| Summary: | In this work, the seed morpho-colorimetric differentiation of the Sardinian endemic species of <i>Anchusa</i> (Boraginaceae) was evaluated. In Sardinia, the <i>Anchusa</i> genus includes the following seven taxa: <i>A. capellii</i>, <i>A. crispa</i> ssp. <i>crispa</i>, <i>A. crispa</i> ssp. <i>maritima</i>, <i>A. formosa</i>, <i>A. littorea</i>, <i>A. montelinasana</i>, and <i>A. sardoa</i>. Seed images were acquired using a flatbed scanner and analyzed using the free software package ImageJ. A total of 74 seed morpho-colorimetric features of 2692 seed lots of seven taxa of <i>Anchusa</i> belonging to 17 populations were extrapolated and used to build a database of seed size, shape, and color features. The data were statistically elaborated by the stepwise linear discriminant analysis (LDA) to compare and discriminate each accession and taxon. In addition, the seed morpho-colorimetric differences among coastal and mountainous taxa were evaluated. Considering the ecological conditions, the LDA was able to discriminate among the <i>Anchusa</i> taxa with a correct identification of 87.4% and 90.8% of specimens for mountainous and coastal plants, respectively. Moreover, the LDA of the 17 populations of <i>Anchusa</i> showed a low separation among species and populations within the coastal group, highlighting how the long-distance dispersal by flotation on the sea water surface and the pollination network may influence the similarity patterns observed. In addition, a misattribution was observed for <i>A. crispa</i> ssp. <i>crispa</i>, which was misclassified as <i>A. crispa</i> ssp. <i>maritima</i> in 14.1% of cases, while <i>A. crispa</i> ssp. <i>maritima</i> was misidentified as <i>A. crispa</i> ssp. <i>crispa</i> in 21.1% of cases, highlighting a close phenotypic relationship between these two taxa. The statistical results obtained through the seed image analysis showed that the morpho-colorimetric features of the seeds provide important information about the adaptation and evolution of <i>Anchusa</i> taxa in Sardinia.
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