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Background  Satellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids ( Paphiopedilum  subgenus  Parvisepalum  and representative species in the other subgenera/sections across the genus). A new satellite repeat in  Paphiopedilum  subgenus  Parvisepalum , SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from  P. armeniacum  (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA.    Results  SatA comprises c. 14.5% of the  P. armeniacum  genome and is specific to subgenus  Parvisepalum . It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin-loop motifs. A new karyotype of  P. vietnamense  (2n = 28) is presented and shows that the chromosome number in subgenus  Parvisepalum  is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven  Paphiopedilum  species of subgenus  Parvisepalum  (2n = 26–28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific.    Conclusions  We conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to  Paphiopedilum  subgenus  Parvisepalum . SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus  Parvisepalum  and indeed of all  Paphiopedilum  could be either 2n = 26 or 28, if  P. vietnamense  is sister to all species in the subgenus as suggested by the ITS data.    Electronic supplementary material  The online version of this article (10.1186/s12864-018-4956-7) contains supplementary material, which is available to authorized users.

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization