| Summary: | Abstract Background Extant lineages of sea spiders (Pycnogonida) exhibit different types of development. Most commonly, pycnogonids hatch as a minute, feeding protonymphon larva with subsequent anamorphic development. However, especially in cold water habitats at higher latitudes and in the deep sea, some taxa have large, lecithotrophic larvae, or even undergo extended embryonic development with significantly advanced postlarval hatching stages. Similar biogeographic trends are observed in other marine invertebrates, often referred to as “Thorson’s rule”. Results To expand our knowledge on the developmental diversity in the most speciose pycnogonid genus Nymphon, we studied the developmental stages of the two tropical representatives N. floridanum and N. micronesicum., We compared classical scanning electron microscopy with fluorescence-based approaches to determine which imaging strategy is better suited for the ethanol-fixed material available. Both species show epimorphic development and hatch as an advanced, lecithotrophic postlarval instar possessing the anlagen of all body segments. Leg pairs 1–3 show a considerable degree of differentiation at hatching, but their proximal regions remain coiled and hidden under the cuticle of the hatching instar. The adult palp and oviger are not anteceded by three-articled larval limbs, but differentiate directly from non-articulated limb buds during postembryonic development. Conclusions Fluorescence imaging yielded more reliable morphological data than classical scanning electron microscopy, being the method of choice for maximal information gain from rare and fragile sea spider samples fixed in high-percentage ethanol. The discovery of epimorphic development with lecithotrophic postlarval instars in two small Nymphon species from tropical shallow-water habitats challenges the notion that this developmental pathway represents an exclusive cold-water adaptation in Nymphonidae. Instead, close phylogenetic affinities to the likewise more direct-developing Callipallenidae hint at a common evolutionary origin of this trait in the clade Nymphonoidea (Callipallenidae + Nymphonidae). The lack of functional palpal and ovigeral larval limbs in callipallenids and postlarval hatchers among nymphonids may be a derived character of Nymphonoidea. To further test this hypothesis, a stable and well-resolved phylogenetic backbone for Nymphonoidea is key.
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