Most anurans possess a tympanic middle ear (TME) that transmits sound waves to the inner ear; however, numerous species lack some or all TME components. To understand the evolution of these structures, we undertook a comprehensive assessment of their occurrence across anurans and performed ancestral character state reconstructions. Our analysis indicates that the TME was completely lost at least 38 independent times in Anura. The inferred evolutionary history of the TME is exceptionally complex in true toads (Bufonidae), where it was lost in the most recent common ancestor, preceding a radiation of >150 earless species. Following that initial loss, independent regains of some or all TME structures were inferred within two minor clades and in a radiation of >400 species. The reappearance of the TME in the latter clade was followed by at least 10 losses of the entire TME. The many losses and gains of the TME in anurans is unparalleled among tetrapods. Our results show that anurans, and especially bufonid toads, are an excellent model to study the behavioural correlates of earlessness, extratympanic sound pathways, and the genetic and developmental mechanisms that underlie the morphogenesis of TME structures.

We evaluate the monophyly and phylogenetic relationships of the Pristimantis leptolophus species group and describe its external morphology, osteology, and some myological characteristics. We also compare the P. leptolophus species group to other related species groups. The P. leptolophus group is not monophyletic due to the inclusion of P. acatallelus, formerly believed to be part of the P. devillei group. The revised P. leptolophus group is composed of nine named species and six unnamed species. Based on our results, we recognize a new species group, the P. boulengeri species group, composed of eight species, many of which were previously assigned to the P. lacrimosus species group.

Vocal sacs are present in most species of frogs and are the product of the interaction of three elements: the gular skin, the superficial submandibular musculature and an internal mucosa derived from the buccal floor. In this paper, we surveyed the structural diversity in the vocal sac of microhylids and related families in 109 exemplar species, including 11 of the 13 currently recognized subfamilies. We defined five characters related to anatomical and histological properties of the m. interhyoideus, as well as the relationship of this muscle and the vocal sac internal mucosa. We describe a vocal sac configuration characterized by highly abundant elastic fibres in association with muscle fibres, a very unusual structure in animal tissues. We discuss the evolution of novel structures in a phylogenetic context and identify new synapomorphies for Microhylidae and internal clades. Furthermore, we comment on the functional implications that these features have in vocal sac inflation.

SUMMARYLysophosphatidylcholine (LPC) is a major bioactive lipid that is enzymatically generated by phospholipase A 2 (PLA 2 ). Previously, we showed that LPC is present in the saliva of the blood-sucking hemipteran Rhodnius prolixus and modulates cell-signaling pathways involved in vascular biology, which aids blood feeding. Here, we show that the saliva of the predator insect Belostoma anurum contains a large number of lipids with LPC accounting for 25% of the total phospholipids. A PLA 2 enzyme likely to be involved in LPC generation was characterized. The activity of this enzyme is 5-fold higher in Belostoma saliva than in other studied hemipterans, suggesting a close association with the predator feeding habits of this insect. Belostoma employs extra-oral digestion, which allows for ingestion of larger prey than itself, including small vertebrates such as amphibians and fish. Therefore, prey immobilization during digestion is essential, and we show here that Belostoma saliva and B. anurum saliva purified LPC have paralytic activity in zebrafish. This is the first evidence that lysophospholipids might play an important role in prey immobilization, in addition to contributing to blood feeding, and might have been an evolutionary acquisition that occurred long before the appearance of hematophagy in this animal group.Key words: Belostoma, extra-oral digestion, lysophosphatidylcholine, paralysis, phospholipase A2, saliva. THE JOURNAL OF EXPERIMENTAL BIOLOGY 33062003), and have described the dual anti-hemostatic activity of LPC that inhibits platelet aggregation and induces NO production in endothelial cells. Belostoma saliva enables different feeding strategies when compared with hematophagous hemipterans from the Reduviidae family. After catching prey, Belostoma inserts its piercing mouthparts to inject saliva and liquefy the prey tissues. Accordingly, Belostoma saliva must contain an array of digestive enzymes. Their saliva is white and viscous, indicating that it may contain an unusual amount of lipids. In the present study, we demonstrate for the first time the presence of lipids and bioactive lysophospholipids in the saliva of a heteropteran predator, B. anurum. We show that these compounds are toxic to neurons in culture, can block neurally evoked twitch contractions, and can paralyze and kill zebrafish (Dario rerio). MATERIALS AND METHODS Belostoma anurum salivaAdult specimens of Belostoma anurum were collected from Picinguaba (São Paulo, Brazil). The belostomatids were kept, under laboratory conditions, in tanks with 200 ml of non-chlorinated tap water and provided with synthetic floats. Aedes aegypti fifth instar larvae and Biomphalaria glabrata served as food. The saliva was obtained from the proboscis by means of electrical stimulation using an isolated pulse stimulator (Model 2100 A-M Systems, Sequim, WA, USA) at the insertion of the first pair of legs to the thorax (50-100V, 3-4Hz, and square pulses of 3ms duration), as previously described (Dan et al., 1993). Lipid analysisSamples of B. anurum s...