The present study is the first to provide a phylogenetic reconstruction of the family Eleotridae based on multiple loci (mtDNA and nuDNA). Based on these results, we inferred phylogenetic hypotheses to shed light on the evolutionary history of freshwater and estuarine eleotrids, encompassing the evolutionary relationships among 48 species.
Miniaturization is a recurrent theme in evolutionary studies since this phenomenon involves processes related to the reduction of body size usually associated with remarkable changes in morphology, physiology, ecology, life history, behavior, and reproductive maturity of organisms25. From a genetic point of view, phylogenetic approaches can greatly contribute to unraveling the evolution of miniature species. For example, the phylogenetic position of the miniature genus Paedocypris, considered one of the smallest groups of vertebrates (standard length of 10–12 mm), was determined based on inferences from mitochondrial DNA (cytochrome b)36, who located Paedocypris as a sister group to the miniature species of the genus Sundadanio, both of which were found to be sister lineages and the other taxon within the family Cyprinidae. Later, 51provided a more consistent phylogenetic signal of this group based on six nuclear genes, where Paedocypris appears as a sister group to all cyprinids. Both reports indicated that the miniaturization processes have taken place independently.
In the case of Eleotridae, previous phylogenetic reconstructions based on mitochondrial genes corroborated Microphilypnus and the non-miniature genus Leptophilypnus as sister groups4,8,9,52 (Fig. 1). Here, we found strong support in the species tree that included the miniature species of Microphilypnus within the Neotropical clade Dormitator / Guavina / Gobiomorus / Hemieleotris (Fig. 2). Although we have no representatives of the miniaturized genus Leptophilypnion in our phylogeny, the miniaturization in Eleotridae has probably arisen, at least, in two moments during their evolution.
Even though the polyphyletic status of Microphilypnus and Leptophilypnus remains unknown, there are no synapomorphies described for both clades so far. Microphilypnus presents a suite of morphological features not found in Leptophilypnus and most other eleotrids such as reduction of pectoral-fin rays (11-15- vs. 15 or more), lateral ethmoid barely ossified and slender in frontal view (vs. ossification and cone-shaped in frontal view), and not ossified scapula in adults (vs. ossified). Leptophilypnus shares with Microphilypnus some reductive features like the slender infraorbital region and the lack of a series of infraorbital papillae absent. Nonetheless, features are likely to have evolved repeatedly in both lineages as the result of independent miniaturization events.
It is noteworthy that miniaturization events are often reported in Gobiiformes, suggesting a trend in this group towards the reduction of body size and loss of some morphological traits associated with miniature forms. Besides, the parallel adaptive evolution to similar microhabitats eventually leads to homoplasy, thus hindering the establishment of reliable phylogenetic relationships based only on morphology. On the other hand, the emergence of miniature and phylogenetically divergent groups supports our hypothesis that the miniaturization processes in Eleotridae represent independent evolutionary pathways.
Unfortunately, the phylogenetic position of Leptophilypnion, a recently described genus of Neotropical miniature eleotrids34, remains obscure. According to morphological traits, Leptophilypnion would be more related to Microphilypnus than to Leptophilypnus, by sharing some features such as the reduction in the number of scales and pectoral fins. Nonetheless, Leptophilypnion is distinguished by the presence of elongated pelvic fin rays, five branchiostegal rays (vs. six in other eleotrids), and additional unusual characters in skeleton34, Therefore, further information is needed to resolve the evolutionary relationships between species of these groups.
Eleotris corresponds to the only genus of Eleotridae that is widely distributed in different biogeographic areas, from the Neotropics, Africa, Indo-Pacific to Oceania. Differently from the reports by4–8, which found a close relationship between Eleotris amblyopsis and Eleotris fusca, the new taxa included in our phylogenetic analyses indicated that Eleotris amblyopsis is the sister species of Eleotris sp. 2, a newly discovered lineage in Nothern coast of Brazil3. We also recovered B. gyrinoides and C. godeffroyi within the clade Eleotris/Erotelis. Both species are distributed in the Indo-Pacific region and have a disjunct range when compared to the Neotropical genera Eleotris and Erotelis. Based on the presence of 10 + 15 vertebrae and pterygiophores in the first dorsal fin arranged in combination 3 (1221), the genera Eleotris, Erotelis and Calumia had been referred to the group “Eleotris”53, which also includes freshwater and estuarine species of Belobranchus from Indo-Pacific. Yet, robust approaches to recover the evolutionary diversification and their relationships with past environmental conditions are required to elucidate the intriguing evolutionary history of such genera of eleotrids. For example, the role of dispersal and/or vicariance events on the distribution and phylogeographic structure of these species should be carefully investigated.
