Unraveling the complex phylogeographic history of freshwater fishes in Lower Central America: A study of the electric fish Brachyhypopomus occidentalis.

Lower Central America (LCA) has a complex biogeographic history shaped by the rise of the Isthmus of Panama and the global climatic oscillations of the Pleistocene. These events have been crucial in structuring biodiversity in LCA, but their consequences for the distribution and partitions of genetic diversity across the region remain to be elucidated. We combined complete mitochondrial genomes and nuclear ultraconserved elements (UCEs) to study the phylogeographic history and population genetic structure of the electric fish Brachyhypopomus occidentalis in LCA. Our results are consistent with the known phylogeographic history of B. occidentalis in LCA, but we update this history in several important ways that help illuminate the phylogeographic history of freshwater fishes in the region. We provide: i) support for three waves of colonization, two of which occurred prior to the final closure of the Panama Isthmus; ii) a more precise understanding of each colonization event, with evidence for a larger footprint of the first event, as well as genetic exchange across the continental divide in subsequent events; and iii) evidence for high levels of previously unrecognized population genetic structure across LCA. This updated model of colonization and diversification of B. occidentalis consists of three waves of dispersal and colonization, which triggered the evolution of geographic breaks in both nuclear and mitochondrial genomes across LCA. These processes are tightly linked to the dynamic uplift of the Isthmus, recent volcanic activity in the region, and the sea-level oscillations of the Pleistocene. These results improve previous phylogeographic inferences regarding the distribution and diversification of freshwater fishes in LCA, and generate testable hypotheses to guide future research exploring the factors shaping biodiversity in the region.

Comparative phylogeography reveals widespread cryptic diversity driven by ecology in Panamanian birds.

Widespread species often harbor unrecognized genetic diversity, and investigating the factors associated with such cryptic variation can help us better understand the forces driving diversification. Here, we identify potential cryptic species based on a comprehensive dataset of COI mitochondrial DNA barcodes from 2,333 individual Panamanian birds across 429 species, representing 391 (59%) of the 659 resident landbird species of the country, as well as opportunistically sampled waterbirds. We complement this dataset with additional publicly available mitochondrial loci, such as ND2 and cytochrome b, obtained from whole mitochondrial genomes from 20 taxa. Using barcode identification numbers (BINs), we find putative cryptic species in 19% of landbird species, highlighting hidden diversity in the relatively well-described avifauna of Panama. Whereas some of these mitochondrial divergence events corresponded with recognized geographic features that likely isolated populations, such as the Cordillera Central highlands, the majority (74%) of lowland splits were between eastern and western populations. The timing of these splits are not temporally coincident across taxa, suggesting that historical events, such as the formation of the Isthmus of Panama and Pleistocene climatic cycles, were not the primary drivers of cryptic diversification. Rather, we observed that forest species, understory species, insectivores, and strongly territorial species-all traits associated with lower dispersal ability-were all more likely to have multiple BINs in Panama, suggesting strong ecological associations with cryptic divergence. Additionally, hand-wing index, a proxy for dispersal capability, was significantly lower in species with multiple BINs, indicating that dispersal ability plays an important role in generating diversity in Neotropical birds. Together, these results underscore the need for evolutionary studies of tropical bird communities to consider ecological factors along with geographic explanations, and that even in areas with well-known avifauna, avian diversity may be substantially underestimated.

