Evolutionary History of B1 Retroposons in the Genus Mus

Short interspersed DNA elements (SINEs) amplify by retroposition either by (i) successive waves of amplification from one or a few evolving master genes or by (ii) the generation of new master genes that coexist with their progenitors. Individual, highly conserved, elements of the B1 SINE family were identified from the GenBank nucleotide database using various B1 subfamily consensus query sequences to determine their integration times into the mouse genome. A comparison of orthologous loci in various species of the genus Mus demonstrated that four subfamilies of B1 elements have been amplifying within the last 1–3 million years. Therefore, B1 sequences are generated by coexisting source genes. Additionally, three B1 subfamilies have been concurrently propagated during subspecies divergence and strain formation in Mus, indicating very recent activity of this retroposon family. The patterns of intra- and interspecies variations of orthologous loci demonstrate the usefulness of B1 integrations as a phylogenetic tool. A single inconsistency in the phylogenetic trends was depicted by the presence of a B1 insert in an orthologous locus exclusively in M. musculus and M. pahari. However, DNA sequence analysis revealed that these were independent integrations at the same genomic site. One highly conserved B1 element that integrated at least 4–6 million years ago suggests the possibility of occasional function for B1 integrations. Received: 25 February 2000 / Accepted: 5 June 2000     

Conservation and variability of sequence-tagged microsatellite sites (STMSs) from chickpea (Cicer aerietinum L.) within the genus Cicer

The conservation of 90 microsatellite-flanking sequences from chickpea in 39 accessions of eight annual and 1 accession of a perennial species of the genus Cicer was investigated. All of the primer sequences successfully amplified microsatellites in related species, indicating the conservation of microsatellite-flanking sequences in chickpea’s relatives. However, the degree of conservation of the primer sites varied between species depending on their known phylogenetic relationship to chickpea, ranging from 92.2% in C. reticulatum, chickpea’s closest relative and potential ancestor, down to 50% for C. cuneatum. A phylogenetic tree revealed that chickpea and the other members of its crossability group were more closely related to the perennial C. anatolicum than to other annual species of the genus. Considerable variation in size and number of amplification products between and within species was observed. Sequence analysis of highly divergent amplification products proved that variation is either due to large differences in the number of microsatellite repeats or to the amplification of a locus unrelated to the one amplified from chickpea. Sequence information and bootstrapping using PAUP suggested that STMSs derived from chickpea may be efficiently and reliably used for synteny studies in chickpea’s crossability group, including C. anatolicum. However, care should be taken when applying these markers to other species of the genus. Considering the data presented here and the known historical record, the age of section Monocicer, including chickpea, is estimated to be about 100,000 years. Received: 13 August 1999 / Accepted: 11 November 1999     

Strong correlation between cross-amplification success and genetic distance across all members of 'True Salamanders' (Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite loci

The unpredictable and low cross-amplification success of microsatellite loci tested for congeneric amphibian species has mainly been explained by the size and complexity of amphibian genomes, but also by taxonomy that is inconsistent with phylogenetic relationships among taxa. Here, we tested whether the cross-amplification success of nine new and 11 published microsatellite loci cloned for an amphibian source species, the fire salamander (Salamandra salamandra), correlated with the genetic distance across all members of True Salamanders (genera Chioglossa, Lyciasalamandra, Mertensiella and Salamandra that form a monophyletic clade within the family of Salamandridae) serving as target species. Cross-amplification success varied strongly among the species and showed a highly significant negative relationship with genetic distance and amplification success. Even though lineages of S. salamandra and Lyciasalamndra have separated more than 30 Ma, a within genus amplification success rate of 65% was achieved for species of Lyciasalamandra thus demonstrating that an efficient cross-species amplification of microsatellite loci in amphibians is feasible even across large evolutionary distances. A decrease in genome size, on the other hand, paralleled also a decrease in amplified loci and therefore contradicted previous results and expectations that amplification success should increase with a decrease in genome size. However, in line with other studies, our comprehensive dataset clearly shows that cross-amplification success of microsatellite loci is well explained by phylogenetic divergence between species. As taxonomic classifications on the species and genus level do not necessarily mirror phylogenetic divergence between species, the pure belonging of species to the same taxonomic units (i.e. species or genus) might be less useful to predict cross-amplification success of microsatellite loci between such species.     

