Isolation and characterization of polymorphic microsatellite markers in Iberolacerta monticola, and cross-species amplification in Iberolacerta galani and Zootoca vivipara

Fourteen polymorphic microsatellite loci are described for the Iberian rock lizard, Iberolacerta monticola. Genetic variation in a sample of 20 individuals from Piornedo (northwestern Spain) was quantified both by the number of alleles per locus, which ranged from six to 13, and by the expected frequency of heterozygotes under random mating (heterozygosity), which ranged from 0.761 to 0.902. Single locus and global exclusion probabilities were also computed, and indicate a high power of these markers for paternity assignments and mating system studies of I. monticola. All the analysed loci were also polymorphic in Iberolacerta galani, but only seven in Zootoca vivipara.     

Genetic differentiation of populations of Iberian rock‐lizards Iberolacerta (Iberolacerta) sensuArribas (1999)

The genetic structure and relationships of five populations of the Iberian group of Iberolacerta (sensu 6 ) were analysed by protein electrophoresis. In this study we confirmed the differentiation between the group of populations from Galicia/Cantabrian/S. Estrela versus the group of populations from de Spanish Central System, Gredos and Guadarrama that are included in the two different species by Arribas (Herpetozoa 9(1/2), 31–56, 1996; Russian J. Herpetol. 6, 1–22, 1999), Iberolacerta (I.) monticola and Iberolacerta (I.) cyreni, respectively. However, the differentiation level is not high enough to clearly prove their specific discrimination. On the other hand, we did not confirm the subspecific discrimination of the Gredos –Iberolacerta (I.) cyreni castiliana, and Guadarrama –Iberolacerta (I.) cyreni cyreni populations, proposed by 5 ). These two populations are genetically almost homogeneous. Interestingly, we found an unexpected high genetic similarity between the Galician and the Serra da Estrela populations, presently included into two different subspecies, Iberolacerta (I.) monticola cantabrica and Iberolacerta (I.) monticola monticola, respectively. Their genetic similarity is even higher than that between the populations of Galicia and the Cantabrian Mountains, which are both included in the same subspecies, cantabrica. This result suggests that the populations of Galicia and Serra da Estrela would have maintained contacts, possibly through the north of Portugal, until relatively recent times. Their separation is thus probably post‐glacial. Some evidence also points to the existence of relatively recent contacts between the population of Serra da Estrela and those of the Central System, particularly, with the neighbouring Peña de Francia population. With the cautions imposed by the reduced sample size of our analysis, the significant differentiation of the populations from Galicia and the Cantabrian Mountains allow us to suggest that this last population may not be the result of a recent expansion of the Galician population as 5 ) suggests, but, more likely, the result of a fragmentation process of a more ancient and wider north‐eastern distribution area of this group of rock‐lizards.     

Novel polymorphic microsatellites for the red‐legged partridge (Alectoris rufa) and cross‐species amplification in Alectoris graeca

The red‐legged partridge, Alectoris rufa, is an endemic species of the southwestern Mediterranean, and the most popular game bird in the Iberian Peninsula. A total of 27 microsatellite loci was isolated from an enriched genomic library of A. rufa. Six perfect GT microsatellites were characterized and optimized in 45 individuals of A. rufa. All loci revealed high levels of polymorphism with a number of alleles that ranged from three to 13. Observed heterozygosity ranged from 0.2 to 0.6. Cross‐species amplification showed that all loci were also polymorphic in rock partridge, Alectoris graeca. The new markers will be useful in determining hybridization between both species of Alectoris.     

The effects of thermal biology and refuge availability on the restricted distribution of an alpine lizard

Aim In an effort to disentangle the ecological processes that confine ectotherms to alpine environments, we studied the thermoregulatory and microhabitat selection behaviours of the rock lizard Iberolacerta cyreni, which is endemic to some mountains of central Spain, and of the wall lizard Podarcis muralis, which is a potential competitor of rock lizards. Location We chose three areas in the Sierra de Guadarrama (central Spain) that differed in their thermal quality [mean deviation of environmental operative temperatures from the lizards’ preferred thermal range (PTR)] and refuge availability: a pine forest (1770 m a.s.l.) in which P. muralis was the only species found, and two mixed shrub and rock sites (1770 and 1900 m a.s.l.) where both species were present. Methods In the field we collected data on refuge availability, sun exposure, body temperature (T_b) and operative temperature (T_e). Thus, we estimated the thermal habitat quality of the areas sampled and the thermoregulation accuracy and effectiveness of both species. Results The pine forest had the lowest thermal quality and refuge availability. The lower‐elevation shrub site offered the best thermal quality, but refuges were much scarcer than at the higher‐elevation site. Both species thermoregulated accurately, because mean deviations of body temperature (T_b) from PTR were considerably smaller than those of T_e. Podarcis muralis had higher T_b values than did I. cyreni, which had similar T_b values at both shrub sites, whereas P. muralis had lower T_b values at higher elevation. Overall, the thermoregulatory effectiveness (extent to which T_b values are closer to the PTR than are T_e values) of both species was similar, but whereas I. cyreni thermoregulated more efficiently at higher elevation, the opposite was true for P. muralis. At the lower‐elevation shrub site, I. cyreni remained closer to refuges than did P. muralis. Main conclusions Our results suggest that the pine forest belt might prevent the expansion of rock lizards towards lower elevations as a result of its low thermal quality and scarcity of refuges, that the thermoregulatory effectiveness of rock lizards in alpine environments depends more on refuge availability than on thermal habitat quality, and that competition with wall lizards is unlikely to explain either the distribution or the thermoregulatory effectiveness of rock lizards.     

Competition with wall lizards does not explain the alpine confinement of Iberian rock lizards: an experimental approach

Interspecific competition can limit the distribution of species along altitudinal gradients. It has been suggested that Western European rock lizards (genus Iberolacerta) are restricted to mountains due to the expansion of wall lizards (Podarcis), but there is no experimental evidence to corroborate this hypothesis. This study examines if interference competition with Podarcis muralis is a plausible explanation for the alpine confinement of Iberian rock lizards Iberolacerta cyreni. In a first experiment, we used an enclosure with four types of microhabitats to investigate whether adult rock and/or wall lizards shifted microhabitat or refuge preferences in the presence of the other species, and to detect aggressive interactions between them. In a second experiment, we staged heterospecific encounters between naïve, laboratory-born juveniles to identify behavioural differences and agonistic interactions. In the enclosure, neither rock nor wall lizards changed their microhabitat preferences in the presence of the other species. Nevertheless, rock lizards increased the diversity of microhabitats and nocturnal refuges used in the single species trials, which had twice the number of conspecifics. Aggressive interactions involved mainly large rock lizard males. Juveniles did not show any interspecific agonistic behaviour, but rock lizards spent more time basking and less time moving. Thus, we found no evidence of competition between both species in terms of habitat shifts or agonistic interactions, although intraspecific interactions seemed to explain the behaviour of adult rock lizards. We conclude that other factors are currently determining the alpine confinement of rock lizards.     

Characterization of seven polymorphic microsatellite loci in the Baja California endemic black‐tailed brush lizard Urosaurus nigricaudus

Seven microsatellite loci were developed for the Baja California endemic black‐tailed brush lizard Urosaurus nigricaudus, using an enriched genomic DNA library. All loci were polymorphic and overall presented high levels of variation. Number of alleles ranged from five to 16 (average 12.14), and observed heterozygosities from 0.535 to 0.923 (average 0.752). Cross‐species amplification was successful and polymorphism was detected for all the loci using the congeners Urosaurus lahtelai and Urosaurus ornatus. These markers will be useful to study fragmented populations of U. nigricaudus on agricultural landscape of the Baja California Peninsula.     

Polymorphic microsatellite loci for species of Australian freshwater cod, <Emphasis Type="Italic">Maccullochella</Emphasis>

The Australian freshwater cod genus, Maccullochella is represented by three species: Murray cod, M. peelii peelii, eastern freshwater cod, M. ikei, and trout cod, M.macquariensis. Seven novel microsatellite loci from M. ikei and six previously published loci from M. peelii peelii were tested on wild populations of Murray, eastern and trout cod. Levels of polymorphism varied between species with 13 loci polymorphic in Murray cod, 9 in trout cod and 7 in eastern cod. Observed heterozygosities ranged from 0.053 to 0.842. This suite of microsatellite loci will facilitate future studies of the genetic status of wild and hatchery bred populations of Maccullochella.     

Microsatellite loci for the southern pine beetle (Dendroctonus frontalis) and cross‐species amplification in Dendroctonus

We developed microsatellite loci for the southern pine beetle (Dendroctonus frontalis). Twelve microsatellite loci were identified. Eight loci were polymorphic and sufficiently variable in 62 individuals (expected heterozygosity ranged from 0.707 to 0.880) to investigate population structure. All loci conformed to HWE except Dfr‐14, which showed heterozygote excess, and no two loci deviated from linkage equilibrium. The loci were tested for cross‐species amplification in four species of Dendroctonus (D. valens, D. terebrans, D. brevicomis, and D. ponderosae). Seven loci were polymorphic in at least one of the species tested.     

Isolation of microsatellites in a parthenogenetic lizard Menetia greyii (Scincidae) and their utility in sexual species of the Menetia greyii complex

Tetra and tri‐nucleotide microsatellite loci were isolated from a parthenogenetic form of the Australian lizard Menetia greyii, using an enrichment method with polymerase chain reaction (PCR)‐based colony screening. Primers for 23 loci were designed and 11 of these loci amplified well in a panel of 10 parthenogenetic individuals from a single population. Seven loci were further characterized and six successfully amplified and were polymorphic in all five sexual species of the Menetia greyii complex they were tested in. These loci will be used to investigate the parental ancestry and clonal diversity of various parthenogenetic forms within the Menetia greyii species complex.     

