Phylogenetic Relationships within GenusLeuciscus(Pisces, Cyprinidae) in Portuguese Fresh Waters, Based on Mitochondrial DNA CytochromebSequences

To investigate phylogenetic relationships amongLeuciscusspecies occurring in Portuguese inland waters, the cytochromebgene was sequenced from representatives of the main rivers. This study supports the recognition of the species level forL. pyrenaicus,including populations from the southern Portuguese drainages (Tejo, Sado, and Guadiana drainages), and forL. carolitertii,including populations from the northern Portuguese drainages. The existence of two new species occurring in the extreme southwestern drainages of Mira and Arade is also suggested. The present results support the monophyly of the Mira and the Arade populations, as well as an early divergence of these two lineages. The present-day distribution ofLeuciscusspecies is seen as a consequence of Pliocene and Pleistocene events, such as river disjunctions and posterior confluence in epicontinental seas and river captures. A mixture of haplotypes was observed in the Mondego and the Tejo drainages, which could be a consequence of ancient river captures, with a possible mitochondrial DNA introgression in the Tejo drainage and a recent introduction by man in the Mondego drainage. The pattern of differentiation among mtDNA haplotypes and their geographic distribution is discussed in terms of evolutionary aspects.     

Genetic structure of the diploid–polyploid fish Squalius alburnoides in southern Iberian basins Tejo and Guadiana, based on microsatellites

Several studies have demonstrated the hybrid origin of Squalius alburnoides , a complex of diploid, triploid and tetraploid fish, with Squalius pyrenaicus as the maternal ancestor and an Anaecypris hispanica -like species as the paternal ancestor. The aim of this study was to assess patterns of genetic diversity and similarity between sympatric forms of the different ploidy levels of S. alburnoides and related parental species in the two largest basins that are part of its southern Iberian distribution, the Tejo and Guadiana, using microsatellites. High levels of genetic diversity were found in populations of S. alburnoides from both basins, reflecting the hybrid origin, the high diversity of forms in these river basins and the different modes of reproduction of the complex. Microsatellites indicated low values of genetic differentiation between the non-hybrid all-male form of S. alburnoides and A. hispanica , supporting the view that these forms share a common ancestor. In some instances, diploid and triploid hybrids from the same river basin were more closely related to each other than to their counterparts in the other basins, suggesting an ancient origin for the complex in these Iberian southern basins.     

Genetic structure of the Iberian chub,Leuciscus pyrenaicus,in the Tejo drainage

The pattern of genetic variation in 38 samples of Leuciscus pyrenaicus, from seven sites in the Tejo drainage sampled on six occasions (over 19 months), were examined electrophoretically at 12 variable loci. Significant levels of spatial subdivision were observed. In general, the genetic distances in a river, were smaller than those between rivers. The differences observed suggest that isolation-by-distance is one important factor responsible for the spatial genetic differentiation. Moreover, the genetic diversity of this species in the Tejo drainage seems to be influenced by habitat heterogeneity, i.e., upland and lowland rivers. In opposition to other cyprinid species inhabiting the same drainage, the disruption of connectivity between populations above and below dams was not confirmed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The genetics of maintenance of an all-male lineage in the Squalius albuvnoides complex

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, withimportant evolutionary implications.     

Contrasting views over a hybrid complex: Between speciation and evolutionary "dead-end"

