Figure 5A is averaged over three months, thus hiding possible 46 47 differences between the start and the end of the interval. Conversely, the reprise of growth 48 49 at the extinction station was delayed, being still absent in April 2010. 50 At Venturina, the first measured length increment for the upstream population (September 51 52 October 2009) was more than twice the growth measured in the other stream (Fig. 5A). At 53 54 this site, the average values of shell increment quickly decreased, which is probably to be 55 again related to the quick ageing of the population inside the mesocosms. 56 57 Specimens moved at the extinction position showed a null growth during the first month 58 59 (PERMANOVA, post hoc tests), followed by a slight reprise in November 2009 and a new, 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 10 of 30
1 2 3 definitive drop, mainly determined by the strong reduction of the experimental replicates. A 4 5 strong mortality indeed affected the specimens moved at the extinction of Venturina, 6 which, on average, after only two months reached almost 80% (Fig. 5B). In the upstream 7 8 position, on the contrary, such a value was attained after seven months. In Bruna stream, 9 10 a different pattern was observed, with an average mortality lower than 30% after 10 11 months in the extinction position and lower than 5% in the upstream position (Fig. 5B). 12 13 14 15 16 3.4 Food items on hard substrates/crop contents 17 The microscopic analysis of the microalgal communities revealed pennate diatoms and 18 For Peer Review 19 cyanobacteria, these two groups being the most common constituents of hard riverbeds. 20 The comparison of periphyton composition between Bruna and Venturina showed opposite 21 22 patterns. In particular, in both sampling datess, diatoms represented the dominant group in 23 24 the Bruna stream with a low occurrence of cyanobacteria. On the contrary, Cyanobacteria 25 appeared the dominant group at Venturina (Chi square test of independence, August 26 27 2009: χ 2= 486.98, df=1, p<0.001; February 2010: χ 2=337.87, df=1, p<0.001; Fig. 6A). 28 29 Similar patterns, although less pronounced, were shown at the Aronna stream and 30 Roselle. 31 32 Despite the consistent quantities of fine detritus, the gut content of M. etrusca showed a 33 34 large amount of macrophyte fragments and a smaller portion of microphytobenthic items 35 (cyanobacteria, diatoms and rare green algae). In particular, Bruna and Venturina had no 36 37 statistical differences between the relative abundance of the microalgal items in the snail’s 38 2 39 gut and in the natural substrates (Chi square test of independence, Bruna: χ =0.62, df=1, 40 p=0.43; Venturina: χ 2=2.17, df=1, p=0.14). On the contrary, the gut content significantly 41 42 differed between the two sites, with specimens from Bruna largely ingesting more 43 44 macrophyte tissues (87.5%) and diatoms (9.7%), whereas in Venturina, blue green algae 45 (48.5%) and macrophytes fragments (47.7%; Fig. 6B; Chi square test of independence, 46 47 χ2=152.43, df=2, p<0.001). 48 49 50 51 4. Discussion 52 53 Our results showed that temperature have a role in shaping life cycles of the isolated 54 55 populations of M. etrusca in Southern Tuscany. Snails needed to cope with heterogeneous 56 57 thermal microhabitats met within different spring systems. However, temperature alone is 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 11 of 30 Submitted to: Invertebrate Biology
1 2 3 not able to fully explain the observed differences in population dynamics among streams 4 5 as observed at Venturina extinction position. 6 The effect of the progressive change in water physico chemical parameters appeared less 7 8 evident due to both the large variability found among sites and the different responses 9 10 obtained in the growth experiments. The most relevant parameters distinguishing between 11 upstream and extinction positions were pH and, partially, NO 3 . The latter was significantly 12 13 higher at Venturina and Roselle, notably the two locations more affected by human activity 14 15 (intensive agriculture, thermal spa and urbanization). 16 Snails moved to the ‘extinction’ position in Bruna exhibited only a delay in the seasonal 17 18 reprise of growth comparedFor to Peeranimals at upstream Review location, however maintaining similar 19 20 rates. At this site, the multivariate analysis did not show significant differences in 21 parameters between upstream and extinction, except for an increase in pH. The slight 22 23 difference in temperature between the two stations could likely be the cause of the 24 25 observed patterns, especially considering the thermoregulatory effect of the springs 26 located on the bottom of the Accesa Lake, whose Bruna is an effluent. On the contrary, 27 28 concerning the extinction at Venturina, despite still relatively elevated temperature (20.1 29 30 31.3 °C), growth was partially suppressed and high mortality was observed. Here, similarly 31 to the extinctions at Roselle and Aronna, the progressive decrease in water temperature 32 33 and dissolved CO 2, which tends to equilibrate with that of the air, determined a significant 34 35 increase of pH and a consequent precipitation of travertine. This phenomenon caused the 36 mesocosms, and the cobbles inside, to be fully coated with a carbonatic film, possibly 37 38 limiting food availability. Similarly, specimens occurring only periodically and at very low 39 40 density in the extinction position of Aronna (possibly dragged off by stream current) were 41 unable to properly retreat inside the shell, due the thick concretion covering the operculum. 