A multiplex panel of microsatellite markers for widespread sub‐Saharan rodents of the genus Mastomys

We isolated and characterized 12 polymorphic microsatellite loci in the sub‐Saharan rodent Mastomys huberti. We tested cross‐species amplification of all these loci in three closely related Mastomys species: M. coucha, M. erythroleucus and M. natalensis. Multiplex panels comprising 11 loci were developed and their application to a set of individuals in each species allowed clear and easy characterization of allele sizes. Statistics from 31 M. huberti coming from one locality in Mali showed no deviation from Hardy–Weinberg equilibrium except for one locus, and no significant linkage disequilibria between loci.     

Structures génétiques comparées de trois espèces de rongeurs africains du genre Mastomys au Sénégal

Résumé Une analyse par électrophorèse des protéines à 20 locus a été réalisée sur trois espèces du genre Mastomys au Sénégal. Malgré l'absence de locus diagnostique, une approche multivariée par analyses factorielles (AFC et AFD) permet néanmoins de reconnaître de façon sûre une des espèces en présence (M. cf. natalensis, 100% d'individus bien classés par l'AFD) et d'attribuer correctement les individus des deux autres espèces (M. erythroleucus et M. huberti) dans 92% et 77% des cas respectivement. Bien qu'écologiquement nettement différenciées, ces deux dernières espèces apparaissent très proches génétiquement (D_Nei=0,12) et montrent par ailleurs de hétérozygoties très elevées. Les faibles distances génétiques entre les trois espèces contrastent avec la forte différenciation chromosomique observée par ailleurs. Chez M. erythroleucus et M. huberti, les distances génétiques observées entre populations continentales ne sont pas corrélées avec les distances géographiques alors que les populations insulaires présentent une nette baisse de variabilité, en relation probable avec leur isolement géographique important. Par ailleurs, chez M. huberti, plusieurs populations s'écartent fortement de la panmixie. Ces tensions génétiques pourraient résulter de phénomènes d'introgression avec M. erythroleucus.

---

Compared genetic structures of three species of African rodents of the genus Mastomys from Senegal. A protein electrophoresis analysis at 20 loci was performed on three species of the genus Mastomys from Senegal. Although no diagnostic locus was found between the three species, they can be readily recognized by multivariate analysis (100% of the M. cf. natalensis, 92% of the M. erythroleucus and 77% of the M. huberti individuals could be correctly classified by discriminant analysis). Although ecologically quite clearly differentiated, M. erythroleucus and M. huberti were found to be genetically very close (D_Nei=0.12) and display very high heterozygosities. On the whole, the small genetic distances between the three species contrast with the high chromosomal differentiation that has been reported in another study. In M. erythroleucus and M. huberti, genetic distances between mainland populations are not correlated with geographic distances, whereas insular populations show an important decrease of variability probably linked with their geographic isolation. In M. huberti, some populations show a strong departure from panmictic equilibrium: these genetical tensions could result from introgression phenomena with M. erythroleucus.

     

Chromosomal characterization of three species of the genus Mastomys in Senegal

Mastomys rats are present as three distinct karyomorphs in Senegal. Specific assignations were accorded to each form on the basis of chromosomal, reproductive and distribution characteristics. Mastomys erythroleucus (2n = 38 , NFa = 52) is ecologically a generalist, M. huberti (2n = 32 , NFa = 44) is restricted to humid biotopes and M. cf natalensrs (2n = 32 , NFa = 54) is strictly commensal and found in Southeastern Senegal.     

Systematics and diversification of Praomys species (Rodentia: Muridae) endemic to the Cameroon Volcanic Line (West Central Africa)

