Material and methods

Extracting the information from GenBank. A new script called Datataxa (^{Maya-Lastra 2019}), that facilitates the extraction of information from GenBank, was written in AutoIt Scripting Language (^{Bennett 2015}). For the extraction of information, an interface was created that recurrently does the following procedures with all names in the main list and then sorts the result into a readable table. Initially, the program searches for the correct spelling and taxonomic synonyms using the Espell utility in the Entrez Application Programming Interface (API, ^{Maglott et al. 2005}). API refers to an interface that allows the inclusion of functions from different applications in new scripts and software in order to utilize of previously developed technologies. In our case, the script makes a connection with the GenBank database through Entrez API. Subsequently, it searches using the Esearch utility in the same API, looking for the GenInfo Identifier (GI) accession number in the Nucleotide GenBank database associated with each species. For each GI accession number, the following information was retrieved using the Efetch utility: nucleotide length, definitions (title found in GenBank), paper titles, journal names, and creation date (GI accessions for each species in Appendix S1 see Supplemental Data with this article). The time required for the extraction of metadata from a list of 6,000 species is commonly six hours, but it can vary depending on the number of accessions found for each species, times and query limits of GenBank (since December 2018, the limitation is 3-10 requests per second), and internet connection speed, to mention a few factors. Other tests showed no apparent limit of the number of species included in the search. To facilitate long time execution, the script automatically saves progress in case of an interruption. In such instances, when it is running again, it restarts from the last saved point. The script can be downloaded at no cost from https://github.com/camayal/Datataxa; detailed instructions on how to use it can also be found in the Github repository. The only input file the user needs to provide is a plain text file with no heading, one species per line, and the generic and specific epithets separated by a “+”. This script is only intended to work with species, and only this functionality was tested. However, it is not limited, and in theory will work for other taxonomic ranks, like genera or families. The output are two different comma separated value (CSV) files, one for each phase, extraction and metasearch.

Classification and analysis using search terms. The entire classification was divided into six categories to determine which species are included in the different types of research. For each category, a list of generalized terms (Table 1) was used to include variations like plural and singular; for diversity studies, a couple of terms were always used, as described in Table 1. Finally, the generalized terms were designed to search in English, Spanish, and Portuguese, and all searches were executed as case-insensitive. The search was performed in the paper titles where each sequence was used (Appendix S2; see Supplemental Data within this article).

Floristic study. Flora del Bajío y de Regiones Adyacentes (FBRA) is one of the most active ongoing floristic projects in Mexico. It was initiated in 1985 by Jerzy Rzedowski and Graciela Calderón, with the aim of publishing an inventory of the wild vascular plants of the region. After five years of intensive collecting to obtain herbarium specimens, the formal preparation and publication of the Flora started in 1991 (^{Calderón de Rzedowski & Rzedowski 1991}). The vascular plant diversity calculated for this region consists of ca. 5,500 species, 1,205 genera, and 185 families (^{Calderón de Rzedowski & Rzedowski 1991}), thus representing 23.5 % of the total Mexican plant diversity. To date, 203 fascicles have been published. Some species included in the FBRA also have been used in phylogenetic, phylogeographic, phylogenomic, barcoding, genetic diversity, and biogeographic studies, in addition to many other studies.

The study area. The geographical delimitation of the Flora del Bajío y de Regiones Adyacentes adheres to Calderón de ^{Rzedowski & Rzedowski (1991)} and corresponds to the traditional Bajío area of central Mexico and some adjacent regions. It includes the entire states Guanajuato and Querétaro, in addition to the northern portion of Michoacán. In total, it covers 60,171 km² (Figure 1), of which 49.8 % has been transformed or fragmented by anthropogenic activities (^{Suárez-Mota et al. 2015}). Three physiographic provinces have shaped the study area: the Trans-Mexican Volcanic Belt, the Mexican Plateau, and the Sierra Madre Oriental (^{Ferrusquia-Villafranca 1993}). The Trans-Mexican Volcanic Belt is a volcanic region that extends east-west across much of the study area (^{Gómez-Tuena et al. 2007}, ^{Ferrari et al. 2012}). The Mexican plateau occurs only in a small north-west portion of the study area, whereas the Sierra Madre Oriental occupies the northeast portion (^{Ferrusquia-Villafranca 1993}). Topographic diversity and ample climatic variation favor the development of both temperate and tropical vegetation in this region (^{Rzedowski 1978}, ¹⁹⁹¹, ^{Calderón de Rzedowski & Rzedowski 1991}). Among the temperate vegetation present are pine forests, fir forests, oak forests, pine-oak forests, and montane cloud forests. The tropical vegetation is represented by tropical dry forests, subdeciduous tropical forests, and xerophytic scrubs (^{Rzedowski 1978}).