The Squalius alburnoides complex, of hybrid origin, comprises diploid, triploid and tetraploid forms and has a widespread distribution in the Iberian waters. The southern populations of this complex, sympatric with S. pyrenaicus, show high genetic variability, diversity of forms and reproductive modes which create pathways that may allow for the establishment of a new species in the future. Here we report a contrasting view over the S. alburnoides complex: in the Mondego River basin (northern Portugal), nuclear "non-hybrid" and tetraploid forms are absent and a clearly impoverished genetic diversity is observed, contributing to a general scarcity of possibilities of generating novel genetic material. Moreover, the bisexual species involved in the maintenance of the complex in this basin (S. carolitertii) exhibits a considerably lower genetic variability, when compared with S. pyrenaicus. The observed differences suggest that, despite being originated by similar hybridization events and maintained by analogous reproductive mechanisms, different populations of the complex were exposed to distinct evolutionary constrains, which in some cases resulted in diversification and speciation while in others led to a compromising situation in terms of evolutionary potential. Additionally, and for the first time all forms were used in the calculation of genetic distances and diversity indices, widening the possibilities of analysis of the complex by allowing the inclusion of a large part of the available data, irrespective of ploidy level.     

Assessing inter‐drainage connections: patterns of genetic diversity in an Iberian cyprinid fish

The phylogeography of Iberian freshwater fish species strongly reflects the geomorphological history of Iberian water drainages and formation of mountain ranges. In particular, the Iberian Central Massif limits two ichthyogeographical districts based on fish distribution assemblages: the Northwest district, which comprises all river basins north of the Central massif, and the Central‐west district, which comprises the Tejo and Sado basins. Despite this scenario, the cyprinid Pseudochondrostoma polylepis occurs in both the Tejo and Mondego rivers (one either side of the Estrela Mountains, in the western end of the Central Massif). By analysing the population structure and demographic history of this straight‐mouth nase, based on both mtDNA and microsatellites, we have found evidence of a relatively recent common origin for both Mondego and Tejo populations. Shared haplotypes and lack of divergent lineages for mitochondrial marker, high levels of admixture and common ancestry at the nuclear level, together with signatures of asymmetrical gene flow from Tejo to Mondego suggest a Mondego colonization through a founder event with an origin in Tejo. We suggest that this colonization might have resulted from the occurrence of river connections/captures between Mondego and Tejo headwater tributaries in the recent past. With this study we reinforce that local‐scale geomorphological events and the adaptation/dispersal ability of a species to new environments can have a major impact on its demographic history, colonization processes, and ultimately its distribution. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 656–669.     

Phylogeography of the cyprinid Squalius aradensis and implications for conservation of the endemic freshwater fauna of southern Portugal

The Iberian cyprinid fauna, characterized by the presence of numerous endemic species, has suffered from significant habitat degradation. The critically endangered Squalius aradensis is restricted to small drainages of southern Portugal, habitats that typically exhibit a characteristic Mediterranean-type heterogeneous hydrological system throughout the year, including alternation of flooding events during winter and complete drought in large river sections during summer. To assess the effect of historical and recent processes on genetic diversity in S. aradensis we examined within- and among-population variability in cytochrome b and six polymorphic microsatellite loci. Estimates of genetic diversity in time and space through the combined use of traditional Phi-/F-statistics, phylogenetic trees, ordination methods and nested clade analysis indicated significant and congruent structuring among populations. Data suggest that the Arade drainage represent the evolutionary centre of the species, with subsequent allopatric fragmentation across drainages. Factors other than isolation by distance strongly affected the within-drainage genetic differentiation observed in these Mediterranean-type drainages, including recent population expansion from a bottleneck event and restricted gene flow imposed by a long-term barrier (brackish water area). Significant correlation was found between S. aradensis allelic diversity and upstream drainage area. The relevance of findings for conservation issues is discussed in relation to local intermittent hydrological conditions, the highly restricted distribution and the critically endangered status of the species.     