42 43 Generally speaking, if the role of hosmoregulatory limitations should be investigated, given 44 45 the brackish/oligohaline environments of M. etrusca ancestors (Esu & Girotti 1989; Stewart 46 et al. 1991; Cipollari et al. 1999; Di Bella et al. 2002), other environmental factors could 47 48 concur in limiting the current distribution of the species. It is known that pattern of 49 50 freshwater distribution along a stream can be predicted by the progressive change of 51 current speed and substrate (Dillon 2000). In particular, similarly to other spring snails 52 53 (Martinez & Thome 2006), M. etrusca seems to prefer moderate currents and hard 54 55 substratum (Aquiloni et al. unpubl. data). 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 12 of 30
1 2 3 At upstream positions, the differences in thermal regimes between streams were reflected 4 5 by differential rates of snails’ growth and mortality. The relatively rapid shell accretion rates 6 (0.03 mm/d, in the first two months) observed for the population dwelling in Venturina (30 7 8 35.1 °C), nearly matched those reported for Pyrgulopsis bruneauensis , a hydrobiid spring 9 10 snail experiencing similar thermal conditions in Idaho (0.03 mm/d at 34 °C for 89 days, for 11 juveniles specimens; Mladenka & Minshall 2001). Growth performance of M. etrusca 12 13 appeared to be enhanced and continuous in hotter environments (Huey & Kingsolver 14 15 1989). Nevertheless, in such conditions, average smaller specimens size and an almost 16 complete lack of age structure was observed. 17 18 M. etrusca at Bruna Forshowed the Peer capability of toleratingReview seasonal drop in temperature (13.7 19 20 °C in the present study, but lower in other years: SIRA, 2011) through 21 reduction/suspension of growth. Our data showed a reduction in shell accretion in October 22 23 2009 – January 2010, with a reprise, at an undefined time, between January and April 24 25 2010. The average shell increment of 0.7% referred to February, March and April 2010, is 26 indeed only an indirect estimate and the reprise of growth can reasonably be hypothesized 27 28 at the achievement of a certain environmental temperature threshold. Such a temporary 29 30 reduction of growth likely appears to be an effect of a general metabolic depression, 31 similarly observed in other overwintering species of freshwater mollusks (Lurman et al. 32 33 2014). In winter, age cohorts structuration appeared less evident and this could be a bias 34 35 due to the tendency, especially of larger animals, to rest below large boulders (pers. 36 observ.): an additional clue of low temperature induced diapause. Such an annual 37 38 dynamics appears to determine longer life cycles, as demonstrated, in our mesocosm 39 40 experiment, by low, discontinuous growth and little mortality. These evidences well 41 matched the observed higher level of age structuration exhibited by the Bruna population, 42 43 where seemingly long standing specimens were found (maximum recorded shell length: 44 45 15.9 mm). Conversely, the high mortality rates of Venturina seem to account for shorter life 46 cycles with faster specimen’s turnover and potentially higher generation rates in hot 47 48 streams. 49 50 Interestingly, we observed a massive amphibious behavior performed by (large) snails 51 inhabiting a headstream area in Roselle, where the highest water temperatures occurred 52 53 (34.6 38.1 °C). Here, differently than in other locations of the same channel, as well as at 54 55 Venturina, a more structured population was observed, despite the warmer conditions. A 56 reduced average body temperature experienced throughout the year by these animals, 57 58 determined by the exploration of a different thermal niche, could account for the observed 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 13 of 30 Submitted to: Invertebrate Biology
1 2 3 population structure. Evidences from population dynamics of M. etrusca naturally exposed 4 5 to different thermal environments seem to verify the general temperature-size rule (e.g. 6 Atkinson et al. 2006), which predict lower temperature causing the majority of ecthoterms 7 8 to growth slower and attaining maturation at larger body size. 9 10 Eventually, temperature also appeared to affect microalgal epilithic communities and, in 11 turn, this had a role in shaping snails’ diet. M. etrusca pattern of periphyton grazing indeed 12 13 appeared to be not a selective one. Blue green algae attained almost half of the total food 14 15 item in the stomach of specimens from Venturina, reflecting the general pattern of 16 cyanobacteria enhancement and diatoms reduction within warmer stream. 17 18 In general, as for theFor congeneric Peer M. praemorsa Review (Heller & Abotbol 1997), leaf litter of 19 20 terrestrial origin appeared one of the most prominent item ingested by M. etrusca . 21 However, if the low trophic values of cyanobacteria are considered (Demott & Müller 22 23 Navarra 1997), sometimes even avoided by certain freshwater snails (Madsen 1992), it is 24 25 possible that the observed differences in diet may have some impact on M. etrusca life 26 cycle. 