Genetic diversity and evolutionary history of the Tyrrhenian treefrog Hyla sarda (Anura: Hylidae): adding pieces to the puzzle of Corsica–Sardinia biota

The Corsica–Sardinia archipelago is a hotspot of Mediterranean biodiversity. Although tempo and mode of arrival of species to this archipelago and phylogenetic relationships with continental species have been investigated in many taxa, very little is known about the current genetic structure and evolutionary history subsequent to arrival. In the present study, we investigated genetic variation within and among populations of the Tyrrhenian treefrog Hyla sarda, a species endemic to the Corsica–Sardinia microplate and the surrounding islands, by means of allozyme electrophoresis. Low genetic divergence (mean D_nei = 0.01) and no appreciable differences in the levels and distribution of genetic variability (H_E: 0.06–0.09) were observed among all but one populations (Elba). Historical demographic and isolation‐by‐distance analyses indicated that this diffused genetic homogeneity could be the result of recent demographic expansion. Along with paleoenvironmental data, such expansion could have occurred during the last glacial phase, when wide and persistent land bridges connected the main islands and a widening of lowland areas occurred. This scenario is unprecedented among Corsica–Sardinia species. Together with the lack of concordance even among the few previously studied species, this suggests either that species had largely independent responses to paleoenvironmental changes, or that most of the history of assembly of the Corsica–Sardinia biota is yet to be written. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 159–167.     

On glacial refugia,genetic diversity,and microevolutionary processes: deep phylogeographical structure in the endemic newt Lissotriton italicus1

We investigated the phylogeographical patterns of Lissotriton italcus, a newt endemic to the Italian peninsula, aiming to determine why hotspots of intraspecific diversity so ‘hot’. We found two main mitochindrial DNA lineages (net sequence divergence of 6.8% at two fragments of total length of 1897 bp): one restricted to part of the Calabrian peninsula (i.e the southernmost portion of the species range) and the other widespread throughout the rest of the species range. Both lineages, which had a parapatric distribution, showed evidence of further subdivisions, with an overall number of eight terminal haplogroups, most of whose times to the most recent common ancestors were estimated at the Late Pleistocene. Analysis of molecular variance suggested that partitioning populations according to the geographical distribution of these haplogroups can explain 97% of the observed genetic variation. These results suggest that L. italicus underwent repeated cycles of allopatric fragmentation throughout the Pleistocene, and that it likely survived the Late Pleistocene paleoenvironmental changes within eight separate refugia. Thus, the current hotspot of intraspecific diversity of L. italicus (within the Calabrian peninsula) has not been moulded by long‐term stability of large populations but rather by multiple events of allopatric fragmentation and divergence. When compared with the patterns recently identified in other species, these results suggest that the occurrence of phases of allopatric divergence (eventually followed by secondary admixture) could be a common, albeit probably underrated feature in the history of formation of hotspots of intraspecific diversity. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 42–55.     

Revision and cladistic analysis of Isoctenus and description of a new neotropical genus (Araneae,Ctenidae, Cteninae)

A cladistic analysis was applied to test the monophyly of the genus Isoctenus. The data matrix comprised 28 taxa scored for 53 morphological and two behavioural characters. The analysis resulted in two equally parsimonious trees of 89 steps. The strict consensus was used to discuss the relationships of Isoctenus and related Cteninae genera. Ctenopsis Schmidt is synonymized with Isoctenus. Isoctenus foliifer Bertkau, I. strandi Mello‐Leitão, I. eupalaestrus Mello‐Leitão, I. janeirus (Walckenaer), I. coxalis (Pickard‐Cambridge), I. corymbus Polotow, Brescovit & Pellegatti‐Franco and I. malabaris Polotow, Brescovit & Ott are maintained in Isoctenus. Four species currently included in Ctenus are transferred to Isoctenus: I. griseolus (Mello‐Leitão) comb. nov., I. taperae (Mello‐Leitão) comb. nov., I. herteli (Mello‐Leitão) comb. nov. and I. minusculus (Keyserling) comb. nov. The following specific names are synonymized: Ctenus sanguineus Walckenaer, C. semiornatus Mello‐Leitão and Ctenopsis stellata Schmidt with Isoctenus janeirus (Walckenaer), Ctenus mourei Mello‐Leitão with Isoctenus herteli (Mello‐Leitão) and Ctenus pauper Mello‐Leitão with Isoctenus strandi Mello‐Leitão. Isoctenus sigma Schenkel, described from French Guiana, is transferred to Ctenus. Four species are newly described: Isoctenus areia sp. nov. from Paraíba, Brazil, I. charada sp. nov. and I. segredo sp. nov. from Paraná, Brazil, and I. ordinario sp. nov. from south and south‐eastern Brazil and north‐eastern Argentina. Isoctenus latevittatus Caporiacco is considered species inquirenda. Parabatinga gen. nov. is proposed to include Ctenus brevipes Keyserling. The following synonymies are established: Ctenus taeniatus Keyserling, C. tatarandensis Tullgren, C. anisitsi Strand, C. atrivulvus Strand, C. mentor Strand, C. brevipes brevilabris Strand, Isoctenus masculus Mello‐Leitão and Ctenus birabeni Mello‐Leitão with Parabatinga brevipes (Keyserling) comb. nov. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 583–614.     

