Protocol used in Evidence supporting cryptic species complexes within two sessile microinvertebrates, Limnias melicerta and L. ceratophylli (Rotifera, Gnesiotrocha) v1

Abstract

Sample collection and culture Sample collection and culture Aquatic plant samples were collected from habitats across the USA and a sediment sample from Australia (Tables S1.1, S1.2). Limnias melicerta and L. ceratophylli were identified and isolated from rehydrated sediments or by removing a piece of vegetation to which they were attached. Species identification was based on tube structure, shape of corona, antennae length, and the number of dorsal nodules [1]. Clonal lineages initiated from single females were cultured in modified MBL media [2] and fed a mixture of the algae Chlorella vulgaris Berijerinck, 1890 (The UTEX Culture Collection of Algae at the University of Texas at Austin [UTEX] strain 30) and Chlamydomonasreinhardtii Dangeard, 1888 (UTEX strain 90). Rotifers in this genus produce tubes of hardened secretions [3]; we added powdered carmine (Alfa Aesar, UK) to lab cultures to provide a supplementary matrix to aid tube construction and to increase their visibility in culture. Voucher specimens were deposited in the UTEP Biodiversity Collections at