27 28 Food availability is known to be a key factor for the development of effective physiological 29 30 response to stressful conditions (e.g. Thomsen et al. 2013), like those potentially met in 31 the warmest streams. Future studies should deepen the actual rates of ingested items 32 33 assimilation, as well as the nutritional value of detritus in the snails’ crop. 34 35 In a conservation perspective, populations dwelling warmer sites seem to hold higher 36 recovery potential from human induced local extinctions, such as those determined by 37 38 water contamination, brock desiccation or riverbed dredging. Some of the studied streams 39 40 indeed fall in urban areas (Venturina, Roselle) and, more in general, all of them are 41 virtually interested by periodic interventions of dams and riverbeds management. On the 42 43 other hand, the risk of bottleneck events appears a serious threaten to the conservation of 44 45 M. etrusca (e.g. Martin et al. 2012), given the present absence of genetic flow among 46 populations. 47 48 It is known that the need to cope with different selective pressures (such as extreme 49 50 temperatures) promotes population responses, ranging from simple phenotypic buffering 51 to truly evolutionary adaptations (e.g. Reusch 2014). This could be the case of the 52 53 amphibious behavior exhibited by a portion M. etrusca population in Roselle (Bartolini and 54 55 Giomi unpubl. data). Such a divergent pattern would indeed make this population an ESU 56 (Evolutionary Significant Unit), thus valuable as conservation target (Crandall et al. 2000). 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 14 of 30
1 2 3 In such a scenario, there is a need of genetic studies, which can quantify the actual level 4 5 of divergence among the Tuscan populations (and relative to other circum Mediterranean 6 entities; Glaubrecht 1993) and consequently establish conservation priorities. Behavioral 7 8 experiments would also be helpful, in order to establish, from an evolutionary point of view, 9 10 the degree of novelty of the amphibious behavior exhibited by a portion of Roselle’s 11 population. Management of isolated populations may actually pose challenges driven by 12 13 contrasting targets and needs. The necessity of guaranteeing appropriate levels of genetic 14 15 variability would require actions such as cross introduction programs (Liu & Hersher 2012), 16 whereas for preserving any evolutionary acquisitions and innovations, the priority would be 17 18 that to maintain populations’For isolation. Peer Review 19 20 M. etrusca represents an intriguing case study for investigating freshwater ectotherms 21 patterns of physiological and behavioral adaptations along temperature clines. In general, 22 23 our study places emphasis on the need of conserving rare and localized populations of 24 25 endemic/isolated ectothermic species, such as those found within spring habitats. Their 26 study seems indeed to hold the potential of clarifying the evolutionary processes and 27 28 adaptive trajectories undertaken by isolated population in response to extreme or limiting 29 30 environments. Furthermore, thermal springs are recognized as valuable model systems to 31 assess responses of lotic communities and species under climate change (Friberg et al. 32 33 2009). 34 35 36 37 Acknowledgments 38 39 40 We wish to thank Dr. Claudio Sili (National Council of Research) for his help in microalgal 41 determinations. Many thanks are due to Dr. Stefano Cannicci and Dr. Folco Giomi for their 42 43 valuable discussion on the experimental setups and comments to an early version of the 44 45 manuscript. FB wish to thanks Dr. Antonio di Franco for useful discussion on statistical 46 analysis of data. 47 48 49 50 51 References 52 53 54 55 Anderson MJ 2001. A new method for non parametric multivariate analysis of variance . 56 Austral. Ecol. 26: 32–46. 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 15 of 30 Submitted to: Invertebrate Biology
1 2 3 Anderson MJ, Gorley RN & Clarke KR 2008. PERMANOVA+ for PRIMER: Guide to 4 5 software and statistical methods. PRIMER E Ltd, Plymouth. 214 pp. 6 Atkinson D, Morley SA & Hughes RN 2006. From cells to colonies: at what levels of body 7 8 organization does the ‘temperature size rule’ apply? Evol. Dev. 8: 202–214. 9 10 Bencini A, Duchi V & Martini M 1977. Geochemistry of thermal springs of Tuscany (Italy). 11 Chem. Geol. 19: 229 252. 12 13 Bhattacharya CG 1967. A simple method of resolution of a distribution into Gaussian 14 15 components. Biometrics. 23: 115–135. 16 Bilgin FH 1973. Studies on the functional anatomy of Melanopsis praemorsa (L.) and 17 18 Zemelanopsis Fortrifasciata (Gray).Peer Proc. Malacol. Review Soc. Lond. 40: 379 393. 19 20 Bouchard RW, Anderson JA 2001. Description and protocol for two quantitative periphyton 21 samplers used for multihabitat stream sampling Documentation of periphyton 22 23 sampling techniques employed by the Central Plains Center for Bioassessment. 24 25 Central Plains Center for Bioassessment, Kansas Biological Survey University of 26 Kansas. 27 28 Celati R, Grassi S, D'Amore F & Marcolini L 1991. The low temperature hydrothermal 29 30 system of Campiglia, Tuscany (Italy): a geochemical approach. 20: 67 81. 31 Cianfanelli S 2010. Melanopsis etrusca . In: IUCN 2011. IUCN Red List of Threatened 32 33 Species. Version 2014.3.
