Molecular systematic of the genus Phoxinus Rafinesque, 1820 (Actynopterigii: Cyprinidae) in the Iberian Peninsula Irene Cobo-Simón, Ignacio Doadrio Departamento de Biodiversidad y Biología Evolutiva. Museo Nacional de Ciencias Naturales, CSIC. C/José Gutiérrez Abascal, 2. 28006 Madrid. E-mail:
[email protected]
ABSTRACT This work provides the first systematic revision of the populations of genus Phoxinus in Spain based on molecular data. Currently, the populations from Spain are denominated as Phoxinus bigerri Kottelat 2007 based only on morphological characters. Our study was based on mitochondrial (Cytochrome b) DNA of 241 individuals from their complete distribution range in Spain. A phylogenetic (Maximum likelyhood ML and Bayesian Inference BI) and a phylogeographical (Haplotypes network) analysis were performed. The results show several well-differentiated phylogroups in the Phoxinus populations. That suggests the possible existence of different species of the genus Phoxinus or genetic introgresion in the Iberian Peninsula. Este trabajo proporciona la primera revisión sistemática de las poblaciones del género Phoxinus en España cib datios moleculares. Actualmente, las poblaciones del género Phoxinus de España son adscritas a la especie Phoxinus bigerri Kottelat, 2007, basándose únicamente en caracteres morfológicos (ver Kottelat, 2007). Nosotros estudiamos 241 individuos del género Phoxinus de toda su área de distribución para un gen mitocondrial (Citocromo b). Un análisis filogenético (Máxima Verosimilitud MV) y filogeográfico (Inferencia Bayesiana IB) nos mostró la existencia de varios linajes diferenciados en la s poblaciones del género Phoxinus de la Península Ibérica. La existencia de diferentes linajes en las mismas localidades sugiere la posible existencia de diferentes especies o introgresión genética en la Península Ibérica.
INTRODUCTION
RESULTS
The genus Phoxinus during these last years have undergone numerous taxonomic changes. Since the early 20th century, it has been accepted that there is a single species in western Europe, Phoxinus phoxinus (L., 1758). A current systematic revision (Kottelat, 2007) based on morphological characters differentiated three European species within the genus: P. bigerri from Adour drainage, France and the upper Ebro drainage, Spain, P. septimaniae from Languedoc, France, and P. strymonicus from Strymon drainage, Greece. Kottelat and Freyhof (2007) warn that the identification of the Iberian minnow populations as P. bigerri is tentative, as Kottelat (2007) did not analyze samples from Iberia. However, in a recent international standardization of common names of Iberian endemic freshwater fishes, Iberian minnow populations have been renamed as Pyrenean minnow P. bigerri Kottelat, 2007 (Leunda et al. 2009). The species is considered to be endemic to the Adour (SW France) and Ebro (NE Spain) basins, as well as some small watersheds in the North of Spain (Kottelat 2007; Kottelat and Freyhof 2007) forming a continuum in its northern distribution, in the Cantabric slope (Doadrio 2001). A recent study (Sánchez-Fernández et al., 2012) include the Atlantic slope of Galicia (Northwest of Spain) within the distribution of P. bigerri. Thus, systematic of genus Phoxinus in the South of Europe in general, and in the Iberian Peninsula in particular, is based only on morphological characters. There are no previous molecular studies that analyze these populations.
The phylogenetic reconstruction and phylogeographical analysis (Figs. 3 and 4) shows three main clades in the iberian Peninsula. Clade A grouped populations of Segre (Ebro basin), Llobregat and Ter rivers (all of them placed in the Northeast of the Iberian Peninsula) with populations from South France rivers on the Mediterranean Slope (Bévéra and Herault rivers). The Clade B also included populations of Segre (Ebro basin), Llobregat and Ter rivers and from South France rivers on the Altlantic Slope (Garonne river). Within of Clade C we observed a well differenciated structure in four subgroups. One of them formed by the rivers from the West of the Cantabric slope (Nansa, Esva, Cares, Pas, Pigüeña and Zardón), another formed by the eastern rivers of the Cantabric slope (Butrón, Barbadún, Agüera, Altube and Asón), a third one formed by Artibai river, also placed in the Cantabric slope, and a last one formed by populations from Ebro, Duero, Nivelle and Baztán basins.
