iNEXT4steps workflow for biodiversity assessment and comparison v1
Authors: Daraghmeh Nauras , Christian El-Khaled Yusuf 
Published: May 2021

In order to assess and compare sample completeness and alpha diversity of samples, Chao et al. (2020) proposed a 4-step-workflow. It is based on their extensive earlier works (e.g., Chao et al. 2014) which make use of the now widely accepted concept of Hill numbers, also known as effective number of equally abundant species qD (Hill 1973, Chao et al. 2014). Here, q denotes the diversity order of a Hill number and determines its sensitivity to species’ relative abundances or frequencies (in case of incidence data, i.e., species presence /absence). Hill numbers based on higher values of q put more emphasis on more commonly occurring species. In their publication, Chao et al. (2020) mention the online tool iNEXT4steps which supposedly offers users to complete the workflow via a web-based application. This tool is an extended and updated version of the earlier iNEXT online tool and R package (Hsieh et al. 2016). Unfortunately, the link provided in the paper does not lead to the new application. We therefore got in touch with the authors and they provided us with a link to the trial version of the iNEXT4steps online tool (see link in section 2 of the protocol). We here present a detailed workflow combining the new iNEXT4steps online tool with the existing iNEXT R package to assess and compare sample completeness, diversity and evenness between habitats as presented. This allows researchers to obtain results and figures as presented in Chao et al. (2020). References: Chao, A., Gotelli, N.J., Hsieh, T.C., Sander, E.L., Ma, K.H., Colwell, R.K. and Ellison, A.M. (2014), Rarefaction and extrapolation with Hill numbers: a framework for sampling and estimation in species diversity studies. Ecological Monographs, 84: 45-67. https://doi.org/10.1890/13-0133.1 Chao, A, Kubota, Y, Zelený, D, et al. Quantifying sample completeness and comparing diversities among assemblages. Ecological Research. 2020; 35: 292– 314. https://doi.org/10.1111/1440-1703.12102 Hill, M.O. (1973), Diversity and Evenness: A Unifying Notation and Its Consequences. Ecology, 54: 427-432. https://doi.org/10.2307/1934352 Hsieh, T.C., Ma, K.H. and Chao, A. (2016), iNEXT: an R package for rarefaction and extrapolation of species diversity (Hill numbers). Methods Ecol Evol, 7: 1451-1456. https://doi.org/10.1111/2041-210X.12613 We further used Microsoft Excel, R (R Core Team 2020) and RStudio (RStudio Team 2020) and the packages ggplot2 (Wickham 2009) and ggpubr (Kassambara 2020). Microsoft Corporation. (2018). Microsoft Excel. Retrieved from https://office.microsoft.com/excel R Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. RStudio Team (2020). RStudio: Integrated Development for R. RStudio, PBC, Boston, MA URL http://www.rstudio.com/. Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis.Springer-Verlag New York. ISBN 978-3-319-24277-4, https://ggplot2.tidyverse.org Kassambara A (2020): ggpubr. https://rpkgs.datanovia.com/ggpubr.