1 2 3 Di Bella L, Carboni MG & Bergamin L 2002. Pliocene pleistocene foraminiferal 4 5 assemblages of the middle and lower Tiber valley: stratigraphy and paleoecology. 6 Geol. Romana. 36: 129 145. 7 8 Di Sabatino A, Cicolani B & Gerecke R 2003. Biodiversity and distribution of water mites 9 10 (Acari, Hydrachnidia) in spring habitats. Freshwater Biol. 48: 2163–2173. 11 Dillon Jr RT 2000. The Ecology of Freshwater Molluscs. Cambridge University Press, 12 13 Cambridge. 509 pp. 14 15 EN 13946 2003. Water quality—Guidance standard for the routine sampling and pre 16 treatment of benthic diatoms from rivers. European Committee for Standardization, 17 18 Brussels. 14 pp.For Peer Review 19 20 Esu D 1980. Neogene freshwater gastropods and their evolution in the western 21 Mediterranean area. Geol. Romana. 19: 231 249. 22 23 Esu D & Girotti O 1989. Late Miocene and Early Pliocene continental and oligohaline 24 25 mollscan faunas of Italy. Boll. Soc. Paleont. Ital. 28: 253 263. 26 Esu D, Girotti O & Kotsakis T 1993. Palaeobiogeographical observations on Villafranchian 27 28 conti nental molluscs of Italy. Scripta Geol. Spec. Issue 2: 102 119. 29 30 Friberg N, Dybkjær JB, Olafsson JS, Gislason GM, Larsen SE & Lauridsen TL 2009. 31 Relationships between structure and function in streams contrasting in temperature 32 33 Freshwater Biol. 54: 2051–2068. 34 35 Gayanilo FC Jr, Sparre P & Pauly D 1996. The FAO ICLARM stock assessment tools 36 (FiSAT) user’s guide. FAO Computerized Information Series (Fisheries). 37 38 Geary DH 1990. Patterns of evolutionary tempo and mode in the radiation of Melanopsis 39 40 (Gastropoda; Melanopsidae). Paleobiology. 16: 492 511. 41 Glaubrecht M 1993. Mapping the diversity: Geographical distribution of the freshwater 42 43 snail Melanopsis (Gastropoda: Cerithioidea: Melanopsidae) with focus in its 44 45 systematics in the Mediterranean Basin. Mitt. Hamb. Zool. Mus. Inst. 90: 41 97. 46 Guerra Merchán A, Serrano F, Garcés M, Gofas S, Esu D, Gliozzi E & Grossi F 2010. 47 48 Messinian Lago Mare deposits near the Strait of Gibraltar (Malaga Basin, Southern 49 50 Spain). Palaeogeogr. Palaeocl. 285: 264–276. 51 Hartog CD 1974. Brackish water classification, its development and problems. Hydrobiol. 52 53 Bull. 8: 15 28. 54 55 Heino J, Virkkala R & Toivonen H 2009. Climate change and freshwater biodiversity: 56 detected patterns, future trends and adaptations in northern regions. Biol. Rev. 84: 57 58 39–54. 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 17 of 30 Submitted to: Invertebrate Biology
1 2 3 Heller J & Abotbol A 1997. Litter shredding in a desert oasis by the snail Melanopsis 4 5 praemorsa . Hydrobiologia. 344: 65 73. 6 Hershler R, Liu H & Sada DW 2007. Origin and diversification of the soldier meadow 7 8 springsnails (Hydrobiidae: Pyrgulopsis ), a species flock in the Northwestern Great 9 10 Basin, United States. J. Mollus. Stud. 73: 167–183. 11 Huey RB & Kingsolver JG 1989. Evolution of thermal sensitivity of ectotherm performance. 12 13 Trends Ecol. Evol. 13: 299 305. 14 15 Liu HP & Hersher R 2012. Phylogeography of ana endangered Western North American 16 springsnail. Conserv. Genet. 13: 299 305. 17 18 Lurman G, Walter For J & Hoppeler Peer HH 2014. Review Seasonal changes in the behaviour and 19 20 respiration physiology of the freshwater duck mussel, Anodonta anatina . J. Exp. Biol. 21 217: 235 243. 22 23 Lydeard C, Cowie H, Ponder WF, Bogan AE, Bouchet P, Clark S, Cummings KS, Frest TJ, 24 25 Gargominy O, Herbert DG, Hershler R, Perez KE, Roth B, Seddon M, Strong EE & 26 Thompson FG 2004. The global decline of nonmarine Mollusks . BioScience. 