Jon Mallatt

Here I quantitatively review the literature on how fish gill morphology is affected by chemical and physical irritants in the surrounding water (e.g. various toxicants, extremes of temperature or pH). I catalogued histopathological gill lesions that were reported, and used statistics to explore how such lesions relate to the irritant-exposure conditions under which they occurred (specifically, to dose and class of irritant, to temperature, and to salinity of the surrounding water). Frequently recorded histopathologic lesions include changes in gill epithelium (lifting, necrosis, hyperplasia, hypertrophy, rupture), bulbing or fusing of gill lamellae, hypersecretion and proliferation of mucocytes, and changes in chloride cells and gill vasculature. I conclude that these lesions are largely nonspecific in nature, as each was detected under many different exposure conditions. The lesions are not entirely independent of exposure conditions, however, as my statistical analysis discerns these trends: (1) Most gill lesion types have been reported more frequently after lethal than after sublethal exposure to irritants. (2) Some lesions were more frequently detected in studies employing heavy metals than in studies using organic toxicants or other irritants; such lesions include necrosis and hypertrophy of gill epithelial cells, plus mucous hypersecretion. (3) Lifting of the branchial epithelium, the most commonly reported lesion, was reported more often in freshwater than in marine fish, suggesting that osmolarity of the ambient water influences this lesion. Little relation was found between recorded lesion frequencies and temperature. Following my statistical analysis, the etiology of irritant-induced gill lesions is considered. The nonspecificity of branchial alterations suggests that they primarily represent stereotyped physiological reactions of gills to stress, and many of them are logically considered defense responses. Some branchial alterations have been considered inflammatory, but I conclude that the literature cannot support that hypothesis. Ultrastructural studies have detected irritant-induced disruptions of branchial epithelial cells, including cytoplasmic vacuolization, autophagosomes and inclusions, loss of microvilli, and abnormal mitochondria and nuclei.

Although the small-subunit ribosomal RNA (SSU rRNA) gene is widely used in the molecular systematics, few large-subunit (LSU) rRNA gene sequences are known from protostome animals, and the value of the LSU gene for invertebrate systematics has not been explored. The goal of this study is to test whether combined LSU and SSU rRNA gene sequences support the division of protostomes into Ecdysozoa (molting forms) and Lophotrochozoa, as was proposed by Aguinaldo et al. (1997) (Nature 387:489) based on SSU rRNA sequences alone. Nearly complete LSU gene sequences were obtained, and combined LSU + SSU sequences were assembled, for 15 distantly related protostome taxa plus five deuterostome outgroups. When the aligned LSU + SSU sequences were analyzed by tree-building methods (minimum evolution analysis of LogDet-transformed distances, maximum likelihood, and maximum parsimony) and by spectral analysis of LogDet distances, both Ecdysozoa and Lophotrochozoa were indeed strongly supported (e.g., bootstrap values >90%), with higher support than from the SSU sequences alone. Furthermore, with the LogDet-based methods, the LSU + SSU sequences resolved some accepted subgroups within Ecdysozoa and Lophotrochozoa (e.g., the polychaete sequence grouped with the echiuran, and the annelid sequences grouped with the mollusc and lophophorates)-subgroups that SSU-based studies do not reveal. Also, the mollusc sequence grouped with the sequences from lophophorates (brachiopod and phoronid). Like SSU sequences, our LSU + SSU sequences contradict older hypotheses that grouped annelids with arthropods as Articulata, that said flatworms and nematodes were basal bilateralians, and considered lophophorates, nemerteans, and chaetognaths to be deuterostomes. The position of chaetognaths within protostomes remains uncertain: our chaetognath sequence associated with that of an onychophoran, but this was unstable and probably artifactual. Finally, the benefits of combining LSU with SSU sequences for phylogenetic analyses are discussed: LSU adds signal, it can be used at lower taxonomic levels, and its core region is easy to align across distant taxa-but its base frequencies tend to be nonstationary across such taxa. We conclude that molecular systematists should use combined LSU + SSU rRNA genes rather than SSU alone.

We investigated evolutionary relationships among deuterostome subgroups by obtaining nearly complete large-subunit ribosomal RNA (LSU rRNA)-gene sequences for 14 deuterostomes and 3 protostomes and complete small-subunit (SSU) rRNA-gene sequences for five of these animals. With the addition of previously published sequences, we compared 28 taxa using three different data sets (LSU only, SSU only, and combined LSU + SSU) under minimum evolution (with LogDet distances), maximum likelihood, and maximum parsimony optimality criteria. Additionally, we analyzed the combined LSU + SSU sequences with spectral analysis of LogDet distances, a technique that measures the amount of support and conflict within the data for every possible grouping of taxa. Overall, we found that (1) the LSU genes produced a tree very similar to the SSU gene tree, (2) adding LSU to SSU sequences strengthened the bootstrap support for many groups above the SSU-only values (e.g., hemichordates plus echinoderms as Ambulacraria; lancelets as the sister group to vertebrates), (3) LSU sequences did not support SSU-based hypotheses of pterobranchs evolving from enteropneusts and thaliaceans evolving from ascidians, and (4) the combined LSU + SSU data are ambiguous about the monophyly of chordates. No tree-building algorithm united urochordates conclusively with other chordates, although spectral analysis did so, providing our only evidence for chordate monophyly. With spectral analysis, we also evaluated several major hypotheses of deuterostome phylogeny that were constructed from morphological, embryological, and paleontological evidence. Our rRNA-gene analysis refutes most of these hypotheses and thus advocates a rethinking of chordate and vertebrate origins.