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Evidence for ancient interspecific gene flow through hybridization has been reported in many animal and plant taxa based on genetic markers. The study of genomic patterns of closely related species with allopatric distributions allow to assess the relative importance of vicariant isolating events and past gene flow. Here, we investigated the role of gene flow in the evolutionary history of four closely related freshwater fish species with currently allopatric distributions in western Iberian rivers - Squalius carolitertii, S. pyrenaicus, S. torgalensis and S. aradensis - using a population genomics dataset of 23 562 SNPs from 48 individuals, obtained through genotyping by sequencing (GBS). We uncovered a species tree with two well differentiated clades: (i) S. carolitertii and S. pyrenaicus; and (ii) S. torgalensis and S. aradensis. By using D-statistics and demographic modelling based on the site frequency spectrum, comparing alternative demographic scenarios of hybrid origin, secondary contact and isolation, we found that the S. pyrenaicus North lineage is likely the result of an ancient hybridization event between S. carolitertii (contributing ~84%) and S. pyrenaicus South lineage (contributing ~16%), consistent with a hybrid speciation scenario. Furthermore, in the hybrid lineage we identify outlier loci potentially affected by selection favouring genes from each parental lineage at different genomic regions. Our results suggest that ancient hybridization can affect speciation and that freshwater fish species currently in allopatry are useful to study these processes.

Downstream of small-scale hydropower plants (SHPs), the intensity, frequency and persistence of hydropeaking events often cause an intolerable stress on fish of all life stages. Rapid increases in flow velocity result in fish avoiding unstable habitats and seeking refuge to reduce energy expenditure. To understand fish movements and the habitat use of native Iberian cyprinids in a high-gradient peaking river, 77 individuals were PIT tagged downstream of Bragado SHP in the North of Portugal. Tagged fish species included Pseudochondrostoma duriense and Squalius carolitertii. Fish positions were recorded manually on two di erent occasions: during hydropeaking events (HP) and without hydropeaking events (NHP). From the 77 tagged fish, we were able to record habitat use for 33 individuals (20 P. duriense and 13 S. carolitertii) in a total of 125 relocations. Fish species were distributed along the river reach with high density in the upstream area in the vicinity of the SHP tailrace, in particular during HP. Fish locations were associated with velocity for P. duriense and S. carolitertii. The latter tended to use faster flowing waters than P. duriense. Our findings on the habitat use in peaking rivers are a valuable tool to help in the selection and design of mitigation measures

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.

Multivariate prey trait analysis is a functional approach to understand predator-prey relationships. Here, seven macroinvertebrate ecological traits have been used for the analysis of trophic ecology of co-occurring age classes of Northern Iberian chub Squalius carolitertii, a cyprinid fish species. The present study identified several key factors in the handling efficiency and habitat utilization for feeding of S. carolitertii that may have a wider application, particularly for other cyprinid species. The results revealed a remarkable similarity in the feeding behaviour among age classes, suggesting a foraging behaviour convergence among them in both prey-handling efficiency and feeding habitat utilization. Nevertheless, some age classes showed clear preferences for particular categories of ecological trait; for example, age 1 showed a clear ability to feed on flattened prey items, whereas ages 2 and 3 were able to feed on preys with different body shape due to their general distribution in the fuzzy principal component analysis (FPCA). Finally, this study shows how multivariate approaches can complement traditional diet analyses, and the method has wide applicability across life-stages of cyprinid species.

Summary The main purpose of this study was to gather swimming performance information for two endemic cyprinids of the Iberian Peninsula to contribute to the optimization of fish ways. Critical swimming speed (Ucrit) was determined for the Tagus nase Pseudochondrostoma polylepis (Steindachner, 1864) and for the bordallo Squalius carolitertii (Doadrio, 1988) in a swimming tunnel. From a total of 80 P. polylepis tested, the mean (± SD) Ucrit observed was 0.78 ± 0.15 ms−1 (c. 3.74 ± 0.93 BL s−1); the 68 S. carolitertii tested presented an Ucrit of 0.54 ± 0.1 ms−1 (c. 4.43 ± 0.74 BL s−1). Significant interspecific differences were found between the Ucrit of the tested cyprinids. Intraspecific comparisons between the Ucrit and the variables of size, sex, condition factor and gonado-somatic index were also made. No sex-or gonad maturation-related differences between the Ucrit were identified, but the robust P. polylepis were found to be stronger swimmers. Water velocities in fish ways for P. polylepis and S. carolitertii should aim, on average, for lower than 0.7 and 0.5 ms−1, respectively.