54: 321 27 28 330. 29 30 Lysne SJ, Perez KE, Brown KM, Minton RL & Sides JD 2008. A review of freshwater 31 gastropod conservation: challenges and opportunities. J. N. Am. Benthol. Soc. 27: 32 33 463–470. 34 35 Madsen H 1992. Food selection by freshwater snails in the Gezira irrigation canals, 36 Sudan. Hydrobiologia. 228: 203 217. 37 38 Martin AP, Echelle AA, Zegers G, Baker S & Keeler Foster CL 2012. Dramatic shifts in the 39 40 gene pool of a managed population of an endangered species may be exacerbated 41 by high genetic load. Conserv. Genet. 13: 349–358. 42 43 Martinez MA & Thome DM 2006. Habitat Usage by the Page springsnail, Pyrgulopsis 44 45 morrisoni (Gastropoda: Hydrobiidae), from Central Arizona. Veliger. 48: 8–16. 46 Mladenka GC & Minshall WG 2001. Variation in the life history and abundance of three 47 48 populations of Bruneau hot springsnails ( Pyrgulopsis bruneauensis ). West. N. Am. 49 50 Naturalist. 61: 204–212. 51 Olden JD, Kennard MJ, Leprieur F, Tedesco PA, Winemiller KO & Garcìa Berthou E 2010. 52 53 Conservation biogeography of freshwater fishes: recent progress and future 54 55 challenges. Divers. Distrib. 16: 496–513. 56 Por FD 1972. Hydrobiological notes on the high salinity waters of the Sinai Peninsula. Mar. 57 58 Biol. 14, 111 119. 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 18 of 30
1 2 3 Reusch TBH 2014. Climate change in the oceans: evolutionary versus phenotypically 4 5 plastic responses of marine animals and plants. Evol. Appl. 7: 104–122. 6 SIRA 2011. Regional Environmental Informative System of Tuscany 7 8 http://sira.arpat.toscana.it/sira/bandat.html. Accessed July 2011. 9 10 Stewart DJ, Ruffell A, Wach G & Cioldring R 1991. Lagoonal sedimentation and fluctuating 11 salinities in the Vectis Formation (Wealden Group, Lower Cretaceous) of the Isle of 12 13 Wight, southern England. Sediment. Geol. 72: 117 134. 14 15 Strayer DL 2006. Challenges for Freshwater Invertebrate Conservation. J N Am Benthol 16 Soc. 25: 271 287. 17 18 Venice System 1959.For The final Peer resolution Review of the symposium on the classification of 19 20 brackish waters. Arch. Oceanogr. Limnol. 11: 243–248. 21 Thomsen J, Casties I, Pansch C, Körtzinger A & Melzner F. 2013. Food availability 22 23 outweighs ocean acidification effects in juvenile Mytilus edulis : laboratory and field 24 25 experiments. Global Change Biol. 19: 1017–1027, 26 Underwood A, Chapman M & Richards S 2002. GMAV 5 for Windows. An Analysis of 27 28 Variance Programme. Centre for Research on Ecological Impacts of Coastal Cities. 29 30 Marine Ecology Laboratories, University of Sydney, Australia. 31 Vinyard GL 1996. Distribution of a thermal endemic minnow, the desert dace ( Eremichthys 32 33 acros ), and observations of impacts of water diversion on its population. Great Basin 34 35 Nat. 56: 360 368. 36 Watson AM & Ormerod SJ 2004. The distribution of three uncommon freshwater 37 38 gastropods in the drainage ditches of British grazing marshes. Biol. Conserv. 118: 39 40 455–466. 41 Woodward G, Perkins DM & Brown LE 2010. Climate change and freshwater ecosystems: 42 43 impacts across multiple levels of organization. Phil. Trans. R. Soc. B. 365: 2093– 44 45 2106. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 19 of 30 Submitted to: Invertebrate Biology
