Appendix: A template for preregistration in taxon delimitation research

Note: This template represents what we consider as a maximalist preregistration effort, i.e. that is used in an ideal situation. The various notes will guide you through the different steps. An example based on a real-world ongoing taxonomic research project, that of the taxonomic revision of the free-living flatworm complex Gyratrix hermaphroditus Ehrenberg, 1831, will be developed throughout the template to illustrate which information is exactly required. As an example, it presents some of the challenges we consider typical for the application of preregistration in taxonomy, such as the extensive use of existing collections, and a lack of clarity on the relevance of taxonomic characters. In practice, the information that must be included for adequate preregistration depends on two fundamental aspects. First, whether research is exploratory or hypothesis-based (in the case of taxonomic revisions). In both cases, confirmatory rules for recognizing taxa must be stated, but in the latter case, a hypothesis must be given as well. Second, whether one is planning to do fieldwork and collect new samples, or planning to collect new data on existing samples, or planning to work with existing data(sets). Some elements are optional, and can be completed when the specific context renders it desirable.

Note: This template is roughly based on the standard template provided by the Open Science Framework (Bowman et al. 2020). Some of the instructions are directly copied from this template. For more information on creating a preregistration and where to store it, please consult the Open Science Framework (https://help.osf.io/article/158-create-a-preregistration). 
A.	Study Information

1.	Title (required):

Note: Provide the working title of your study. This may or may not be the title that you submit for the publication of your final manuscript. The title should be a specific and informative description of your project. 

Example: Detangling the species complex: Gyratrix hermaphroditus Ehrenberg, 1831 species complex

2.	Description: 

Note: This is the abstract of the preregistration. Briefly summarize the research for readers to get a general idea of what this registration is about.

Example: Gyratrix hermaphroditus Ehrenberg, 1831, although never formally split, has for long known to be a species complex that comprises substantial phenotypic and genotypic variation, and there is agreement that it can reasonably be split into several species. Recent research by Tessens et al. (2021) has shown that diagnosable morphotypes can be delimited within the complex, and that these morphotypes are monophyletic according to a molecular phylogeny. However, with regard to species delimitation, the authors applied multiple taxonomic methods, based on morphology and genetic species delimitation. This resulted in possible taxonomies that recognize 14, 62 and 78 species, leaving the best manner to proceed with a formal split in doubt. Here, we aim to offer and justify a formal taxonomic revision of the species complex, starting with the diagnosable morphotypes defined by Tessens et al., by testing their consistency with additional material from new locations, and by developing a yardstick for ascribing species status to newly discovered morphotypes in the future. 

3.	Authors (required):

Note: Cite all authors that will be involved in the research process, not only the authors contributing to the preregistration. 

4.	Research aims (required):

Note: Elaborate the exact research aims of your study, providing as much background as is needed for the interpretation of the information cited in the preregistration. It serves to provide context to what follows. State the exact research questions or objectives in as concise and concrete a manner as possible.

Example: Our concrete research aims consist in (a.) assessing the consistency of the morphotypes within G. hermaphroditus as defined by Tessens et al. (2021) with new material; (b.) developing a yardstick for the recognition of new, unknown morphotypes; (c.) delimiting new, unknown morphotypes in the additional material used; and (d.) providing a formal taxonomic revision of G. hermaphroditus along the different recognized morphotypes. 

5.	Study type(s) (required):

o	Discovery of new diversity 
o	Exploratory taxonomic revision
o	Taxonomic revision with detailed hypothesis
o	Other:…

Note: Specify the type of research in which your study fits best. The most straightforward types of taxonomic research are, on the one hand, research efforts aimed at the discovery of unknown diversity, for example concerning a particular taxonomic group in a particular region, and, on the other hand, the revision or testing of an existing taxonomy with a new method or data, in which the existing taxonomy serves as a detailed hypothesis. However, in taxonomy, there appear to be many intermediate forms, in which revisions are conducted, for example, without a detailed hypothesis in mind, although there might be intuitions on splitting or lumping, or in which existing taxa and newly described taxa are combined. For such cases, the category of ‘exploratory taxonomic revisions’ was introduced. Although this category does not start with detailed hypotheses, any hypothesis, however vague, requires preregistration in this category as well.

Example: Our example falls in the intermediate category of ‘exploratory taxonomic revisions’, combining the revision of known diversity along new data, with exploratory research (e.g. the description of new morphotypes).

6.	Data type(s) (required): 

o	Collection of new samples through fieldwork
o	Collection of new data from existing material
o	Use of existing data
o	Other:…

Note: Specify all sources of data that are used in your study. This is important to determine which methodological information should be given, for instance about fieldwork, but on the other hand also which possible pre-existing biases must be taken into account when using existing material or data. Existing material concerns studied or unstudied museum samples, herbarium material, etc. Existing genetic sequences count as existing data. Of course, more than one data type can be used. In that case, check all boxes that apply. 

Example: We will make use of both new data collected from existing material, and existing data.

7.	Hypotheses (required for taxonomic revisions):

Note: List here the hypotheses you have, with as much detail as possible. If you simply think that an existing taxonomic situation will be upheld, or will be rejected, state that. If you have specific ideas (for example that taxon x will have to be split in three), state that as well. 
 
Example: We expect that the morphotypes of Gyratrix hermaphroditus as described by Tessens et al. (2021) show consistent, diagnosable morphological differences, and will for that reason, as detailed below, merit recognition as formal species. We also expect to encounter other morphotypes not yet described, for which that will be the case. 
B.	General Taxonomic Approach

Note: Given the variety of approaches that exist in taxonomy, both on the conceptual and on the methodological levels, the main aim of preregistration in taxonomy is to fix a method, limiting the room for conscious or unconscious deviations, and to create transparency. First, you are asked to describe the overall taxonomic approach of your study, which will be detailed in the remainder of the preregistration. Ideally, this general description breaks down into a conceptual and a methodological part, but the latter counts as a minimal requirement. 

8.	Species (or other taxon) concept (optional):

Select the species concept that fits your study best:

o	Biological Species Concept
(Interbreeding natural populations reproductively isolated from other such groups / all individuals capable of producing fertile offspring.)
o	Evolutionary/General Lineage/Unified Species Concept
(Population or metapopulation-level lineages that evolve separately from other such lineages.)
o	Genotypic Cluster Species Concept
(Groups of individuals or populations that have no intermediates when they are in, or come into, contact.)
o	Phylogenetic Species Concept, diagnosability version
(The smallest group of populations diagnosable by a unique combination of character states.)
o	Phylogenetic Species Concept, monophyly version
(The least inclusive monophyletic taxon worthy of formal recognition because of its biological relevance.)
o	Other:…

Note: Species concepts, definitions of species, have been argued to play an important role in structuring taxonomic research. If your study design is guided by a certain species concept, select or cite it here. This step is not strictly required to meet the objectives of preregistration, but it counts as good taxonomic practice to be explicit on the concept of species used. 

Example: We follow a Phylogenetic Species Concept, diagnosability version. 

9.	 General approach to species delimitation (required):

Note: Describe here the general approach to species or taxon delimitation your study will follow. If you follow a specific species concept, describe how that species concept will be operationalized. 

Example: Our taxonomic approach builds on the morphologically diagnosable clades supported as monophyletic in the molecular phylogeny of Tessens et al. (2021), which are treated as representative of good candidates for species status. Based on additional material and additional data, such as morphometric measurements, we will test these candidates and describe them as formal species accordingly. Based on these results, we will use them as yardstick to describe other such diagnosable groups within the species complex based on similarly divergent morphotypes not yet included in existing phylogenies. 
C.	Sample/data collection
Note: In this section, you will be asked to describe how you plan to collect data and samples for new data. The elements that require completion depend on the data types that were selected above. For all selected data types, the required elements need to be completed. If multiple data sources of the same type are used that follow a different methodology (for example multiple fieldwork campaigns), elements need to be completed multiple times. 

10.	[If relevant] Existing data (required)

10.1 Scenario of existing data use:

Note: The aim of preregistration is to provide as much methodological information before any analysis is performed. Of course, when using existing data, part of the work has already been done. Therefore, creating a research plan in which existing data will be used (for confirmatory research) presents challenges to avoid biases. Please select the description that best describes your situation. 

