Species-specific Microsatellite Markers Research Articles

Is catheter-related candidemia a polyclonal infection?

Diagnosis of catheter-related candidemia (CRC) requires the simultaneous isolation of Candida spp. from both blood and catheter samples. We previously observed that in most CRC cases, the genotype of the yeast found in catheter samples is also recovered from blood. However, it is not clear whether CRC is a polyclonal infection. We prospectively studied 20 patients with CRC caused by Candida albicans, C. parapsilosis, or C. glabrata to analyze whether their infections were polyclonal. As many as 10 colonies per sample (n = 475) isolated from blood (n = 220) and catheter (n = 255) specimens were studied using species-specific microsatellite markers. Genotyping always revealed matches between the Candida spp. from blood and catheter samples. However, 15% of patients had a polyclonal pattern of infection or catheter colonization that was species specific. An additional genotype was found exclusively in the catheters of two patients infected with C. albicans, whereas an additional genotype was noted in the blood culture of a patient infected with C. parapsilosis. Considering only the presence of different genotypes in blood samples, 5% of patients had polyclonal infections. We conclude that most cases of CRC are caused by a single genotype.

Short Communication Cross-species transferability of eastern white pine (Pinus strobus) nuclear microsatellite markers to five Mexican white pines

We examined cross-species transferability and usefulness of six nuclear microsatellite markers developed in consubgeneric eastern white pine (Pinus strobus) with regard to ecologically and commercially important Mexican white pine species of conservation genetics concern: Pinus chiapensis (Mart.) Andresen, P. flexilis James, P. strobiformis Engelm., P. ayacahuite Ehrenb. Ex Schltdl, and P. ayacahuite var. veitchii (Roezl) G.R. Shaw. Four to six microsatellite loci were found to be polymorphic in different species, with moderate to high informativeness in a relatively small number of samples (PIC/HE=0.25-0.93). This successful transfer sidesteps the time- and resource-consuming development of species-specific microsatellite markers, and will facilitate population and conservation genetic studies and genetic resource management of the less studied Mexican white pines.

Migration and dispersal may drive to high genetic variation and significant genetic mixing: the case of two agriculturally important, continental hoverflies (Episyrphus balteatus and Sphaerophoria scripta)

Population structure of pests and beneficial species is an important issue when designing management strategies to optimize ecosystem services. In this study, we investigated for the first time the population structure at a continental scale of two migratory species of hoverflies providing both pest regulation and pollination services [Episyrphus balteatus and Sphaerophoria scripta (Diptera: Syrphidae)]. To achieve this objective, we used two sets of 12 species-specific microsatellite markers on a large-scale sampling from all over Europe. Our findings showed a high level of genetic mixing resulting in a lack of genetic differentiation at a continental scale and a great genetic diversity in the two species. All the pairwise FST values between European localities were less 0.05 in the two species. These low values reflect a large-scale genetic mixing probably caused by the existence of frequent migratory movements in the two species. Mantel tests revealed isolation-by-distance pattern on the East-West axis, but not on the North-South axis. This isolation-by-distance pattern confirms the existence of North-South migratory movements in both directions and suggests an important step by step dispersal. Population features shown by this study are common in invasive species and pests, but are not often observed in beneficial species. They reflect great colonization abilities and a high adaptive potential when dealing with a changing environment. Our results highlight the two studied species as particularly interesting beneficial insects for pollination and pest predation in the current context of global change.

Development of twenty microsatellite loci from the Tibetan ground tit (Parus humilis)

