This survey allows the evaluation of multiple ESU scenarios.

The first scenario always represents the null hypothesis, which assumes a single Evolutionarily Significant Unit (ESU). This one-ESU model must be tested first and serves as the baseline against which all alternative scenarios involving multiple ESUs are compared.

When selecting scenarios that include multiple ESUs (i.e. not the default single ESU), all ESUs must be mutually distinct and supported by evidence. Each ESU must have a clear evidentiary basis for inclusion in the scenario being tested. If any two ESUs cannot be distinguished from one another, they should be merged, and a revised scenario should be considered for testing.

For each question there are three possible answers: ‘Yes’, ‘No’ and ‘No data’.

If you answer each question you need to keep the following in mind:

The evidence within the question has been tested/evaluated -> answer can be ‘Yes’ or ‘No’.

Genetic evidence

Evidence through (multiple can apply): Documented heritable differences in chromosome number, ploidy, or chromosome structure between candidate ESUs. Because some species can have natural variation in karyotype within a single ESU, hybrids between individuals with different ploidy should be rare or non-existent, be unviable, or have significantly reduced relative fitness (e.g., lower reproductive success/reduced body condition) for this line of evidence to be scored.

Examples of Suitable data:
· Evidence of heritable differences from cytogenetic analysis and structural variant detection software coupled with evidence of no/rare/unfit hybrids between proposed units.
· Evidence of no/rare/unfit hybrids.

• Karyotype variation
• Difference in ploidy levels between units
• Structural variation of chromosomes between units

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Evidence through (multiple can apply): Genetic evidence of reproductive isolation, due to highly restricted or no gene flow between focal ESUs that may have fueled evolutionary distinctiveness.

Examples of Suitable data:
· Genetic data that can be tested for reciprocal monophyly (where no alleles are shared between subpopulations or ESUs = no Admixture). Please note, that with reciprocal monophyly the picture detected is strongly dependent on data type.
· Estimated divergence times between lineages, benchmarked against outgroups that include sister species/clade.
· Estimates of ancient gene flow during divergence, e.g., low gene flow indicating deep divergence.
· Evidence of outbreeding depression or stable hybrid zones when reproductively isolated populations become connected.
· Genetic differentiation (F ST or similar) for genetic variants identified as deleterious (currently based on genomic data with inferred effect prediction from reference genomes).

• Reciprocal Monophyly
• Molecular Estimate of Divergence Time

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Evidence through (multiple can apply): This refers to the evolutionary or adaptive uniqueness present within a single species. Specifically, where there is robust evidence of a focal population(s) harboring unique local adaptation(s). Adaptations are driven by selection including environmental (e.g. differences in the local climate or conditions) and sexual selection (e.g. mate choice), and can be recent (e.g. to climate change, pests/parasites, pollution). Separate ESUs should only be recognized if local adaptation is coupled with some level of restricted (limited but not necessarily absent) gene flow between the focal subpopulation and other subpopulations within the species that do not harbor the same form of the local adaptation i.e., an ESU cannot be defined if all focal populations show the same local adaptation.

Examples of Suitable data:
· Robust genomic evidence of local adaptation in focal subpopulation(s) (i.e., FST outliers, Gene environment associations “GEA”, ratio of non-synonymous to synonymous substitutions “dN/dS”.
· Evidence of hybrid zones where candidate ESUs make geographic contact that consist of individuals with intermediate phenotypes.

• Genomic signals of local adaptation
• Evidence from hybrid zones

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Recorded biological evidence

Evidence through (multiple can apply): Consistent heritable differences in life history or ecologically/species important traits (e.g., body size, color, breeding time, use of spawning grounds) observed in common garden experiment/common environment. Please note, that artificially bred or selected populations (non-natural trait levels by humans (e.g., antler size, unique coat colors etc.)) are not included here.

Examples of Suitable data:
· Distinctive “body types”, i.e., morphological or functional ecomorphs, that reflect morphological adaptations of a species to its (local) niche.
· Statistically supported heritable differences in phenotypes (e.g., traits measured in a common garden experiment).
· Statistically significant evidence of local adaptation in traits demonstrated by quantitative genetic analyses (e.g., common garden experiments/population crosses/animal models).

• Heritable differences in focal traits observed in experiments or confirmed by analytical tests

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Evidence through (multiple can apply): Candidate ESUs show consistent differences in traits that are locally transmitted but not robustly shown to be heritable (acquired behavioral or phenotypic differences; environmentally modified traits)
Examples of Suitable data:
· Different heritable phenotypes (e.g., from a common garden experiment) that are statistically significant between ESUs.

• Cultural or learnt behavioral differences unique or specific between units
  
• Acquired or transmitted traits (e.g., migration pattern, methylation differences, body size differences)

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Evidence through (multiple can apply): A population that is considered unique based on indigenous, local, or traditional knowledge. Information must be gathered according to the IUCN ILK framework (https://www.iucnredlist.org/resources/ilk). The IUCN ILK framework ensures that Indigenous and Local Knowledge is gathered ethically, accurately, and collaboratively. By combining scientific methods with participatory ILK approaches such as: Community Consultations & Participatory Workshops, Semi-Structured Interviews & Storytelling, Participatory Mapping & Seasonal Calendar, Indigenous-Led Biodiversity Monitoring, Review of Indigenous & Local Records.
Examples of Suitable data:
· Traditional knowledge (e.g., local differences in bird song or behavior, migration patterns and corridors between populations). Note that traditional knowledge evidence that is compiled and statistically analyzed, should be considered under the line of evidence: Is there evidence of (inherited) phenotypic differences?
· Compiled knowledge (e.g., assembled accounts from communities across the species range).

• Recorded differences based on traditional and local knowledge

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