Multilocus phylogeography of the common midwife toad, Alytes obstetricans (Anura, Alytidae): Contrasting patterns of lineage diversification and genetic structure in the Iberian refugium.

Recent investigations on the evolutionary history of the common midwife toad (Alytes obstetricans) revealed high levels of geographically structured genetic diversity but also a situation where delineation of major historical lineages and resolution of their relationships are much more complex than previously thought. We studied sequence variation in one mitochondrial and four nuclear genes throughout the entire distribution range of all recognized A. obstetricans subspecies to infer the evolutionary processes that shaped current patterns of genetic diversity and population subdivision. We found six divergent, geographically structured mtDNA haplogroups diagnosing population lineages, and varying levels of admixture in nuclear markers. Given the timeframe inferred for the splits between major lineages, the climatic and environmental changes that occurred during the Pleistocene seem to have shaped the diversification history of A. obstetricans. Survival of populations in allopatric refugia through the Ice Ages supports the generality of the "refugia-within-refugia" scenario for the Iberian Peninsula. However, lineages corresponding to subspecies A. o. almogavarii, A. o. pertinax, A. o. obstetricans, and A. o. boscai responded differently to Pleistocene climatic oscillations after diverging from a common ancestor. Alytes o. obstetricans expanded northward from a northern Iberian refugium through the western Pyrenees, leaving a signal of contrasting patterns of genetic diversity, with a single mtDNA haplotype north of the Pyrenees from SW France to Germany. Both A. o. pertinax and A. o. boscai are widespread and genetically diverse in Iberia, the latter comprising two divergent lineages with a long independent history. Finally, A. o. almogavarii is mostly restricted to the north-eastern corner of Iberia north of the Ebro river, with additional populations in a small region in south-eastern France. This taxon exhibits unparalleled levels of genetic diversity and little haplotype sharing with other lineages, suggesting a process of incipient speciation.

Copy number polymorphism in the α-globin gene cluster of European rabbit (Oryctolagus cuniculus).

Comparative genomic studies have revealed that mammals typically possess two or more tandemly duplicated copies of the α-globin (HBA) gene. The domestic rabbit represents an exception to this general rule, as this species was found to possess a single HBA gene. Previous electrophoretic surveys of HBA polymorphism in natural populations of the European rabbit (Oryctolagus cuniculus) revealed extensive geographic variation in the frequencies of three main electromorphs. The variation in frequency of two electromorphs is mainly partitioned between two distinct subspecies of European rabbit, and a third is restricted to the hybrid zone between the two rabbit subspecies in Iberia. Here we report the results of a survey of nucleotide polymorphism, which revealed HBA copy number polymorphism in Iberian populations of the European rabbit. By characterizing patterns of HBA polymorphism in populations from the native range of the European rabbit, we were able to identify the specific amino-acid substitutions that distinguish the previously characterized electromorphs. Within the hybrid zone, we observed the existence of a second HBA gene duplicate, named HBA2, that mostly represents a novel sequence haplotype, which occurs in higher frequency within the hybrid zone, and thus appears to have arisen in hybrids of the two distinct subspecies. Although this novel gene is also present in other wild Iberian populations, it is almost absent from French populations, which suggest a recent ancestry, associated with the establishment of the post-Pleistocene contact zone between the two European rabbit subspecies.

The genomic legacy from the extinct Lepus timidus to the three hare species of Iberia: contrast between mtDNA, sex chromosomes and autosomes.

Extensive interspecific genetic introgression is often reported, and appraising its genomic impact can serve to determine whether it results from selection on specific loci or from demographic processes affecting the whole genome. The three species of hares present in the Iberian Peninsula harbour high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species now extinct in the region. This could result from the invasive replacement of L. timidus by the temperate species during deglaciation but should then have left traces in the nuclear genome. We typed single nucleotide polymorphisms (SNPs) discovered by sequencing 10 autosomal loci, two X-linked and one Y-linked in species-wide samples of the four taxa. Based on lineage-diagnostic SNPs, we detected no trace of L. timidus sex chromosomes in Iberia. From the frequencies of inferred haplotypes, autosomal introgression into L. granatensis appeared mostly sporadic but always widespread instead of restricted to the north as mtDNA. Autosomal introgression into Iberian L. europaeus, inhabiting the Pyrenean foothills, was hardly detectable, despite quasi-fixation of L. timidus mtDNA. L. castroviejoi, endemic to the Cantabrian Mountains and fixed for L. timidus mtDNA, showed little traces of autosomal introgression. The absence of sex-chromosome introgression presumably resulted from X-linked hybrid male unfitness. The contrasting patterns between the autosomes and mtDNA could reflect general gender asymmetric processes such as frequency-dependent female assortative mating, lower mtDNA migration and higher male dispersal, but adaptive mtDNA introgression cannot be dismissed. Additionally, we document reciprocal introgression between L. europaeus and both L. granatensis in Iberia and L. timidus outside Iberia.

