Pedro Galán

Radically different phylogeographies and patterns of genetic variation in two European brown frogs, genus Rana

We reconstruct range-wide phylogeographies of two widespread and largely co-occurring Western Palearctic frogs, Rana temporaria and R. dalmatina. Based on tissue or saliva samples of over 1000 individuals, we compare a variety of genetic marker systems, including mitochondrial DNA, single-copy protein-coding nuclear genes, microsatellite loci, and single nucleotide polymorphisms (SNPs) of transcriptomes of both species. The two focal species differ radically in their phylogeographic structure, with R. temporaria being strongly variable among and within populations, and R. dalmatina homogeneous across Europe with a single strongly differentiated population in southern Italy. These differences were observed across the various markers studied, including microsatellites and SNP density, but especially in protein-coding nuclear genes where R. dalmatina had extremely low heterozygosity values across its range, including potential refugial areas. On the contrary, R. temporaria had comparably high range-wide values, including many areas of probable postglacial colonization. A phylogeny of R. temporaria based on various concatenated mtDNA genes revealed that two haplotype clades endemic to Iberia form a paraphyletic group at the base of the cladogram, and all other haplotypes form a monophyletic group, in agreement with an Iberian origin of the species. Demographic analysis suggests that R. temporaria and R. dalmatina have genealogies of roughly the same time to coalescence (TMRCA ~3.5 mya for both species), but R. temporaria might have been characterized by larger ancestral and current effective population sizes than R. dalmatina. The high genetic variation in R. temporaria can therefore be explained by its early range expansion out of Iberia, with subsequent cycles of differentiation in cryptic glacial refugial areas followed by admixture, while the range expansion of R. dalmatina into central Europe is a probably more recent event.

Colonization of spoil benches of an opencast lignite mine in northwest Spain by amphibians and reptiles

Abstract Colonization by reptiles and amphibians of the spoil benches of the Meirama opencast lignite mine in northwest Spain was studied over the 10 years following the start of the revegetation process. At Meirama, spoil benches are initially fertilized and hydroseeded with a pasture mix, but are subject to little subsequent management and are gradually colonized by scrub vegetation characteristic of the region. Herpetofauna censuses were carried out (1) yearly on a single 2 ha plot over the 6 years following hydroseeding, and (2) in a single year on ten 2 ha plots which had been hydroseeded between 0 and 10 years previously. In addition, censuses were carried out on three undisturbed ‘control’ plots close to the mine. Fifteen species (nine amphibians and six reptiles) were detected in one or more of the spoil-bench plots. Recolonization is particularly rapid after the first 2–3 years post-hydroseeding, and is clearly closely related to habitat development due to revegetation and natural succession. The first reptile species to colonize the study plots was Podarcis bocagei (first year), and the first amphibians Alytes obstetricans and Rana perezi (second year). Species composition was most similar to that in control plots in the oldest (ten-year-old) spoil plots.

Reproductive characteristics of the Pyrenean high-mountain lizards: Iberolacerta aranica (Arribas, 1993), I. aurelioi (Arribas, 1994) and I. bonnali (Lantz, 1927)

The three lacertid lizards species of the Iberolacerta genus, which have recently been described or recognised as different species, are reptiles that live strictly at the highest altitudes in Europe, from elevations of near 1900 up to more than 3000 m a.s.l. in the Pyrenees (Spain, Andorra, France). In this paper, the reproductive cycle and reproductive characteristics of these species are described for the first time. Data were obtained from field studies carried out in different high-mountain Pyrenean locations from 1989–2002. In addition we also conducted hatching studies in laboratory. Due to the harsh climatic conditions in the high mountains the activity cycle of these species is very short, just over 4 months, from mid-May to late September or beginning of October. This affects their reproductive cycle (i.e., only one annual egg-clutch is produced), as well as other reproductive characteristics, as the existence of a very advanced embryonic development at oviposition, which is interpreted as an advanced stage in the tendency towards viviparity, and diverse life history characteristics as a very little annual growth which greatly delays sexual maturity to 4 years in males and 4–5 years in females. Clutch size correlates significantly with female snout-vent length (SVL) in all three species. The average clutch size is 2.53 eggs in I. aurelioi (the smallest species), 3.03 in I. bonnali and 3.44 in I. aranica (the biggest species). Notwithstanding the differences in egg number the three species have a similar egg volume. The incubation period in the laboratory is very short with an average of 30–36 days among the species. The low reproductive potential observed in these three species and revealed in this paper is a strongly threatening factor which, together with their extremely reduced distribution area, endangers these endemic, rare and very threatened species.

