# Transcriptome Landscape Variation in the Genus _Thymus_
## 3 Results and Discussion
### Orthogroups Identified, Phylogenetic Analysis and Finding Genes under Positive Selection  
Our understanding of the phylogenetic relationships of Lamiaceae has improved in recent years, with new phylogenetic studies placing Lamiaceae within a large clade called \"core Lamiales\" [94] that comprises more than 7000 species assigned to 236 genera [2]. However, the relationships among most genera and species have remained unresolved. In the present study, a large-scale phylogenetic reconstruction of _Thymus_ species, which has been investigated so far mainly using small-scale molecular data [95], was carried out using 592 single-copy genes as follows. An orthogroup is the set of genes that have evolved from a single gene of the last common ancestor of all the species via speciation [96]. To identify the orthogroups for the five _Thymus_ species, with _S. splendens and M. spicata_ as outgroups, protein sequences obtained from assembled and annotated transcriptome data were used with OrthoFinder [34]. The total number of orthogroups with all species present was 592. These were used for a subsequent phylogenetic analysis to define evolutionary relationships between these species. Relationships found in our ML tree for the most part agree with the Bayesian tree (Figure 6). These phylogenetic trees strongly support a close genetic relationship between _T. daenensis_ and _T. vulgaris_ as sisters, as well as between this clade and a clade composed of _T. pubescens_ and _T. lancifolius_. Interestingly, the close relationship between _T. daenensis_ and _T. vulgaris_ is strongly supported by morphological and oil content similarity. _T. daenensis_ is characterized by a high percentage of thymol, and _T. vulgaris_ displays a mixture of thymol and g-terpinene as major constituents [97]. Divergence time estimation resulting from a Bayesian analysis suggests that _Thymus_ originated during the late Pleistocene (2.24 MYA), then diverged and dispersed. This estimate falls into the range of ages estimated by [PERSON] et al. (2012) [98]. Phylogenetic analysis of 121 accessions of the tribe  
Figure 5: Maximum likelihood (ML) phylogeny of putative TPS genes in _Thymus_ species compared to other species of TPS subfamilies. Mentheae based on cpDNA and nrDNA suggest that _Thymus_ originated during the Mid-Pliocene (3.06 MYA, between 2.40 and 3.72 MYA) [98]. In a ML framework, divergence dates generally resulted in an increase in the estimated ages of younger nodes. The ML tree indicated that _Thymus_ originated during the Tortonian (10.2 MYA). This difference is due to the methodology used for time calibration [99], molecular substitution [100], and clock model choice [101]. Orthogroups shared between _Thymus_ species ranged from 18,412 between Tdae and Tper, and 21,071 between Tian and Tvul. There were also 727 single-copy orthogroups in our species comparison that were retained for positive selection analysis. Using the branch-site model, 55, 49, 29, and 54 genes were identified as being under positive selection in Tdae, Tian, Tper, and Tpub, respectively. Positively selected genes were further used to identify functional categories according to their assigned gene ontologies. The distribution of classification of PSGs showed that in all species (Figure S3), biological  
Figure 6: Phylogenetic relationships among five _Thymus_ species inferred from 592 single-copy orthogroups. (**A**) Maximum likelihood tree. (**B**) Bayesian tree. process (GO:0008150) had the most hits. The second most abundant GO category was related to metabolic processes (GO: 0008152). The unigenes subjected to positive selection were further annotated in the Swiss-Prot database (Table S7). Our search for gene matches from this study with other research revealed remarkable similarities. A protein involved in the pathway protein ubiquitination, namely F-box protein, was found to be common among all species. Indeed, it has been indicted that some F-box proteins are under selection and show a strong tendency of positive selection [102; 103].