Twenty-three polymorphic microsatellite markers for the Caribbean endemic Zenaida dove, Zenaida aurita, and its conservation in related Zenaida species

Twenty-three polymorphic microsatellite loci, six dinucleotidic loci and 17 tetranucleotidic loci, were developed for the Zenaida dove (Zenaida aurita), a bird species endemic to the Caribbean Islands. From a set of 30 individuals captured at one single location in Barbados, we obtained 20 loci that did not deviate from Hardy–Weinberg Equilibrium. Number of alleles per locus ranged between 2 and 11 (average 7.05) and the expected heterozygosity per locus, He ranged between 0.321 and 0.881 (average 0.712). This gives an exclusionary power for parental analysis of 0.9999 and 1.0000, knowing the genotype of one social parent, or both, respectively. Such results indicate that these 20 loci will be useful for both studying population genetics and mate choice patterns in Z. aurita. All 20 loci amplified in four other Zenaida species, the Galápagos dove, Z. galapagoensis, the eared dove, Z. auriculata, the mourning dove, Z. macroura, the Pacific dove, Z. meloda, with 30–96% being polymorphic.     

Microsatellite loci for great white herons and great blue herons (Aves,Ardeidae, Ardea herodias)

We used an enrichment technique to isolate 60 microsatellite loci in Ardea herodias. We developed primers for 17 loci, screened for variation in A. herodias and attempted to amplify these loci in three closely related species (A. alba, A. cinerea and A. cocoi). Fifteen loci were polymorphic in A. herodias. Observed heterozygosities ranged from 0.10 to 0.81. Two loci appeared to be monomorphic in A. herodias, but exhibited variation in product size among species within the genus. Our ability to amplify polymorphic products in closely related species suggests that these markers may be useful in other herons.     

Development of microsatellite loci for Pelophylax plancyi and cross-amplification in other ranid species

Thirteen polymorphic microsatellite loci were obtained from the gold-striped pond frog, Pelophylax plancyi. The number of alleles per locus ranged from 5 to 13 with an average of 8.38. The observed and expected heterozygosities (H _O and H _E) ranged from 0.533 to 0.890 and from 0.620 to 0.897, with averages of 0.686 and 0.751, respectively. In order to assess interspecific amplification, all primer pairs were tested with the same PCR conditions on three other Pelophylax species: Pelophylax hubeiensis, Pelophylax fukienensis, Pelophylax nigromaculata and two species from the genus Hylarana and Hoplobatrachus, respectively. All of these loci can be amplified successfully in the Pelophylax species, with eight loci amplified in all species tested.     

Blood Parasite Infection Intensity Covaries with Risk‐Taking Personality in Male Carpetan Rock Lizards (Iberolacerta cyreni)

Identifying evolutionary and developmental mechanisms underlying consistent between‐individual differences in behaviour is the main goal in ‘animal personality studies’. Here, we explored whether activity and risk‐taking varied consistently between individuals and correlated to various – potentially fitness linked – male traits in Carpetan rock lizards (Iberolacerta cyreni). Lizards showed significant consistency within both behaviours, implying the presence of activity and risk‐taking personalities. However, there were no correlation between activity and risk‐taking, neither on the between‐ nor on the within‐individual levels, implying the absence of a behavioural syndrome. We found a strong link between the intensity of blood parasite (Haemogregarinidae) infection and risk‐taking: lizards with higher infection intensity took more risk. While we cannot distinguish cause from causative in the parasite intensity – risk‐taking correlation – our results are in line with the asset protection hypothesis predicting that individuals with lower future reproductive value should focus on the current reproductive event and take higher risk.     

Characterization,multiplex conditions,and cross‐species utility of tetranucleotide microsatellite loci for Blanchard's cricket frog (Acris crepitans blanchardi)

We have isolated and characterized 17 tetranucleotide microsatellite loci for Blanchard's cricket frog (Acris crepitans blanchardi), an anuran common in the central USA. Sixteen loci were organized into four multiplex amplification reactions. These loci were highly polymorphic when screened in 55 individuals from two distant populations, with 11–48 alleles per locus (average = 24.8). Observed and expected heterozygosities ranged from 0.18 to 0.97 and from 0.17 to 0.96, respectively. Nine loci were also polymorphic in Acris crepitans crepitans, with seven polymorphic in Acris gryllus. Five loci amplified in all three taxa. These loci will be useful for population‐ and species‐level investigations of this widespread group.     