The Tibetan ground tit Parus humilis is a sexually monomorphic passerine bird endemic to the Tibetan plateau. It inhabits the alpine meadows at elevations of 2500–5500 m across the plateau. This species is particularly interesting for its phylogenetic position. It was initially assigned to the family Corvidae (crows and jays) based on lifestyle and morphology (Hume 1871), and recent evidence of nuclear and mitochondrial genes along with osteology suggested it to be a ground-adapted tit in the family Paridae (tits and chickadees) (James et al. 2003). Moreover, the ground tits have also been explored with respect to phylogeography using mtDNA sequences (Yang et al. 2006). In terms of social behaviour, this species is a socially monogamous, facultatively cooperative breeder (Du and Lu 2009; Lu et al. 2011; Oh 2011), which is uncommon in high-altitude birds because most cooperative species occur in the tropical or southern temperate regions (Jetz and Rubenstein 2011). It has been shown that there is a difference in social system among different ground tit populations (Johannessen et al. 2011). Ground tits are obligate primary cavity-nesters, excavating burrows under the earth for breeding and roosting (Ke and Lu 2009). Ground tit burrows may provide nest sites for several secondary cavity-nesters (Zeng and Lu 2009a, b; Li and Lu 2012). Therefore, the birds are a keystone species for biodiversity in the species-poor, vulnerable alpine meadow ecosystems. Despite the significant implications of ground tits for studies of systematics, evolutionary ecology and conservation biology, no species-specific microsatellite markers are available, which limits our ability to make further investigations into these fields. In response to such a need, we

Exploiting the transcriptome of Euphrates Poplar, Populus euphratica (Salicaceae) to develop and characterize new EST-SSR markers and construct an EST-SSR database.

BackgroundMicrosatellite markers or Simple Sequence Repeats (SSRs) are the most popular markers in population/conservation genetics. However, the development of novel microsatellite markers has been impeded by high costs, a lack of available sequence data and technical difficulties. New species-specific microsatellite markers were required to investigate the evolutionary history of the Euphratica tree, Populus euphratica, the only tree species found in the desert regions of Western China and adjacent Central Asian countries.Methodology/Principal FindingsA total of 94,090 non-redundant Expressed Sequence Tags (ESTs) from P. euphratica comprising around 63 Mb of sequence data were searched for SSRs. 4,202 SSRs were found in 3,839 ESTs, with 311 ESTs containing multiple SSRs. The most common motif types were trinucleotides (37%) and hexanucleotides (33%) repeats. We developed primer pairs for all of the identified EST-SSRs (eSSRs) and selected 673 of these pairs at random for further validation. 575 pairs (85%) gave successful amplification, of which, 464 (80.7%) were polymorphic in six to 24 individuals from natural populations across Northern China. We also tested the transferability of the polymorphic eSSRs to nine other Populus species. In addition, to facilitate the use of these new eSSR markers by other researchers, we mapped them onto Populus trichocarpa scaffolds in silico and compiled our data into a web-based database (http://202.205.131.253:8080/poplar/resources/static_page/index.html).ConclusionsThe large set of validated eSSRs identified in this work will have many potential applications in studies on P. euphratica and other poplar species, in fields such as population genetics, comparative genomics, linkage mapping, QTL, and marker-assisted breeding. Their use will be facilitated by their incorporation into a user-friendly web-based database.

Development of polymorphic microsatellite loci for the rattlesnake species Crotalus atrox, C. cerastes, and C. scutulatus (Viperidae: Crotalinae) and cross-species amplification of microsatellite markers in Crotalus and Sistrurus species

We provide details on 46 microsatellite loci for Crotalus and Sistrurus rattlesnake species. We isolated 14, five, and four novel polymorphic species-specific microsatellite markers for Crotalus atrox, C. scutulatus, and C. cerastes, respectively. We observed seven to 36 alleles per locus. Additionally, we provide data on the cross-species amplification of previously published microsatellite markers for Crotalus and Sisturus species. Subsets of novel and cross-species amplified microsatellite markers were used to genotype populations of C. atrox, C. cerastes, and C. scutulatus in Southern Arizona. The numbers of alleles per locus in the combined marker sets range from three to 38 (C. atrox: 8–37, C. scutulatus: 3–35, C. cerastes: 4–38). Many novel and existing microsatellite markers cross-species amplified successfully. Marker sets of 20 or more polymorphic microsatellites provide high resolution for population genetics studies. Markers used in this study will likely be informative in additional Crotalus species.

