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The morphological variation in the annual killifish Austrolebias luteoflamulatus was studied using meristic and pigmentation characters analysed with univariate and multivariate analysis, and morphometric characters analysed with a geometric morphometry approach (thin plate spline). The results showed that morphological variation in this species was associated with a geographical pattern. In the case of meristic and pigmentation characters, the strongest distinction occured in males, while females were more similar among different drainages. Males from Rocha (eastern Uruguay) exhibited the greatest differences from the others. The number of dorsal fin rays and bars on the flanks were the characters that contributed most to this segregation. When morphometrics were considered, the three populations analysed could be distinguished well from each other in both sexes. Some of the characters that contributed to this pattern were dorsal fin origin, position of eyes and length of snout. Since pigmentation characteristics and some characters of the dorsal fin in males were involved in the differences observed, sexual selection could have played an important role in the differentiation of populations of this species. Since Austrolebias luteoflamulatus has a restricted geographical distribution, conservation of this species should include consideration of these populations individually since they might represent different evolutionary units.