Population genetic structure in the Iberian cyprinid fish Iberochondrostoma lemmingii (Steindachner, 1866): disentangling species fragmentation and colonization processes

Interplay between the complex geography, hydrogeomorphological history, past climatic changes, and anthropogenic pressures is likely responsible for the current diversity and species' distribution of freshwater fishes in the Iberian Peninsula. To further disentangle the evolutionary processes promoting the diversification of endemic Iberian Cyprinids through time and space, we explored the patterns of genetic diversity of the Iberian arched‐mouth nase, Iberochondrostoma lemmingii (Steindachner, 1866), using molecular markers rendering at different timescales: the mitochondrial gene cytochrome b and seven microsatellite loci. Both markers showed significant differentiation of populations though the relative genetic distances among populations were different between markers. Mitochondrial DNA results indicate the isolation of hydrographic basins as the main driver of population differentiation, with Tejo as the centre of diversification. The results also support connections between Tejo, Guadiana, and Guadalquivir, with levels of divergence suggesting an earlier severance of Guadalquivir, whereas Guadiana and Tejo maintained connections until a more recent past. Establishment of more peripherial populations in small southern basins (Quarteira and Almargem) could have been ruled by founder events. However, the analysis of present‐day genetic configuration suggested by microsatellite data implies, for the first time, the involvement of other factors in the evolution of arched‐mouth Iberian nase populations. Relative low genetic distances between inter‐basin populations (Tejo and Guadiana) and the lack of concordance between differentiation and geography suggest a possible influence of human‐mediated translocations in the population genetic patterns of I. lemmingii. High intra‐basin differentiation levels were found within Tejo and Guadiana and may be associated with factors intrinsic to the species (e.g. low dispersal capability) or natural and/or artificial barriers to gene flow. The low vagility of the species appears to be an important factor influencing the evolutionary processes shaping the phylogeographical patterns of I. lemmingii, which could be relevant for the conservation of this threatened species. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 559–572.     

Evolutionary and biogeographical patterns within Iberian populations of the genus Squalius inferred from molecular data

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.     

Spatial genetic variation and habitat association of <Emphasis Type="Italic">Rhinichthys cataractae</Emphasis>, the longnose dace,in the Driftless Area of the upper Mississippi River basin

The Driftless Area of the upper Mississippi River drainage is a unique geographic region because of its complex geological history and the influence of recent, intensive human activities. The longnose dace, Rhinichthys cataractae, is a relatively common, small freshwater fish that is distributed in swift, cool streams within the region. The aim of this study was to determine the spatial genetic differentiation of the longnose dace and define the broad scale environmental variables that shape the distribution of the species in the southwestern portion of the Driftless Area. Genotypic data from seven microsatellite loci were analyzed for 276 individuals from 15 localities representing major drainages within the region in northeast Iowa. Broad scale environmental variables including hydrologic, soil, and climatic factors were evaluated to construct an ecological niche model (ENM) to predict the suitability of habitat for the species within the region. Results of the genetic analyses revealed two distinct, but somewhat admixed genetic clusters of longnose dace in Iowa. The genetic differentiation between localities and between drainages was low to moderate with some evidence of isolation by distance. Most of the variation was observed by differences between individuals within local populations. The ENM generated largely reflected the known distribution of the species in Iowa with a decreasing probability of suitable habitat from northern to southern drainages. Geologic factors played a key role in the model. The distribution and population structure of the longnose dace in the northeast Iowa revealed that isolation by distance, historical processes and the underlying geology are primarily responsible for the observed spatial distribution of genetic variation.     

Genetic divergence and phylogeographic relationships among european perch (Perca fluviatilis) populations reflect glacial refugia and postglacial colonization

