Morphology, nuclearSNPsand mate selection reveal thatCOIbarcoding overestimates species diversity in a Mediterranean freshwater amphipod by an order of magnitude

Abstract

DNA sequence information has revealed many morphologically cryptic species worldwide. For animals, DNA‐based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, a growing amount of evidence indicate that mitochondrial markers alone can lead to misleading species diversity estimates due to mito‐nuclear discordance. Therefore, reports of putative species based solely on mitochondrial DNA should be verified by other methods, especially in cases where COI sequences are identical for different morphospecies or where divergence within the same morphospecies is high. Freshwater amphipods are particularly interesting in this context because numerous putative cryptic species have been reported. Here, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within Echinogammarus sicilianus. We used an integrative approach combining DNA barcoding with mate selection observations, detailed morphometrics and genome‐wide double digest restriction site‐associated DNA sequencing (ddRAD‐seq). Within a relatively small sampling area, we detected twelve COI MOTUs (divergence = 1.8–20.3%), co‐occurring in syntopy at two‐thirds of the investigated sites. We found that pair formation was random and there was extensive nuclear gene flow among the ten MOTUs co‐occurring within the same river stretch. The four most common MOTUs were also indistinguishable with respect to functional morphology. Therefore, the evidence best fits the hypothesis of a single, yet genetically diverse, species within the main river system. The only two MOTUs sampled outside the focal area were genetically distinct at the nuclear level and may represent distinct species. Our study reveals that COI‐based species delimitation can significantly overestimate species diversity, highlighting the importance of integrative taxonomy for species validation, especially in hyperdiverse complexes with syntopically occurring mitochondrial MOTUs.

Integrative taxonomic revision of the genera Nesticella and Howaia in Japan with the description of five new species (Araneae, Nesticidae, Nesticellini)

“…The taxonomic revision of the Japanese species is reported in the “Taxonomic account” section. The use of a single mitochondrial gene marker for species delimitation may overestimate the number of putative cryptic species (e.g., see Hupało et al 2022 ). This is probably the case with the bPTP analysis where several clades are resolved as split into putative species, often in contrast with previous taxonomic studies and with the results of the other methods used.…”

A new deep-sea species of Halirages Boeck, 1871 (Crustacea: Amphipoda: Calliopiidae) inhabiting sponges

“…Relying solely on genetic distances in the mitochondrial and strictly maternally inherited COI can overestimate species diversities in evolutionary old and thus genetically highly diverse and divergent species. For example, for the freshwater amphipod Echinogammarus sicilianus Karaman & Tibaldi, 1972, multiple genetically highly divergent lineages (exceeding 10% genetic distance in COI) were identified (Hupało et al 2022). However, nuclear data suggest that many of these divergent lineages are not reproductively isolated and thus belong to a single biological species (Hupało et al 2022).…”

Comparative phylogeography of Ponto-Caspian amphipods throughout the invaded Baltic and native NW Black Sea donor ranges – can introduction mode affect genetic diversity?

“…One the other hand, the investigated nuclear marker exhibited high levels of heterozygosity, likely reflecting the high heterozygosity and large genomes commonly encountered in amphipods (Rees et al, 2007; Kao et al, 2016; Jeffery et al, 2017). Nevertheless, given that we sequenced only one nuclear marker, these patterns should be studied further using reduced representation genomic approaches based on single nucleotide polymorphisms (SNPs) which have been proven useful in amphipods (Hupalo et al, 2022). It is likely that a genomic approach might still reveal a certain loss of nuclear genetic variation relative to the native range, but not at the same magnitude as observed for the mitochondrial genome.…”