High levels of population subdivision in a morphologically conserved Mediterranean toad (Alytes cisternasii) result from recent, multiple refugia: evidence from mtDNA, microsatellites and nuclear genealogies.

Pleistocene glaciations often resulted in differentiation of taxa in southern European peninsulas, producing the high levels of endemism characteristic of these regions (e.g. the Iberian Peninsula). Despite their small ranges, endemic species often exhibit high levels of intraspecific differentiation as a result of a complex evolutionary history dominated by successive cycles of fragmentation, expansion and subsequent admixture of populations. Most evidence so far has come from the study of species with an Atlantic distribution in northwestern Iberia, and taxa restricted to Mediterranean-type habitats remain poorly studied. The Iberian Midwife toad (Alytes cisternasii) is a morphologically conserved species endemic to southwestern and central Iberia and a typical inhabitant of Mediterranean habitats. Applying highly variable genetic markers from both mitochondrial and nuclear genomes to samples collected across the species' range, we found evidence of high population subdivision within A. cisternasii. Mitochondrial haplotypes and microsatellites show geographically concordant patterns of genetic diversity, suggesting population fragmentation into several refugia during Pleistocene glaciations followed by subsequent events of geographical and demographic expansions with secondary contact. In addition, the absence of variation at the nuclear beta-fibint7 and Ppp3caint4 gene fragments suggests that populations of A. cisternasii have been recurrently affected by episodes of extinction and recolonization, and that documented patterns of population subdivision are the outcome of recent and multiple refugia. We discuss the evolutionary history of the species with particular interest in the increasing relevance of Mediterranean refugia for the survival of genetically differentiated populations during the Pleistocene glaciations as revealed by studies in co-distributed taxa.

The limits of mtDNA phylogeography: complex patterns of population history in a highly structured Iberian lizard are only revealed by the use of nuclear markers.

Phylogeographic analyses based on the sole use of the mitochondrial DNA (mtDNA) molecule reveal only a small part of the evolutionary history of a species or a set of related species. In this study, we have combined the application of slow- and fast-evolving nuclear markers (proteins and microsatellites, respectively) together with the analysis of two-gene genealogies to further understand the history of the Iberian endemic Schreiber's green lizard, Lacerta schreiberi, a species for which a well established phylogeographical scenario is available. In sharp contrast with the observation of four divergent and almost allopatric mtDNA clades, our nuclear data revealed how two groups of populations diverged, persisted and began to admix along the mountains of the Iberian Central System. In addition, the combination of mtDNA and nuclear data showed how the core area of the species distribution responded to ice ages, first by relatively old processes of population expansion to the south followed by episodes of contraction that are at the origin of present-day isolates, and more recently by a postglacial expansion to the Iberian Northwest where new habitats were made available after climatic amelioration. Taken together with recently published results for a variety of other organisms, our results suggest that complex processes of fragmentation, expansion and admixture can only be properly addressed through the use of several and complementary types of molecular markers. Finally, we also suggest that southern European refugia are both hotspots and melting pots of genetic diversity.

Reduced introgression of the Y chromosome between subspecies of the European rabbit (Oryctolagus cuniculus) in the Iberian Peninsula.

The role of the Y chromosome in speciation is unclear. Hybrid zones provide natural arenas for studying speciation, as differential introgression of markers may reveal selection acting against incompatibilities. Two subspecies of the European rabbit (Oryctolagus cuniculus) form a hybrid zone in the Iberian Peninsula. Previous work on mitochondrial DNA (mtDNA), Y- and X-linked loci revealed the existence of two divergent lineages in the rabbit genome and that these lineages are largely subspecies-specific for mtDNA and two X-linked loci. Here we investigated the geographic distribution of the two Y chromosome lineages by genotyping two diagnostic single nucleotide polymorphisms in a sample of 353 male rabbits representing both subspecies, and found that Y chromosome lineages are also largely subspecies-specific. We then sequenced three autosomal loci and discovered considerable variation in levels of differentiation at these loci. Finally, we compared estimates of population differentiation between rabbit subspecies at 26 markers and found a surprising bimodal distribution of F(ST)values. The vast majority of loci showed little or no differentiation between rabbit subspecies while a few loci, including the SRY gene, showed little or no introgression across the hybrid zone. Estimates of population differentiation for the Y chromosome were surprisingly high given that there is male-biased dispersal in rabbits. Taken together, these data indicate that there is a clear dichotomy in the rabbit genome and that some loci remain highly differentiated despite extensive gene flow following secondary contact.

