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Fluorochrome staining with chromomycin A 3 (CMA) was used to characterize and compare the CMA banding patterns of chromosomes in 14 accessions of 12 species of major Citrus species. All accessions had 2n = 18 chromosomes. These chromosomes were classified into seven types based on the number and position of CMApositive bands: A: two telomeric and one proximal band, B: one telomeric and one proximal band, C: two telomeric bands, D: one telomeric band, E: without bands, F: one proximal band, and Dst: type D with a satellite chromosome. Each accession possessed two to six types of chromosomes and unique CMA banding patterns. The CMA banding patterns were 2B + 8D + 8E in C. medica,micrantha, 2B + 1C + 11D + 3E + 1F in C. macroptera, and 3B + 1C + 8D + 3E + 2F + 1Dst in C. hystrix.

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Fluorochrome staining with chromomyscin A 3 (CMA) was used to characterize and compare the CMA banding patterns of chromosomes of 17 species from 13 genera of Aurantioideae, which is one of the seven subfamilies of Rutaceae. All species used in this study had 2n = 18 chromosomes. These chromosomes were classified into five types based on the number and position of CMA-positive bands; B: one telomeric and one proximal band, C: two telomeric bands, D: one telomeric band, E: without bands and Dst: type D with a satellite chromosome. Each species possessed two or four types of chromosomes and unique CMA banding patterns. The CMA banding patterns were 6D + 12E in Clausena anisata (Willd.) Hook. f.,1C + 3D + 14E in Murraya koenigii (L.) Spreng., 1C + 11D + 6E in Murraya paniculata (L.) Jack, 1B + 1C + 10D + 6E in Merrillia caloxylon (Ridl.) Swing., 1C + 9D + 7E + 1Dst in Paramignya lobata Burkill, 2C + 5D + 11E in Severinia buxifolia (Poir.) Tenore, 5D + 13E in Hesperethusa crenulata (Roxb.) Roem., 16D + 2E in Citropsis gabunensis (Engl.) Swing. et M. Kell, 14D + 4E in Citropsis schweinfurthii (Engl.) Swing. et M. Kell, 8D + 10Ein Atalantia monophylla DC., 1C + 3D + 14E in Atalantia ceylanica (Arn.) Oliv., 2D + 16E in Atalantia roxburghiana Hook. f., 18E in Swinglea glutinosa (Blanco) Merr., 2D + 16E in Aegle marmelos (L.) Corr., 4D + 14E in Afraegle paniculata (Schum.) Engl., 2B + 2D + 13E + 2Dst in Feronia limonia (L.) Swing. and 3B + 9D + 6E in Feroniella oblata Swing. The advanced genera in each tribe and subtribe such as Merrillia and Feroniella showed heterochromatin (CMA-positive bands) rich chromosome configuration in general. Swinglea glutinosa without heterochromatin rich chromosome, which is considered to be the most primitive chromosome configuration, seems to be the key genus for clarifying the evolution of the tribe Citreae.

Highlights• This is the first report of genetic diversity of the few remaining populations of C. sativa in the southern limits of its distribution in Eurasia.• Low genetic diversity and high genetic differentiation among small isolated populations of C. sativa with low geographical distance represent serious genetic erosion indicators in the Hyrcanian forest, even hinting at an ongoing extinction vortex. AbstractCastanea sativa Mill. is one of the most endangered tree species in Iran where it is represented by small fragmented populations in the north of the country. 18 simple sequence repeat (SSR) loci (10 nuclear and 8 chloroplastic) were used to evaluate the genetic diversity and population structure of C. sativa from the Hyrcanian forest. For nuclear SSR, the number of alleles detected per locus ranged from 1 to 5 and observed heterozygosity (H O ) was between 0.125 and 1.000. Analysis of molecular variance (AMOVA) indicated a high level of variation within populations (84%) and low levels between populations (16%). Based on structure analysis, the four studied populations were divided into two main clusters that have genetic distance F st = 0.3. The Shafaroud population was separated in the first cluster, Siyahmazgi, Qalehroudkhan and Veysroud were placed in the second cluster. The UPGMA analysis confirmed the results of Structure analysis, separating the Shafaroud population from the others. The 8 chloroplast SSR loci used to screen the populations showed no polymorphism. In General, low nuclear genetic diversity, no polymorphism in cpDNA and considerable genetic differentiation among populations in short geographical distance represent a serious genetic erosion threat for C. sativa in the Hyrcanian forest, even hinting at an ongoing extinction vortex. Therefore, due to significant decline in genetic diversity, it is essential to introduce constraints protection upon the areas of distribution of all four populations of this species in Iran.