The size and shape of red blood cells (RBCs) provide key information on life‐history strategies in vertebrates. However, little is known about how RBC shape evolved in response to environmental factors, body size and the role of evolutionary rate. Here, we analysed RBC morphometrics in a set of Teleostei (bony fishes) and Elasmobranchii (sharks and rays) species testing the hypothesis that phylogenetic relationship explains species occupation of morphospace. We collected data on cell and nucleus area and volume, nucleus:cytoplasm ratio and shape factor for 65 species belonging to 28 orders. Then, we built phylomorphospaces separately for bony fish and sharks and rays. To test whether phylogenetic relationships predicted phenotypic similarity, we calculated multivariate phylogenetic signal. We also estimated the evolutionary rate of RBC shape for each node and tip using ridge regression. Finally, we tested whether habitat and body size influenced RBC shape using a PGLS. We found a significant phylogenetic signal in RBC shape for bony fish, but not sharks and rays. Saltwater teleost species were more clustered than freshwater ones in the phylomorphospace, suggesting clade disparity. Accordingly, the rate of evolution was highly heterogeneous, with significant decrease in Acanthopterygii. Neither habitat nor body size influenced RBC shape. In conclusion, RBC shape seems to have evolved in fishes in response to multiple selective pressures independent of life‐history characters.

The size and shape of Red Blood Cells (RBC) can provide key information on life history strategies in vertebrates. However, little is known about how RBC shape evolved in response to environmental factors and the role of phylogenetic relationship. Here, we analyzed RBC morphometrics in a continental radiation of fishes testing the hypothesis that phylogenetic relationship determines species occupation of morphospace. We collected blood samples of five specimens of 15 freshwater fish species from six orders and used basic stereological methods to measure cell and nucleus area, perimeter, and diameter, cell and nucleus volume, nucleus:cytoplasm ratio, and shape factor of 50 cells per specimen. Then, we conducted a phylogenetic Principal Components Analysis using a dated phylogeny and built a phylomorphospace. To test if the phylogenetic relationship predicted the phenotypic similarity of species, we calculated multivariate phylogenetic signal. We also estimated the evolution rate of RBC shape for each node and tip using ridge regression. Finally, we tested if the position in the water column influenced RBC shape using a phylogenetic GLS. RBC shape seems to have evolved in a non-stationary way because the distribution pattern of species in the phylomorphospace is independent of the phylogeny. Accordingly, the rate of evolution for shape was highly heterogeneous, with an increase in the genus Pygocentrus. Water column position does not influence RBC shape. In conclusion, RBC shape seem to have evolved in response to multiple selective pressures independent of life history characters.

Hematological parameters can provide key information to an animal health status. However, this information is usually hard to obtain. Here, we described hematological parameters of Leptodactylus podicipinus in the Brazilian Pantanal. We measured red blood cell morphometrics, erythrogram, and leukogram. We also tested for phylogenetic signal in erythrogram and leukogram of 48 frog species from 15 families, testing if body size explains their variation. Lymphocytes were the most abundant leukocytes (> 60%) in Leptodactylus podicipinus, followed by neutrophils (∼10%). Given that L. podicipinus is an abundant and widely distributed species in central Brazil, knowing its hematological pattern can help stablish a baseline and improve its use as a bioindicator of environmental degradation. Mean corpuscular hemoglobin and value contributed more to the phylomorphospace of erythrogram, in which Leptodactylus spp. and Hypsiboas raniceps had lower values of these variables, while Bufotes viridis and Hyla arborea had high values. The phylogenetic signal was spread throughout the dimensions of leukogram phylomorphospace. The variables that most contributed to it were total leukocytes counts, lymphocytes and neutrophils. We also found a moderate phylogenetic signal for both the erythrogram and leukogram. Accordingly, body size accounted for a low proportion of variation in both the leukogram (4.7%) and erythrogram (0.57%). By combining phylogenetic comparative methods with hematological parameters, our results add a new perspective on the evolution of blood cell physiology in frogs.

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