Evidence for contrasting modes of selection at interacting globin genes in the European rabbit (Oryctolagus cuniculus).

In hybrid zones between genetically differentiated populations, variation in locus-specific rates of introgression may reflect adaptation to different environments or adaptation to different genetic backgrounds. The European rabbit, Oryctolagus cuniculus, is well-suited to studies of such hybrid zone dynamics because it is composed of two genetically divergent subspecies that hybridize in a zone of secondary contact in central Iberia. A species-wide survey of allozyme variation revealed a broad range of locus-specific divergence levels (F(ST) ranged from 0 to 0.54, mean F(ST)=0.16). Interestingly, the two loci that fell at opposite ends of the distribution of F(ST) values, haemoglobin alpha-chain (HBA) and haemoglobin beta-chain (HBB), encode interacting subunits of the haemoglobin protein. The contrasting patterns of spatial variation at these two loci could not be reconciled under a neutral model of population structure. The HBA gene exhibited higher-than-expected levels of population differentiation, consistent with a history of spatially varying selection. The HBB gene exhibited lower-than-expected levels of population differentiation, consistent with some form of spatially uniform selection. Patterns of linkage disequilibrium and allele frequency variation do not appear to fit any simple model of two-locus epistatic selection.

Myb-Ets fusion oncoprotein inhibits thyroid hormone receptor/c-ErbA and retinoic acid receptor functions: a novel mechanism of action for leukemogenic transformation by E26 avian retrovirus.

The E26 and avian erythroblastosis virus (AEV) avian retroviruses induce acute leukemia in chickens. E26 can block both erythroid and myeloid differentiation at an early multipotent stage. Moreover, E26 can block erythroid differentiation at the erythroid burst-forming unit/erythroid CFU (BFU-E/CFU-E) stage, which also corresponds to the differentiation stage blocked by AEV. AEV carries two oncogenes, v-erbA and v-erbB, whereas E26 encodes a single 135-kDa Gag-Myb-Ets fusion oncoprotein. v-ErbA is responsible for the erythroid differentiation arrest through negative interferences with both the retinoic acid receptor (RAR) and the thyroid hormone receptor (T3R/c-ErbA). We investigated whether Myb-Ets could block erythroid differentiation in a manner similar to v-ErbA. We show here that Myb-Ets inhibits both RAR and c-ErbA activities on specific hormone response elements in transient-expression assays. Moreover, Myb-Ets abrogates the inactivation of transcription factor AP-1 by RAR and T3R, another feature shared with v-ErbA. Myb-Ets also antagonizes the biological response of erythrocytic progenitor cells to retinoic acid and T3. Analysis of a series of mutants of Myb-Ets reveals that the domains of the oncoprotein involved in these inhibitory activities are the same as those involved in oncogenic transformation of hematopoietic cells. These data demonstrate that the Myb-Ets oncoprotein shares properties with the v-ErbA oncoprotein and that inhibition of ligand-dependent RAR and c-ErbA functions by Myb-Ets is responsible for blocking the differentiation of hematopoietic progenitors.

Mechanism for mutational inactivation of the tumor suppressor Smad2.

Transforming growth factor beta (TGF-beta) is a potent natural antiproliferative agent that plays an important role in suppressing tumorigenicity. In numerous tumors, loss of TGF-beta responsiveness is associated with inactivating mutations that can occur in components of this signaling pathway, such as the tumor suppressor Smad2. Although a general framework for how Smads transduce TGF-beta signals has been proposed, the physiological relevance of alterations of Smad2 functions in promoting tumorigenesis is still unknown. Here, we show that expression of Smad2.P445H, a tumor-derived mutation of Smad2 found in human cancer, suppresses the ability of the Smads to mediate TGF-beta-induced growth arrest and transcriptional responses. Smad2.P445H is phosphorylated by the activated TGF-beta receptor at the carboxy-terminal serine residues and associates with Smad3 and Smad4 but is unable to dissociate from the receptor. Upon ligand-induced phosphorylation, Smad2.P445H interacts stably with wild-type Smad2, thereby blocking TGF-beta-induced nuclear accumulation of wild-type Smad2 and Smad2-dependent transcription. The ability of the Smad2.P445H to block the nuclear accumulation of wild-type Smad2 protein reveals a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Participatory dam systems modelling: a case study of the transboundary Guadiana River in the Iberian Peninsula.