We used the widely distributed freshwater fish, perch (Perca fluviatilis), to investigate the postglacial colonization routes of freshwater fishes in Europe. Genetic variability within and among drainages was assessed using mitochondrial DNA (mtDNA) D-loop sequencing and RAPD markers from 55 populations all over Europe as well as one Siberian population. High level of structuring for both markers was observed among drainages and regions, while little differentiation was seen within drainages and regions. Phylogeographic relationships among European perch were determined from the distribution of 35 mtDNA haplotypes detected in the samples. In addition to a distinct southern European group, which includes a Greek and a southern Danubian population, three major groups of perch are observed: the western European drainages, the eastern European drainages including the Siberian population, and Norwegian populations from northern Norway, and western side of Oslofjord. Our data suggest that present perch populations in western and northern Europe were colonized from three main refugia, located in southeastern, northeastern and western Europe. In support of this, nested cladistic analysis of mtDNA clade and nested clade distances suggested historical range expansion as the main factor determining geographical distribution of haplotypes. The Baltic Sea has been colonized from all three refugia, and northeastern Europe harbours descendants from both eastern European refugia. In the upper part of the Danube lineages from the western European and the southern European refugia meet. The southern European refugium probably did not contribute to the recolonization of other western and northern European drainages after the last glaciation. However, phylogenetic analyses suggest that the southern European mtDNA lineage is the most ancient, and therefore likely to be the founder of all present perch lineages. The colonization routes used by perch probably also apply to other freshwater species with similar distribution patterns.     

Nucleotide sequence data confirm diagnosis and local endemism of variable morphospecies of Andean astroblepid catfishes (Siluriformes: Astroblepidae)

Phylogenetic analysis based on nuclear and mitochondrial DNA sequences was used to test the validity of morphospecies of catfishes of the family Astroblepidae inhabiting the southern‐most limit of their Andean distribution in the upper Ucayali and upper Madre de Dios river basins. Population samples of morphospecies designated a priori on the basis of morphological features were further diagnosed by the presence of unique and unreversed molecular synapomorphies, thereby confirming species validity for seven of nine cases. Although each are distinguished by unique combinations of morphological features, two morphospecies (designated F and H) cannot be diagnosed on the basis of apomorphic changes in molecular sequence that did not also occur in other astroblepid morphospecies or outgroup taxa. Further, one morphospecies (species G) was recovered as nested within the assemblage of populations sampled from morphospecies F, whose morphological diagnosis does not involve unique or apomorphic characters. In contrast, the absence of corroborating molecular apomorphies for species H, otherwise recognized by distinctive and uniquely derived morphological characters, suggests a history of rapid divergence and insufficient time for fixation of genetic differences. Species sharing syntopic distributions were not recovered as sister groups, and in some cases species distributed in adjacent river drainage basins were not more closely related to one another than to species distributed in more distant drainages. Three independent instances were observed of sister‐group relationships involving species distributed in both the Apurimac and Urubamba rivers (Ucayali drainage). These observations combine to suggest that the current distribution of astroblepid species in the southern region may have arisen via a complex history involving both divergence between and dispersal amongst drainage basins that is probably repeated numerous times throughout the Andean distribution of the group. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 162 , 90–102.     

Genetic variation and population subdivision of the endangered Iberian cyprinid Chondrostoma lusitanicum

Products of 24 presumptive enzyme loci were used to analyse the consequences on genetic structure and variation of the Chondrostoma lusitanicum population decline within the Tejo basin. This rare cyprinid is endemic to the Iberian Peninsula and has a very restricted distribution. Five samples from the Tejo catchment were compared with a sample from a small basin, the Samarra, that has not suffered obvious anthropogenic pressures and where the fish is still abundant. Heterozygosity and polymorphism were higher overall in the Samarra. Several lines of evidence indicate a high degree of population subdivision within the Tejo basin. In fact, about half of the total gene diversity detected in the Tejo population was due to differences among samples. This differentiation appeared to be caused by genetic drift and possibly differential local selection, coupled with reduced gene flow among localities. The accelerated process of habitat degradation occurring in the lowland streams of the Tejo basin will lead to the inevitable reduction of intraspecies genetic diversity.     