Figure 1 Map showing the area of the Flora del Bajío y de Regiones Adyacentes (represented by the polygon in red). 

Data obtainment and database curation. In order to use data that is as accurate and current as possible, we included only the 2,558 species previously published in treatments of the FBRA. Although not a complete representation of the diversity of the region, it constitutes nearly half of the species present and is a sufficiently representative sample of the total flora. All 203 published floristic treatments of the FBRA were reviewed in order to prepare a list of families, genera, and species (Appendix S3; see Supplemental Data with this article). Searches of taxonomic and nomenclatural updates were conducted using the “Taxonomy name/id Status Report Page” (^{NCBI Resources Coordinators 2016}). All names on the main list that are no longer in use were removed. It is worth mentioning that the biggest families (Asteraceae, Fabaceae, Orchidaceae, and Poaceae) have only been partially treated, due the large number of species belonging to each of them.

Results

After taxonomic and nomenclatural updates of the names on the list, we obtained a total of 2,558 species, 799 genera, and 186 families for the region (Appendix S3; see Supplemental Data with this article). Of the species, 1,575 have a least one accession number in the GenBank database (Table 2; Appendix S1, S3), that represents 61.57 % of the total species studied in FBRA from the region. A total of 1,322 of the 1,575 species have been used in phylogenetic studies, and this figure represents 83.94 % of the species in GenBank and 51.68 % of the total species treated in FBRA (Table 2). Barcoding studies represent the second place in Genbank with 326 species, followed by biogeographic studies with 298, 41 for phylogenomic studies, 34 for phylogeographic studies, and finally only 34 species for diversity studies (Table 2).

In conjunction with the floristic treatments, one genus, 173 species, four subspecies, and five varieties have been described as new to science from the region (Appendix S4; see Supplemental Data with this article). Of these, only 26 species have been included in phylogenetic studies, seven in biogeographic studies, and one in a barcoding analysis. This value (19.8 %) is significantly lower than the overall percentage of species included in each study.

Of all the families studied in the FBRA, only eight lack species from the region that have sequences in GenBank, these being: Theaceae (five spp.), Ebenaceae and Eriocaulaceae (three spp.), Mayacaceae, Platanaceae, Pterostemonaceae, Thymelaeaceae, and Xyridaceae (with one species each). On the other hand, 77 families have a GenBank accession for all species that occur in the region. Crassulaceae and Malvaceae are two speciose families that have 48 and 46 % of the species with at least one entry in GenBank. Some other families lack a good representation of species with regard to the total included in GenBank. These are Aristolochiaceae (1 of 7 species), Dioscoreaceae (3 of 12), Geraniaceae (2 of 14), Linaceae (2 of 9), Phrymaceae (1 of 5), and Sterculiaceae (4 of 16) (Appendix S5; see Supplemental Data with this article).

Discussion

The extensive API system integrated in GenBank makes this repository not only a public platform to store data but also an important source to obtain data. In this case, an open source script was designed to extract the information and organize it in a way that demonstrates the importance and representation of the species found in floristic projects like FBRA. However, it can also be applied to other floristic projects or different sets of taxa of interest. This script mainly uses the Entrez system to access information other than the DNA sequences, which separates it from other programs and scripts that automatically extract data from GenBank. For our purposes, the focus was on the title of the cited paper for each sequence in order to infer the use of the data in the different disciplines analyzed. A future possibility could be explored using other information like abstract and keywords (retrieved from other public databases), by employing deep learning techniques that refine the search and classification.

The use of molecular-based phylogenies has improved our understanding of taxonomic relationships (^{Ulloa-Ulloa et al. 2017}). Phylogenetic and phylogeographic studies, based on molecular DNA sequences, are sometimes necessary for the quality and accuracy of floristic work. In the case of FBRA, the correct taxonomic position of Velascoa recondita Calderón & Rzed. (Crossosomataceae), a newly described genus and species with unusual floral morphology endemic to the FBRA region (^{Calderón de Rzedowski & Rzedowski 1997}), was only possible through a phylogenetic analysis using the chloroplast gene rcbL (^{Sosa & Chase 2003}). The phylogenetic analysis of the Crossosomataceae found that V. recondita is the sister species to Apacheria chiricahuensis C.T. Mason, another monotypic genus endemic to Arizona and New Mexico (^{Mason 1975}, ^{Sosa & Chase 2003}).