Knowledge of food resource partitioning between sympatric fish species is critical for understanding the fish communities functioning. Four sympatric fish species (Salmo trutta, Pseudochondrostoma duriense, Squalius carolitertii and Barbus bocagei) were captured in August 2010 in the River Tormes (Avila, Central Spain) in order to study food resource partitioning between fish species such us as a possible mechanism enabling their coexistence, using a fuzzy principal component analysis (FPCA). Diet comparison among species shows that detritus are present in P. duriense, B. bocagei and S. carolitertii, although in different occurrence, showing that these species present typically omnivorous feeding habits. Regarding prevalent food, in all species, benthic prey constituted the most important prey in abundance terms, and terrestrial invertebrates were only consumed by S. trutta, S. carolitertii and P. duriense. High overlap values (Schoener's index from 0.8 up to 1) were found, but it may not indicate competition, since species can adopt different strategies to overcome competence. Thus, prey traits analyses suggest that differences in macrohabitat use, drift behaviour of prey and prey size are important adaptive features that may reduce the inter-specific competition in the fish community and permit the food partitioning that allows coexistence.

Over the last two years (2008-2010) we performed a monitoring project to update the Spanish freshwater fish fauna. We have collected in 785 sampling localities homogeneously distributed throughout Spain each year. We find as the most important result of this study the occurrence of several species that constitute new records for some rivers or basins in Spain: Lampetra planeri, Petromyzon marinus, Barbus haasi, Iberochondrostoma lemmingii, Squalius alburnoides, Squalius carolitertii, Squalius malacitanus, Cobitis calderoni, Cobitis paludica, and Salaria fluviatilis. Finally we corroborate the existence of Barbatula barbatula in the Duero basin and the assignation of the northeastern populations of this genus to B. quignardi.Entre los años 2008 y 2010 hemos realizado un proyecto de monitorización para actualizar la base de datos de la ictiofauna continental española. Se ha llevado a cabo en 785 estaciones de muestreo cada año, repartidas homogéneamente por toda España. Entre los datos más sobresalientes se incluyen nuevas citas para algunas cuencas y ríos de España de Lampetra planeri, Petromyzon marinus, Barbus haasi, Iberochondrostoma lemmingii, Squalius alburnoides, Squalius carolitertii, Squalius malacitanus, Cobitis calderoni, Cobitis paludica y Salaria fluviatilis. También se ha confirmado la presencia de Barbatula barbatula en la cuenca del Duero y la adscripción de las poblaciones de este género del Noreste de la Península Ibérica a B. quignardi.

Hybridization, ploidy level and genomic constitution may be important to respond to different environments, by producing different phenotypes and thus reducing competitive interaction. Through geometric morphometrics, we examined variation in body size and shape among biotypes of the Squalius alburnoides hybrid complex and their sperm donor (Squalius carolitertii). Results showed that S. carolitertii is significantly larger in size than the biotypes of the complex. No significant relationship was observed between ploidy and body size among S. alburnoides biotypes. Significant variation in body shape was found between S. carolitertii and S. alburnoides, and between tetraploids and the other biotypes. These differences in biotypes may reduce resource competition, highlighting the potential importance of resource availability favouring one biotype over another. In S. alburnoides, the adaptation to different trophic niches through modification of trophic morphology, body shapes, and feeding behaviour, may result from an increase in ploidy and genomic constitution. This adaptation may account also for the formation and maintenance of this nonsexual complex.

The congeneric freshwater fish Squalius carolitertii and S. torgalensis inhabit different Iberian regions with distinct climates; Atlantic in the North and Mediterranean in the South, respectively. While northern regions present mild temperatures, fish in southern regions often experience harsh temperatures and droughts. Previous work with two hsp70 genes suggested that S. torgalensis is better adapted to harsher thermal conditions than S. carolitertii as a result of the different environmental conditions. We present a transcriptomic characterisation of these species' thermal stress responses. Through differential gene expression analysis of the recently available transcriptomes of these two endemic fish species, comprising 12 RNA-seq libraries from three tissues (skeletal muscle, liver and fins) of fish exposed to control (18 °C) and test (30 °C) conditions, we intend to lay the foundations for further studies on the effects of temperature given predicted climate changes. Results showed that S. carolitertii had more upregulated genes, many of which are involved in transcription regulation, whereas S. torgalensis had more downregulated genes, particularly those responsible for cell division and growth. However, both species displayed increased gene expression of many hsps genes, suggesting that they are able to deal with protein damage caused by heat, though with a greater response in S. torgalensis. Together, our results suggest that S. torgalensis may have an energy saving strategy during short periods of high temperatures, re-allocating resources from growth to stress response mechanisms. In contrast, S. carolitertii regulates its metabolism by increasing the expression of genes involved in transcription and promoting the stress response, probably to maintain homoeostasis. Additionally, we indicate a set of potential target genes for further studies that may be particularly suited to monitoring the responses of Cyprinidae to changing temperatures, particularly for species living in similar conditions in the Mediterranean Peninsulas.

