Underestimated diversity: Cryptic species and phylogenetic relationships in the subgenus Cobalius (Coleoptera: Hydraenidae) from marine rockpools.

Marine rockpools are isolated patches of habitat in the supratidal environment (the so-called splash zone), at the transition between sea and land, found along the rocky shores worldwide and characterized by harsh conditions for life. Nonetheless, few specialized invertebrates successfully colonized this peculiar environment. Among them several members of the water beetles Ochthebius Leach, 1815, subgenus Cobalius Rey (1886), which are found almost exclusively in supratidal and upper-most intertidal marine rockpools from the eastern Atlantic Ocean (Cape Verde, Canary Islands and Morocco, Madeira, Azores) throughout the whole Mediterranean basin. The subgenus Cobalius before 2020 was considered to include ten valid species, based on morphological differences. In late 2020, four additional new species were described. However, recent molecular phylogenetic studies have uncovered further cryptic diversity suggesting the presence of multiple undetected species within this group, highlighting that the species boundaries remain unclear and systematics and taxonomy are in need of revision. In this study we provide a molecular phylogeny based on DNA sequence data from mitochondrial and nuclear genes obtained from ten described species belonging to the subgenus Cobalius, and extensive taxon sampling, in order to better understand the phylogenetic relationships within this genus and to infer the biogeographic processes behind its diversification. We also used a molecular clock to define a time window for diversification of distinct clades within the subgenus, and explore aspects of its evolutionary history. Finally, we used three species delimitation methods (PTP, GMYC and ABGD) to clarify taxonomy and validate species boundaries. Our phylogenetic and biogeographic results identified sixteen independent lineages grouped in four main clades and the possible origin of Cobalius was estimated to be in the Early Miocene (~22 Mya) in W Mediterranean area. Moreover, species delimitation methods suggest there are between 16 and 24 putative species, most of them diverged during the Late Miocene, Pliocene and Pleistocene (6.0-0.11 Mya).