Genetic structure of brown trout, salmo trutta l., at the southern limit of the distribution range of the anadromous form

Genetic variation at 33 protein loci was investigated in 41 wild brown trout populations from four river basins in Galicia (northwest Spain) to analyse the amount and distribution of genetic diversity in a marginal area, located in the distribution limit of the anadromous form of this species. The genetic diversity detected within populations (H between 0 and 6%) lies within the range quoted for this species in previous reports. The Mino, the most southern river basin analysed, showed a significantly lower genetic diversity and the highest genetic differentiation among the river basins studied. The hierarchical gene diversity analysis showed high population differentiation in a restricted area (GST = 27%), mostly due to differences among populations within basins (GSC = 22%). The reduction of GST observed when the isolated samples were excluded from the analysis (GST = 17%) showed the importance of habitat fragmentation on the heterogeneity detected. Gene flow among populations was comparatively evaluated by three indirect methods, which in general revealed low figures of absolute number of migrants per generation, slightly higher than 1. The gene flow among basins reflected a positive relationship with geographical distance. This trend was confirmed by the significant correlation observed between geographical and genetic distances, including all population pairs, which suggests a component of isolation by distance in brown trout genetic structure. Nevertheless, the nonsignificant intrabasin correlation demonstrates the complexity of genetic relationships among populations in this species. The model of genetic structure in brown trout is discussed in the light of the results obtained.     

Paleobiogeography of an Iberian endemic species,Luciobarbus sclateri (Günther, 1868) (Actinopterygii,Cyprinidae), inferred from mitochondrial and nuclear markers

Since the Cenozoic Era, the southern Iberian Peninsula has undergone a series of complex geological and climatic changes that have shaped the hydrographic configuration of the freshwater network, influencing the present‐day distribution of primary freshwater species and favoring a high level of local endemicity. The cyprinid species Luciobarbus sclateri (Günther, 1968) is an endemic species confined to the southern Iberian Peninsula and characterized by a complex evolutionary history. Previous studies linked the structure of L. sclateri populations to the effects of climate change during glaciations and were not able to explain the genetic discordance found between nuclear and mitochondrial markers. The results of this study show that the structure of L. sclateri populations is a reflection of diversification processes linked to the geological history of the region. Thus, we found three main mitochondrial phylogroups: the first one corresponding to small basins in southern Iberian Peninsula, a second one in eastern Iberian Peninsula, corresponding to Segura population, and a third one including the rest of the basins where the species is distributed. The southern group began diverging in the Pliocene as result of tectonic dynamics characterized by the emersion of the basins around the Strait of Gibraltar. The other two groups began diverging with the formation of the current Iberian hydrographic system during Pleistocene. So, the isolation of the hydrographic basins was the main factor driving intraspecific differentiation, followed by recent secondary contacts, admixture, and re‐isolation of the populations.     

Southern Mexican minnows of the genus Notropis (Actinopterygii, Cyprinidae): genetic variation, phylogenetic relationships and biogeographical implications

Allozyme analyses were performed to determine patterns of genetic variation and phylogenetic relationships within the genus Notropis in southern Mexico. Products of 28 gene loci were resolved in 24 geographic samples belonging to four putative species. These species represent the southern limit of the Cyprinidae in North America. Five loci were found monomorphic and 11 were diagnostic among species when the outgroup, Phoxinus phoxinus, was not taken into account. Four groups were identified, but these do not correspond perfectly to the four nominal species. Notropis sallaei was identified by allozyme analyses being the most basal among southern Mexican Notropis and the most genetically divergent. Notropis imeldae, was also diagnosed on the basis of allozymic variation, however a population sample representing the Balsas drainage was genetically divergent and is considered as an undescribed new species (N. n. sp.). No genetic differences were found between the samples of N. boucardi and N. moralesi. Therefore, we recommend that N. moralesi should be considered as a junior synonym of N. boucardi. A broad geographic sampling strategy was employed across all the distribution range of N. boucardi representing rivers from three different drainages, which cover both Mexican slopes. The slight divergence found among the headwater populations of N. boucardi permitted us to predict a model of paleohydrographic relationships of these three drainages. Two alternative hypotheses are postulated to explain the current distribution pattern of populations of N. boucardi in southern Mexico.     