Modelling tools have been widely used to investigate best management practices. But in contrast to the plethora of modelling studies, the practical implementation of outcomes is comparatively small. There is an urgent need to implement results and to show the practical validation of the concept developed, especially against the context of water stress mitigation. The participative development of modelling studies as a joint effort of stakeholders and modellers is seen as a key to achieve a wider identification, acceptance, trust and applicability of results. Participatory planning in the water sector is also increasingly requested in water management, where tasks have been for clarified decades through different institutional arrangements and national laws. Stakeholder involvement in water resources management have been limited to what was long time seen as participation, merely information on action to be taken. In the last decade the need for participation has been reflected in different ways. In Europe, the implementation of the Water Framework Directive (WFD) under Art. 14, requires all the European countries to involve stakeholders in decision making processes on water resource management. The aim of this paper is to present and discuss the research framework and possible results of investigating dam modelling through participatory systems modelling. We introduce a structured approach to use participatory modelling (PM) for stimulating the integration of modelling and decision making, also as a way of implementation of some articles of the WFD. The results and the framing of this paper are part of the AQUASTRESS Project. The conceptual modelling has been developed by a multidisciplinary research team, local stakeholders and local experts. Some results are discussed and recommendations made.

Differential roles of JNK and Smad2 signaling pathways in the inhibition of c-Myc-induced cell death by TGF-beta.

The transforming growth factor beta (TGF-beta) plays an important role in constraining cellular proliferation, but it is also a potent inducer of programmed cell death or apoptosis. Here, we demonstrate that TGF-beta can have an opposite effect, acting as a survival factor to prevent c-Myc-induced cell death in Rat-1 fibroblasts. However, in marked contrast to TGF-beta, Smad2, which is a critical intracellular mediator of the TGF-beta signaling pathway, functions as an antagonist to induce increased cell death. The protective activity of TGF-beta was associated with the activation of c-Jun N-terminal Kinase (JNK) and was not linked to the ability of TGF-beta to promote cell cycle progression. Expression of dominant-interfering forms of various components of the JNK signaling pathway, including Rac1, Cdc42, mitogen-activated protein kinase kinase 4 (MKK4), and c-Jun, abolished TGF-beta-mediated cell survival. Furthermore, overexpression of the constitutively activated mutant RacL61F37A, which selectively stimulates JNK cascade but not G1 cell cycle progression or actin polymerization, was sufficient to prevent apoptosis induced by c-Myc. These findings describe a differential effect of two separated signaling pathways of TGF-beta and indicate for the first time that Smad2 can act as antagonist to suppress TGF-beta-dependent cell survival. Oncogene (2000) 19, 1277 - 1287.

c-erbA alpha/T3R and RARs control commitment of hematopoietic self-renewing progenitor cells to apoptosis or differentiation and are antagonized by the v-erbA oncogene.

In AEV-transformed erythroleukemic cells the v-erbA gene product is likely to antagonize the function of triiodothyronine (T3) and retinoic acid (RA) receptors and thereby to block cell differentiation. We have thus investigated the effects of T3 and RA on normal early erythrocytic progenitor cells. Here we show: (1) that either RA or T3 play an essential role during the early commitment to erythrocytic differentiation, (2) that both T3 and RA induce death by apoptosis and a strong inhibition of self-renewal in progenitor cells grown in the absence of differentiation-inducing agents and (3) that the v-erbA oncogene renders erythrocytic progenitor cells insensitive to apoptosis and to self-renewal inhibition induced by RA or T3. The behaviour of a non-transforming mutant of v-erbA suggests that this v-erbA-induced protection is related to its transforming potential.

Estimation of gene diversity at the b locus of the constant region of the immunoglobulin light chain in natural populations of European rabbit (Oryctolagus cuniculus) in Portugal, Andalusia and on the Azorean Islands.