Especies extendidas frecuentemente tiene diversidad genética no reconocida, y investigando los factores asociados con esta variación críptica puede ayudarnos a entender las fuerzas que impulsan la diversificación. Aquí, identificamos especies crípticas potenciales basadas en un conjunto de datos de códigos de barras de ADN mitocondrial de 2,333 individuos de aves de Panama en 429 especies, representando 391 (59%) de las 659 especies de aves terrestres residentes del país, además de algunas aves acuáticas muestreada de manera oportunista. Adicionalmente, complementamos estos datos con secuencias mitocondriales disponibles públicamente de otros loci, tal como ND2 o citocroma b, obtenidos de los genomas mitocondriales completos de 20 taxones. Utilizando los números de identificación de código de barras (en ingles: BINs), un sistema taxonómico numérico que proporcina una estimación imparcial de la diversidad potencial a nivel de especie, encontramos especies crípticas putativas en 19% de las especies de aves terrestres, lo que destaca la diversidad oculta en la avifauna bien descrita de Panamá. Aunque algunos de estos eventos de divergencia conciden con características geográficas que probablemente aislaron las poblaciones, la mayoría (74%) de la divergencia en las tierras bajas se encuentra entre las poblaciones orientales y occidentales. El tiempo de esta divergencia no coincidió entre los taxones, sugiriendo que eventos históricos tales como la formación del Istmo de Panamá y los ciclos climáticos del pleistoceno, no fueron los principales impulsores de la especiación. En cambio, observamos asociaciones fuertes entre las características ecológicas y la divergencia mitocondriale: las especies del bosque, sotobosque, con una dieta insectívora, y con territorialidad fuerte mostraton múltiple BINs probables. Adicionalmente, el índice mano-ala, que está asociado a la capacidad de dispersión, fue significativamente menor en las especies con BINs multiples, sugiriendo que la capacidad de dispersión tiene un rol importamente en la generación de la diversidad de las aves neotropicales. Estos resultos demonstran la necesidad de que estudios evolutivos de las comunidades de aves tropicales consideren los factores ecológicos en conjunto con las explicaciones geográficos.

Interactions among Escovopsis, Antagonistic Microfungi Associated with the Fungus-Growing Ant Symbiosis.

Fungi in the genus Escovopsis (Ascomycota: Hypocreales) are prevalent associates of the complex symbiosis between fungus-growing ants (Tribe Attini), the ants' cultivated basidiomycete fungi and a consortium of both beneficial and harmful microbes found within the ants' garden communities. Some Escovopsis spp. have been shown to attack the ants' cultivated fungi, and co-infections by multiple Escovopsis spp. are common in gardens in nature. Yet, little is known about how Escovopsis strains impact each other. Since microbe-microbe interactions play a central role in microbial ecology and evolution, we conducted experiments to assay the types of interactions that govern Escovopsis-Escovopsis relationships. We isolated Escovopsis strains from the gardens of 10 attine ant genera representing basal (lower) and derived groups in the attine ant phylogeny. We conducted in vitro experiments to determine the outcome of both intraclonal and interclonal Escovopsis confrontations. When paired with self (intraclonal interactions), Escovopsis isolated from lower attine colonies exhibited antagonistic (inhibitory) responses, while strains isolated from derived attine colonies exhibited neutral or mutualistic interactions, leading to a clear phylogenetic pattern of interaction outcome. Interclonal interactions were more varied, exhibiting less phylogenetic signal. These results can serve as the basis for future studies on the costs and benefits of Escovopsis coinfection, and on the genetic and chemical mechanisms that regulate the compatibility and incompatibility observed here.

Hidden biodiversity in Neotropical streams: DNA barcoding uncovers high endemicity of freshwater macroinvertebrates at small spatial scales.

Aquatic macroinvertebrates play a crucial role in freshwater ecosystems, but their diversity remains poorly known, particularly in the tropics. This "taxonomic void" limits our understanding of biodiversity patterns and processes in freshwater ecosystems, and the scale at which they operate. We used DNA barcoding to estimate lineage diversity (and the diversity of unique haplotypes) in 224 specimens of freshwater macroinvertebrates at a small spatial scale within the Panama Canal Watershed (PCW). In addition, we compiled available barcoding data to assess macroinvertebrate diversity at a broader spatial scale spanning the Isthmus of Panama. Consistently across two species delimitation algorithms (i.e., ABGD and GMYC), we found high lineage diversity within the PCW, with ~ 100-106 molecular operational taxonomic units (MOTUs) across 168 unique haplotypes. We also found a high lineage diversity along the Isthmus of Panama, but this diversity peaked within the PCW. However, our rarefaction/extrapolation approach showed that this diversity remains under-sampled. As expected, these results indicate that the diversity of Neotropical freshwater macroinvertebrates is higher than previously thought, with the possibility of high endemicity even at narrow spatial scales. Consistent with previous work on aquatic insects and other freshwater taxa in this region, geographic isolation is likely a main factor shaping these patterns of diversity. However, other factors such as habitat variability and perhaps local adaptation might be reshaping these patterns of diversity at a local scale. Although further research is needed to better understand the processes driving diversification in freshwater macroinvertebrates, we suggest that Neotropical streams hold a high proportion of hidden biodiversity. Understanding this diversity is crucial in the face of increasing human disturbance.