Sequence variations at a complex microsatellite locus in rice and its conservation in cereals

In an attempt to study changes associated with microsatellites in rice, the DNAs of cultivated rice, including indica and japonica varieties, and wild rice genotypes were amplified by the polymerase chain reaction with primers flanking the (GATA) _n and (AC) _n repeats at a microsatellite-containing locus OS1E6 (Genebank accession number AFO16647) previously reported from a PstI rice (var. Malkolam) genomic library in pUC18. Eight alleles of varying sizes were obtained which were cloned and sequenced. Sequencing data indicated that the size variations of the different alleles were due to differences in the repeat number as well as to sequence variations in the region flanking the microsatellite motifs. In order to study the presence of this complex microsatellite-containing locus of rice in different cereals, their DNAs were amplified using primers flanking the OS1E6 locus. It was found that this locus was present in the various cereal genotypes analyzed, indicating its conservation across different cereal members. Received: 10 March 2000 / Accepted: 14 April 2000     

Evolution of a long-range repeat family in Chromosome 1 of the genus Mus

Copy numbers and variation of a clustered long-range repeat family on Chromosome (Chr) 1 have been studied in different species of the genus Mus. The repeat sequence was present in all, as inferred from cross-hybridization with probes derived from the Mus musculus repeat family. Copy numbers determined by dot blot hybridization were very low, from three to six per haploid genome in M. caroli, M. cervicolor, and M. cookii. These species form one branch of the phylogenetic tree in the genus Mus. In the other group of phylogenetically related species—M. spicilegus, M. spretus, M. musculus and M. macedonicus—copy numbers ranged from 6 to 1810 per haploid genome. The repeat cluster is cytogenetically visible as a fine C-band in M. macedonicus and as a C-band positive homogeneously staining region (HSR) in several populations of M. m. domesticus and M. m. musculus. When cytogenetically visible, the clusters contained from 179 to 1810 repeats. Intragenomic restriction fragment length polymorphisms (RFLPs), which reflect sequence variation among different copies of the long-range repeat family, increased with higher copy numbers. The high similarity of the RFLP pattern among genomes with C-band positive regions in Chr 1 of M. m. musculus, M. m. domesticus, and M. macedonicus points to a close evolutionary relationship of their Chr 1 repeat families.     

Isolation of simple and compound polymorphic tetranucleotide microsatellites for the neotropical leaflitter frog Eleutherodactylus ockendeni (Leptodactylidae)

Few studies of population structure and genetic diversity exist for frogs in the Amazon of South America, an area renowned for exceptionally high species richness. We isolated seven highly variable tetranucleotide microsatellite loci for the neotropical leaflitter frog, Eleutherodactylus ockendeni using an enrichment method. Three of the repeats are simple, three are compound and one is imperfect. We screened all loci with 175 individuals from one geographical area in the upper Napo of Ecuador and found high polymorphism in all loci (> 14 alleles/locus). These markers are suitable for population genetics studies of E. ockendeni and perhaps other leaflitter frogs of the same genus.     

Phylogenetic affinities and species limits within the genus Megadontomys (Rodentia: Cricetidae) based on mitochondrial sequence data

The evolutionary history of the genus Megadontomys, a group of mice allopatrically distributed along the cool‐humid forest in the highlands of México, is controversial. In this study, we examined phylogenetic relationships within the genus using sequences data from the complete mitochondrial cytochrome b gene. This information also allowed us to corroborate species limits, geographic boundaries of taxonomic entities and assess genetic variation within each taxon. The results of the phylogenetic analyses based on maximum likelihood, Bayesian inference and maximum parsimony were largely congruent in that M. nelsoni and M. thomasi were more closely related relative to M. cryophilus. These results are concordant with previous studies based on morphology and allozyme variation. However, testing of the alternative hypothesis of a closer evolutionary affinity between M. nelsoni–M. cryophilus did not produce a significantly less likely tree. The lack of unambiguous support towards one of these previously proposed contending hypotheses is congruent with the alternative scenario of an almost simultaneous diversification of the three species. Application of the phylogenetic species concept and the genetic species concept supports the recognition of three distinct taxonomic entities at the specific level. M. nelsoni inhabits the Sierra Madre Oriental (Hidalgo, Veracruz, and Puebla) including the Sierra Mazateca (Oaxaca); M. cryophilus is restricted to the Sierra de Juárez (Oaxaca); and M. thomasi occurs in portions of the Sierra Madre del Sur (Guerrero) and the Sierra Mixteca (Oaxaca). Our data show that M. thomasi is formed by two genetically distinct lineages that potentially may represent distinct Evolutionary Significant Units.     