1 2 3 Tables 4 5 Table 1 Average (av.), maximum (max), minimum (min) and standard deviation (sd) 6 7 values of physico chemical water parameters for upstream (Ups) and extinction 8 (Ext) positions of the considered streams. Unit of measure for ionic concentrations 9 10 is mg/L. 11 12 13 14 Bruna Aronna Roselle Roselle - Venturina 15 Parameter Ups Ext Ups Ext Ups Ext Amph Ups Ext 16 17 18 T (°C) av. For18.3 17.3 Peer 20.6 15Review 35.8 22.2 36.6 33.5 26.6 19 max 25.7 25 22.7 21.9 36.5 27.3 38.1 35.2 31.3 20 min 13.7 12.9 19 8.5 34.6 18.2 34.6 30 20.1 21 ds 5.7 5.7 1.7 7 0.8 4 0.5 2.4 5.6 22
23 pH av. 7.4 7.8 7.4 8 7.3 7.6 - 7.3 7.9 24 25 (20 °C) max 7.6 7.9 7.5 8.4 7.5 7.7 - 7.6 8 26 min 7.1 7.7 7.2 7.8 7.2 7.4 - 6.8 7.7 27 ds 0.2 0.1 0.1 0.2 0.1 0.1 - 0.4 0.1 28 29 TDS av. 1938 1831 2084 1736 2638 2546 1627 1685 30 max 2065 2015 2144 2045 2678 2580 1772 1739 31 32 min 1755 1523 1952 1381 2598 2509 1368 1639 33 ds 135 223 89 315 35 30 178 44 34 + 35 Na av. 12 12.8 11.5 13.5 28 31 - 28.5 29.3 36 max 13 14 12 17 29.5 34 - 31 31 37 min 11 12 11 11 26 29 - 27 28 38 39 ds 0.8 1 0.6 2.6 1.5 2.2 - 1.9 1.5 40 + 41 K av. 3 3 3.8 3 3.5 4.7 - 4.6 4.6 42 max 3.2 3.2 6 3.4 4.3 7 - 5 5 43 min 2.8 2.7 3 2.4 3.1 3.5 - 4.3 4.3 44 ds 0.2 0.2 1.5 0.4 0.5 1.6 - 0.3 0.3 45
46 Mg 2+ av. 98 93 112 95 122 116 - 80 78 47 48 max 106 101 122 111 125 118 - 83 81 49 min 90 78 105 72 118 114 - 78 76 50 ds 7 10 7 19 3 2 - 2 2 51 52 2+ Ca av. 405 379 442 355 575 556 - 364 352 53 max 426 418 465 423 592 566 - 379 365 54 55 min 364 313 407 289 564 545 - 352 341 56 ds 29 50 25 74 12 9 - 12 13 57 + 58 NH 4 av. 0.04 0.02 0.03 0.06 0.05 0.04 0.05 0.02 59 max - - - - 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 20 of 30
1 2 3 Bruna Aronna Roselle Roselle - Venturina Parameter 4 Ups Ext Ups Ext Ups Ext Amph Ups Ext 5 6 min - - - - 7 ds - - - - 8 - 9 F av. 1.1 1.1 1.3 1.2 2.1 2 - 1.5 1.5 10 max 1.2 1.2 1.5 1.4 2.4 2 - 1.6 1.8 11 min 1 0.9 1.2 1 2 1.9 - 1.4 1.3 12 13 ds 0.1 0.1 0.1 0.2 0.2 0.1 - 0.1 0.2 14 - 15 Cl av. 17.3 17.5 16 25.5 34.9 36.3 - 36.5 39 16 max 19 19 18 49 38.5 40 - 40 46 17 min 16 16 14 15 32 34 - 33 34 18 ds For1.5 1.3Peer 1.6 16Review 3.1 2.9 - 3.5 6 19
20 NO - av. 2.5 2.2 2.8 2.1 2.8 4.8 - 2.2 4.2 21 3 22 max 2.8 2.4 3 3.2 3.5 7.9 - 2.5 6.4 23 min 1.9 1.7 2.6 1.2 1.8 3.2 - 2 2.6 24 ds 0.4 0.4 0.2 0.9 0.7 2.2 - 0.2 1.7 25 26 2- SO 4 av, 1156 1094 1238 1063 1609 1554 - 889 969 27 max 1217 1199 1289 1271 1664 1586 - 1020 1004 28 29 min 1030 879 1133 721 1572 1510 - 621 937 30 ds 88 151 71 256 39 32 - 181 35 31 - 32 HCO 3 av, 243 228 257 179 260 242 - 221 209 33 max 280 260 268 262 265 257 - 227 234 34 min 226 217 242 102 257 232 - 216 183 35 36 ds 25 21 11 81 3 11 - 4 21 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 21 of 30 Submitted to: Invertebrate Biology
1 2 3 Table 2 Results of the two way multivariate permutational analysis of variance 4 5 (PERMANOVA) conducted on √(x + 1) transformed physico chemical water 6 parameters. Factors are: Site (‘Si’; four levels: Bruna, Aronna, Venturina, Roselle; 7 8 orthogonal and fixed, Position (‘Po’; two levels: upstream and extinction; orthogonal 9 10 and fixed). The degrees of freedom, df, variance, MS, value of pseudo F ratio and 11 probability levels, P, are shown. Number of replicates: 4. 12 13 14 15 Source df SS MS pseudo-F P 16 17 Site (Si) 3 215.86 71.95 17.898 0.001 18 PositionFor (Po) Peer1 35.61 Review35.61 4.4437 0.037 19 20 SixPo 3 24.04 8.01 1.9933 0.007 21 Res 24 96.49 4.02 22 Total 31 372.00 23 24 25 Table 3 Results of the two way analysis of variance (ANOVA) conducted on two main 26 27 physico chemical parameters. Factors are: Site (‘Si’; four levels: Bruna, Aronna, 28 29 Venturina, Roselle; orthogonal and fixed, Position (‘Po’; two levels: upstream and 30 extinction; orthogonal and fixed). The degrees of freedom, df, variance, MS, value 31 32 of F ratio and probability levels, P, are shown. Number of replicates: 4. 33 34 35 - 36 Source DF pH NO 3 37 MS F P MS F P 38