DUERO, EBRO, NIVELLE AND BAZTÁN BASINS
A)
The aim of this study is to offer nuclear and mitochondrial molecular data to our better comprehension of the taxonomy of the genus Phoxinus in the Iberian Peninsula.
METHODS
B)
CLADE C
-A mitochondrial gene (Cythocrome b) of 241 individuals was studied, covering almost the entire known geographical range of Phoxinus genus in the Iberian Peninsula and the South of France (Figure 2). -A phylogenetic reconstruction was performed using Bayesian Inference (BI) and Maximum Likelihood (ML) methods, with Phoxinus erythrogaster as outgroup (Figure 3). -Genetic distances between groups were obtained based on the phylogenetic reconstruction. -An haplotypes network was performed for the phylogeographical analysis (Figure 4).
A)
Eastern rivers C)
CANTABRIC SLOPE
Western rivers D)
B)
Artibai river
C)
CLADE A. NOTHEASTERN IBERIAN PENINSULA AND MEDITERRANEAN FRENCH RIVERS CLADE B. NORTHEASTERN IBERIAN PENINSULA AND ATLANTIC FRENCH RIVERS Phoxinus erythrogaster (Outgroup)
Figure 1. A) Specimen of Phoxinus bigerri on its left side. B) Specimen of Phoxinus bigerri in a river. C) Male with breeding tubercles Figura 1. A) Ejemplar de Phoxinus bigerri sobre su lado izquierdo. B) Ejemplar de Phoxinus bigerri en su hábitat. C) Macho con tubérculos nupciales.
Figure 3. Phylogenetic reconstruction of Phoxinus based on sequences of Cytochrome b by Maximum Likelyhood (ML) and Bayesian Inference (BI). Posterior probabilities from ML are shown over the nodes. Bootstrap from Bayesian Inference is shown above nodes. A) Individual from Pisuerga river (Duero basin). B) Individual from Oka river (Eastern of Cantabric slope). C) Individual from Nansa river (Western of Cantabric slope). D) Individuals from Artibai river (Cantabric slope). Figura 3. Reconstrucción filogenética del género Phoxinus basada en secuencias del Citocromo b mediante Máxima verosimilitud (MV) e Inferencia Bayesiana (IB). Las probabilidades posteriores (MV) se muestran sobre los nodos. Los soportes de bootstrap (IB) se muestran bajo los nodos. A) Ejemplar del río Pisuerga (Cuenca del Duero). B) Ejemplar del río Oka (Este de la Vertiente Cantábrica). C) Ejemplar del río Nansa (Oeste de la Vertiente Cantábrica). D) Ejemplar del río Artibai (Vertiente Cantábrica).
B)
CLADE C
CLADE A
Artibai river Eastern cantabric slope rivers
A)
Duero, Ebro, Nivelle and Baztán basins Figure 2. A) Range of distribution of genus Phoxinus in the Iberian Peninsula and the South of France (blue). B) Sample sites of the individuals of genus Phoxinus that were studied in this work. Figura 2. A) Área de distribución del género Phoxinus en la Península Ibérica y el Sur de Francia (azul). B) Localidades donde fueron colectados los individuos del género Phoxinus estudiados en este trabajo.
CLADE B
Western Cantabric slope rivers Figure 4. Haplotypes network based on Cytochrome b gene of Phoxinus samples obtained in this work. Figura 4. Red de haplotipos basada en el gen del Citocromo b de los ejemplares de Phoxinus estudiados en este trabajo.
CONCLUSIONS Phoxinus specimens show the formation of three Iberian clades, clade A, B and C. Within of Clade C we observed a well differenciated structure in four subgroups. Genetic distances among haplogroups is high and a geographical structure of different haplotypes can be observed.That suggests the possible existence of different species of the genus Phoxinus in the Iberian Peninsula . However, in northeastern rivers different haplogroups appear in simpatry. These results are preliminary and the incorporation of more phylogenetic analysis of this sequences is needed, as well as morphological analysis and the study of nuclear genes of the Iberian populations, to better elucidate the systematic of the group.
AKNOWLEDGEMENTS The authors thank L. Alcaraz for their help in laboratory work. We are also grateful to M. Casal, M. Cobo, D. Corona, M. Esteve, E. G. González and S. Perea for their interest and suggestions. This study was funded by project C GL2010-15231BOS.
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