Genetic diversity analysis of a world-wide collection of <Emphasis Type="Italic">Ascochyta rabiei</Emphasis> isolates using sequence tagged microsatellite markers

A previously characterized compound microsatellite locus ARMS1, containing penta- and decameric repeat units, has been reported to reveal genetic diversity in Ascochyta rabiei (Pass.) Labr. isolates. Therefore, 37 isolates of Ascochyta rabiei collected from different states of India and 38 isolates from fifteen other countries in the world were examined for their diversity at this locus. Twenty-six alleles on the basis of size (228--451 base pairs) were detected in the world isolates examined, while 15 alleles (287--418 base pairs) were observed in isolates from the Indian subcontinent. To the best of our knowledge, this study is the first to demonstrate diversity in representative Ascochyta rabiei isolates from different parts of the world at the ARMS1 locus.     

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.     

Universal primers for a large cryptically simple cpDNA microsatellite region in Aristolochia (Aristolochiaceae)

We provide a new and valuable marker to study species relationships and population genetics in order to trace evolutionary, ecological, and conservational aspects in the genus Aristolochia. Universal primers for amplification and subsequent sequencing of a chloroplast microsatellite locus inside the trnK intron are presented. Utility of the primers has been tested in 32 species representing all clades of Aristolochia, including population studies within the A. pallida complex, A. clusii and A. rotunda. The microsatellite region is characterized as a (A_nT_m)_k repeat of 22–438 bp containing a combination of different repeats arranged as ‘cryptically simple’.     

Identification and characterization of a novel conserved DNA repeat

Homozygous In(10)17Rk mice contain a paracentric inversion within Chromosome (Chr) 10 and exhibit a pygmy phenotype, suggesting that the distal inversion breakpoint is within the pygmy (pg) locus. In order to obtain the pygmy gene by positional cloning procedures, In(10)17Rk DNA was subjected to RFLP analysis with single-copy probes derived from the wild-type pygmy locus. This analysis identified a DNA polymorphism in the DBA/2J mouse strain on which the In(10)17Rk mutation was originally induced. A detailed characterization of this polymorphism revealed the presence of a novel, tandemly repeated DNA element. Copy number estimation experiments indicate that there are approximately 100,000 copies of this element in the haploid DBA/2J genome. PCR typing studies revealed the presence of the repeat at the pygmy locus of 6 of the 18 Mus domesticus strains analyzed. The absence of the repeat from the pygmy locus of 12 strains of the M. domesticus species and from the M. caroli, M. spretus, M. castaneus, and M. molossinus species suggests that the repeat could serve as a strain-specific hybridization probe in genetic mapping studies. Finally, the novel tandem DNA repeat is conserved in both rat and human genomes as indicated by Southern hybridization experiments.     

Allelic variation,fragment length analyses and population genetic models: a case study on Drosophila microsatellites*

The allelic variation of 16 microsatellite loci from selected species of the Drosophila melanogaster and D. obscura group was determined. Intra‐ and interspecific sequence comparisons allowed discrimination of mutations affecting the repetitive microsatellite from those affecting the flanking regions. The hypotheses that slippage needs a minimum number of repeats in order to become efficient with respect to microsatellite variability, and of an increased mutation rate with increased length of the microsatellite are supported by the results of our analyses. There is in particular at the interrupted complex microsatellite locus BICOID in the species of the D. obscura group, extensive variation in the flanking regions in addition to length and sequence variation of the repetitive microsatellite. The allelic variation at this locus can hardly be explained by slippage alone. Estimates of microsatellite variability by fragment length analyses will pick up only a minor fraction of allelic variation at such loci, and conclusions that are based on the stepwise mutation model will not hold.     

