Nor was it only from the Medicean faction that the Duke's tranquillity was threatened. Whilst his fortunes were yet in suspense, he was warned by Castiglione, then diplomatic resident at Rome for his brother-in-law the Duke of Mantua, that Ascanio Colonna was agitating certain vague pretensions on the duchy of Urbino, through his mother Agnesina di Montefeltro. The nature of these claims, which were from time to time revived, is not very intelligible. All authorities make Giovanna, wife of the Prefect, older than Agnesina, wife of Fabrizio Colonna, both being daughters of Duke Federigo. Thus, even supposing Francesco Maria's title irretrievably annulled, by the deprivations he had successively sustained from Julius II. and Leo X., if the old investitures did confer any rights upon females, his nephew Sigismondo Varana, grandson of Giovanna, would have excluded the Colonna. Ascanio's intrigues were, however, neutralised by the dexterity of Castiglione, and the influence of the Duke of Mantua, until Francesco Maria's cordial reconciliation with the Church and the Emperor had rendered his position secure.[293] Even the Medici thereupon refused to promote the pretender's views, and his only adherent was Gian Maria Varana, who, having within a few weeks succeeded in recovering possession of Camerino, sought so to occupy the Duke of Urbino as to prevent his espousing the cause of Sigismondo, its rightful lord. The latter also looked for support to his wife's uncle, Cardinal Prospero Colonna, whilst the interests of his competitor were backed by Cardinal Innocenzo Cibò, his brother-in-law. But ere these respective claims could be tested, they were sadly set at rest by the death of "poor dear but ill-starred Sigismondo," as he is called by Castiglione, who was set upon and slain on the 24th of June by a band of assassins, whilst riding with five attendants near La Storta. This foul deed, in accordance with the wild habits of that age, and the fratricidal tendencies of the Varana family, was imputed to Ascanio Colonna at the instigation of Giovanni Maria, uncle of the victim. [Footnote 293: However these pretensions may have originated, they derived a _quasi_ warrant in 1525, from a conditional investiture of the duchy for three generations, granted by Clement VII. to Ascanio "in case it should happen to lapse to the Holy See," Agnesina being there mentioned as eldest sister. Charles V. was vainly solicited by Ascanio to render this condition eventual, or by some other means to make good his possession, and the claim did not drop until 1530. Nor was it the only one vamped up on account of Duke Guidobaldo's unfruitful marriage. In 1505 the Prince of Salerno seems to have made similar pretensions through his mother, a sister still younger than Agnesina; and in order to dispose of these, Julius II. is said to have offered him his own daughter Felice, a union which however did not take place.]