o	Registration prior to any human observation of the data: At the time of submission, the data exist but have not yet been quantified, constructed, observed, or reported by anyone - including individuals that are not associated with the proposed study. For example, data that have been collected by non-human collectors and are inaccessible.
o	Registration prior to accessing the data: As of the date of submission, the data exist, but have not been accessed by you or your collaborators. Commonly, this includes data that has been collected by another researcher or institution.
o	Registration prior to analysis of the data: At the time of submission, the data exist and you have accessed it, though no analysis has been conducted related to the research plan (including calculation of summary statistics). A common situation for this scenario is when a large dataset exists that is used for many different studies over time, or when a dataset is randomly split into a sample for exploratory analyses, and the other section of data is reserved for later confirmatory data analysis.
o	Registration following analysis of the data: At the time of submission, you have accessed and analyzed some of the data relevant to the research plan. This includes preliminary analysis of variables, calculation of descriptive statistics, and observation of data distributions. Please see cos.io/prereg for more information. 

Example: Our study falls in the category of ‘Registration following analysis of the data’, because it builds heavily on the data published by Tessens et al. (2021), a study to which some co-authors of this preregistration have contributed. 

10.2 Rationale of existing data use (optional):

Note: Explain here why you will be using existing data, and what measures have been taken so that the principles and objectives of preregistration can be safeguarded. For instance, to assure that you are unaware of any patterns or summary statistics in the data. This may include an explanation of how access to the data has been limited, who has observed the data, or how you have avoided observing any analysis of the specific data you will use in your study. For example, an appropriate instance of using existing data would be collecting a sample size much larger than is required for the study, using a small portion of it to conduct exploratory analysis, and then registering one particular analysis that showed promising results. After registration, conduct the specified analysis on that part of the dataset that had not been investigated by the researcher up to that point. 

Example: Our study builds on the work by Tessens et al. (2021) and makes use of their data and non-published data related to that work as collected by coauthors of that study. These data, therefore, are known and fully analyzed, and serve, in the context of this study, as the starting point for the elaboration of the research question and aim, and for the setting of hypotheses. These data will not be used to test our hypotheses on the consistency of the morphotypes described by these authors. That will be done with new data collected from existing material. 

10.3 Data collection (optional):

Note: Describe how existing data will be collected, which datasets will be used and where they will be obtained. For example, if existing DNA sequences or trait data are used, describe where they come from.

Example: All data referred to above will be used, no specific procedure of data collection applies.

10.4 Sampling strategy (optional):

Note: If relevant, describe any procedure you will use to make samples of data. What will be the sample size? How many units will be analyzed? If your data collection procedures do not give you full control over your exact sample size, specify how you will decide when to terminate your data collection. 

Example: Not applicable

11.	[If relevant] Existing material for new data

Note: When using existing material for the collection of new data, similar challenges arise as with the use of existing data. The exact nature of the data, and the way in which they will be obtained, will be specified further in the preregistration. 

11.1 Scenario of existing material use:

o	Registration prior to any human analysis of the material: At the time of submission, the material exists but has not yet been observed, or studied by anyone - including individuals that are not associated with the proposed study. For example, field samples that have not yet been looked at. 
o	Registration prior to accessing the material: At the time of submission, the material exists, but has not been accessed by you or your collaborators. Commonly, this includes material that has been collected by another researcher or institution, such as herbarium material from another collection. 
o	Registration prior to analysis of the material: At the time of submission, the material exists and you have accessed it, though no analysis has been conducted related to the research plan (including calculation of summary statistics). 
o	Registration following analysis of the material: At the time of submission, you have accessed and analyzed some of the material relevant to the research plan. This includes preliminary analysis of characters. 
	
Example: Our example makes use of material that has been collected through the years in our lab collection, which has been collected, prepared, but not analyzed from a taxonomic viewpoint. Our research therefore falls in the category of ‘Registration prior to analysis of the material’. 

11.2 Rationale of existing material use (optional):

Note: Explain here why you will be using existing material, and what measures have been taken so that the principles and objectives of preregistration can be safeguarded. For instance, to ensure no sampling biases of existing collections are imported in your study, or no bias arises from prior interaction with existing material.

Example: We want to make use of a large reference collection of G. hermaphroditus with specimens obtained throughout field excursions across the years and in many areas, as additional material to test putative species boundaries, their consistency and diagnosability. While this collection is subject to sampling bias, it is the largest in the world and sufficiently large for credible use in taxonomic work.

11.3 Material collection:

Note: Describe how existing material will be collected, which material will be used and where it will be obtained. 

Example: We plan to use all material identified as belonging to the G. hermaphroditus complex available in the lab collection. 