Missoup, A.D., Nicolas, V., Wendelen, W., Keming, E., Bilong Bilong, C.F., Couloux, A., Atanga, E., Hutterer, R. & Denys, C. (2012). Systematics and diversification of Praomys species (Rodentia: Muridae) endemic to the Cameroon Volcanic Line (West Central Africa). —Zoologica Scripta, 41, 327–345. Our integrative approach combines two mitochondrial genes (16S and cyt b gene), two nuclear genes (exon 10 GHR and exon 1 IRBP) and craniometrical data to test the status and to infer phylogenetic relationships of the three Praomys Cameroon Volcanic Line endemics (P. hartwigi, P. morio and P. obscurus). The taxonomic rank of the principal genus group is assessed and the mode of diversification of species of the P. tullbergi complex in Afrotropical forests is discussed based on estimates of times to the most recent common ancestors and on tree topologies. This study documents for the first time the molecular and morphometrical distinctiveness of P. hartwigi and P. morio within the P. tullbergi species complex. Further studies including specimens of P. hartwigi from all its distribution range are needed to conclude on the status of P. obscurus. The monophyly of the genus Praomys is refuted. Times to the most recent common ancestors of major clades within the P. tullbergi species complex are estimated for the last 2.5 Mya and during the last 1 or 2 Mya for different species or forms. The lowland forest refuge hypothesis might well explain the diversification of P. misonnei, P. rostratus and P. tullbergi in the guineo‐congolese forest block. The isolation of montane forests could have facilitated the divergence between the two montane forest forms P. hartwigi and P. obscurus and between populations of P. morio from the continent and those from the island of Bioko. Praomys populations (species) that inhabit the Cameroon Volcanic Line Praomys probably originated as lowland forms subsequently specialized to highland conditions.     

The impact of the Congo River and its tributaries on the rodent genus Praomys: speciation origin or range expansion limit?

We studied the taxonomy, distribution, and ecology of species within Praomys, a common rodent genus present in rainforests and montane forests in sub‐Saharan Africa. The taxonomy of the group is problematic, and for the sampled region of Kisangani (Democratic Republic of Congo) no prior genetic study has been published. We used a combination of molecular (cyt b sequencing) and craniometric techniques (canonical analyses of skull measurements) for the species identification of a total of 654 specimens. We confirm the presence of Praomys minor in the region, up to now only known from the type and paratype specimens. At least seven species are present in the Kisangani region, and two clades occur along both banks of the Congo River. The present‐day distribution of the genus seems to be influenced by large‐scale rainforest fragmentation related to drier periods in geological history. The Congo River could in this case constitute a modern barrier to gene flow when the climate enabled rainforest expansion. The tributaries of the Congo River play no role in limiting Praomys species distribution, apart from the Aruwimi River for Praomys jacksoni s.l. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 983–1002.     

Diversity,dynamics and reproduction in a community of small mammals in Upper Guinea,with emphasis on pygmy mice ecology

As part of a large survey on reservoirs of Lassa fever in Guinea, three villages were investigated in high endemic zone, close to Sierra Leone border. Biodiversity of the small mammal community is presented in this study through a standardized trapping in houses, cultivations and forest. Identification of the small mammals was based on morphology and by molecular technique for sibling species. Of the 1123 specimens collected in 2003–2005, we identified seventeen species (thirteen Muridae, four Soricidae), leading to high diversity (Shannon index = 1.6–1.8) and high equitability (evenness index = 0.7–0.8) in cultivations and forest. In houses conversely, the rodent community was dominated by Mastomys natalensis (95–98%), leading to low diversity and equitability. Dynamics and reproduction were investigated in two species of pygmy mice, Mus mattheyi and Mus minutoides, two species of Praomys, P. daltoni and P. rostratus, and in Mastomys erythroleucus. The pygmy mice were abundant in cultivations in early rainy season, and reproduced from rainy to dry season. Praomys daltoni was also found more abundant in cultivations and seemed to reproduce between rainy and dry season, whereas P. rostratus preferred forest and cultivations in late rainy season, and reproduced throughout the year. Finally, M. erythroleucus was more abundant in forest in dry season, and seemed to reproduce from late rainy to dry season. This species had a low occurrence (6.5%) in the Faranah’s zone, and probably lived at its southern limit in Guinea. The presence of other Murinae, such as M. natalensis, Praomys spp as possible competitors in the same habitats, is discussed. For the first time, this study relates population biology of pygmy mice with molecular identification.     

Molecular and morphometric variation in two sibling species of the genus Praomys (Rodentia: Muridae): implications for biogeography

