The two-toned pygmy squid ( Idiosepius pygmaeus, Streenstrup, 1881) is a small, tropical cephalopod that inhabits seagrass meadows and is known to attach to blades of seagrass using a specialised adhesive organ. A global decline in seagrass habitat due to anthropogenic disturbances may threaten the species that rely on seagrass meadows for shelter, foraging and other crucial activities. It is unclear if I. pygmaeus can utilise alternative habitats in the absence of seagrass. Here, we test the effect of habitat type, material and composition type, and the presence of conspecifics on the settlement preferences of I. pygmaeus. Individuals each underwent three separate experiments: 1) testing the preference between their known habitat, Zostera muelleri subsp. Capricorni and potential substitute, Sargassum, 2) testing the preference to attach to a natural or artificial structure of similar shape and size, and 3) testing the preference of identical habitats, one with a conspecific adhered to and one without. We found that I. pygmaeus did not have a strong preference for either Zostera or Sargassum. However, a strong preference was found for attachment to the natural structure over the artificial one. There was also strong evidence for the use of conspecific cues in habitat choice, with 75% of individuals selecting the habitat that contained a conspecific. As habitat loss is the leading cause of species extinction, the knowledge of habitat preferences for I. pygmaeus is vital in order to assess their risk of population decline. The findings of this study suggest that I. pygmaeus could utilise an alternative habitat in the presence of seagrass meadow reduction, which is under threat from human activity.

Globally, amphibian species have suffered drastic population declines over the past 40 years. Hundreds of species are now listed as Critically Endangered, with many of these considered “possibly extinct”. Most of these species are stream-dwelling frogs inhabiting remote, montane areas, where traditional surveys limit our ability to find remnant populations. Environmental DNA (eDNA) monitoring could revolutionize surveys for missing and endangered amphibian populations by enabling sampling of downstream sections to screen the vast upstream catchments. However, this is dependent on quantifying downstream detection probability and distances. Here we tested this in two endangered stream frogs (Litoria nannotis and L. lorica) that co-occur in a remote stream catchment, and for which we know precise downstream distributional limits from traditional surveys. Importantly, the last populations of L. lorica persist in this catchment; one small (~1,000 frogs) and one very small (~100 frogs). We conducted eDNA screening at set distances downstream from the populations (up to 22.8 km) using precipitation from two water volumes and via filtering, during moderately high and low flow conditions. During high flow, we reliably detected both species as far as 22.8 km downstream using the larger water volumes and filtering techniques. We could only detect the very small population of L. lorica immediately downstream. Detection success was higher at the sites further downstream for both species during high flow compared to low flow. Our downstream detection distances (> 20 km) suggest eDNA is a valuable tool for detecting rare stream amphibians. We provide recommendations on optimal survey techniques.