The minimal gene diversity at a locus of the antibody constant region, as estimated in natural populations of rabbit, revealed levels of heterozygosity similar to those reported for the major histocompatibility complex in human and murine populations. Sera of 416 wild rabbits were collected on the Iberian peninsula and on three islands of the Azorean archipelago and analyzed for the occurrence of the serological markers of the b locus of the immunoglobulin light chain. All four serotypes present in domestic rabbits were found in Portugal. They represented less than 50% of the gene pool. In Andalusia this was less than 15% and on the Azorean islands less than 10%. The pronounced and systematic hierarchy in allele frequencies, previously found in populations from the more recent distribution area of the species, was not observed. On the peninsula, the frequencies of the "domestic" alleles were similar, averaging 10%. The Portuguese sample revealed a total heterozygosity of at least 87%. This high value was supported by at least 11 serologically different alleles, none of them occurring at frequencies above 20%. These data are in agreement with an Iberian origin of the European rabbit and strongly suggest the coalescence of b locus allelic lines drawn from Iberian and western populations. The role of balancing selection in the evolution of the b locus polymorphism was further emphasized.

Cellular and subcellular expression of Golf/Gs and Gq/G11 alpha-subunits in rat pancreatic endocrine cells.

We studied the cellular and subcellular localization of Galpha-subunits in pancreas by immunocytochemistry. Golfalpha and G11alpha were specifically localized in islet insulin B-cells and glucagon A-cells, respectively. Gsalpha and Gqalpha labeling was more abundant in B-cells. The presence of Golfalpha in B-cells was confirmed by in situ hybridization. In B-cells, Golfalpha and Gsalpha were found in the Golgi apparatus, plasma membrane (PM) and, remarkably, in mature and immature insulin secretory granules, mainly at the periphery of the insulin grains. Gqalpha was detected on the rough endoplasmic reticulum (RER) near the Golgi apparatus. In A-cells, the Galpha-subunits were mostly within the glucagon granules: G11alpha gave the strongest signal, Gsalpha less strong, Gq was scarce, and Golf was practically absent. Gqalpha and Gsalpha immunoreactivity was detected in acinar cells, although it was much weaker than that in islet cells. The cell-dependent distribution of the Galpha-subunits indicates that the stimulatory pathways for pancreatic function differ in acinar and in islet B- and A-cells. Furthermore, the G-protein subunits in islet cell secretory granules might be functional and participate in granule trafficking and hormone secretion.

Stimulatory transducing systems in pancreatic islet cells.

We have determined the cellular distribution of different alpha subtypes of G proteins and adenylyl cyclase (AC) isoforms in endocrine, exocrine, and established pancreatic cell lines. VIP, PACAP, and tGLP-1 receptor proteins are expressed to varying extents in A and B cells, whereas the expression of G alpha subunits is cell specific. Thus, G(olf) alpha is detected in normal rodent B cells and immortalized pancreatic B cell lines, whereas Gs alpha is more ubiquitously expressed. The cellular density of AC isoforms labeling (I, II, III, IV, V/VI) is also islet cell-specific and their distribution is age- and species-dependent. The identification of numerous signaling molecule subtypes, together with the discovery of their specific subcellular distribution, will help the functional characterization of their intraregulatory pathways, leading to the extrusion of insulin or glucagon secretory granules, and those leading to differentiation and apoptosis of islet cells.

Synaptic inputs on rat brainstem motoneurones in organotypic slice culture.

To study the formation of target specific afferents on brain stem motoneurones of the rat, we used an organotypic co-culture of embryonic rat (E18) brain stem explants containing the facial or hypoglossal motor nuclei together with a tongue explant. The brain stem explants also contained known dorsal premotor structures such as lateral reticular nuclei and vestibular or spinal trigeminal nuclei. In cultures maintained in vitro for over 3 weeks, silver impregnation studies identified neurones in the dorsal sensory structures with axons arborizing within the motor nucleus. A double fluorescent labelling procedure demonstrated that axons originating from dorsal sensory regions come in close contact with identified motoneurones. Electrical stimulation of neurones in the dorsal regions induced monosynaptic and polysynaptic EPSPs and spikes in identified motoneurones together with muscle contraction. This work demonstrates that premotor structures in slice cultures develop organotypic functional synaptic connections with embryonic brain stem motoneurones.