The rodent genus Praomys is widely distributed in the African tropics. The species are cryptic, rendering the species taxonomy unclear. There are differences of opinion concerning the specific status of Praomys misonnei and Praomys tullbergi, and their geographical distribution. We sequenced the cytochrome b and/or the 16S gene of 221 specimens from 12 countries in order to evaluate the genetic variability within these two species, and to precisely determine their geographical distribution. Morphological and morphometrical analyses on the sequenced specimens were also performed to find criteria useful for the identification of museum specimens. Our results confirm that P. misonnei and P. tullbergi are two valid species that can be separated by molecular data. However, no single discrete morphological character or simple metric measurement can be used to discriminate them. The percentage of misclassified individuals in multivariate discriminant analysis is relatively high (10%). The two species have allopatric distributions: P. tullbergi occurs in West Africa, from eastern Guinea to western Ghana, and P. misonnei is widely distributed from eastern Ghana to western Kenya. Within P. misonnei we identified three or four major geographical clades: a West Central African clade, an East African clade, a Nigerian clade, and a possible West African clade. Within P. misonnei, high geographical morphometrical variability was also identified. The role of both rivers and Pleistocene forest refugia in promoting speciation within the genus Praomys is discussed. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160 , 397–419.     

Daily rhythms of body temperature and heat production of sibling Mastomys species from different ecosystems--the response to photoperiod manipulations

We compared body temperature (T_b) and metabolic rates, measured as oxygen consumption (VO₂), daily rhythms of two sibling species of the genus Mastomys. We also studied their responses to long day (16L: 8D, LD) and short day (8L: 16D, SD) photoperiod manipulations at a constant ambient temperature of 26 + 1 °C. We noted significant differences in T_b and VO₂ daily rhythm patterns, under SD and LD-acclimation between the sibling species. These differences explain adaptation to the climatic conditions that prevail in the different ecosystems where these species live. To the best of our knowledge, this is the first time that physiological differences between the two siblings are measured by using chronobiological methods.     

Novel microsatellite markers for Dalechampia scandens (Euphorbiaceae) and closely related taxa: application to studying a species complex

We developed novel microsatellite markers for D alechampia scandens L. (Euphorbiaceae). The target plants belong to a distinct, but undescribed, species in the D . scandens species complex, characterized by small resin‐producing glands. In total, 110 alleles over 36 novel markers were identified across 39 individuals from three populations. The number of alleles varied from one to seven, with an average of 3.06 ± 0.26 alleles per locus. The developed markers, along with previously developed ones for a large‐glanded D . scandens species, were tested for amplification in 11 additional species of the genus D alechampia. Four markers did not produce any detectable allele in 37 individuals from two populations of the large‐glanded species. Average expected heterozygosity across all small‐ and large‐glanded specific loci was 0.36 and 0.15, for the small and large glanded populations, respectively. Cross‐species amplification showed that 89% of all markers were successfully amplified in at least one of the 11 other D alechampia species. These microsatellite markers may be useful for detecting undescribed species in the D . scandens species complex, and can be used for comparative analyses of genetic structure, mating system and phylogeography of other D alechampia species.     

Species Identification of Multimammate Rats of the Genus <Emphasis Type="Italic">Mastomys</Emphasis> (Rodentia: Muridae) in Eastern Africa Using PCR Typing of Cytochrome <Emphasis Type="Italic">b</Emphasis> Gene Fragments

The multimammate rats of the genus Mastomys are widespread throughout sub-Saharan Africa. They are the major agricultural pests and reservoirs of many infections dangerous to humans. A simple and accurate species identification of multimammate rats is crucial in ecological and epidemiological studies; however, it is complicated by the absence of pronounced morphological differentiation between Mastomys species. We describe a simple molecular assay based on PCR typing of the cytochrome b gene fragments as a method that allows fast genotyping of a large number of samples without sequencing to distinguish Mastomys species of East Africa.     

Isolation and characterization of microsatellite markers in Ijima's leaf warbler,Phylloscopus ijimae (Aves: Sylviidae)

Microsatellite markers were isolated from Ijima's leaf warbler, Phylloscopus ijimae. From an enriched genomic library and using the fast isolation by AFLP of sequences containing repeats (FIASCO) method, 10 polymorphic loci were obtained. Four to 12 alleles were identified in an analysis of 23 Ijima's leaf warblers, with the degree of heterozygosity ranging from 0.35 to 0.87. The markers were tested in four species of Phylloscopus and two other non‐Phylloscopus species of Sylviidae. Some loci were successfully amplified and showed polymorphism in Phylloscopus species, whereas amplification in the non‐Phylloscopus species was less successful.     

