Geometric morphometric analysis of shape variation in the cone scales of two closely related larch species, Larix dahurica Laws. (=Larix gmelinii (Rupr.) Rupr) and L. cajanderi Mayr, was carried out. The data on the taxonomy and distribution of L. dahurica and L. cajanderi are contradictory. The taxonomic status of L. cajanderi has been confirmed by the genetic and morphological studies performed in Russia and based on considerable evidence, but the species has not been recognized internationally, being considered as a synonym of Larix gmelinii var. gmelinii. In the systematics of larch, morphological characters of the generative organs are mainly used as diagnostic markers, among the most important being the shape variation of the cone scales. The aim of this study was to test geometric morphometrics as a tool for analyzing differentiation of L. dahurica and L. cajanderi in the shape of their cone scales. Characterization of shape variations in cone scales using geometric morphometric methods consists in digitizing points along an outline of scales followed by analysis of partial warps, describing individual differences in coordinates of the outline points. We studied the populations of L. dahurica from Evenkia and the Trans-Baikal region and six L. cajanderi populations from Yakutia and Magadan Oblast. In each population, we analyzed samples of 100-150 cones collected from 20-30 trees. Scales taken from the middle part of the cones were scanned using an Epson Perfection V500 Photo. On the scanned images, outline points were placed with a TPSDig program (Rolf, 2010), using angular algorithm (Oreshkova et al., 2015). The data were processed and analyzed using Integrated Morphometrics Programs (IMP) software (http://www.canisius.edu/~sheets/ morphsoft.html, Sheets, 2001), following the guidelines on geometric morphometrics in biology (Pavlinov, Mikeshina, 2002; Zelditch et al., 2004). Initial coordinates of the scale landmarks were aligned with the mean structure for L. dahurica and L. cajanderi cone scales using Procrustes superimposition in the CoordGen6 program. PCA based on covariances of partial warp scores was applied to reveal directions of variation in the shape of the cone scales. The relative deformations of the cone scales (PCA scores) were used as shape variables for statistical comparisons of these two larch species with canonical discriminant analysis. Morphotypes of the cone scales were distinguished in L. dahurica populations by pairwise comparison of samples from trees in the TwoGroup6h program using Bootstrap resampling-based Goodall’s F-test (Sheets, 2001). Samples from the trees in which the cone scales differed significantly (p < 0.01) were considered to belong to different morphotypes. Morphotypes distinguished in L. dahurica populations were compared with the morphotypes that we had previously determined in L. cajanderi populations. The composition and the frequency of occurrence of morphotypes were used to determine phenotypic distances between populations (Zhivotovskii, 1991). Multidimensional scaling matrix of the phenotypic distances was applied for ordination of larch populations. In this research, we revealed differentiation of L. dahurica and L. cajanderi using geometric morphometric analysis of the shape variation of cone scales. The results of PCA of partial warp scores exposed four principal components, which account for 90% of total explained variance in the shape of the cone scales in the two larch species. Graphical representations of these shape transformations in the vector form characterized directions of shape variability in scales corresponding to the maximum and minimum values of four principal components (See Fig. 2). PCA-ordination of the larch populations revealed some difference in the shape variation of the cone scales in L. dahurica and L. cajanderi (See Fig. 3). The results of canonical discriminant analysis of relative deformations of scales showed differentiation of the populations of the two larch species (See Fig. 4). Eleven morphotypes were identified in L. dahurica cones from Evenkia and nine morphotypes in the Ingoda population, three of the morphotypes being common for both populations (See Fig. 5). The shape of L. dahurica cone scales varied from spatulate to oval and their apical margins from weakly sinuate to distinctly sinuate. The Trans-Baikal population was dominated by scales with obtuse (truncate) and rounded apexes. The obtained morphotypes were compared with 25 cone scale morphotypes previously distinguished in the Yakut and the Magadan L. cajanderi populations (See Fig. 3). Four similar morphotypes of cone scales were revealed in the North-Yeniseisk population of L. dahurica and the Yakut populations of L. cajanderi. The differences between them in the populations of the two larch species were nonsignificant (p > 0.01). All morphotypes of cone scales from the Ingoda population of L. dahurica differed significantly from L. cajanderi cone scale morphotypes. The results of multidimensional scaling phenotypic distance matrix calculated based on the similarity of morphotypes of L. dahurica and L. cajanderi populations were consistent with the results of their differentiation based on relative deformations of scales obtained using canonical discriminant analysis (See Fig. 4 and Fig. 7). In spite of the differences in the shape of the cone scales between the North-Yeniseisk and the Trans-Baikal populations of L. dahurica, they both differed from L. cajanderi populations. Thus, phenotypic analysis confirmed differentiation of these two larch species. Despite the similarities between a number of morphotypes, the Yakut L. cajanderi populations were differentiated from L. dahurica populations. Significant differences were noted between intraspecific groups: between L. cajanderi populations from Okhotsk-Kolyma Upland and Yakutia and between L. dahurica populations from Evenkia and the Trans-Baikal region (See Fig. 4). The similarities between species and intraspecific differences may be attributed to the ongoing processes of hybridization and species formation in the region where the ranges of the larches overlap with the ranges of L. czekanowskii Szafer and L. dahurica×L. cajanderi hybrids. Geometric morphometrics can be used as an effective tool for analyzing differentiation of L. dahurica and L. cajanderi in the shape of their cone scales.
Novel neuroanatomical integration and scaling define avian brain shape evolution and development