Utility of a set of microsatellite primers developed for the massasauga rattlesnake (Sistrurus catenatus) for population genetic studies of the timber rattlesnake (Crotalus horridus)

I tested six microsatellite DNA primer pairs developed for the massasauga rattlesnake (Sistrurus catenatus) on a sample population of the timber rattlesnake (Crotalus horridus). It had been speculated in a previous publication that cross‐species amplification would not be worthwhile across the two rattlesnake genera. However, for this primer set (the only one currently published for the genus Sistrurus), successful amplification at each locus was accomplished for all loci with an annealing temperature of 57 °C and locus‐specific buffer conditions. Each locus was polymorphic, with the number of alleles per locus ranging from two to 12. Significant heterozygote deficits were detected for three loci (Scu01, Scu05 and Scu07). For Scu01, all individuals were homozygous for the same allele except one female who was homozygous for a different allele. This same female was also homozygous for a rare allele at Scu07. When this female was removed from the data set, the number of observed heterozygotes at Scu01 and Scu07 did not differ significantly from random expectations. However, a large heterozygote deficit persisted at Scu05 (despite subsampling), suggesting that this locus may not be useful for population genetic studies of timber rattlesnakes. Despite some limitations, this set of primers may be a useful complement to those already developed for the genus Crotalus. Moreover, the results of this study seem to provide new justification for further studies of cross‐species amplification of microsatellite loci across the two rattlesnake genera.     

Evolution of Microsatellite Alleles in Four Species of Mice (Genus Apodemus)

Microsatellite length variation was investigated at a highly variable microsatellite locus in four species of Apodemus. Information obtained from microsatellite allele sequences was contrasted with allele sizes, which included 18 electromorphs. Additional analysis of a 400-bp unique sequence in the flanking region identified 26 different haplotype sequences or ``true' alleles in the sample. Three molecular mechanisms, namely, (1) addition/deletion of repeats, (2) substitutions and indels in the flanking region, and (3) mutations interrupting the repeat, contributed to the generation of allelic variation. Size homoplasy can be inferred for alleles within populations, from different populations of the same species, and from different species. We propose that microsatellite flanking sequences may be informative markers for investigating mutation processes in microsatellite repeats as well as phylogenetic relationships among alleles, populations, and species. Received: 3 November 1999 / Accepted: 2 May 2000     

Isolation and characterization of microsatellite markers for Casearia sylvestris Sw. (Salicaceae), a neotropical medicinal tree

Casearia sylvestris Sw. is a widespread neotropical tree utilized in popular medicine. Recent research ranked Casearia as one of the most promising genus in the search of drugs against cancer. Despite its wide distribution and pharmacological importance, no microsatellite markers have yet been developed for this genus. In this study, we provide 10 polymorphic microsatellite loci specifically designed for C. sylvestris, used to analyse 90 individuals distributed in two populations from São Paulo state, Brazil. On average, 12.3 alleles per locus were identified, showing the ability of the markers to detect microsatellite polymorphism in this species.     

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.     

Multilocus phylogeny reveals unexpected diversification patterns in Asian wolf snakes (genus Lycodon)

The diverse group of Asian wolf snakes of the genus Lycodon represents one of many poorly understood radiations of advanced snakes in the superfamily Colubroidea. Outside of three species having previously been represented in higher‐level phylogenetic analyses, nothing is known of the relationships among species in this unique, moderately diverse, group. The genus occurs widely from central to Southeast Asia, and contains both widespread species to forms that are endemic to small islands. One‐third of the diversity is found in the Philippine archipelago. Both morphological similarity and highly variable diagnostic characters have contributed to confusion over species‐level diversity. Additionally, the placement of the genus among genera in the subfamily Colubrinae remains uncertain, although previous studies have supported a close relationship with the genus Dinodon. In this study, we provide the first estimate of phylogenetic relationships within the genus Lycodon using a new multi‐locus data set. We provide statistical tests of monophyly based on biogeographic, morphological and taxonomic hypotheses. With few exceptions, we are able to reject many of these hypotheses, indicating a need for taxonomic revisions and a reconsideration of the group's biogeography. Mapping of color patterns on our preferred phylogenetic tree suggests that banded and blotched types have evolved on multiple occasions in the history of the genus, whereas the solid‐color (and possibly speckled) morphotype color patterns evolved only once. Our results reveal that the colubrid genus Dinodon is nested within Lycodon—a clear finding that necessitates the placing of the former genus in synonymy with the latter.     

