2.3 Usage
The main menu presents the initial options to verify the availability of data for a species, determine divergence times, a timeline, a time tree, validate the data, or print the citation for Time Tree, as summarized in Figure 2 . The data availability option brings up a submenu to specify if availability should be verified for a single species or species list. When checking a single species, the availability is printed to the screen as the species name followed by either ‘Available’ or ‘Not Available’. This function was designed to ensure data availability prior to further data retrieval steps. For a list of species, the species name and availability are printed on screen and stored to export results in a CSV file.
The timeline and time tree options will bring up a similar submenu that provides the option to retrieve information for an individual species, individual taxon, or a list of species. As before, individual species will result in a single output. The timeline retrieval function will retrieve an image in the JPEG format that illustrates the evolutionary history for the species highlighting the major time points where a kingdom, order, class, genus, and species first emerged. As an example, a timeline was retrieved for the Lazuli bunting, Passerina amoena [Say, 1823]. Input given as a list will retrieve individual images for each species in the list. The time tree option has similar functionality except that input can either be a larger taxonomic group, such as family or genus containing several congenic species, or a list of species as this output provides a time calibrated tree displaying both the interrelatedness of species as well as the timescale along which the branches diverged. As an example, two time trees were generated for the genus Catharus and Malurus respectively. The individual diversification rate (r ) was calculated for each genus in R (R Core Team, 2020) using the geiger (version 2.0) package (Harmon et al. , 2008) with the Magallon-Sanderson equation (Magallón and Sanderson, 2001).
The Divergence time submenu provides the option to check for the divergence time between two species e.g., the Neotropical Swainson’s thrush, Catharus ustulatus [Nuttall, 1840], and the Australasian Superb fairywren, Malurus cyaneus [Ellis, 1782]. The result will be printed to the screen as the name of ‘Taxon a’ and ‘Taxon b’ followed by the mean divergence time in millions of years ago (MYA) e.g., 35 MYA. The list option takes a text file with a list of species names and iterates through the list to determine the divergence time between each species in the list. The results are stored in a ‘dataframe’ object which can be exported as a CSV file in the form of a three column vectorized matrix with the names for ‘Taxa 1’ in the first column, the names of ‘Taxa 2’ in the second column, and the divergence times between them in the third column. Additional functionality is provided in the main menu to validate data. This option can be used to check output files for missing values, as well as retrieve and replace such values, in case a server error occurred.
RESULTS
Examples of timelines and time tree data retrieved from the Time tree resource are illustrated in Figure 3 and Figure 4 . The timeline was retrieved for the Neotropical bird the Lazuli bunting. The left panel indicates the major geologic timescales in Eons and Eras while the right indicates the main divergence times when specific taxa emerged. This includes the current Phanerozoic Eon as well as the subdivisions from the Paleozoic Era, which lasted until approximately 250 MYA, and the Mesozoic which lasted until 65 MYA. This period was marked by the emergence of the first birds (class: Aves) approximately 110 MYA. The specific species, a bunting in the family Cardinalidae, emerged from a common ancestor shared with other cardinals in the Cenozoic, while species of the genus Passerina first emerging 4 MYA and subsequently diverged to form species and subspecies.
Time trees were generated for two genera of birds that each contain several congenic species (11-12 species) but have different evolutionary histories in terms of the divergence times within their genus. The first represents the genus Catharus for which 12 species are included in the time tree. These species diverged over a period spanning approximately 4.73 million years and have an absolute diversification rate of 0.38. By comparison, the second tree for the genus Malurus includes a similar number of species, however, these species diverged over a period of approximately 9 million years and have a diversification rate (r = 0.19) of nearly half the rate observed in Catharus .
PAReTT was furthermore used to retrieve data availability and divergence times for a list of forty bird species, as well as a time tree for seventy-six species, in a recent review and meta-analysis on clock genes as candidate genes in migration studies, published in Biological Reviews . This study found a significant relationship between divergence times and the observed genetic distance in two candidate genes, illustrating high heritability of genotypes within lineages and a lack of selection. This illustrates the significance in studying divergence time data in relation to genetic data in molecular studies. The results from the benchmarking tests, performed in tandem, showed an average time for submitting data and retrieving the result ranged from fifteen to thirty seconds and memory consumption remained low, ranging from <100 to 400 Megabytes (Supplementary table 1 ).
DISCUSSION
Time is an important variable in the study of evolutionary processes as it relates both to large scale geographic remodelling that occurred over millions of years and the timelines for the emergence and diversification of taxonomic and phylogenetic lineages. Time is also a key factor in determining the diversification rate within lineages which may be vastly different even between families in the same class and order. The Time tree resource provides relevant data that can easily be incorporated into molecular studies tracking diversification rates as well as studies comparing the heritability of specific genotypes as ancestrally inherited or currently undergoing active selection. The resource does, however, still lack options to facilitate batch retrieval for lists of species. Here, we illustrate the use of a newly scripted PYTHON package called PAReTT that provides an added layer of functionality to the Time Tree resource by enabling the batch retrieval of data for lists of species in a format that can enhance molecular and ecological studies.