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Habitat loss and degradation are causing collapses in freshwater fish in the Mediterranean region, where habitat restoration actions are still hampered by poor understanding of fish habitat selection and fitness. Here, we combined field surveys and laboratorial experiments to investigate how water velocity, body size and intra-specific interactions influence habitat selection and foraging success by the highly endangered Mira chub Squalius torgalensis. Velocity negatively affected habitat selection and fitness of chub via its negative effects on prey capture rate. Small chub occupied lower velocity ranges than large chub, and both captured the most prey at the range of velocities selected in the stream. Size-based intra-specific interactions also affected capture success, with small chub capturing proportionally less prey in the presence of large chub. Our results suggest that, during base-flow conditions, restoring low and moderate velocities up to 26 cm/s will help ensure suitable habitat and improve the fitness of small and large chub occurring in interacting groups. Integrated approaches uncovering factors directly related to habitat selection and individual fitness should guide habitat restoration for fish in Mediterranean streams and may help identify critical habitat features for other endangered species.

Future climate change scenarios predict threatening outcomes to biodiversity. Available empirical data concerning biological response of freshwater fish to climate change remains scarce. In this study, we investigated the physiological and biochemical responses of two Iberian freshwater fish species (Squalius carolitertii and the endangered S. torgalensis), inhabiting different climatic conditions, to projected future scenarios of warming (+3°C) and acidification (ΔpH=-0.4). Herein, metabolic enzyme activities of glycolytic (citrate synthase - CS, lactate dehydrogenase - LDH) and antioxidant (glutathione S-transferase, catalase and superoxide dismutase) pathways, as well as the heat shock response (HSR) and lipid peroxidation were determined. Our results show that, under current water pH, warming causes differential interspecific changes on LDH activity, increasing and decreasing its activity in S. carolitertii and in S. torgalensis, respectively. Furthermore, the synergistic effect of warming and acidification caused an increase in LDH activity of S. torgalensis, comparing with the warming condition. As for CS activity, acidification significantly decreased its activity in S. carolitertii whereas in S. torgalensis no significant effect was observed. These results suggest that S. carolitertii is more vulnerable to climate change, possibly as the result of its evolutionary acclimatization to milder climatic condition, while S. torgalensis evolved in the warmer Mediterranean climate. However, significant changes in HSR were observed under the combined warming and acidification (S. carolitertii) or under acidification (S. torgalensis). Our results underlie the importance of conducting experimental studies and address species endpoint responses under projected climate change scenarios to improve conservation strategies, and to safeguard endangered freshwater fish.

Movement may critically influence population persistence in the face of disturbance. However, data on individual movements into and out of refugia in response to disturbance remain limited, leading to an incomplete understanding of the ecological significance of mobility. Using individually marked chubs Squalius torgalensis in a seasonally drying Mediterranean stream, we quantified the rate, direction and magnitude of movements out of dry season pools, and of movements displayed across the stream over the period of hydrological connection. Recapture rate was lower in individuals marked in dry season pools (3.3%; n = 894) than in individuals marked during hydrological connection (13.0%; n = 522). Nearly half the recaptures (46.7%; n = 30) of chubs marked in dry season pools corresponded to movements that were generally longer than 100 m, overpassing the average distance among isolated pools. The frequency and distances travelled out of dry season pools were similar in upstream and downstream directions. Only 20.6% of recaptures of individuals marked during hydrological connection corresponded to movements, with more frequent and larger movements in the upstream direction. Body size was related to movement probability only for chubs marked during hydrological connection. Our results highlight considerable individual variability in movement and underscore the importance of understanding the consequences of this variation and how it may be maintained in fish populations facing increasing fragmentation and intermittency of riverscapes. Copyright © 2014 John Wiley & Sons, Ltd.

We hypothesized that distribution and microhabitat use by imperilled chub Squalius torgalensis in the Torgal stream, Portugal, during low flows, were related to spatial patchiness in physical resources and shifts in ontogenetic preferences. We mapped fish abundance and sampled microhabitat use and availability via snorkelling. We used the coefficient of dispersion in abundance, and spatial autocorrelation analyses to characterize chub distribution, and Hurdle models to relate chub presence and abundance to habitat characteristics. We showed that chub displayed an aggregated distribution, apparently in association with patchily distributed and autocorrelated physical resources, such as debris, roots and aquatic vegetation. Microhabitat use generally was unrelated to velocity, and ontogenetic differences in microhabitat use were not substantial. Nevertheless, sometimes small chub preferred low-velocity patches with coarse substrata, debris and roots, whereas large chub preferred deeper patches with roots and aquatic vegetation. Results suggest that, in low flow conditions, chub respond to resource patchiness by congregating in favourable microhabitats, and that maintenance of mosaics of patches with variable substrata, cover and depth may be important for the persistence of fish in Mediterranean streams.

In this study, the description of the karyotypes of the endangered chubs Squalius aradensis (Coelho, Bogutskaya, Rodrigues and Collares-Pereira, 1998) and Squalius torgalensis (Coelho, Bogutskaya, Rodrigues and Collares-Pereira, 1998) is presented by means of conventional (Giemsa-staining, Chromomycin A3 (CMA3)-fluorescence, Silver-impregnation (Ag-NORs)) and molecular (fluorescence in situ hybridization (FISH) with 18S rDNA probe) protocols. These endemic sister-species have an allopatric but adjacent distribution in the most southwestern part of the Iberian Peninsula. Diploid chromosome number was invariably 2n = 50 and karyotypes of both species were grossly similar, composed of metacentric and submetacentric elements with a reduced number of acrocentric pairs. Sequential staining using FISH with an 18S rDNA probe, CMA3 and Ag-NORs treatments revealed consistent positive signals located at the end of the short arms of a submetacentric chromosome pair, likely homologous in both species. While providing useful cytogenetic comparative data against other members of the genus Squalius Bonaparte, 1837, the work aimed to draw attention towards the conservation of two narrow-range and highly confined fish species.

Factors shaping individual behavior remain poorly understood for European cyprinids. We examined the effects of extrinsic (temperature and year) and intrinsic factors (body size and shoal size) on the daily activity of chub Squalius torgalensis, a critically endangered cyprinid found in the Torgal stream, Portugal. We quantified chub behaviors while snorkeling, using instantaneous sampling of focal individuals, in spring 2009 and 2010. We used Principal Component Analysis to describe individual behavior and assessed competing models relating individual behavior to explanatory variables using Akaike's Information Criterion. Year had a strong influence on chub behavior, with individuals spending more time drift foraging and orienting in 2009, whereas in 2010 they spent more time benthic foraging and cruising. Shoal size did not influence behaviors in 2009, but in 2010 individuals in small shoals foraged benthically and searched with greater frequency than those in large shoals. Individuals increased cruising and decreased searching as they grew in length, but the opposite trend was observed as temperature increased. Our results indicate that both extrinsic and intrinsic factors influence daily activity of chub, with individual behaviors varying between years, and depending upon shoal size, body size and temperature. This suggests that chub display high levels of individual behavioral flexibility, which may be important for fish inhabiting harsh and highly variable Mediterranean streams. Conservation efforts for this species will benefi tb y integrating behavioral information into management strategies.

Freshwater species with small distribution ranges are vulnerable to extinction, especially when they exhibit small effective population sizes. Squalius torgalensis, Cyprinidae, is an endemic critically endangered fish species from southwestern Portugal, confined to the intermittent streams of the Mira drainage. Assessment of genetic diversity and population structure levels was conducted through the analysis of both mitochondrial (cytochrome b) and nine nuclear microsatellite markers. Remarkable low levels of genetic diversity for both markers were observed (He < 0.38, π < 0.001). These results may be related with the restricted distribution range and dispersion patterns of the species together with demographic fluctuations associated with the intermittent features of the river system. Population structure analyses suggest the existence of two incipient divergent populations; one comprising the tributaries located upstream and the other comprising downstream tributaries of the Mira River. Data also supports the occurrence of high gene flow mainly from downstream to upstream, failing to reflect isolation of populations due to seasonal drought. Obtained results should be incorporated in future management plans for the species. These should be centred around the current connection between populations and on the maintenance of suitable habitat patches, not only for reproduction, but also for summer refuges.

This article documents the public availability of transcriptomic resources for (i) the stellate sturgeon Acipenser stellatus, (ii) the flowering plant Campanula gentilis and (iii) two endemic Iberian fish, Squalius carolitertii and Squalius torgalensis.

Chub Squalius torgalensis and nase Chondrostoma lusitanicum, in a Mediterranean stream, showed important differences in life-history traits and population dynamics. Both species reached mean maturity at age 2 years. Chub lived up to age 5 years, spawned in March to June, grew at a maximum rate of 0.59 mm mm -1 year -1 and showed a low reproductive allocation, with fecundity and egg size increasing with body size. Nase lived up to age 4 years, spawned in January to April, grew at a maximum rate of 0.46 mm mm -1 year -1 and showed a high reproductive allocation, with egg size independent of body size. Both chub and nase showed moderate fluctuations in population size during 1991-1998, but differed in factors driving density at age. Density of age 1 year juvenile chub decreased following severe summer droughts and proportionate survival prevailed thereafter. Density of age 2 year adult nase decreased following severe spring floods, but neither environmental nor parental stock effects were detected for juveniles and older fishes. The results illustrated the interplay between life history and environmental variability in driving fish population dynamics, with impacts of both summer droughts and spring floods being contingent on species-specific patterns of spawning and reproductive investment.

Current knowledge on the biological responses of freshwater fish under projected scenarios of climate change remains limited. Here, we examine differences in the protein configuration of two endemic Iberian freshwater fish species, Squalius carolitertii and the critically endangered S. torgalensis that inhabit in the Atlantic-type northern and in the Mediterranean-type southwestern regions, respectively. We performed protein structure modeling of fourteen genes linked to protein folding, energy metabolism, circadian rhythms and immune responses. Structural differences in proteins between the two species were found for HSC70, FKBP52, HIF1α and GPB1. For S. torgalensis, besides structural differences, we found higher thermostability for two proteins (HSP90 and GBP1), which can be advantageous in a warmer environment. Additionally, we investigated how these species might respond to projected scenarios of 3 climate change warming, acidification (ΔpH = -0.4), and their combined effects. Significant changes in gene expression were observed in response to all treatments, particularly under the combined warming and acidification. While S. carolitertii presented changes in gene expression for multiple proteins related to folding (hsp90aa1, hsc70, fkbp4 and stip1), only one such gene was altered in S. torgalensis (stip1). However, S. torgalensis showed a greater capacity for energy production under both the acidification and combined scenarios by increasing cs gene expression and maintaining ldha gene expression in muscle. Overall, these findings suggest that S. torgalensis is better prepared to cope with projected climate change. Worryingly, under the simulated scenarios, disturbances to circadian rhythm and immune system genes (cry1aa, per1a and gbp1) raise concerns for the persistence of both species, highlighting the need to consider multi-stressor effects when evaluating climate change impacts upon fish. This work also highlights that assessments of the potential of endangered freshwater species to cope with environmental change are crucial to help decision-makers adopt future conservation strategies. © 2017 Jesus et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Tese de doutoramento, Biologia (Ecologia), Universidade de Lisboa, Faculdade de Ciências, 2014 Submitted by Amelia Janeiro (ajaneiro@reitoria.ul.pt) on 2014-12-22T11:08:01Z No. of bitstreams: 1 ulsd69393_td_Joana_Martins.pdf: 2047367 bytes, checksum: ecaafa7f37153b05b00267ff90b94417 (MD5) Made available in DSpace on 2014-12-22T13:02:10Z (GMT). No. of bitstreams: 1 ulsd69393_td_Joana_Martins.pdf: 2047367 bytes, checksum: ecaafa7f37153b05b00267ff90b94417 (MD5) Previous issue date: 2014 Fundação para a Ciência e a Tecnologia (FCT)

The captive breeding of highly endangered species is considered to be an indispensable instrument when the natural habitats of those species are extremely degraded and/or when the effective population sizes are so reduced that there is an eminent risk of extinction (Bentsen and Olesen 2002; Maitland and Morgan 2002; Faria et al. 2010). Several authors agree that captive breeding programs do not completely solve conservation problems and should only be adopted as a safeguard against species extinction until the natural habitats are restored and the animals can be reintroduced into their original habitats under appropriate conditions (Johnson and Jensen 1991; Philippart 1992; Caughley and Gunn 1996; Naslund 1998; Snyder et al. 1999; Berejikian 2000; Bobori et al 2001; Maitland and Morgan 2002; Schonhuth et al. 2003), a strategy known as supportive breeding (Wang and Ryman 2001). This strategy has been applied in several countries when it is urgent to preserve fish populations in considerable risk of extinction (Cambray 1997; Arkush and Siri 2001; Schonhuth et al. 2003; CIP El Palmar 2006; Cardoso and Carrapato 2008; Kitanishi et al. 2013). Indeed, a vast array of factors is threatening freshwater fishes at a global scale (Duncan and Lockwood 2001). The Mediterranean populations are among the most threatened (Hermoso and Clavero 2011) due to the introduction of alien species, impoundment of rivers, habitat degradation and fragmentation, summer droughts and water quality deterioration (Collares-Pereira and Cowx 2004). In the Iberian Peninsula there is a consistent spatial relationship between the incidence of threats and the level of imperilment of species, whereby the presence of invasive species and the water extraction are the main drivers of native fish declines (Clavero et al. 2010). With the tendency for global warming, the typical summer droughts in Iberia are expected to become more intense and extended in time. This could pose an additional extinction risk factor for the Portuguese native cyprinid fish species, around 67 % of which are already considered to be ‘‘Threatened’’, ‘‘Vulnerable’’ or ‘‘Critically endangered’’ according to the Portuguese Red Book [Cabral et al. 2005; Electronic Supplementary Material (ESM) Table S1]. These species are small cyprinids with no economical value, which show restricted geographic ranges and low levels of genetic diversity (Sousa-Santos et al. 2013). A pioneer project of supportive breeding of critically endangered endemic fish species started in 2007 with nine populations belonging to six endangered and critically endangered cyprinid species endemic to the Iberian Peninsula: Achondrostoma occidentale, Iberochondrostoma almacai, Iberochondrostoma lusitanicum, Squalius pyrenaicus, Squalius aradensis and Squalius torgalensis (Fig. 1; Table 1). In this paper we describe the implementation and major results of this project and discuss the advantages of the adopted breeding scheme. Also, potential disadvantages of supportive breeding programs targeting endangered fish species are discussed. Ex situ conditions and the adoption of a ‘‘naturalistic approach’’ of reproduction. Following the research Electronic supplementary material The online version of this article (doi:10.1007/s10228-013-0383-6) contains supplementary material, which is available to authorized users.