39 40 Site (Si) 3 0.0772 2.09 0.128 0.2102 3.71 0.025 41 Po (Po) 1 1.6313 24.79 0.016 0.2052 0.85 0.425 42 SiXPo 3 0.0658 1.78 0.178 0.2419 4.27 0.015 43 RES 24 0.0369 0.0567 44 TOT 31 45
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 22 of 30
1 2 3 Table 4 Results of the two way analysis of variance (ANOVA) conducted on snails density 4 5 and average shell lengths. Factors are Site (‘Si’; four levels: Bruna, Aronna, 6 Venturina, Roselle; orthogonal and fixed, Transect (‘Tr’; two levels; random and 7 8 nested in ‘Si’). The degrees of freedom, df, variance, MS, value of F ratio and 9 10 probability levels, P, are shown. Number of replicates: 8. 11 12 13 Density (ind/m2) Size (SL) 14 Source df MS F P MS F P 15 16 17 Si 3 152254.28 8.1 0.036 3.48 11.0 0.020 18 Ti(Si)For 4 Peer18847.41 1.0Review 0.440 0.31 0.8 0.530 19 Res 24 19337.55 0.38 20 21 Total 31 22 23 24
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 23 of 30 Submitted to: Invertebrate Biology
1 2 3 Table 5 Battacharya determinations of M. etrusca age cohorts at different streams, based 4 5 on summer and winter populations’ samplings. Computations are based on 0.5 mm 6 shell length intervals. For each age cohort, the mean shell length (‘Mean’) the 7 8 relative satandard deviation (‘S.D.’), the total number of observations (‘n’) and the 9 10 Separtaion Index (‘S.I.’) are reported. S.I. values smaller than 2 are showed in 11 italics. 12 13 14 15 JULY 2009 FEBRUARY 2010 16 Age Site 17 Cohort Mean S.D. n S.I. Mean S.D. n S.I. 18 For Peer Review 19 I 2.91 0.71 244 n.a. 1.9 0.56 1036 n.a. 20 II 6.01 0.75 130 4.25 4.64 1.71 362 2.41 21 Bruna 22 III 8.13 0.67 48 2.99 11.1 1.17 46 4.49 23 IV 10.87 0.84 57 3.63 24 25 I 4.17 1.48 548 n.a 2.57 0.66 431 n.a 26 Valpiana II 7.85 1.31 77 2.32 4.27 1.14 418 1.93 27 28 29 I 2.15 0.52 30 n.a. 2.82 0.69 421 n.a 30 Roselle- II 5.63 1.15 81 4.17 6.91 1.66 142 2.99 31 Amph III 8.23 1.11 101 2.3 10.99 0.46 23 2.46 32 33 34 I 3.17 0.86 1809 n.a. 1.38 0.73 1876 n.a 35 II 6.76 0.66 103 4.72 3.91 1.01 217 2.75 Roselle 36 III 6.55 0.71 29 2.37 37 38 39 I 3.87 0.86 1987 n.a. 2.9 0.87 2220 n.a. 40 Venturina II 5.77 0.90 143 2.16 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 24 of 30
1 2 3 Table 6 Results of the three way multivariate permutational analysis of variance 4 5 (PERMANOVA) carried out on M. etrusca shell length increments during the first 6 month of growth experiment. Factors are Site (‘Si’; two levels: Bruna and Venturina; 7 8 orthogonal and fixed), Position (‘Po’; two levels: upstream and extinction; orthogonal 9 10 and fixed), Mesocosm (‘Me’; four levels; random and nested in ‘Si’ and ‘Po’). The 11 degrees of freedom, df, variance, MS, value of pseudo F ratio and probability levels, 12 13 P, are shown. Number of replicates: 30. 14 15 16 Source df SS MS pseudo-F P 17 18 For Peer Review 19 Si 1 0.172 0.172 7.587 0.024 20 Po 1 0.973 0.973 42.792 0.001 21 SixPo 1 0.888 0.888 39.077 0.001 22 Me(SixPo) 11 0.281 0.026 1.830 0.048 23 24 Res 332 4.639 0.014 25 Total 346 7.643 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 25 of 30 Submitted to: Invertebrate Biology
1 2 3 Figures 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 1 Present and past distribution of Melanopsis etrusca in Southern Tuscany, back to 41 42 early XX century. The species dwells the first few hundred meters of streams fed 43 44 by thermo mineral springs. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 26 of 30