Identification of Ensis siliqua Samples and Establishment of the Catch Area Using a Species-Specific Microsatellite Marker

European Council Regulation 104/2000 states that fishery products must be labeled to indicate commercial designation of species, the production method, and the catch area. Therefore, traceability of seafood implies knowledge of the species offered to retail and their origin. Ensis siliqua is a bivalve intensively fished in Europe and sold in fresh and canned forms. Although several published methods clearly differentiate Ensis genus species, none of those assess the origin of the commercial samples. In the present study, a microsatellite marker (Esi-UDC3055F) was developed to establish the catch area of E. siliqua samples. Amplification yielded a fragment of 275 or 302 base pairs, depending on whether they were Iberian or Irish populations. The usefulness of this method was also assessed in commercial samples. The results of this study provide a reliable methodology for the identification of catch area in European E. siliqua commercial samples. The coupling of this methodology with existing techniques for razor clam species identification provides a powerful tool for traceability and labeling enforcement.

Successful Determination of Larval Dispersal Distances and Subsequent Settlement for Long-Lived Pelagic Larvae

Despite its importance, we still have a poor understanding of the level of connectivity between marine populations in most geographical locations. Taking advantage of the natural features of the southeast coast of New Zealand's North Island, we deployed a series of settlement stations and conducted plankton tows to capture recent settlers and planktonic larvae of the common intertidal gastropod Austrolittorina cincta (6–8 week larval period). Satellite image analysis and ground truthing surveys revealed the absence of suitable intertidal rocky shore habitat for A. cincta over a 100 km stretch of coastline between Kapiti Island to the south and Wanganui to the north. Fifteen settlement stations (3 replicates×5 sites), which were used to mimic intertidal habitat suitable for A. cincta, were deployed for two months around and north of Kapiti Island (at 0.5, 1, 5, 15, 50 km). In addition, we also conducted plankton tows at each settlement station when the stations were first deployed to collect A. cincta larvae in the water column. On collection, all newly settled gastropods and larvae in the plankton samples were individually isolated, and a species-specific microsatellite marker was used to positively identify A. cincta individuals. Most of the positively identified A. cincta settlers and larvae were collected at the first three sampling stations (<5 km). However, low numbers of A. cincta settlers and larvae were also recorded at the two more distant locations (15 and 50 km). Dispersal curves modeled from our data suggested that <1% of gastropod larvae would travel more than 100 km. While our data show that most larvae are retained close to their natal populations (<5 km), a small proportion of larvae are able to travel much larger geographic distances. Our estimates of larval dispersal and subsequent settlement are one of only a few for marine species with a long-lived larva.

Genetic Diversity and Population Structure of Chinese White Poplar (Populus tomentosa) Revealed by SSR Markers

An understanding of allelic diversity and population structure is important in developing association studies and constructing core collections for tree breeding. We examined population genetic differentiation in the native Populus tomentosa by genotyping 460 unrelated individuals using 20 species-specific microsatellite markers. We identified 99 alleles with a mean of 4.95 observed alleles per locus, indicating a moderate level of polymorphism across all individuals. A model-based population structure analysis divided P. tomentosa into 11 subpopulations (K = 11). The pattern of individual assignments into the subsets (K = 3) provided reasonable evidence for treating climatic zones as genetic regions for population genetics. The highest level of genetic variation was found in the southern region (i.e., N = 93, N (P) = 11, H (E) = 0.445, F = -0.102), followed by the northeastern and northwestern regions. Thus, the southern region is probably the center of the current species distribution. No correlation was found between population genetic distance and geographic distance (r = 0.0855, P = 0.3140), indicating that geographical distance was not the principal factor influencing genetic differentiation in P. tomentosa. These data provide a starting point for conserving valuable natural resources and optimizing breeding programs.

Fast and Cost-Effective Mining of Microsatellite Markers Using NGS Technology: An Example of a Korean Water Deer Hydropotes inermis argyropus