Our data revealed a close phylogenetic relationship between the genera Guavina and Dormitator, which has also been reported in the previous studies4,8,9. Morphological evidence also supports these results inasmuch as both genera share one unambiguous synapomorphy first two hemal spines curved, arched; see53. On the other hand, D. latifrons and D. maculatus were not recovered as sister species, thus differing from previous reports5,8,52. The inclusion of new species in this study, i.e., D. lebretonis and D. cubanus resulted in a close relationship between species from the Atlantic Ocean, following the same trend observed in the diversification of Eleotris, in which the Atlantic species (D. maculatus, D. cubanus and D. lebretonis) form a monophyletic group. Therefore, our results corroborate the previous inference by39.
According to the present molecular analyses, Gobiomorus is paraphyletic in relation to H. latifasciata, as also indicated by8. Gobiomorus dormitor (Western Atlantic) and G. polylepis (Eastern Pacific) are also sister species, representing a didactic example of geminate species that diverged after the formation of the Isthmus of Panama. The origin of Eleotridae dates to Eocene (55.6 My), but their ancestral area remains unknown because sister groups to this family were not included in this study. Our phylogenetic analysis successfully recovered the species from Eastern Pacific and Western Atlantic (Erotelis armiger / E. smaragdus; Guavina guavina / G. micropus; Gobiomorus polylepis / G. dormitor; Leptophilypnus fluviatilis / L. panamesis) as sister groups, similarly to the results obtained by52. The time-calibrated phylogeny showed that the lineages diverged before the formation of the Isthmus of Panama 3.1 My;54. However, the most recent speciation events (1.4 Ma) occurred between D. cubanus, endemic to Cuba, and D. lebretonis from Eastern-Central Atlantic.
Regarding the miniaturized clade Microphilypnus, the estimate of divergence time of the clade Dormitator / Guavina was approximately 16.6 Mya. 55considered M. ternetzi, Dormitator and Eleotris as marine-derived taxa, representing an endemic remnant of ancient radiations to Neotropical freshwater habitats. This result is in accordance with the origin of freshwater lineages from marine ancestors driven by sea level fluctuations in South America coast during Cretaceous-Eocene55,56,57. Thacker Many clades, such as Plagioscion (Sciaenidae), Jurengraulis and Anchovia (Engraulidae), and Pseudotylosurus (Belonidae), have probably evolved from marine lineages by the connections formed between the Caribbean Sea and the Upper Amazon basin during this period via Los Llanos basin and Pebas Lake in Venezuela58,59,60,61,62.
Similarly, the paraphyletic status of Gobiomorphus in relation to Hemieleotris herein observed agrees with other reports. However, we suggest caution before the synonymization of these lineages since unambiguous synapomorphies for the clade Hemieleotris and G. polylepis /G. dormitor have not been described. In this context, subsequent radiations throughout the South American basins determined a profusion of morphologically and ecologically distinct species not seen in marine habitats63. Exploring this biogeographic background is highly recommended to understand the miniaturization process in Eleotridae. In fact, smaller body sizes in freshwater taxa when compared to marine forms have been widely reported, with explanations ranging from the advantages of reduced size in offering greater maneuverability in structured environments64 to the reduction of energetic demands in size-constrained or complex microhabitats28.
In summary, these data provide the most complete hypothesis for Eleotridae phylogeny to date, because it includes representatives from several biogeographical regions. Our results were based on evolutionary information from mitochondrial and nuclear genes, and then, revealed a novel phylogenetic relationship from previous studies based only on mtDNA. The miniaturization does not seem to be a frequent event in Eleotridae, because the miniature taxa evolved in at least two genera (Microphilypnus and Leptophilypnion). As a result, we propose that miniaturization is an evolutionary process in the genus Microphilypnus with a strongly supported sister group relationship between Microphilypnus and the neotropical genus Guavina, Dormitator and Gobiomorus. As the position of Leptophilypnion was not established in the phylogeny, we cannot affirm the close relationship between the miniature taxa. Thus, more extensive taxonomic and geographical sampling and analysis based on multi loci may reveal whether this event is exclusively part of a clade. The non-miniature genus Leptophilypnus was often considered to be a sister group of the Microphilypnus, however, our results are consistent with the hypothesis that both lineages evolved independently.