OSTEOBLASTIC CELLS FROM RAT LONG BONE II: ADHESION TO SUBSTRATA AND INTEGRIN EXPRESSION IN PRIMARY AND PROPAGATED CULTURES

Propagation in vitro of rat tibial osteoblasts (ROB) is accompanied by increased expression of the early osteogenic marker alkaline phosphatase (AP) and maturation of the osteogenic phenotype. In order to establish the pattern of the integrin expressed in ROB during progression to the mature osteoblastic phenotype, we have used biosynthetic, immunoblotting and immuno-histochemical assays. We immunoprecipitated from osteoblasts, expanded for 1.5- and 7.5-doubling, α5β1, αvβ3, α3β1, α6β1 and α1β1 integrin heterodimers; furthermore β5, α2 and α4 chains were detected by immunoblots and indirect immunofluorescence. αv, α1, α6 subunits in most cells, and β3 and β1 subunits in a minority, were found to be associated with adhesion plaques in osteoblasts of 1.5-, 4.5- and 7.5-doubling grown in the presence of FCS, while all other subunits stained diffusely all the cells. Adhesion to fibronectin (FN), laminin (LN), collagen type I (COL I) and III (COL III) by ROB at different doubling (1.5–11) was dependent on substratum concentration, and after 2.5h at 55nm 60% of the cells adhered to all substrata. Arg-Gly-Asp-Ser (RGDS) containing peptides inhibited adhesion of cells differentially, according to substratum; no dependence on extent of progation in vitro was observed. In conclusion, ROB cultured in vitro for 1.5- to 11-doubling had an unchanged pattern of expression of integrin subunits, heterodimer association and cellular distribution. Adhesion specificity and affinity were also unchanged. These results suggest that the phenotypic maturation, detected as an increase in AP expression, is not accompanied by major changes in the potential for cell—matrix interactions, and does not correspond to changes in the type of integrin subunits expressed by osteoblasts.     

Molecular evidence for allopolyploid speciation and a single origin of the western Mediterranean orchid Dactylorhiza insularis (Orchidaceae)

The hybrid origin of the western Mediterranean orchid Dactylorhiza insularis was demonstrated by genetic markers. Allozyme data showed that throughout its range D. insularis has an allotriploid constitution and reproduces apomictically. The parental species of D. insularis were identified as D. romana andD. sambucina; they contributed 2 alleles and 1 allele, respectively, at the allozyme loci studied. The maternal species of D. insularis was D. romana , as inferred from cpDNA ( trn L(UAA) intron). High genetic similarities were found when comparing present populations of D. romana and D. sambucina with their respective genomes 'frozen' in D. insularis. Dactylorhiza insularis showed fixed (or nearly fixed) heterozygosity at 11 out of the 19 loci studied, and poor genetic variation: eight multilocus genotypes were detected at allozyme level. No multilocus genotype differs from the most similar one by more than one allele substitution. All D. insularis individuals showed the same cpDNA haplotype (I) , regardless of their geographic origin and multilocus genotype. The I haplotype is similar, but not identical to that found in D. romana (R). No recurrent formation of D. insularis was observed in hybrid zones between D. romana and D. sambucina , where diploid sexual hybrids (F_1; F_n, backcrosses) were detected. Available data agree with a single origin for D. insularis , which possibly occurred in the present postglacial, when D. romana and D. sambucina , expanding from their glacial refugia, came into contact. The genetic homogeneity found between D. romana and D. markusii , both from their locus classicus , indicates that the latter is a junior synonym of D. romana; on the other hand, D. romana and D. sambucina are well differentiated species ( D_Nei = 0.59).     

Genetic variation and its evolutionary implications in a Mediterranean island endemic lizard

The peculiar bioclimatic and geographic features of Corso–Sardinian islands may provide an ideal scenario for investigating microevolutionary processes, given their large heterogeneity of environments, which could affect dispersal and gene flow among populations, as well as processes of local adaptation. The genetic variation and differentiation among populations of the endemic lizard Archaeolacerta bedriagae were studied by allozyme electrophoresis at 20 presumptive loci. The genetic structure of this species is characterized by relatively high levels of polymorphism and low differentiation among populations. The pattern of genetic differentiation cannot be explained by genetic drift as a function of geographic distance. Genetic distance data show that genetic variation is distributed into three geographically coherent population groups and suggest a recent (Late Pleistocene) origin for the observed geographic fragmentation. The analysis of environmental correlates of allozymic variation indicates a strong correlation of the Idh-1 locus with climatic variables. The frequency of the Idh-1¹⁰⁶ allele is negatively correlated with annual temperature, and positively correlated with annual precipitation. In addition, the observed heterozygosity at this locus decreases towards more arid climatic regimes. The results obtained support the assumption of differential selection acting on Idh-1 allozymes under diverse climates. An association between Idh-1 allozymes and local bioclimatic regimes was also observed for the sympatric lizard Podarcis tiliguerta , suggesting a key role for such selective agents on Idh-1 polymorphism in these two Corso–Sardinian lacertids.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 661–676.