Phylogenetic Assessment of Freshwater Mussels Castalia ambigua and C. inflata at an Ecotone in the Paraguay River Basin, Brazil Shows That Inflated and Compressed Shell Morphotypes Are the Same Species

Abstract

The phylogeny and taxonomy of freshwater mussels of the genus Castalia in South America is complicated by issues of morphological plasticity and limited molecular genetic data. We present field data on the distributions of the nominal Castalia ambigua and C. inflata in the upper Paraguay River basin in Brazil based on original occurrence data at 23 sample sites and on historical records. The upper basin has distinct highland and lowland regions, the latter including the Pantanal wetland, where “C. ambigua” occurs in the highlands and “C. inflata” occurs in both regions. At Baixo Stream in the highlands, we observed individuals with shell morphologies of either C. ambigua or C. inflata, and also individuals with intermediate shell morphology. DNA sequence variation in the upland Baixo Stream and two representative lowland populations were screened. Two mitochondrial and three nuclear genes were sequenced to test hypotheses regarding the number of species-level phylogenetic lineages present. Reported individual DNA sequences from Amazon-basin C. ambigua and other Castalia and outgroup species were included in the analysis as outgroups. Individuals from the Paraguay River basin exhibited 17 haplotypes at the mitochondrial cytochrome oxidase I (COI) gene and nine at mitochondrial 16S rRNA. Analysis of haplotype networks and phylogenetic trees of combined COI + 16S rRNA sequences among individuals with the respective shell morphologies supported the hypothesis that C. ambigua and C. inflata from the Paraguay River basin belong to the same species and one phylogenetic lineage. No variation was observed at the nuclear 18S rRNA internal transcribed spacer, 28S rRNA, or H3NR histone genes among individuals used in this study. Across all markers, less variation was observed among Paraguay basin populations than between Paraguay and Amazon basin populations. Our results collectively suggest that: (1) “C. ambigua”, “C. inflata”, and morphologically intermediate individuals within the upper Paraguay drainage represent one phylogenetic lineage, (2) a phylogeographic divide exists between Castalia populations occurring in the Paraguay and Amazon River basins, and (3) the evolutionary and taxonomic uncertainties that we have identified among Castalia species should be thoroughly assessed across their distribution using both morphological and molecular characters.

Relative importance of the environment and sexual dimorphism in determining shell shape in the Amazonian freshwater mussel Castalia ambigua (Unionida: Hyriidae) along a hydrological gradient

“…Mussels may have variation in shell features that are di cult to locate and quantify (Froufe et al, 2016), and shape analysis using outlines may deal effectively with these more subtle changes (Haines & Crampton, 2000). Shell shape variation in South American freshwater mussels has been generally evaluated using traditional linear descriptors (Mansur & Valer, 1992;Mansur et al, 1997;Olivera-Hyde et al, 2020). However, these approaches may mask ecophenotypic responses: e.g.…”

Conspicuous shell shape plasticity across lake-stream habitats in a freshwater mussel (Pyganodon grandis)

“…Importantly, an underlying process explaining this ecophenotypic pattern is unknown. Many integrative studies using morphometric, molecular phylogenetic, and/or population genetic tools have failed to find genetic correlation with ecophenotypic variation (Baker et al 2003; Cyr et al 2007; Inoue et al 2013; Olivera-Hyde et al 2020; Zieritz et al 2010) leading to phenotypic plasticity as the default explanation for all intraspecific morphological variation in freshwater mussels (Inoue et al 2013; Zieritz et al 2010; Hornbach et al 2010; Jeratthitikul et al 2019; Sheldon 2017; Wu et al 2022). Although these molecular studies provide compelling evidence supporting phenotypic plasticity, they do not sequence markers involved in shell production and thus genetic mechanisms (e.g.…”

Experimental evidence associates burrowing behavior of Castalia ambigua (Bivalvia: Hyriidae) with shell shape and density

“…South America has approximately 168 native species of freshwater mussels, with species of the genus Castalia Lamarck, 1819 (Unionida: Hyriidae) distributed across the main river basins (Olivera‐Hyde et al, 2020 ). In particular, the species C. ambigua is found in the Amazon, Paraguay, and Paraná River basins and has a restricted distribution in the Uruguay River basin (Pereira et al, 2014 ).…”

“…In addition, this species is mainly associated with areas of low hydrodynamic energy in river margins with poorly compacted sandy substrates and low silt deposition (Simeone et al, 2021c ). Molecular and linear morphometric analyses demonstrated that C. ambigua in the upper Paraguay River has phenotypic variation expressed as two morphotypes: one laterally compressed and another inflated (Olivera‐Hyde et al, 2020 ). In the eastern Amazon, Simeone et al ( 2022 ) using outline morphometric analyses, observed two co‐occurring morphotypes of C. ambigua in the Caeté River (Figure 1 ), that are shaped by sexual dimorphism in habitats with reduced hydraulic energy (Reynolds number of 855 ± 94.6): the first morphotype has an elongate posterior margin and is laterally compressed (male specimens), and the second has rounded anterior–posterior margins and is laterally inflated (female specimens).…”