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.

The iberian chub (Squalius carolitertii) is an endemic species whose biology is yet largely unknown and which is catalogued as being endangered. Therefore more information about its biology is essential to develop appropriate management strategies. The Estorâos River population has been studied from December 1995 to March 2002 through regular electro-fishing surveys. Additionally, a fish trap installed in the river from November 2001 to July 2002 allowed for the collection of information on population movements and reproduction. The results revealed some fluctuations in the density estimates, though the Squalius carolitertii population in the Estorâos River is quite low. Six age classes (0+ to 5+) were identified. Iberian chub growth is isometric and can reach a final length of 33.5 cm. Sexual dimorphism is evident, with males maturing earlier and at smaller sizes. An increase of the Iberian chub movements was detected from middle April onwards. These movements were related to the beginning of the reproductive period and were mainly correlated with the rise of water temperature. Our results indicate that the population characteristics do not differ substantially from those of other endemic chub populations in the Iberic peninsula. El bordallo (Squalius carolitertii) es una especie cuya biología es en gran parte desconocida y que está catalogada en peligro de extinción. El estudio de su biología es importante para la gestión de sus poblaciones y de su hábitat. La población del río Estorâos (Cuenca del río Límia) ha sido estudiada de Diciembre de 1995 a Marzo de 2002 mediante muestreos con pesca eléctrica. Un sistema de redes utilizado de Noviembre de 2001 a Julio de 2002 permitió la obtención de información sobre los movimientos y parámetros reproductivos de la población. Los datos indican fluctuaciones de la densidad, a pesar de la población presentar valores muy bajos. Seis clases de edad (0+ a 5+) han sido identificadas. El crecimiento de Squalius carolitertii es isométrico y puede llegar a una talla final de 33,5 cm. El dimorfismo sexual es evidente y los machos maduran más temprano y con tallas inferiores a de las hembras. Un creciente de movimientos del bordallo ha sido encontrado a mediados del mes de Abril, lo que está de acuerdo con la fecha del período reproductivo y también con el incremento de la temperatura del agua. Nuestros datos indican que esta población no es muy distinta de otras poblaciones de bordallos endémicos de la península Ibérica.

– Microhabitat use of Iberian nase Chondrostoma polylepis (Steindachner 1865) and Iberian chub Squalius carolitertii (Doadrio 1988) was studied in three small streams of the Lima basin, north-west Portugal. Measurements were made for 210 and 248 individuals, respectively, distributed in two size classes, based on fish ages, during winter, spring, summer and autumn 1998. Both species moved to shallow and slow-flowing microhabitats in summer and autumn, but this mainly reflected seasonal variation in microhabitat availability. Larger individuals of both species used deeper and lower percentage cover areas than smaller conspecifics, however significant ontogenetic differences were only found for water velocity. Different patterns of nonrandom microhabitat use were displayed by both cyprinids. Larger I. nase were in deeper and more discovered waters than those available in the habitat, whereas I. chub was over-represented in slow-flowing areas. There was no evidence of interspecific competition for microhabitat, therefore it is unlikely that spatial resource was limited during the present study. Resumen 1. La utilizacion de micro-habitats por Chondrostoma polylepis y Squalius carolitertii fue estudiada en tres rios de la cuenca del Rio Lima (noroeste de Portugal). Se tomaron medidas de 210 individuos de Ch. polylepis y de 248 individuos de S. carolitertii, distribuidos en dos clases de tamanos (basados en determinaciones de edad), en las cuatro estaciones del ano 1998. 2. Ambas especies se movieron hacia micro-habitats someros y de caudal lento en verano y en otono pero esto, esencialmente refleja variaciones estacionales en la disponibilidad de micro-habitats. Los individuos mayores de ambas especies utilizaron areas mas profundidad con menor porcentaje de cubierta vegetal. Sin embargo, solo encontramos diferencias ontogeneticas significativas para la velocidad del agua. Patrones diferentes de uso no al azar de micro-habitats fueron evidentes para ambos ciprinidos. Los individuos mayores de Ch. polylepis utilizaron aguas mas profundas y descubiertas que las disponibles en el habitat mientras que S. carolitertii aparecio sobre-representado en areas de flujo lento. 3. No encontramos evidencia de competencia inter-especifica para los micro-habitats. Es, por ello, poco probable que los recursos espaciales fueron limitantes durante el periodo de estudio.