BackgroundMicrosatellites, a special class of repetitive DNA sequence, have become one of the most popular genetic markers for population/conservation genetic studies. However, its application to endangered species has been impeded by high development costs, a lack of available sequences, and technical difficulties. The water deer Hydropotes inermis is the sole existing endangered species of the subfamily Capreolinae. Although population genetics studies are urgently required for conservation management, no species-specific microsatellite marker has been reported.MethodsWe adopted next-generation sequencing (NGS) to elucidate the microsatellite markers of Korean water deer and overcome these impediments on marker developments. We performed genotyping to determine the efficiency of this method as applied to population genetics.ResultsWe obtained 98 Mbp of nucleotide information from 260,467 sequence reads. A total of 20,101 di-/tri-nucleotide repeat motifs were identified; di-repeats were 5.9-fold more common than tri-repeats. [CA]n and [AAC]n/[AAT]n repeats were the most frequent di- and tri-repeats, respectively. Of the 17,206 di-repeats, 12,471 microsatellite primer pairs were derived. PCR amplification of 400 primer pairs yielded 106 amplicons and 79 polymorphic markers from 20 individual Korean water deer. Polymorphic rates of the 79 new microsatellites varied from 2 to 11 alleles per locus (He: 0.050–0.880; Ho: 0.000–1.000), while those of known microsatellite markers transferred from cattle to Chinese water deer ranged from 4 to 6 alleles per locus (He: 0.279–0.714; Ho: 0.300–0.400).ConclusionsPolymorphic microsatellite markers from Korean water deer were successfully identified using NGS without any prior sequence information and deposited into the public database. Thus, the methods described herein represent a rapid and low-cost way to investigate the population genetics of endangered/non-model species.

Mining functional microsatellites in legume unigenes.

Highly polymorphic and transferable microsatellites (SSRs) are important for comparative genomics, genome analysis and phylogenetic studies. Development of novel species-specific microsatellite markers remains a costly and labor-intensive project. Therefore, interest has been shifted from genomic to genic markers owing to their high inter-species transferability as they are developed from conserved coding regions of the genome. This study concentrates on comparative analysis of genic microsatellites in nine important legume (Arachis hypogaea, Cajanus cajan, Cicer arietinum, Glycine max, Lotus japonicus, Medicago truncatula, Phaseolus vulgaris, Pisum sativum and Vigna unguiculata) and two model plant species (Oryza sativa and Arabidopsis thaliana). Screening of a total of 228090 putative unique sequences spanning 219610522 bp using a microsatellite search tool, MISA, identified 12.18% of the unigenes containing 36248 microsatellite motifs excluding mononucleotide repeats. Frequency of legume unigene-derived SSRs was one SSR in every 6.0 kb of analyzed sequences. The trinucleotide repeats were predominant in all the unigenes with the exception of C. cajan, which showed prevalence of dinucleotide repeats over trinucleotide repeats. Dinucleotide repeats along with trinucleotides counted for more than 90% of the total microsatellites. Among dinucleotide and trinucleotide repeats, AG and AAG motifs, respectively, were the most frequent. Microsatellite positive chickpea unigenes were assigned Gene Ontology (GO) terms to identify the possible role of unigenes in various molecular and biological functions. These unigene based microsatellite markers will prove valuable for recording allelic variance across germplasm collections, gene tagging and searching for putative candidate genes.

Comparative genetic diversity of wild and hachery populations of Korean threadsail filefish Stephanolepis cirrhifer using cross-species microsatellite markers

The threadsail filefish Stephanolepis cirrhifer is a highly commercial fisheries resource in Korea that suffers intensive anthropogenic pressure across much of its range. For basic information about its current genetic status in relation to stock enhancement, the level and distribution of genetic variation between a wild and a hatchery-bred population were investigated using 10 microsatellite markers developed for Thamnaconus modestus. High levels of polymorphism were observed between the two populations. A total of 95 different alleles were found at all loci, with some alleles being unique. The allelic variability ranged from six to 13 in the wild population and from five to 13 in the hatchery one. The average observed and expected heterozygosities were estimated to be 0.72 and 0.80 in the wild sample and 0.70 and 0.79 in the hatchery one, respectively. These results showed similar genetic variability in the hatchery population, as compared with the wild population and significant genetic differentiation between the wild population and the hatchery samples (FST = 0.016, P < 0.05). Genetic drift in the intensive breeding practices for stock enhancement has probably promoted differentiation between populations. Significant deviations from Hardy-Weinberg equilibrium were detected in both populations. Our results indicate that further studies using species-specific microsatellite markers will be necessary for a more reliable assessment of genetic diversity of the species.

