RAPD-PCR molecular analysis of the threatened Cabrera’s vole populations in the Iberian Peninsula

Optimal management and conservation programs of the threatened Cabrera’s vole require investigating potential molecular genetic markers in the genomic background, if the few remaining fragile populations are to be conserved. A collection of 30 Cabrera’s vole representing four populations in Spain and Portugal was characterized by 134 RAPD-PCR markers. Molecular analyses did not detect low level of the genetic diversity or population bottlenecks in all studied populations, in discordance with the expectation of low level of viability of the Cabrera’s vole. The results described Cabrera’s vole populations as a single genetic unit with slightly restricted gene flow. Phylogenetic reconstruction suggested genetic differentiation between Northern and Southern Cabrera’s vole populations, with the basal branches formed by the southern populations, which may be an evidence of the southern origin of Iberian vole ancestral population. To our knowledge, this is the first study on the genetic diversity of Microtus cabrerae, which may have further application for the conservation programs of this threatened species of Iberian vole.Keywords: Microtus cabrerae, RAPD-PCR, Spain, Portugal, gene flow, genetic diversity, bottleneck, conservationAfrican Journal of Biotechnology Vol. 12(26), pp. 4065-407

Molecular-genetic approaches to species identification of platyhelminthes of the genus <i>Ligophorus</i> (Monogenea) parasitising flathead mullet

Mugil cephalus L., 1758 (flathead mullet) is a valuable commercial fish and a promising object of artificial breeding in the Black Sea and the Sea of Azov, and the study of its parasite fauna is important for fishery and mariculture. Monogeneans of the genus Ligophorus are common ectoparasites dwelling on the gills of mullets. Two representatives of this genus parasitise flathead mullet in the Azov-Black Sea region, namely Ligophorus mediterraneus Sarabeev, Balbuena et Euzet, 2005 and Ligophorus cephali Rubtsova, Balbuena, Sarabeev, Blasco-Costa et Euzet, 2006. Morphological identification of these species requires spending much time and a high level of experience in monogenean taxonomy. For quick and correct species identification of these parasites, we have developed a genotyping approach based on the polymerase chain reaction of allele-specific gene sites for various Monogenea species. A fragment of the 28S ribosomal gene, which includes conserved and variable sites, was chosen as a genetic marker. Three approaches were used as follows: amplified fragment length analysis, allelespecific PCR with endpoint detection and allele-specific real-time PCR using SYBR Green intercalating dye. The first approach was by obtaining PCR products of different lengths that were specific either to L. mediterraneus or to L. cephali. This approach was implemented due to the presence of several variable sites located at a distance from each other. The PCR mixture contained three primers: one forward and two reverse. The forward primer was complementary to the conserved site, which did not differ between species. Reverse primers were speciesspecific and, for each species, they were complementary to different DNA regions located 100 bp apart. As a result, L. mediterraneus was characterized by shorter amplicons than L. cephali. For the second and third approaches, a pair of primers was designed according to the following principle: the forward primer was complementary to both species, since it was selected for the conserved gene region. Reverse primers were species-specific and were designed for the 28S variable region. The two parasite species were distinguished by three-point mutations. Thus, one pair of primers was complementary to L. mediterraneus, the other, to L. cephali. The amplified fragment length analysis and the allele-specific real-time PCR demonstrated 100 % coincidence of genotyping results compared with Sanger sequencing. The developed genotyping protocols can be used not only to distinguish two species of Ligophorus from flathead mullet in ecological studies and veterinary practice but also for further development of similar approaches for other monogeneans, among which there are many pathogenic species

Comparative phylogeography of four Apodemus species (Mammalia: Rodentia) in the Asian Far East: evidence of Quaternary climatic changes in their genetic structure

The phylogeography of four Apodemus species (Apodemus agrarius, Apodemus peninsulae, Apodemus latronum, and Apodemus draco) was studied in the Far East of Asia, based on sequences of the mitochondrial DNA cytochrome b gene. The results obtained show the existence of many different genetic lineages within the studied Apodemus species, suggesting the isolation and differentiation of populations in multiple refuge areas. Higher genetic diversities in some regions such as Yunnan, Sichuan (China), and eastern Russia suggest these areas are potential refuges for these species. The existence of such complex genetic structures could be linked to the presence of many biogeographic barriers (Himalaya Mountains, Tien-shan Mountains, Altai Mountains, Tibetan Plateau, Gobi desert, Yunnan Guizhou Plateau, Dzungaria basin, and others) in these regions, which were probably reinforced during the Quaternary climate changes. These barriers also played an important role concerning the low dispersal abilities of the two studied Apodemus species adapted to forest habitats (A. latronum and A. draco) with respect to colonizing regions other than China. © 2010 The Linnean Society of London

Restoration of the Genus <i>Paraunisaccoides</i> Martin, 1973 (Digenea: Haploporidae) and Description of <i>P. elegans</i> n. sp. and <i>Unisaccus halongi</i> n. sp. from Mugilid Fish in Vietnam

We restore the genus Paraunisaccoides (Haploporidae), synonymised earlier with the genus Skrjabinolecithum. Adult worms, detected in Vietnamese mullet fish, were highly similar to trematodes described as P. lobolecithum via digestive and genital system structures and relative organ arrangement. Differences are expressed as absence and presence of pads on the hermaphrodite duct, respectively, and the disjunction of some metric parameter values, namely body, ovary and eggs. Ribosomal DNA sequences, based on the phylogenetic analysis of Haploporidae, indicates that new worms represent a sister clade to Unisaccus tonkini. Genetic divergence between new worms and Skrjabinolecithum species can be interpreted as intergeneric. Based on morphological and molecular data, we recognise Paraunisaccoides as a valid genus within Waretrematinae and worms from Vietnam as a new species of this genus, P. elegans n. sp. Other worms detected in Vietnamese mugilids are morphologically similar to representatives of Paraunisaccoides и Skrjabinolecithum. However, molecular-based phylogenetic analysis showed that these trematodes are closely related to Unisaccus tonkini; the genetic divergence between them is at the interspecific level, despite considerable differences in vitellarium structure as intergeneric character. Accepting the priority of molecular results, we include these new worms into the genus Unisaccus as new species, Unisaccus halongi n. sp