Squalius alburnoides is a complex of minnows common to the Iberian Peninsula, consisting of two distinct forms. The most common form is comprised of diploid and polyploid asexual hybrids heterozygous for several diagnostic allozyme loci contributed by Squalius pyrenaicus or Squalius carolitertii and a missing ancestor. The other form is diploid and homozygous for the allele contributed by the missing ancestor at these same loci. Present results from analyses of sex ratio and cytochrome b sequences are not consistent with the evolutionary distinctiveness of this non-hybrid form and suggest that it represents an all-male lineage imbedded within an almost all-female complex. This all-male lineage allowed preservation of the ancestral paternal nuclear genome after the paternal ancestor became extinct in all or most drainages, with important evolutionary implications.

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.

Background. The northern Iberian chub Squalius carolitertii (Doadrio, 1988) is a small endemic cyprinid inhabiting the rivers of the Iberian Peninsula. The knowledge of feeding patterns is essential to understand the ecological role of fish populations, helping to the development of conservation and management plans. The aim of the present study was to analyze the ontogenetic dietary shifts and food selection of S. carolitertii, contributing to knowledge of the feeding behaviour of this fish species. Materials and methods. Diet composition of S. carolitertii was compared to benthos and drift composition in a river of Central Spain (Ávila, River Tormes) using selectivity indices of Ivlev and Savage. The age of 57 S. carolitertii collected in August 2010 was determined by scale reading and by length frequency analyses (LFA) with the Petersen method. Maximum length of benthos, drift and prey invertebrates was measured for each item to establish whether prey-size selection depends upon the size-frequency distribution of available prey. Results. Detritus were found in 33 fish (57.9% of occurrence). Nymphs of Baetis spp. were the most abundant prey (46.6%) and were identified in the 49.1% of the stomachs. Moreover, Baetis spp. was selected positively from the benthos and drift by all age classes. Abundant potential prey items such as Epeorus spp. in the benthos and Simuliidae in the drift were negatively selected. Individuals without detritus in the gut contained more animal prey items than individuals with a dominance of detritus, and the frequency of occurrence of detritus decreased with the age. Mean prey size increased with fish size (r = 0.646, P < 0.001). Conclusion. Age-related diet shifts occur at three different levels: (1) frequency of occurrence of detritus decreases with fish age; (2) prey selection varied with fish age; and (3) mean prey size increased as fish size increased. The rejection of Epeorus spp. and Simuliidae suggests that other factors, apart of prey abundance, including sitespecific prey accessibility, prey size, energetic selection criteria and prey preference of fishes play an important role in feeding behaviour of S. carolitertii. Prey-size selection is probably dependent on the size-frequency distribution of the available prey. This work has been partially supported by the project 10PXIB2111059PR of the Xunta de Galicia and the project MIGRANET of the Interreg IV B SUDOE (SouthWest Europe) Territorial Cooperation Programme (SOE2/P2/E288) SI

There is a need to quantify the multivariate quality of a recreational fishery at the site scale to better communicate the relative quality among sites to the public and anglers. Borrowing on the general approach of multimetric indices of biotic integrity (IBIs), we developed fishery quality indices (FQIs) from species quality indices (SQIs) based on measures of fish abundance and size structure for northern and central Portuguese streams. Our FQIs showed regional patterns indicating a range in fishery quality. Higher coldwater FQI scores were mostly found in the northwestern (Minho and Lima), northeastern Douro, and northern Tagus basins. Higher warmwater FQI scores occurred in the eastern Tagus basin. The species that contributed the most to warmwater FQI scores were largemouth bass Micropterus salmoides, pumpkinseed Lepomis gibbosus, the cyprinid Luciobarbus bocagei, chubs Squalius carolitertii and S. pyrenaicus, and nases Pseudochondrostoma duriense and P. polylepis. The chubs, nases, and brown trout Salmo trutta contributed the most to coldwater FQI scores. As expected, our indices were correlated with river size and with disturbance at the catchment, segment, and site scales. Regression models for separate coldwater and warmwater FQIs were stronger than those for the individual SQIs and for an all-site FQI. The correlation was positive between the coldwater FQI and a coldwater IBI but negative between the warmwater FQI and warmwater IBIs. The proposed FQIs offer a quantitative approach for assessing relative fishery quality among sites and for making regional assessments given an appropriate study design. The component SQIs and SQI metrics of the FQIs can be disassociated to determine the population and species characteristics most affected by various environmental variables.

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.