Ontogenetic and sexual variation in the coloration of the lacertid lizards Iberolacerta monticola and Podarcis bocagei. Do the females prefer the greener males

Changes in the coloration of the lacertid lizards Iberolacerta monticola and Podarcis bocagei with age in populations from NW Spain are described. The onset of sexual maturity in P. bocagei males involves a change in the ventral (yellow) and dorsal (green) colorations, which is different from immature males (dorsally brownish in color). In I. monticola males, the ventral coloration also changes to a deep green when they reach maturity, while the dorsal coloration remains brownish as in the immature specimens. In this species, the green dorsal coloration is acquired gradually after maturity. Only the oldest individuals have a predominantly green dorsal coloration. The differences between the two species in the time males take to acquire the green dorsal coloration could be related to their different longevity. The coloring is acquired gradually in the most long-lived species ( I. monticola ). A field study was carried out on the behaviour of adult males of I. monticola during the reproductive period. The males with green dorsal coloration were seen to pair with females significantly more frequently than those with the brownish dorsal color. The increase in the green dorsal coloration (conspicuous) with the size and age of the males of this species would appear to have a clear function as an intersexual or intrasexual signal.

Discordant Patterns of Nuclear and Mitochondrial Introgression in Iberian Populations of the European Common Frog (Rana temporaria)

Amphibians often show complex histories of intraspecific and interspecific genetic introgression, which might differ in mitochondrial and nuclear genes. In our study of the genetic differentiation of the European common frog, Rana temporaria (159 specimens from 23 populations were analyzed for 24 presumptive allozyme loci; 82 specimens were sequenced for a 540-bp fragment of the mitochondrial 16S rRNA gene), multilocus correspondence analysis (CA) and Bayesian assignment tests of the nuclear data were concordant in identifying 2 population groups corresponding to 1) the Pyrenees in the east and 2) the Galicia and Asturias regions in the west, the latter corresponding to the subspecies R. temporaria parvipalmata. Geographically intermediate populations were genetically intermediate in the allozyme CA and, less clearly in the Bayesian assignment, with mitochondrial haplotypes exclusively belonging to the parvipalmata group. This indicates different degrees of introgression in the mitochondrial and nuclear genomes. Although Pyrenean high-altitude populations are morphologically distinct from low-altitude populations, these 2 groups were not separate clusters in any analysis. This suggests that the morphological differences may be due to fast adaptation to elevational gradients, likely under maintenance of gene flow, and that the underlying genetic changes are not detectable by the analyzed markers. We argue that a parsimonious explanation for the observed pattern along the east-west axis in northern Spain may be competition between invading and resident populations, with no need to invoke selection. However, in order to conclusively rule out selective processes, additional and finer scale data are required to test for asymmetric mating preference/behaviour, sex-biased gene flow, or sex-biased survival of potential hybrids.

Structure of a population of the lizard Podarcis bocagei in northwest Spain: variations in age distribution, size distribution and sex ratio

Mark-recapture techniques were used to investigate population size, age distribution, size distribution and sex ratio in a population of the lacertid lizard Podarcis bocagei in an abandoned gravel pit in northwest Spain. The study was carried out over a 2-year period. Despite relatively high maximum longevity, the population age distribution was characteristic of small, short-lived lizard species (i.e., there was a relatively high proportion of immature individuals). Population size declined over the study period, largely because of a drop in the number of immature animals: this may be partially attributable to density-dependent factors, but was probably due largely to a decline in habitat favourability as a result of colonisation of the study site by vegetation. The sex ratio was significantly female-biased in all cohorts studied, not only among adults but also among juveniles and sub-adults. However, sex ratio at hatching (as investigated by laboratory hatching of clutches laid by captured pregnant females) did not differ significantly from one-to-one. There was no difference found in survival probabilities between males and females. The observed bias in sex ratio must therefore be attributed to between-sex differences in net emigration.

Reproductive characteristics of a lowland population of an alpine lizard: Lacerta monticola (Squamata, Lacertidae) in north-west Spain

We studied the reproductive ecology of a population of the lacertid lizard Lacerta monticola at a lowland location in A Corun a (NW Spain) from 1997 to 2002. The timing of the reproductive cycle was examined based on mark-recapture records of individual lizards in the e eld. The characteristics of the eggs and hatchlings were obtained from clutches laid in the laboratory by pregnant females that were temporarily removed from the study area. Our results indicate that mating took place between late March and July. The smallest female withsigns of sexual maturity had a snout- vent length (SVL) of 52.4 mm and the smallest male was 50.7 mm. Sexual maturity was attained at the age of 2 years by 41% of the individuals, while the remaining 59% of the lizards matured at the age of 3 years. The laying period occurred between June and the beginning of August considering all years. About 61% of the reproductive females produced a single clutch annually, while 39% of the females produced two clutches per year. Females that produced two clutches were generally larger (average SVL D 70.9 mm) than those that laid a single annual clutch (average SVL D 62.6 mm). The mean clutch size was 6.4 eggs (range 4-9). Both clutch size and clutch mass increased signie cantly with female SVL. The mean egg mass in a clutch decreased signie cantly with clutch size. Hatching occurred between August and September. Hatching success in the laboratory was 71.7%. The mean SVL was higher in female hatchlings than in males.