11.4 Sampling strategy (optional):

Note: If relevant, describe any procedure you will use to take samples of existing material. What will be the sample size? How many units will be analyzed? If your material collection procedures do not give you full control over your exact sample size, specify how you will decide when to terminate your material collection. 

Example: Not applicable

12.	[If relevant] Fieldwork strategy:

12.1 Location of fieldwork (required): 

Note: Describe here where fieldwork will be conducted, in as precise a manner as possible. 

Example: Not applicable

12.2 Sampling methodology (required):

Note: Describe here how sampling will be conducted, for example what samples exactly are sought, how they will be collected, how they will be stored, etc.

Example: Not applicable

12.3 Sample size (optional):

Note: If relevant, describe here how many samples you aim to collect, or what will count as a stopping rule to end the sampling effort.

Example: Not applicable
D.	Analysis Plan

Note: In this section, the details of the actual taxonomic analysis will be expounded. 

13.	Characters (required)

Note: Describe each character type that you will use, through existing data, or through observation or analysis of existing or new material. Here, it is particularly important to be as precise as possible. For example, 'morphometrics,' 'morphological traits,' ‘mtDNA' and 'mating calls' are too vague. Of course, in various taxonomic approaches the relevance of characters is not always clear from the onset. For this reason, it is advisable to include all characters that are potentially relevant. Other characters can be added to the preregistration throughout the research process, should that appear to be necessary.

Example: Our analysis will focus on morphological characters traditionally used in rhabdocoel taxonomy. These include in particular characters related to (sclerotized parts of) the reproductive organs, such as the general form of the male copulatory organ, form and length of the stylet, sheath and stalk, and proportions between them; the presence and absence of distal hooks, flags, and other protruding structures; and the form and size of the potentially sclerotized vagina. Other potentially relevant characters include the presence or absence of eyes, and the coloration of the mesenchyme tissue. This will be linked to molecular data obtained from 18S, 28S, ITS1 and ITS2 ribosomal DNA genes. 

14.	Transformations (optional):

Note: If you plan on transforming, centering, recoding character data, or will require a coding scheme for categorical variables, describe that process.

Example: Not applicable

15.	Character analysis (required):

Note: Describe here any analysis that will be performed on your characters, particularly if multiple characters are used. Be as complete as possible. 

Example: We do not plan any quantitative or statistical analysis of characters. 

16.	Inference criteria (required):

Note: Describe here the criteria you set for species (or other taxon) recognition. Which criteria must the characters or the output of the analysis meet for the recognition? Note that this is probably the most difficult aspect of preregistering taxonomic research, in particular in an exploratory context. In many contexts, patterns of diversity remain unknown, and a degree of flexibility is required in assessing them and translating them into a species classification. The criteria described here should be as precise as possible in the context of the research in question. They can probably be more precise in explicit hypothesis-based revisions than in exploratory work. 

Example: Groups will be recognized as species if samples show consistent, diagnosable differences in the studied morphological characters, primarily of the stylets of the male copulatory organ, to an extent similar to or greater than differences between the known monophyletic morphotypes. Previously unknown characters might play a role in the assessment of new species. 

17.	 Data exclusion (optional)

Note: Describe here how you will determine what samples or data, if any, to exclude from your analysis. How will outliers be handled? Will you use any awareness check?

Example: Not applicable

18.	Missing data (optional)

Note: Describe here how you will deal with incomplete or missing data. 

Example: Not applicable (accessed 13/04/2023)
E.	Other

19.	Other (Optional):

Note: If there is any additional information that you feel needs to be included in your preregistration, please enter it here. Literature cited, disclosures of any related work such as replications or work that uses the same data, or other context that will be helpful for future readers would be appropriate here. 

References

Bowman, S., DeHaven, A.C., Errington, T.M., Hardwicke, T.E., Mellor, D.T., Nosek, B.A. & Soderberg, C.K. (2020) OSF Prereg Template.  https://10.31222/osf.io/epgjd 
Tessens, B., Monnens, M., Backeljau, T., Jordaens, K., Van Steenkiste, N., Breman, F.C., Smeets, K. & Artois, T. (2021) Is “everything everywhere”? Unprecedented cryptic diversity in the cosmopolitan flatworm Gyratrix hermaphroditus. Zoologica Scripta 50, 837–851. https://doi.org/10.1111/zsc.12507