Genome size, cell size, and the evolution of enucleated erythrocytes in attenuate salamanders

Within the salamander family Plethodontidae, five different clades have evolved high levels of enucleated red blood cells, which are extremely unusual among non-mammalian vertebrates. In each of these five clades, the salamanders have large genomes and miniaturized or attenuated body forms. Such a correlation suggests that the loss of nuclei in red blood cells may be related, in part, to the interaction between large genome size and small body size, which has been shown to have profound morphological consequences for the nervous and visual systems in plethodontids. Previous work has demonstrated that variation in both the level of enucleated cells and the size of the nuclear genome exists among species of the monophyletic plethodontid genus Batrachoseps. Here, we report extensive intraspecific variation in levels of enucleated red blood cells in 15 species and provide measurements of red blood cell size, nucleus size, and genome size for 13 species of Batrachoseps. We present a new phylogenetic hypothesis for the genus based on 6150 bp of mitochondrial DNA sequence data from nine exemplar taxa and use it to examine the relationship between genome size and enucleated red blood cell morphology in a phylogenetic framework. Our analyses demonstrate positive direct correlations between genome size, nucleus size, and both nucleated and enucleated cell sizes within Batrachoseps, although only the relationship between genome size and nucleus size is significant when phylogenetically independent contrasts are used. In light of our results and broader studies of comparative hematology, we propose that high levels of enucleated, variably sized red blood cells in Batrachoseps may have evolved in response to rheological problems associated with the circulation of large red blood cells containing large, bulky nuclei in an attenuate organism.     

The cytomorphic system of anucleate non-mammalian erythrocytes

Summary Cytomorphic structure was studied in erythrocytes ofBatrachoseps salamanders, a genus unique among non-mammalian vertebrates because most of the erythrocytes are anucleate. These anucleate erythrocytes are highly flattened, quite variable in size, and generally elliptical. All of them were found to contain marginal bands of microtubules (MBs), as observed in phase contrast and darkfield after Triton lysis. The MBs of larger cells typically twisted into figure-8 forms upon lysis. Whole mounts of the lysed anucleate cells consisted only of the MB plus a trans-MB network of material (TBM), as observed by electron microscopy. If lysis was carried out in the presence of 0.5 M KCl, all of the MBs circularized immediately and none were twisted. The network (TBM) was now missing, suggesting that it is needed for maintenance of MB ellipticity and plays a role in MB twisting. Small numbers of living anucleate erythrocytes were constricted in their mid-region, and others were pointed at one end. Correspondingly pointed MBs were observed after lysis, exhibiting a range of forms compatible with the mechanism proposed byEmmel (1924) in which the anucleate erythrocytes arise by amitotic division of nucleated ones.The results show that these erythrocytes retain the typical nonmammalian cytomorphic system, and are thus unlike those of adult mammals. The network component (TBM) is present even though nuclei are absent, making it unlikely that it functions simply to position the nucleus. The observations are consistent with the hypothesis that the flattened, elliptical shape of non-mammalian vertebrate erythrocytes is generated by TBM tension applied across the faces of the MB frame, and that excessive tension induced by lysis (without KCl) produces MB twisting.     

Extreme population subdivision throughout a continuous range: phylogeography of Batrachoseps attenuatus (Caudata: Plethodontidae) in western North America

Low-vagility species with deep evolutionary histories are key to our understanding of the biogeographical history of geologically complex areas, such as the west coast of North America. We present a detailed study of the phylogeography of the salamander Batrachoseps attenuatus (Caudata: Plethodontidae) using sequences of the mitochondrial gene cob from 178 individuals sampled from throughout the species' range. Sequences of three other mitochondrial genes (16S, cox1, nad4) and a nuclear gene (RAG-1) were used to investigate the deeper evolutionary history of the species. We found high levels of genetic diversity and deep divergences within a mostly continuous distribution, with five genetically well-differentiated and geographically structured mitochondrial DNA clades. Significant association between geographical and genetic distances within these clades suggests demographic stability, whereas Fu's FS tests suggest demographic expansions in three of them. Mantel tests identify two biogeographical barriers, the San Andreas Fault and the Sacramento-San Joaquin Delta, as important in the diversification of lineages. The timing of the main splitting events between intraspecific lineages was estimated by applying relaxed molecular clock methods combining several mutation rates and a fossil calibration. The earliest splitting events are old (Pliocene/Miocene), with more recent (Pleistocene) subdivisions in some clades. Disjunct populations distributed along the western foothills of the Sierra Nevada colonized this area relatively recently from a single refugium east of San Francisco Bay. The combination of fine-scale, comprehensive sampling with phylogenetic, historical demographic and hypothesis-based tests allowed delineation of a complex biogeographical scenario with general implications for the study of codistributed taxa.     

GEOGRAPHIC VARIATION AND PHENOTYPIC PLASTICITY OF NUMBER OF TRUNK VERTEBRAE IN SLENDER SALAMANDERS,BATRACHOSEPS (CAUDATA: PLETHODONTIDAE)

To understand the evolutionary significance of geographic variation, one must identify the factors that generate phenotypic differences among populations. I examined the causes of geographic variation in and evolutionary history of number of trunk vertebrae in slender salamanders, Batrachoseps (Caudata: Plethodontidae). Number of trunk vertebrae varies at many taxonomic levels within Batrachoseps. Parallel clines in number occur along an environmental gradient in three lineages in the Coast Ranges of California. These parallel clines may signal either adaptation or a shared phenotypically plastic response to the environmental gradient. By raising eggs from 10 populations representing four species of Batrachoseps, I demonstrated that number of trunk vertebrae can be altered by the developmental temperature; however, the degree of plasticity is insufficient to account for geographic variation. Thus, the geographic variation results largely from genetic variation. Number of trunk vertebrae covaries with body size and shape in diverse vertebrate taxa, including Batrachoseps. I hypothesize that selection for different degrees of elongation, possibly related to fossoriality, has led to the extensive evolution of number of trunk vertebrae in Batrachoseps. Analysis of intrapopulational variation revealed sexual dimorphism in both body shape and number of trunk vertebrae, but no correlation between these variables in either sex. Females are more elongate than males, a pattern that has been attributed to fecundity selection in other taxa. Patterns of covariation among different classes of vertebrae suggest that some intrapopulational variation in number results from changes in vertebral identity rather than changes in segmentation.