Application of microsatellite markers developed for arvicoline species in a population genetic study of the root vole <Emphasis Type="Italic">Microtus oeconomus</Emphasis>

Using a root vole Microtus oeconomus (Pallas, 1776) population in NE Poland we applied 31 microsatellite markers previously developed for root voles and closely related species, with the aim to improve the population genetic tools in this species. Here we present 16 polymorphic microsatellite markers grouped into four sets suitable for simultaneous amplification and genetically sex identification in M. oeconomus. The number of alleles per locus in 227 individuals varied from 7 to 26 with a low frequency of null alleles, expected heterozygosity ranged from 0.758 to 0.927, and observed heterozygosity from 0.722 to 0.947. Two loci showed significant deviation from Hardy-Weinberg equilibrium (p<0.05) and all loci showed independent inheritance. We expect these markers to be useful for studies of genetic population structure and kinship of M. oeconomus populations.     

Mus in Morocco: a Quaternary sequence of intraspecific evolution

North Africa is an intricate biogeographical region at the crossroads of immigration waves from tropical Africa and Asia. Species confined between various barriers (Atlas Mountains, arid environments such as the Sahara in the south, water masses such as the Mediterranean Sea in the north, and the Atlantic Ocean in the west) were generally forced to adapt locally to environmental changes instead of tracking their habitat by shifting their distribution area. The present study aims at providing first insight into the evolution of the genus Mus, and more specifically of the western Mediterranean species Mus spretus in this area. The study relies on the abundant Late Pleistocene and Middle Holocene fossil assemblage from the El Harhoura 2 cave (Rabat‐Témara, Morocco). This exceptional record was studied using geometric morphometrics applied to first upper and lower molars, constituting the most informative and best preserved fossil remains for such small rodents. Two main issues were addressed. (1) Geometric morphometrics was used to clarify taxonomic status and phylogenetic relationships among fossil and modern species in this area. Morphometric analysis revealed good discrimination of most modern and fossil species but failed to document intermediate forms tracing anagenetic evolution. Not mutually exclusive, the occurrence of complex processes of morphological evolution in this genus such as parallel evolution and the action of stabilizing selection may make it difficult to translate patterns of morphological evolution into phylogenetic conclusions. (2) The record was shown to document a sequence of intraspecific evolution of M. spretus. The morphology of the molars through the fossil record of El Harhoura 2 was surprisingly stable despite extensive modern variation. The limited temporal variation largely failed to correlate to palaeoenvironmental proxies. The mouse fossil record at El Harhoura 2 thus presents an intriguing case of morphological stasis despite extensive environmental changes. This long‐term stability may have been recently perturbed by anthropogenic factors including landscape changes and introduction of various competitors and predators, leading to a size reduction. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 599–621.     

Field tests of cis-regulatory variation at the prairie vole avpr1a locus: association with V1aR abundance but not sexual or social fidelity

The neuropeptide vasopressin and its receptor V1aR are broadly implicated in social behavior and play a central role in several key aspects of male mating tactics in voles. In the prairie vole, a microsatellite in the cis-regulatory region of the gene encoding V1aR (avpr1a) provides a potential genetic basis for individual variation in neural phenotype and behavior; recent studies found that allele length predicts V1aR expression and male social attachment in the laboratory. Here, we explore the relationship between avpr1a microsatellite length, V1aR neural phenotype, and field measures of monogamy and fitness in male prairie voles. We found significant effects of allele length on V1aR expression in structures integral to pairbond formation. These effects did not, however, translate to differences in mating tactics or reproductive success. Together, these data suggest that, while length polymorphism in the avpr1a microsatellite influences neuronal phenotype, this variation does not contribute significantly to male reproductive success and field behavior. We propose that previously reported behavioral effects may be mediated primarily by sequence variation at this locus, for which allele length is an imperfect proxy. By combining genetic, neuronal and ecological approaches, these data provide novel insights into the contribution of genotype to natural diversity in brain and behavior.