On the other hand, floristic studies are an essential tool for any evolutionary study (^{Funk 2006}), being sometimes the only updated repository where morphological descriptions and identification keys can be found. Such studies support the correct application of names to samples in an evolutionary study (^{Palmer & Richardson 2012}). For this reason, a complete descriptive Flora can influence the results and quality of the phylogenetic hypotheses.

For the FBRA, currently 1575 species have sequence records in GenBank, making that study an important repository for nomenclature, morphological descriptions and in some cases keys to their proper identification.

Despite the importance of having a better understanding of land plant phylogeny and evolution, at a regional level we do not even know the total number of species present in a small area like that covered by the FBRA. Furthermore, although we understand the phylogenetic relationships of the orders and families of Angiosperms, for the most of the 416 families recognized by ^{APG et al. (2016)}, we do not know their internal (genus and species level) phylogenetic relationships.

For the FBRA, having 51.68 % of species included in a phylogenetic analysis can be an important state of the knowledge of the evolutionary relationships at a regional level. Currently, half of the species documented in the FBRA lack a tested evolutionary relationship. This impact can be being mitigated by the high percentage of genera included in some phylogenetic analysis, with least 84.35 % of the genera having been found in the GenBank repository. Nevertheless, genera like Velascoa show that the proper inclusion in an evolutionary study can change the understanding of their evolutionary history and taxonomy.

The low values in barcoding studies for species from the region show a potential concern about the correct interpretation and implementation of these kind of techniques in a broad study. In the region, only 12.74 % of the species were included in a barcoding study. With next-generation sequencing costs in constant decline, sequencing projects nowadays are taxonomically broad (^{Chan & Ragan 2013}). For example, the objective of the 1KP initiative is to sequence complete plant genomes (https://sites.google.com/a/ualberta.ca/onekp/ accessed February 20, 2019). However, we are still very far from sequencing the complete genomes of the 383,671 vascular species in the world (^{Lughadha et al. 2016}), showing a worrying panorama, where the number species sequenced is currently increasing, whereas the number of species treated in a floristic study is decreasing. It is necessary to include species never studied before to try to have a complete and robust evolutionary history of vascular plants. Our study region includes 2,558 species that have been formally included in the FBRA, and our results indicate that only 41 species (Table 2) have been used for phylogenomic studies. Our study case is an example of the impact of floristic projects for a particular region where most of its species have been used in phylogenetic studies, but it reveals that the number of species used for phylogenomic studies continues to be very low.

The goal of the Flora del Bajío y de Regiones Adyacentes is to contribute to our knowledge of the species of the area and to provide morphological descriptions and keys to the correct identification of these species. Additionally, it aims to document the total number of species for the region, their distribution, and the level of endemism.

It could be possible that in the upcoming years, all the floristic knowledge generated worldwide could be deposited on the Biodiversity CyberBank an initiative proposed by ^{Wen et al. (2015)}, and it will be free to use, as is GenBank today. This information will be essential for phylogenetic, biogeographic, phylogeographic, phylogenomic, and diversity studies, but also crucial for policy makers involved in the conservation strategies of the flora of a particular region. It is expected that as time passes, the use of the Flora in various studies will increase, and although this is not yet reflected in the statistics, it will be clearer when the Flora del Bajío y de Regiones Adyacentes has been completed.

Of all families included in this metadata extraction, Crassulaceae and Malvaceae are two interesting groups that need to have their sampling efforts increased. Despite the other largest families having included a high percentage of species in GenBank, these two families have the fewest species with an entry in GenBank.

Some other low represented families like Aristolochiaceae, Dioscoreaceae, Geraniaceae, Linaceae, Phrymaceae, and Sterculiaceae sensu FBRA also require more effort in future molecular evolutionary studies to try to have a better sample.

In conclusion, our new script worked well to download metadata sequence information from GenBank. Although the script was developed and tested with a checklist of plants from the Flora of the Bajío region, it can be used with any list of species to answer various questions involving the metadata that usually are present but unused in accessions.

Supplementary Material

Appendix S1. List of the species with Genbank accession by type of study (“X” indicates found in that particular study, whereas “-” indicates not found).

Appendix S2. List of species with GenBank accession by type of study; the order of the species corresponds to the order of the published treatments.

Appendix S3. List of the species treated in the published FBRA treatments.

Appendix S4. List of new taxa described from research conducted for the FBRA.

Appendix S5. Number of species per family by category.