Origins and biogeography of the chub, genus Squalius (formerly within the genus Leuciscus), in the Iberian Peninsula were inferred from comparison between patterns of geographic distribution and phylogenetic relationships among populations belonging to 14 European Squalius species. The phylogeny recovered was based on the complete sequence of the mitochondrial cytochrome b gene. Squalius species were grouped into three major clades. The basal clade included species distributed across the ancient Paratethys Sea. The second clade included species from Central and East Europe and the northern areas of the Mediterranean basin towards Minor Asia. The third clade included species from the Mediterranean Peninsulas (Iberian, Italy, and Balkans). The Iberian Squalius species do not constitutes a monophyletic group. Our data indicate that the Iberian Peninsula was colonized at least twice by two different monophyletic lineages, a meridional group and a Central Europe group. The amount of species diversity found in the Iberian Peninsula and the phylogenetic relationships among these species, together with their geographic distribution, suggest that the Central Europe lineage colonized the Iberian Peninsula at a latter time. Our data indicate that the northeastern Iberian lineage is phylogenetically close to Greek populations of Squalius cephalus, while the second lineage formed a monophyletic group including Squalius pyrenaicus, Squalius carolitertii, Squalius aradensis, and Squalius torgalensis. The speciation process that generated these species and the geographic structure of their populations, principally in S. pyrenaicus, can be attributed to paleogeographical events like the ancient endorrheism and the development of hydrographic basins.

Common name: Escalo do Arade (Portuguese). Conservation status: Critically Endangered according to the Portuguese Red Data Book (Cabral et al. 2005). Identification: Small cyprinid with 131mm of maximum standard length and 35–42 canaliculate scales on the lateral line, 7–8.5 scales above the lateral line, 2.5–3 scales below the lateral line, 8 dorsal and 7–8 anal fin rays. Pharyngeal teeth are two-rowed (2.5–5.2); vertebral formulae are 21+17 or 21+16; preopercular–mandibular cephalic sensory canal communicates with infraorbital canal; and fourth and fifth infraorbitals are large and completely fused (Coelho et al. 1998). Illustration: Marcos Oliveira. Distribution: This species is endemic to Portugal and occurs in the rivers Arade, Seixe, Algibre, Bordeira, Aljezur, Alvor and Quarteira. It was isolated from its sister species, Squalius torgalensis, around 5.13 MYago due the uplift of the Caldeirao Mountains (Sousa-Santos et al. 2007). Abundance: It is estimated that the number of mature individuals is higher than 10.000 but strong seasonal fluctuations may occur (Cabral et al. 2005). Habitat and ecology: Generalist species, inhabiting a broad range of habitats outside the reproductive season—in spring migrations towards higher velocity areas with coarse substrata seem to occur (Santos and Ferreira 2008). Reproduction: Breeding season is expected to be similar to that of S. torgalensis: March to June. Extensive hybridization with Squalius alburnoides occurs in the Quarteira River (Sousa-Santos et al. 2006). Threats: Dams, proliferation of introduced exotic species, water scarcity, habitat degradation and loss of water quality. Conservation: Some efforts are being conducted to the ex situ reproduction of this species. Conservation recommendations: Habitat restoration of some stretches followed by restocking with fish produced in ex-situ breeding programmes. Mesquita et al. (2005) suggested that conservation efforts should pay particular attention to isolated populations and smaller drainages, where low levels of genetic diversity were found (as a Environ Biol Fish DOI 10.1007/s10641-009-9550-z