Gating of action potential propagation by an axonal A-like potassium conductance in the hippocampus: a new type of non-synaptic plasticity.

Synaptic plasticity is usually considered as the main form of activity-dependent functional plasticity in the mammalian brain. Short- and long-term regulation of synaptic transmission have been shown in various types of excitatory synapses including cortical and hippocampal synapses. In this review, we discuss a novel form of non-synaptic plasticity that involves voltage-gated K+ conductances in CA3 pyramidal cell axons. With experimental and theoretical arguments, we show that axons cannot only be considered as a simple structure that transmit reliably the action potential (AP) from the cell body to the nerve terminals. The axon is also able to express conduction failures in specific axonal pathways. We discuss possible physiological conditions in which these axonal plasticity may occur and its incidence on hippocampal network properties.

Developing rat brainstem motoneurones in organotypic culture express calcium permeable AMPA-gated receptors.

Recent studies suggest that calcium permeable excitatory amino acid receptors may play an important role in many developmental processes including dendritic differentiation, synaptogenesis and activity dependent plasticity or excitotoxicity related disorders. In this work, we investigate the existence of calcium permeable AMPA receptors in embryonic rat motoneurones in organotypic slice culture, which display an early sensitivity to AMPA and Kainate. We used excitatory amino acids induced cobalt uptake and show that developing motoneurones express Ca2+ permeable AMPA receptors. We demonstrate, as already described for other neuronal types in acute slices of adult animals, that the cobalt loading of motoneurones is not suppressed by blockade of synaptic activity by TTX. It is not induced by NMDA stimulation and does not result from the activation of voltage dependent calcium channels. It is specifically suppressed by the non-competitive AMPA antagonist GYKI 53784 (LY303070) and enhanced by the AMPA-receptor desensitization blocker Aniracetam. We conclude that cobalt loading results from the specific activation of AMPA receptors. We further show that, when cobalt loading is induced by threshold doses of Glutamate agonists, cobalt-sulfide deposits are found specifically in primary dendrites, dendritic spines and localized spots on the somatic and peripheral dendritic membrane. We suggest that this particular pattern of staining, different from the Golgi-like staining obtained with excitotoxic doses, may offer new information regarding the membrane density and distribution of calcium permeable AMPA receptors.

Genetic polymorphism of rabbit (Oryctolagus cuniculus) tissue acid phosphatases (ACP2 and ACP3).

Rabbit (Oryctolagus cuniculus) red cell and tissue acid phosphatases were studied by means of horizontal starch gel electrophoresis and isoelectric focusing followed by enzyme blotting. Red cell acid phosphatase 1 (ACP1) is monomorphic while tissue acid phosphatase 2 (ACP2) is polymorphic in a wild rabbit population, with two alleles: ACP2*1 (0.96) and ACP2*2 (0.04). A third locus homologous of human acid phosphatase 3 (ACP3) is characterized by the presence of three alleles (ACP3*1, ACP3*2 and ACP3*3). ACP3*1 is the most common allele and was detected in all populations, ACP3*2 was found in domestic breeds and in a wild population from Southern France, whereas ACP3*3 is typical of Portuguese wild rabbits. The geographical distribution of ACP3*2 and ACP3*3 is in agreement with the subspecific level of differentiation of the rabbit species in O. cuniculus cuniculus and O. c. algirus. The comparative study of the acid phosphatase activity in red cells of several mammalian species, including humans, suggests that ACP3 activity in erythrocytes exists only in rabbit.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2010-31 January 2011.

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterraneus, Kirengeshoma palmata, Lysimachia japonica, Macrolophus pygmaeus, Microtus cabrerae, Mytilus galloprovincialis, Pallisentis (Neosentis) celatus, Pulmonaria officinalis, Salminus franciscanus, Thais chocolata and Zootoca vivipara. These loci were cross-tested on the following species: Acanthina monodon, Alytes cisternasii, Alytes maurus, Alytes muletensis, Alytes obstetricans almogavarii, Alytes obstetricans boscai, Alytes obstetricans obstetricans, Alytes obstetricans pertinax, Cambarellus montezumae, Cambarellus zempoalensis, Chorus giganteus, Cobitis tetralineata, Glossina fuscipes fuscipes, Glossina pallidipes, Lysimachia japonica var. japonica, Lysimachia japonica var. minutissima, Orconectes virilis, Pacifastacus leniusculus, Procambarus clarkii, Salminus brasiliensis and Salminus hilarii.