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Plastid genomes (plastomes) have long been recognized as highly conserved in their overall structure, size, gene arrangement and content among land plants. However, recent studies have shown that some lineages present unusual variations in some of these features. Members of the cactus family are one of these lineages, with distinct plastome structures reported across disparate lineages, including gene losses, inversions, boundary movements or loss of the canonical inverted repeat (IR) region. However, only a small fraction of cactus diversity has been analysed so far.

Here, we investigated plastome features of the tribe Opuntieae, the remarkable prickly pear cacti, which represent one of the most diverse and important lineages of Cactaceae. We assembled de novo the plastome of 43 species, representing a comprehensive sampling of the tribe, including all seven genera, and analysed their evolution in a phylogenetic comparative framework. Phylogenomic analyses with different datasets (full plastome sequences and genes only) were performed, followed by congruence analyses to assess signals underlying contentious nodes.

Plastomes varied considerably in length, from 121 to 162 kbp, with striking differences in the content and size of the IR region (contraction and expansion events), including a lack of the canonical IR in some lineages and the pseudogenization or loss of some genes. Overall, nine different types of plastomes were reported, deviating in the presence of the IR region or the genes contained in the IR. Overall, plastome sequences resolved phylogenetic relationships within major clades of Opuntieae with high bootstrap values but presented some contentious nodes depending on the dataset analysed (e.g. whole plastome vs. genes only). Congruence analyses revealed that most plastidial regions lack phylogenetic resolution, while few markers are supporting the most likely topology. Likewise, alternative topologies are driven by a handful of plastome markers, suggesting recalcitrant nodes in the phylogeny.

Our study reveals a dynamic nature of plastome evolution across closely related lineages, shedding light on peculiar features of plastomes. Variation of plastome types across Opuntieae is remarkable in size, structure and content and can be important for the recognition of species in some major clades. Unravelling connections between the causes of plastome variation and the consequences for species biology, physiology, ecology, diversification and adaptation is a promising and ambitious endeavour in cactus research. Although plastome data resolved major phylogenetic relationships, the generation of nuclear genomic data is necessary to confront these hypotheses and assess the recalcitrant nodes further.

In the past three decades, several studies have predominantly relied on a small sample of the plastome to infer deep phylogenetic relationships in the species-rich Melastomataceae. Here, we report the first full plastid sequences of this family, compare general features of the sampled plastomes to other sequenced Myrtales, and survey the plastomes for highly informative regions for phylogenetics.

Genome skimming was performed for 16 species spread across the Melastomataceae. Plastomes were assembled, annotated and compared to eight sequenced plastids in the Myrtales. Phylogenetic inference was performed using Maximum Likelihood on six different data sets, where putative biases were taken into account. Summary statistics were generated for all introns and intergenic spacers with suitable size for polymerase chain reaction (PCR) amplification and used to rank the markers by phylogenetic information.

The majority of the plastomes sampled are conserved in gene content and order, as well as in sequence length and GC content within plastid regions and sequence classes. Departures include the putative presence ofrps16andrpl2pseudogenes in some plastomes. Phylogenetic analyses of the majority of the schemes analyzed resulted in the same topology with high values of bootstrap support. Although there is still uncertainty in some relationships, in the highest supported topologies only two nodes received bootstrap values lower than 95%.

Melastomataceae plastomes are no exception for the general patterns observed in the genomic structure of land plant chloroplasts, being highly conserved and structurally similar to most other Myrtales. Despite the fact that the full plastome phylogeny shares most of the clades with the previously widely used and reduced data set, some changes are still observed and bootstrap support is higher. The plastome data set presented here is a step towards phylogenomic analyses in the Melastomataceae and will be a useful resource for future studies.

Putatively single‐copy nuclear (SCN) loci, which are identified using genomic resources of closely related species, are ideal for phylogenomic inference. However, suitable genomic resources are not available for many clades, including Melastomataceae. We introduce a versatile approach to identify SCN loci for clades with few genomic resources and use it to develop probes for target enrichment in the distantly relatedMemecylonandTibouchina(Melastomataceae).

We present a two‐tiered pipeline. First, we identified putatively SCN loci using MarkerMiner and transcriptomes from distantly related species in Melastomataceae. Published loci and genes of functional significance were then added (384 total loci). Second, using HybPiper, we retrieved 689 homologous template sequences for these loci using genome‐skimming data from within the focal clades.

We sequenced 193 loci common toMemecylonandTibouchina. Probes designed from 56 template sequences successfully targeted sequences in both clades. Probes designed from genome‐skimming data within a focal clade were more successful than probes designed from other sources.

Our pipeline successfully identified and targeted SCN loci inMemecylonandTibouchina, enabling phylogenomic studies in both clades and potentially across Melastomataceae. This pipeline could be easily applied to other clades with few genomic resources.