Is Oryza malampuzhaensis Krish. et Chand. (Poaceae) a valid species? Evidence from morphological and molecular analyses

Phylogenetic relationship between O. malampuzhaensis Krish. et Chand. (2n = 4x = 48; Poaceae, Oryzeae), a South Indian endemic wild rice with a disputed taxonomic identity, and eight other species belonging to the O. officinalis complex of the genus Oryza was examined using 62 morphological characters and 445 random amplified polymorphic DNA (RAPD) markers. Multivariate and cluster analyses using both the data sets clearly separated all accessions of O. malampuzhaensis into a distinct group. Genetic distances between O. malampuzhaensis and other species in O. officinalis complex were comparable with the distance between any other two taxa with species rank in this complex. Case-by-case taxonomic treatment of O. malampuzhaensis in relation to other species examined is presented. A taxonomic key for the discrimination of O. malampuzhaensis from other species in the O. officinalis complex has been constructed. Based on the present results, we strongly argue to restore the species rank to O. malampuzhaensis, as originally proposed by Krishnaswamy and Chandrasekharan (1958).     

Isolation and characterization of microsatellite markers from malaria vector,Anopheles culicifacies

Anopheles culicifacies, an important vector in the Indian subcontinent is a complex of five sibling species of which four are vectors. We describe the isolation of 31 microsatellite markers from the recently recognized isomorphic species A of which 13 were characterized in sympatric populations of Anopheles culicifacies isomorphic species A and B. The allele frequencies ranges from two to 12 in species A and two to seven in species B. Species A being a vector, and that these markers can be used in closely related species, makes the isolation of these markers important to study population structure of all sibling species in this complex.     

Microsatellite markers for the large blue butterflies Maculinea nausithous and Maculinea alcon (Lepidoptera: Lycaenidae) and their amplification in other Maculinea species

We developed microsatellite markers for Maculinea nausithous and Maculinea alcon, two of five species of endangered large blue butterflies found in Europe. Two separate microsatellite libraries were constructed. Eleven markers were developed for M. nausithous and one for M. alcon. The primers were tested on both species as well as on the three other European Maculinea species. The number of alleles per locus ranged from two to 14. These markers will be useful tools for population genetic studies of Maculinea species.     

Genetic Divergence Detected by ISSR Markers and Characterization of Microsatellite Regions in Mytilus Mussels

The wide distribution of microsatellites in mussels of the Mytilus edulis complex (Mytilidae) enables the analysis of inter-simple-sequence repeat (ISSR) markers. The aim of this investigation was to assess genetic differentiation in six sampling localities distributed along the European Atlantic coast to expose the potential of these markers in genetic studies requiring the detection of low polymorphism and as a source of sequences for developing microsatellite markers. We detected low genetic structuring within each member of the Mytilus edulis complex. Nei and Li distances and AMOVA clustered the individuals studied into two groups. On the basis of these results two sampling localities coming from the M. edulis × M. galloprovincialis mosaic hybrid zone in Western Europe were assigned to one species. On the other hand, mussels of a sampling locality in the Baltic Sea were not significantly different from a pure M. edulis locality supporting an extensive introgression of M. edulis in these individuals. Finally, 148 microsatellites were found in the sequences of 51 ISSR markers, and two polymorphic microsatellite markers were developed.     

Two new species in the Chaetoceros socialis complex (Bacillariophyta): C. sporotruncatus and C. dichatoensis,and characterization of its relatives,C. radicans and C. cinctus

The diatom genus Chaetoceros is one of the most abundant and diverse phytoplankton in marine and brackish waters worldwide. Within this genus, Chaetoceros socialis has been cited as one of the most common species. However, recent studies from different geographic areas have shown the presence of pseudo‐cryptic diversity within the C. socialis complex. Members of this complex are characterized by curved chains (primary colonies) aggregating into globular clusters, where one of the four setae of each cell curves toward the center of the cluster and the other three orient outwards. New light and electron microscopy observations as well as molecular data on marine planktonic diatoms from the coastal waters off Chile revealed the presence of two new species, Chaetoceros sporotruncatus sp. nov. and C. dichatoensis. sp. nov. belonging to the C. socialis complex. The two new species are similar to other members of the complex (i.e., C. socialis and C. gelidus) in the primary and secondary structure of the colony, the orientation pattern of the setae, and the valve ultrastructure. The only morphological characters that can be used to differentiate the species of this complex are aspects related to resting spore morphology. The two newly described species are closely related to each other and form a sister clade to C. gelidus in molecular phylogenies. We also provide a phylogenetic status along with the morphological characterization of C. radicans and C. cintus, which are genetically related to the C. socialis complex.     

Miocene divergence,phenotypically cryptic lineages,and contrasting distribution patterns in common lichen‐forming fungi (Ascomycota: Parmeliaceae)

Despite the recent advancements in recognizing diversity in lichen‐forming fungi, assessing the timing of diversification remains largely unexplored in these important fungal symbionts. To better understand the evolutionary processes driving diversification in common lichen‐forming fungi, we investigated the phylogeny and biogeography of the broadly distributed Melanelixia fuliginosa/M. glabratula group, using molecular data from six nuclear markers. Phylogenetic analyses of individual gene alignments and combined data provide strong evidence for five species‐level lineages within this species complex. Three of these lineages correspond to the previously described species M. fuliginosa, M. glabratula, and M. subaurifera. The remaining two lineages, ‘M. sp. 1’ and ‘M. sp. 2’, merit species recognition based on genealogical concordance. Both M. glabratula and M. subaurifera had broad intercontinental distributions, sharing identical haplotypes among intercontinental populations. Based on the current sampling, M. fuliginosa s.s. was represented exclusively by European material and was not collected in North America. ‘M. sp. 1’ was represented by collections from Scotland and Spain; and ‘M. sp. 2’ was represented by collections in California, USA. Environmental factors driving the contrasting distribution patterns in this group remain unknown. Divergence times estimated using a coalescence‐based multilocus species‐tree approach suggest that diversification within the M. fuliginosa/M. glabratula group occurred exclusively during the Miocene. The results of the present study indicate that phenotypically cryptic lichen‐forming fungal species‐level lineages may be relatively ancient and do not necessarily reflect recent divergence events. Furthermore, diagnosable phenotypic differences may be absent even millions of years after the initial divergence. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●●, ●●–●●.     

Molecular identification of four cryptic species of Mastomys (Rodentia,Murinae)

Multimammate rats (genus Mastomys) are abundant in many regions throughout sub-Saharan Africa, and are of high economical and sanitary importance as agricultural pests as well as reservoir/vectors of human diseases. In pest management and in epidemiological studies, unequivocal species identification of individuals collected in the field is crucial. However, the discrimination among most of the Mastomys species is often difficult, if not impossible, on the basis of external characters. Karyology provides unambiguous specific assignations, but is not suitable for population studies involving large numbers of individuals because it requires fresh material and/or quick transfer from the field to the laboratory. The purpose of this study was to search for molecular markers allowing a clear discrimination of field collected individuals on the basis of ethanol-preserved samples. Using sequences of the cytochrome b region of mitochondrial DNA, two molecular tests based on species-specific primers (test 1) and restriction sites generating species-specific profiles (test 2), were designed and evaluated for species identification on a large number of karyotypically or electrophoretically unambiguously determined individuals. The tests clearly discriminate the four most widespread species. They are easy to perform on a small piece of ear or tail taken from live animals, and can probably be adapted to identify museum specimens.     

Two highly validated multiplexes (12-plex and 8-plex) for species delimitation and parentage analysis in oaks (Quercus spp.)

Multiplex PCR is a fast and cost‐effective technique allowing increased genotyping throughput of microsatellites. We developed two multiplexes for Quercus petraea and Q. robur, a 12‐plex of EST‐SSRs (eSSRs) and an 8‐plex of genomic SSRs (gSSRs). We studied the origin of allele calling errors at the human reader and software levels. We showed that the robustness of allele identification can be improved by binning on raw peak sizes prior to genetic data analysis. We checked through simulation the power of these markers for species delimitation and hybrid detection. The resolution achieved with all 20 markers was greatly improved compared to that of previous studies based on a subset of the markers. Preliminary PCR tests suggest that these multiplexes might be useful to study other oak species as well. The strategy used for multiplex microsatellite development (from PCR conditions to the definition of allele calling rules) should be broadly applicable.     

Polymorphic microsatellite markers for Mexican salamanders of the genus Ambystoma

We screened a partial genomic library enriched for microsatellites and characterized nine loci for the Mexican species of Ambystoma for studies of population structure. We tested marker variability in two metamorphic (A. granulosum, A. altamirani) and two paedomorphic (A. andersoni, A. mexicanum) species of the A. tigrinum complex. Our microsatellites were developed from pooled genomic DNA from three species, and may work on all species in the A. tigrinum complex in Mexico. These markers will be important for studies of conservation genetics in this radiation.