An integrative taxonomy approach is used to study the diversity of the Brazilian aquatic pipid frog Pipa carvalhoi, evaluating whether within this nominal species two or more lineages are evolving independently. To test these competing scenarios, we analysed morphological (tadpoles and adults), molecular (16S rRNA) and karyological data for up to 109 specimens to check for corroborating patterns among characters evolving under different selective pressures. Three well-supported haplogroups were recovered and their distributions match major hydrographic basins: clade I occurs in the North-eastern Atlantic river basin, clade II in the São Francisco and East Atlantic river basins, and clade III in the South-eastern Atlantic river basin. A bPTP species delimitation analysis recovered three candidate species, congruent with the clades recovered in the molecular phylogenetics. Genetic distances among the three clades of P. carvalhoi were higher than the expected value for interspecific divergence in the genus recovered using LocalMinima. Despite the lack of differences with conventional cytogenetic markers, in situ fluorescence hybridization of microsatellite repeats shows clade-specific differences in the accumulation and distribution of the microsatellite motif (GACA)4 among karyotypes. The morphology of adults from different localities was similar, whereas the morphometric characters of tadpoles revealed morphological clusters that partially match the genetic structure. Our results show a strong genetic structure that indicates that P. carvalhoi is actually three cryptic species, apparently related to hydrographic basins. Additional data on nuclear molecular markers, fine-scale geographic distribution, and possibly other sources (acoustic and internal morphology) are needed to further test and potentially describe these lineages as distinct species.

中文翻译：

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水线以下：通过综合分类法揭示水生蛙类（Pipa carvalhoi）的神秘多样性

综合分类方法用于研究巴西水生蛙皮蛙 Pipa carvalhoi 的多样性，评估在这个名义物种中是否有两个或多个谱系独立进化。为了测试这些竞争场景，我们分析了多达 109 个样本的形态学（蝌蚪和成虫）、分子（16S rRNA）和核学数据，以检查在不同选择压力下进化的特征之间的确证模式。恢复了三个支持良好的单倍群，它们的分布与主要的水文盆地相匹配：进化枝 I 出现在东北大西洋河流域，进化枝 II 出现在圣弗朗西斯科和东大西洋流域，进化枝 III 出现在东南大西洋河流盆地. bPTP 物种定界分析恢复了三个候选物种，与分子系统发生学中恢复的进化枝一致。P. carvalhoi 的三个进化枝之间的遗传距离高于使用 LocalMinima 恢复的属中种间差异的预期值。尽管与传统的细胞遗传标记没有差异，但微卫星重复序列的原位荧光杂交显示了微卫星基序 (GACA)4 在核型之间的积累和分布的进化枝特异性差异。来自不同地区的成虫形态相似，而蝌蚪的形态特征揭示了与遗传结构部分匹配的形态簇。我们的结果显示了一个强大的遗传结构，表明 P. carvalhoi 实际上是三个隐秘物种，显然与水文盆地有关。