1 2 3 4 4 5 6 7 8 9 2 10 11 2+ 12 Mg SO 2- 13 4 2- TDF HCO 3 14 Ca 2+ 15 16 0 17 - pH 18 F- NO 3
PC2 23.8% PC2 For Peer Review 19 Temperature 20 K+ 21 Na + Cl - 22 2 23 24 25 26 27 28 4 29 30 6 4 2 0 2 4 31 32 PC1 43.5 % 33 Upstream Upstream Upstream Upstream 34 Bruna Aronna Roselle Venturina Extinction Extinction Extinction Extinction 35 36 37 Figure 2 Two dimensional scatter plot (PCA) of first and second principal components for 38 39 40 physico chemical water parameters of the four study sites. For each stream, 41 42 upstream and extinction positions were considered. Vectors of the linear 43 44 correlations between individual variables are superimposed on the graph. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 27 of 30 Submitted to: Invertebrate Biology
1 2 3 12000 * 4 * 5 2 6 10000 A 7 8 8000 9 10 11 6000 12 13 4000 14 15 2000 16 Number Number of snailsm 17 18 0 For Peer Review 19 Aronna Bruna Roselle Venturina 20 21 10 22 9 * 23 * 24 8 B 25 7 26 27 6 28 5 29 4 30 31 3 32 2 33
34 length shell (mm) Av. 1 35 0 36 Aronna Bruna Roselle Venturina 37 38 39 Figure 3 Average values of M. etrusca density per square meters (A) and shell length (B) 40 41 measured at upstream positions of different streams. *: statistically significant 42 43 44 differences with a probability level of 95%. Error bars are ± 1 SE. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 28 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Figure 4 Frequency distribution of M. etrusca shell length classes of 0.5 mm at different 52 53 sites, with superimposed the age cohorts (solid lines and vertical bars) as 54 55 determined by Battacharya computation. (A: Bruna; B: Roselle ‘amphibians’; C: 56 57 Aronna; D: Venturina; E: Roselle). 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Page 29 of 30 Submitted to: Invertebrate Biology
1 2 3 4 Bruna - Upstream 1 5 i Bruna - Extinction 6 8 7 8 × SL 7 A
9 1 Venturina - Upstream 10 6 11 Venturina - Extinction 12 5 13 14 4 15 16 3 17 18 2 For Peer Review 19 20 1
21 increment × % SL mo 22 0 23 Sept-Oct Oct-Nov Nov-Jan Jan-Apr Apr-Jul 24 B 25 100 26 27 28 80 29 30 31 60 32 33 34 40 35 36 37 20 38
39 Mortality (% specimens) 40 0 41 Sept-Oct Oct-Nov Nov-Jan Jan-Apr Apr-Jul 42 Sept Oct Oct Nov Nov Jan Jan Apr Apr Jul 43 44 45 Time interval 46 47 48 Figure 5 Results of individual growth and mortality inside the mesocosms at Bruna and 49 50 Venturina, from September 2009 until July 2010. A) Average monthly shell length 51 52 increments estimated from the considered time intervals. B) Average cumulative 53 54 55 percentage mortality. Error bars indicate 99% confidence intervals. 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB Submitted to: Invertebrate Biology Page 30 of 30
1 2 3 Diatoms Cyanobacteria 4 A 5 100% 6 7 8 75% 9 10 50% 11 12 13 25% 14 15 % of phytobenthic groups phytobenthic of % 16 0% 17 SWSWSWSW 18 ForBruna PeerAronna Review Roselle Venturina 19 20 Legend 21 22 Diatoms Bruna Fossa Calda B 23 24 Cyanobacteria 25 26 Chlorophycaea 27 28 Macrophytes fragments 29 30 31
32 33 34 Figure 6 A) Semi quantitative estimate of summer and winter abundances of phytobenthic 35 36 organisms found on hard substrates at upstreams position of different streams. B) 37 38 Relative abundance of main food items found inside the crop of M. etrusca 39 40 specimens (n=10) collected at Bruna and Venturina in summer 2010. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 An international journal of the American Microscopical Society and the Division of Invertebrate Zoology/SICB