Karyological Characterization of the Endemic Iberian Rock Lizard, Iberolacerta monticola (Squamata, Lacertidae): Insights into Sex Chromosome Evolution

Rock lizards of the genus Iberolacerta constitute a promising model to examine the process of sex chromosome evolution, as these closely related taxa exhibit remarkable diversity in the degree of sex chromosome differentiation with no clear phylogenetic segregation, ranging from cryptic to highly heteromorphic ZW chromosomes and even multiple chromosome systems (Z1Z1Z2Z2/Z1Z2W). To gain a deeper insight into the patterns of karyotype and sex chromosome evolution, we performed a cytogenetic analysis based on conventional staining, banding techniques and fluorescence in situ hybridization in the species I. monticola, for which previous cytogenetic investigations did not detect differentiated sex chromosomes. The karyotype is composed of 2n = 36 acrocentric chromosomes. NORs and the major ribosomal genes were located in the subtelomeric region of chromosome pair 6. Hybridization signals of the telomeric sequences (TTAGGG)n were visualized at the telomeres of all chromosomes and interstitially in 5 chromosome pairs. C-banding showed constitutive heterochromatin at the centromeres of all chromosomes, as well as clear pericentromeric and light telomeric C-bands in several chromosome pairs. These results highlight some chromosomal markers which can be useful to identify species-specific diagnostic characters, although they may not accurately reflect the phylogenetic relationships among the taxa. In addition, C-banding revealed the presence of a heteromorphic ZW sex chromosome pair, where W is smaller than Z and almost completely heterochromatic. This finding sheds light on sex chromosome evolution in the genus Iberolacerta and suggests that further comparative cytogenetic analyses are needed to understand the processes underlying the origin, differentiation and plasticity of sex chromosome systems in lacertid lizards.

Mitochondrial uniformity in populations of the treefrog Hyla molleri across the Iberian Peninsula

Based on DNA sequences of fragments of the mitochondrial COI and 16S rRNA genes we provide a first assessment of genetic variability of Iberian treefrog populations that have been historically allocated to Hyla arborea, partly as subspecies H. a. molleri. Our data from 147 specimens and 33 populations confirm strong divergence between these frogs and Central European H. arborea but relatively low differentiation across their range, supporting their status as a separate species, H. molleri. Preliminary phylogeographic data indicate a possible weak genetic differentiation of populations from the northern coast of the region of Galicia. We suggest inclusion of nuclear markers and an extension of the sampling into the coastal regions of Asturias and Cantabria, as well as the identification of the contact zone between H. molleri and H. arborea in either the Spanish Basque country or in France, as priorities for future research on this species.

Evolutionary dynamics of two satellite DNA families in rock lizards of the genus Iberolacerta (Squamata, Lacertidae): different histories but common traits.

Satellite DNAs compose a large portion of all higher eukaryotic genomes. The turnover of these highly repetitive sequences is an important element in genome organization and evolution. However, information about the structure and dynamics of reptilian satellite DNA is still scarce. Two satellite DNA families, HindIII and TaqI, have been previously characterized in four species of the genus Iberolacerta. These families showed different chromosomal locations, abundances, and evolutionary rates. Here, we extend the study of both satellite DNAs (satDNAs) to the remaining Iberolacerta species, with the aim to investigate the patterns of variability and factors influencing the evolution of these repetitive sequences. Our results revealed disparate patterns but also common traits in the evolutionary histories of these satellite families: (i) each satellite DNA is made up of a library of monomer variants or subfamilies shared by related species; (ii) species-specific profiles of satellite repeats are shaped by expansions and/or contractions of different variants from the library; (iii) different turnover rates, even among closely related species, result in great differences in overall sequence homogeneity and in concerted or non-concerted evolution patterns, which may not reflect the phylogenetic relationships among taxa. Contrasting turnover rates are possibly related to genomic constraints such as karyotype architecture and the interspersed organization of diverging repeat variants in satellite arrays. Moreover, rapid changes in copy number, especially in the centromeric HindIII satDNA, may have been associated with chromosomal rearrangements and even contributed to speciation within Iberolacerta.