Species delimitation in a range-restricted group of cascudinhos (Loricariidae: Epactionotus) supports morphological and genetic differentiation across coastal rivers of southern Brazil

Epactionotus species are known for inhabiting the rocky-bottom stretches of fast-flowing rivers in a limited geographic area along the Atlantic coast of southern Brazil. These species are endemic to single coastal river drainages (two neighbouring drainages for Epactionotus bilineatus) isolated from each other by the coastal lacustrine environments or the Atlantic Ocean. E. bilineatus is from the Maquiné and Três Forquilhas River basins, both tributaries of the Tramandaí River system, whereas E. itaimbezinho is endemic to the Mampituba River drainage and Epactionotus gracilis to the Araranguá River drainage. Recent fieldwork in the Atlantic coastal drainages of southern Brazil revealed new populations in the Urussanga, Tubarão, d'Una and Biguaçu River drainages. Iterative species delimitation using molecular data (cytochrome c oxidase subunit I) and morphology (morphometrics and meristics) was applied to evaluate species recognition of isolated populations. With regard to new data, the genus was re-diagnosed, the status of Epactionotus species/populations was re-evaluated, formerly described species were supported and population structure was recognized. As for the newly discovered populations, both morphological and molecular data strongly support the population from the Biguaçu River drainage, in Santa Catarina State, as a new species. Molecular data revealed strong per-basin population structure, which may be related to species habitat specificity and low or no dispersal among drainages.     

Lack of mtDNA variation among remote middle Volga and upper Ural brown trout suggests recent and rapid recolonization

Brown trout populations from the middle Volga and upper Ural drainages in the Russian Federation were analyzed with respect to their inter‐relationships and phylogenetic status using complete mtDNA control region sequences and 10 microsatellite loci. Among 56 individuals from eight populations (six from the middle Volga and two from the upper Ural drainages) only one haplotype was detected: Iran1 from the Danube phylogenetic lineage. This haplotype is very frequent in the southern Caspian basin in Iran. Given the high diversity of haplotypes in northern Iran, in contrast to the complete domination of the Iran1 haplotype in our study, it seems likely that Iran1 reached the middle Volga and upper Ural drainages as a consequence of colonization from the southern Caspian basin. In contrast to the mtDNA analysis, a more pronounced differentiation was observed among the studied populations on the basis of microsatellite DNA data. Based on this result, it is proposed that each population should be treated as a distinct management unit in the context of future conservation activities.     

Genetic structure and signature of population decrease in the critically endangered freshwater cyprinid <Emphasis Type="Italic">Chondrostoma lusitanicum</Emphasis>

The endemic and critically endangered cyprinid Chondrostoma lusitanicum has a very restricted distribution range. In order to estimate genetic diversity, characterize population structure and infer the demographic history, we examined six microsatellite loci and cytochrome b (mtDNA) sequences from samples taken throughout C. lusitanicum’s geographical range. Estimates of genetic diversity were low in all samples (average He < 0.35). The microsatellite data pointed to a major difference between northern (Samarra and Tejo drainages) and southern (Sado and Sines drainages) samples. This separation was not so clear with mtDNA, since one sample from the Tejo drainage grouped with the southern samples. This could be related with ancestral polymorphism or with admixture events between northern and southern sites during the late Pleistocene. Nevertheless, both markers indicate high levels of population differentiation in the north (for microsatellites F _ST >  0.23; and for mtDNA Φ_ST > 0.74) and lower levels in the south (F _ST < 0.05; Φ_ST < 0.40). With microsatellites we detected strong signals of a recent population decrease in effective size, by more than one order of magnitude, starting in the last centuries. This is consistent with field observations reporting a severe anthropogenic-driven population decline in the last decades. On the contrary mtDNA suggested a much older expansion. Overall, these results suggest that the distribution of genetic diversity in C. lusitanicum is the result of both ancient events related with drainage system formation, and recent human activities. The potential effect of population substructure generating genetic patterns similar to a population decrease is discussed, as well as the implications of these results for the conservation of C. lusitanicum. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.