Tempo and mode of allopatric divergence in the weakly electric fish Sternopygus dariensis in the Isthmus of Panama.

Spatial isolation is one of the main drivers of allopatric speciation, but the extent to which spatially-segregated populations accumulate genetic differences relevant to speciation is not always clear. We used data from ultraconserved elements (UCEs) and whole mitochondrial genomes (i.e., mitogenomes) to explore genetic variation among allopatric populations of the weakly electric fish Sternopygus dariensis across the Isthmus of Panama. We found strong genetic divergence between eastern and western populations of S. dariensis. Over 77% of the UCE loci examined were differentially fixed between populations, and these loci appear to be distributed across the species' genome. Population divergence occurred within the last 1.1 million years, perhaps due to global glaciation oscillations during the Pleistocene. Our results are consistent with a pattern of genetic differentiation under strict geographic isolation, and suggest the presence of incipient allopatric species within S. dariensis. Genetic divergence in S. dariensis likely occurred in situ, long after the closure of the Isthmus of Panama. Our study highlights the contribution of spatial isolation and vicariance to promoting rapid diversification in Neotropical freshwater fishes. The study of spatially-segregated populations within the Isthmus of Panama could reveal how genetic differences accumulate as allopatric speciation proceeds.

Mitogenomics of Central American weakly-electric fishes.

Electric fishes are a diverse group of freshwater organisms with the ability to generate electric organ discharges (EODs) that are used for communication and electrolocation. This group (ca. 200 species) has originated in South America, and six species colonized the Central American Isthmus. Here, we assembled the complete mitochondrial genomes (mitogenomes) for three Central American electric fishes (i.e. Sternopygus dariensis, Brachyhypopomus occidentalis, and Apteronotus rostratus), and, based on these data, explored their phylogenetic position among Gymnotiformes. The three mitogenomes show the same gene order, as reported for other fishes, with a size ranging from 16,631 to 17,093 bp. We uncovered a novel 60 bp intergenic spacer (IGS) located between the COII and tRNALys genes, which appears to be unique to the Apteronotidae. Furthermore, phylogenetic relationships supported the traditional monophyly of Gymnotiformes, with the three species positioned within their respective family. In addition, the genus Apteronotus belongs to the early diverging lineage of the order. Finally, we found high sequence divergence (13%) between our B. occidentalis specimen and a sequence previously reported in GenBank, suggesting that the prior mitogenome of B. occidentalis represents a different South American species. Indeed, phylogenetic analyses using Cytochrome b gene across the genus placed the previously reported individual within B. bennetti. Our study provides novel mitogenome resources that will advance our understanding of the diversity and phylogenetic history of Neotropical fishes.

Molecular Ecological Insights into Neotropical Bird-Tick Interactions.

In the tropics, ticks parasitize many classes of vertebrate hosts. However, because many tropical tick species are only identifiable in the adult stage, and these adults usually parasitize mammals, most attention on the ecology of tick-host interactions has focused on mammalian hosts. In contrast, immature Neotropical ticks are often found on wild birds, yet difficulties in identifying immatures hinder studies of birds' role in tropical tick ecology and tick-borne disease transmission. In Panama, we found immature ticks on 227 out of 3,498 individually-sampled birds representing 93 host species (24% of the bird species sampled, and 13% of the Panamanian land bird fauna). Tick parasitism rates did not vary with rainfall or temperature, but did vary significantly with several host ecological traits. Likewise, Neotropical-Nearctic migratory birds were significantly less likely to be infested than resident species. Using a molecular library developed from morphologically-identified adult ticks specifically for this study, we identified eleven tick species parasitizing birds, indicating that a substantial portion of the Panamanian avian species pool is parasitized by a diversity of tick species. Tick species that most commonly parasitized birds had the widest diversity of avian hosts, suggesting that immature tick species are opportunistic bird parasites. Although certain avian ecological traits are positively associated with parasitism, we found no evidence that individual tick species show specificity to particular avian host ecological traits. Finally, our data suggest that the four principal vectors of Rocky Mountain Spotted Fever in the Neotropics rarely, if ever, parasitize Panamanian birds. However, other tick species that harbor newly-discovered rickettsial parasites of unknown pathogenicity are frequently found on these birds. Given our discovery of broad interaction between Panamanian tick and avian biodiversity, future work on tick ecology and the dynamics of emerging tropical tick-borne pathogens should explicitly consider wild bird as hosts.