High genetic diversity and connectivity in a common mesopelagic fish of the Southern Ocean: The myctophid Electrona antarctica

Many marine pelagic fish species are characterized by subtle but complex genetic structures and dynamics, depending on the balance between current-mediated larval dispersal and adult active homing behavior. The circumantarctic continuous hydrodynamics of the Southern Ocean is a prime example of a system with a potentially great homogenizing effect among distant populations. We tested this hypothesis by analyzing the contemporary genetic relatedness among populations of a common and endemic mesopelagic fish of the Southern Ocean, Electrona antarctica. Seven newly developed species-specific microsatellite markers were used to investigate patterns of neutral genetic variation in 11 geographically widespread samples ( n=400) collected between 2006 and 2007. We detected a very high level of genetic diversity, but a striking lack of genetic differentiation on a circumantarctic scale, indicating large effective population sizes complemented with high levels of admixture. These findings underscore the large scale homogenizing effect of the Southern Coastal Current, leading to a high level of connectivity of our model species in the Southern Ocean, which is congruent with its huge biomass and central role in marine food webs. As an important Antarctic marine living resource this species may as such be managed on a circumantarctic level, although the demographic stability of this stock should be estimated urgently.

Fine-scale spatial genetic structure and within population male-biased gene-flow in the grasshopper Mioscirtus wagneri

Dispersal is a life history trait that plays a key role in population dynamics, determining gene flow and influencing the size, structure and persistence of populations. For these reasons, the study of the genetic consequences of dispersal can be considered a central topic in both conservation and population genetics. In this study we examine the patterns of fine-scale genetic structure within two populations of the grasshopper Mioscirtus wagneri (Orhoptera: Acrididae). For this purpose, we have used seven species-specific microsatellite markers to type 266 individuals from two populations (Pena Hueca and El Salobral) located in Central Spain. We have found subtle genetic differentiation between some sampling patches and significant kinship structures up to 25 m distance which were particularly patent for females. In Pena Hueca locality, patterns of isolation-by-distance at both the patch scale and the individual level have also revealed an association between genetic differentiation/similarity and geographical distance in females but not in males. Overall, these data suggest a fine-scale spatial genetic substructure in the studied populations which seems to be mainly driven by female philopatry. Such pattern of within population genetic structure together with the inferred restricted dispersal distances is likely to contribute to reduce effective population sizes and inter-population gene flow. This can erode genetic variability and limit the colonization ability of this orthoptera, factors which can ultimately compromise the long-term persistence of their small size and isolated populations.

Comparative population structure of Plasmodium malariae and Plasmodium falciparum under different transmission settings in Malawi

BackgroundDescribed here is the first population genetic study of Plasmodium malariae, the causative agent of quartan malaria. Although not as deadly as Plasmodium falciparum, P. malariae is more common than previously thought, and is frequently in sympatry and co-infection with P. falciparum, making its study increasingly important. This study compares the population parameters of the two species in two districts of Malawi with different malaria transmission patterns - one seasonal, one perennial - to explore the effects of transmission on population structures.MethodsSix species-specific microsatellite markers were used to analyse 257 P. malariae samples and 257 P. falciparum samples matched for age, gender and village of residence. Allele sizes were scored to within 2 bp for each locus and haplotypes were constructed from dominant alleles in multiple infections. Analysis of multiplicity of infection (MOI), population differentiation, clustering of haplotypes and linkage disequilibrium was performed for both species. Regression analyses were used to determine association of MOI measurements with clinical malaria parameters.ResultsMultiple-genotype infections within each species were common in both districts, accounting for 86.0% of P. falciparum and 73.2% of P. malariae infections and did not differ significantly with transmission setting. Mean MOI of P. falciparum was increased under perennial transmission compared with seasonal (3.14 vs 2.59, p = 0.008) and was greater in children compared with adults. In contrast, P. malariae mean MOI was similar between transmission settings (2.12 vs 2.11) and there was no difference between children and adults. Population differentiation showed no significant differences between villages or districts for either species. There was no evidence of geographical clustering of haplotypes. Linkage disequilibrium amongst loci was found only for P. falciparum samples from the seasonal transmission setting.ConclusionsThe extent of similarity between P. falciparum and P. malariae population structure described by the high level of multiple infection, the lack of significant population differentiation or haplotype clustering and lack of linkage disequilibrium is surprising given the differences in the biological features of these species that suggest a reduced potential for out-crossing and transmission in P. malariae. The absence of a rise in P. malariae MOI with increased transmission or a reduction in MOI with age could be explained by differences in the duration of infection or degree of immunity compared to P. falciparum.