Causes and Evolutionary Consequences of Population Subdivision of an Iberian Mountain Lizard,Iberolacerta monticola

Aim

The study of the factors that influence population connectivity and spatial distribution of genetic variation is crucial for understanding speciation and for predicting the effects of landscape modification and habitat fragmentation, which are considered severe threats to global biodiversity. This dual perspective is obtained from analyses of subalpine mountain species, whose present distribution may have been shaped both by cyclical climate changes over ice ages and anthropogenic perturbations of their habitats. Here, we examine the phylogeography, population structure and genetic diversity of the lacertid lizard Iberolacerta monticola, an endemism considered to be facing a high risk of extinction in several populations.

Location

Northwestern quadrant of the Iberian Peninsula.

Methods

We analyzed the mtDNA variation at the control region (454 bp) and the cytochrome b (598 bp) loci, as well as at 10 nuclear microsatellite loci from 17 populations throughout the distribution range of the species.

Results

According to nuclear markers, most sampling sites are defined as distinct, genetically differentiated populations, and many of them show traces of recent bottlenecks. Mitochondrial data identify a relatively old, geographically restricted lineage, and four to six younger geographically vicariant sister clades, whose origin may be traced back to the mid-Pleistocene revolution, with several subclades possibly associated to the mid-Bruhnes transition. Geographic range fragmentation of one of these clades, which includes lowland sites, is very recent, and most likely due to the accelerated loss of Atlantic forests by human intervention.

Main Conclusions

Altogether, the data fit a “refugia within refugia” model, some lack of pattern uniformity notwithstanding, and suggest that these mountains might be the cradles of new species of Iberolacerta. However, the changes operated during the Holocene severely compromise the long-term survival of those genetic lineages more exposed to the anthropogenic perturbations of their habitats.     

Ten new microsatellite loci for the yellowhammer (Emberiza citrinella) and their cross‐species applicability among related taxa

We describe isolation and characterization of the first microsatellite loci specifically developed for a very common European passerine bird, the yellowhammer Emberiza citrinella. Nine out of our 10 loci are polymorphic within the species E. citrinella. Number of alleles ranged from two to 21 per locus and observed heterozygosity between 0.20 and 0.91. Four primer pairs also yielded reproducible results in other species of Emberizidae. These loci comprise a set of molecular markers for various applications, from moderately polymorphic loci suitable for population studies to highly polymorphic loci for pedigree analysis in Emberizidae.     

Isolation and characterization of microsatellites in Leuciscus cephalus (Cypriniformes,Cyprinidae) and cross‐species amplification within the family Cyprinidae

Thirteen polymorphic microsatellites were isolated from Leuciscus cephalus, a widespread cyprinid species with great ecological tolerance. Together with the cross‐species amplification of six additional loci originally published for three cyprinid fish species, we optimized a multiplex panel for L. cephalus allowing the genotyping of 19 polymorphic loci. Number of alleles and heterozygosity per locus in a sample of 20 fish individuals ranged from two to 16 and from 0.05 to 0.90, respectively. These primers will be useful in determining the population structure of L. cephalus. In addition, successful cross‐amplification was obtained for several species of Cyprinidae.     

Microsatellite loci in the Propertius duskywing,Erynnis propertius (Lepidoptera: Hesperiidae), and related species

Fifteen polymorphic dinucleotide microsatellite loci were characterized for Erynnis propertius using an enrichment protocol. The number of alleles varied from nine to 28 for a sample of 24 individuals. Observed heterozygosities ranged from 0.25 to 0.96. Homozygote excess was detected for 10 loci. Twelve markers successfully amplified in related Erynnis species and eight loci were polymorphic in at least one other species.     

Microsatellite primers for three Western Atlantic Farfantepenaeus prawn species

Microsatellite loci were identified for three closely related penaeid species, Farfantepenaeus subtilis, F. paulensis and F. sp., from genomic libraries enriched for CA repeats. Seven out of nine highly polymorphic loci detected were amplified across all three species. Between four and 64 alleles were recorded per locus (average = 36). The average observed heterozygosity ranged from 0.094 to 0.897 (mean = 0.613), while the expected heterozygosity ranged from 0.091 to 0.985 (mean = 0.822).     

Isolation, characterization and cross-species amplification of polymorphic microsatellite loci in Asclepias (Apocynaceae)

Ten polymorphic loci were isolated and characterized from the milkweed, Asclepias syriaca L., of North America. These loci successfully cross-amplified in A. exaltata L. Polymorphism ranged from two to 16 alleles per locus per species in 68 individuals of A. syriaca and 56 individuals of A. exaltata. Expected heterozygosities ranged from 0.017 to 0.851 and significant deviations from Hardy–Weinberg equilibrium were found for two and three loci in A. syriaca and A. exaltata, respectively. No linkage disequilibrium was detected. These markers should prove useful for assessing population genetic structure and interspecific gene flow in these and other species of Asclepias.