Extreme mitogenomic divergence between two syntopic specimens of Arremon aurantiirostris (Aves: Emberizidae) in central Panama suggests possible cryptic species.

We report the complete mitochondrial genome of two specimens of Orange-billed Sparrow Arremon aurantiirostris from Colón Province, in central Panama. The two specimens were collected on the same day, and at the same locality; however, they showed substantial divergence (6.3% average pairwise divergence among coding genes). A survey of ND2 sequence variation across Panama suggests that this divergence is the result of geographic differentiation and secondary contact. This high level of mitochondrial divergence among co-occurring individuals raises the possibility of multiple biological species in Orange-billed Sparrows. Our results are yet another demonstration that much remains to be discovered regarding avian biodiversity in Panama and throughout the Neotropics.

Complete mitochondrial genomes of the New World jacanas: Jacana spinosa and Jacana jacana.

The New World jacanas, Jacana spinosa (Mexico to Panama and also the West Indies) and Jacana jacana (Panama and South America), are polyandrous freshwater waders that are common throughout the Neotropics. These two species hybridize narrowly at their contact zone in Panama, and as part of a study of the hybrid zone dynamics, we present complete, annotated mitochondrial genomes for both species. The two species have very similar mitochondrial genomes, showing identical gene orders, and differing in size in only two RNA features and the control region, and among protein-coding genes, the two genomes had average uncorrected pairwise divergence of 1.8%, ranging from 0.7% for ND4L and 3.6% for ATP8. However, control region divergence is high (∼ 16%). These mitochondrial genome sequences may be useful tools for understanding jacana hybridization dynamics, especially regarding potential mitonuclear incompatibilities.

Mitochondrial genome organization of the Ochre-bellied Flycatcher, Mionectes oleagineus.

We sequenced and compared the mitogenome organization of two specimens of suboscine tyrant flycatcher Mionectes oleagineus from western and eastern Panama, representing distinct mtDNA clades. These samples show identical gene arrangement and vary in size by less than 5 base pairs. Both depict a non-standard avian gene order with an extra non-coding region (e.g. the remnant CR2), which differs in one base pair between them. Small size differences are also found on the control region and the 16S rRNA. Average uncorrected pairwise divergence among protein-coding genes (PCGs) was 2.8, ranging from 1.9% for COXIII and ND6 to 3.2% for ND2 and ATP6, respectively. These mitogenomes may be useful for understanding the evolutionary dynamics of gene order in bird mitochondrial genomes.

Extreme sequence divergence between mitochondrial genomes of two subspecies of White-breasted Wood-wren (Henicorhina leucosticta, Cabanis, 1847) from western and central Panamá.

Prior studies of mitochondrial variation in White-breasted Wood-Wrens (Henicorhina leucosticta) have suggested that populations in South American and Mesoamerica might represent multiple species. Here we report the complete mitochondrial genomes from two individuals of H. leucosticta, representing the Panamanian subspecies pittieri and alexandri. The two sequences were 16,721 and 16,726 base pairs in size with both genomes comprised of the usual 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes, and one displacement loop region in the standard avian order. Uncorrected pairwise divergence between mitogenome features was high, with the highest divergence occurring in protein-coding genes (average = 8.2%), followed by control region (6.7%). RNA features had lower pairwise divergences (average tRNA = 4.3%, average rRNA = 2.3%). The protein-coding ATPase 6 gene had a different stop codon between these two specimens. The high level of sequence variation between these subspecies suggests that Mesoamerican H. leucosticta might be comprised of multiple species. We urge a full phylogeographic survey of this widespread Neotropical forest bird.