A species-specific microsatellite marker to discriminate European Atlantic salmon, brown trout, and their hybrids

Atlantic salmon and brown trout frequently co-occur in the same habitats and juveniles of both species are difficult to discriminate. Hybridization between the two taxa has also been widely documented especially in endangered populations hence the need for species-specific molecular markers. Here we show that the microsatellite marker SsAD486 has two species-specific alleles allowing the identification of Atlantic salmon, brown trout, and their hybrids throughout the European range of these species. Since this marker is polymorphic in Atlantic salmon populations from Western Atlantic, it could also help discriminating between North American and European salmon.

Parentage assignment in Haliotis midae L.: a precursor to future genetic enhancement programmes for South African abalone

The establishment and evaluation of family lines using pedigree information provides an advanced understanding of the variability that exists for complex, economically valuable traits and is a necessary step in the execution of an effective breeding programme. The aim of this study was to assign parentage to mass-spawned Haliotis midae juveniles using species-specific microsatellite markers. Screening of wild abalone individuals revealed that the nine loci selected complied with the minimum requirements for parentage analyses: a null allele frequency <5% as well as a high number and frequency of alleles per locus. A total of 598 individuals were genotyped (198 breeding individuals and 400 F1 progeny) from two farms, with parentage results yielding 91% and 90% successful assignment for Farms A and B respectively. This study, therefore, provided the necessary pedigree information required for controlled breeding of individual adult abalone and indicated the usefulness of the panel of microsatellite markers selected for parentage assignment.

Characterization of highly informative cross‐species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.

Comparative Genetic Diversity of Wild and Captive Populations of the Bare-Faced Curassow (Crax fasciolata) Based on Cross-Species Microsatellite Markers: Implications for Conservation and Management

The bare-faced curassow (Crax fasciolata) is a large Neotropical bird that suffers anthropogenic pressure across much of its range. A captive population is maintained for conservation management, although there has been no genetic screening of stocks. Based on the six microsatellite markers developed for Crax globulosa, the genetic variability of C. fasciolata and possible differences between a wild and a captive population were investigated. Only three loci were polymorphic, with a total of 27 alleles. More than half of these alleles were private to the wild (n = 8) or captive (n = 7) populations. Significant deviations from Hardy-Weinberg equilibrium were restricted to the captive population. Despite the number of private alleles, genetic drift has probably promoted differentiation between populations. Our results indicate that wild C. fasciolata populations are genetically impoverished and structured, but species-specific microsatellite markers will be necessary for a more reliable assessment of the species' genetic diversity.

Population genetics ofMioscirtus wagneri, a grasshopper showing a highly fragmented distribution

The genetic consequences of population fragmentation and isolation are major issues in conservation biology. In this study we analyse the genetic variability and structure of the Iberian populations of Mioscirtus wagneri, a specialized grasshopper exclusively inhabiting highly fragmented hypersaline low grounds. For this purpose we have used seven species-specific microsatellite markers to type 478 individuals from 24 localities and obtain accurate estimates of their genetic variability. Genetic diversity was relatively low and we detected genetic signatures suggesting that certain populations of M. wagneri have probably passed through severe demographic bottlenecks. We have found that the populations of this grasshopper show a strong genetic structure even at small geographical scales, indicating that they mostly behave as isolated populations with low levels of gene flow among them. Thus, several populations can be regarded as independent and genetically differentiated units which require adequate conservation strategies to avoid eventual extinctions that in highly isolated localities are not likely to be compensated for with the arrival of immigrants from neighbouring populations. Overall, our results show that these populations probably represent the 'fragments' of a formerly more widespread population and highlight the importance of protecting Iberian hypersaline environments due to the high number of rare and endangered species they sustain.