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The topographical, geological, climatic and biodiversity complexity of Mesoamerica has made it a primary research focus. The Mesoamerican highlands is a region with particularly high species richness and within-species variation. The Cinnamon-bellied Flowerpiercer, Diglossa baritula (Wagler, 1832), is a species endemic to the Mesoamerican highlands, with three allopatric subspecies currently recognized. To characterize divergence within this species, we integrated genomics, morphology, coloration and ecological niche modeling approaches, obtained from sampling individuals across the entire geographic distribution of the species. Our results revealed a clear genomic divergence between the populations to the east versus the west of the Isthmus of Tehuantepec. In contrast to the genomic results, morphology and coloration analyses showed intermediate levels of differentiation, indicating that population groups within D. baritula have probably been under similar selective pressures. Our morphology results indicated that the only sexually dimorphic morphological variable is the wing chord, with males having a longer wing chord than females. Finally, ecological data indicated that there are differences in ecological niche within D. baritula. Our data suggest that D. baritula could contain two or more incipient species at the intermediate phase of the speciation continuum. These results highlight the importance of the geographical barrier of the Isthmus of Tehuantepec and Pleistocene climatic events in driving isolation and population divergence in D. baritula. The present investigation illustrates the speciation potential of the D. baritula complex and the capacity of Mesoamerican highlands to create cryptic biodiversity and endemism.

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Sex determination systems and genetic sex differentiation across fishes are highly diverse but are unknown for most Cypriniformes, including Rio Grande silvery minnow (Hybognathus amarus). In this study, we aimed to detect and validate sex-linked markers to infer sex determination system and to demonstrate the utility of combining several methods for sex-linked marker detection in nonmodel organisms. To identify potential sex-linked markers, Nextera-tagmented reductively amplified DNA (nextRAD) libraries were generated from 66 females, 64 males, and 60 larvae of unknown sex. These data were combined with female and male de novo genomes from Nanopore long-read sequences. We identified five potential unique male nextRAD-tags and one potential unique male contig, suggesting an XY sex determination system. We also identified two single-nucleotide polymorphisms (SNPs) in the same contig with values of FST, allele frequencies, and heterozygosity conforming with expectations of an XY system. Through PCR we validated the marker containing the sex-linked SNPs and a single nextRAD-tag sex-associated marker but it was not male specific. Instead, more copies of this locus in the male genome were suggested by enhanced amplification in males. Results are consistent with an XY system with low differentiation between sex-determining regions. Further research is needed to confirm the level of differentiation between the sex chromosomes. Nonetheless, this study highlighted the power of combining reduced representation and whole-genome sequencing for identifying sex-linked markers, especially when reduced representation sequencing does not include extensive variation between sexes, either because such variation is not present or not captured.

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Genetic differentiation between and within natural populations is the result of the joint effects of neutral and adaptative processes. In addition, the spatial arrangement of the landscape promotes connectivity or creates barriers to gene flow, directly affecting speciation processes. In this study, we carried out a landscape genomics analysis using NextRAD data from a montane forest specialist bird complex, the Mesoamerican Chestnut-capped/Green-striped Brushfinch of the genus Arremon. Specifically, we examined population genomic structure using different assignment methods and genomic differentiation and diversity, and we tested alternative genetic isolation hypotheses at the individual level (e.g., isolation by barrier, IBB; isolation by environment, IBE; isolation by resistance, IBR). We found well-delimited genomic structuring (K = 5) across Mesoamerican montane forests in the studied group. Individual-level genetic distances among major montane ranges were mainly explained by IBR hypotheses in this sedentary Neotropical taxon. Our results uncover genetic distances/differentiation and patterns of gene flow in allopatric species that support the role of tropical mountains as spatial landscape drivers of biodiversity. IBR clearly supports a pattern of conserved niche-tracking of suitable habitat conditions and topographic complexity throughout glacial-interglacial dynamics.

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Mosquito susceptibility to Plasmodium spp. infection is of paramount importance for malaria occurrence and sustainable transmission. Therefore, understanding the genetic features underlying the mechanisms of susceptibility traits is pivotal to assessing malaria transmission dynamics in endemic areas. The aim of this study was to investigate the susceptibility of Nyssorhynchus darlingi-the dominant malaria vector in Brazil-to Plasmodium spp. using a reduced representation genome-sequencing protocol. The investigation was performed using a genome-wide association study (GWAS) to identify mosquito genes that are predicted to modulate the susceptibility of natural populations of the mosquito to Plasmodium infection. After applying the sequence alignment protocol, we generated the variant panel and filtered variants; leading to the detection of 202,837 SNPs in all specimens analyzed. The resulting panel was used to perform GWAS by comparing the pool of SNP variants present in Ny. darlingi infected with Plasmodium spp. with the pool obtained in field-collected mosquitoes with no evidence of infection by the parasite (all mosquitoes were tested separately using RT-PCR). The GWAS results for infection status showed two statistically significant variants adjacent to important genes that can be associated with susceptibility to Plasmodium infection: Cytochrome P450 (cyp450) and chitinase. This study provides relevant knowledge on malaria transmission dynamics by using a genomic approach to identify mosquito genes associated with susceptibility to Plasmodium infection in Ny. darlingi in western Amazonian Brazil.

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The Antarctic Circumpolar Current (ACC) dominates the open-ocean circulation of the Southern Ocean, and both isolates and connects the Southern Ocean biodiversity. However, the impact on biological processes of other Southern Ocean currents is less clear. Adjacent to the West Antarctic Peninsula (WAP), the ACC flows offshore in a northeastward direction, whereas the Antarctic Peninsula Coastal Current (APCC) follows a complex circulation pattern along the coast, with topographically influenced deflections depending on the area. Using genomic data, we estimated genetic structure and migration rates between populations of the benthic bivalve Aequiyoldia eightsii from the shallows of southern South America and the WAP to test the role of the ACC and the APCC in its dispersal. We found strong genetic structure across the ACC (between southern South America and Antarctica) and moderate structure between populations of the WAP. Migration rates along the WAP were consistent with the APCC being important for species dispersal. Along with supporting current knowledge about ocean circulation models at the WAP, migration from the tip of the Antarctic Peninsula to the Bellingshausen Sea highlights the complexities of Southern Ocean circulation. This study provides novel biological evidence of a role of the APCC as a driver of species dispersal and highlights the power of genomic data for aiding in the understanding of the influence of complex oceanographic processes in shaping the population structure of marine species.

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Extant species in the order Crocodylia are remnants of an ancient lineage of large-bodied archosaur reptiles. Despite decades of systematic studies, phylogenetic relationships among members of the genus Crocodylus (true crocodiles) in the Neotropics are poorly understood. Here we estimated phylogenomic relationships among the four extant Crocodylus species in the Americas. Species-tree reconstructions using genotypic data from 17,538 SNPs collected for 33 individuals spanning six Crocodylus species (four ingroup and two outgroup) revealed novel relationships for all Neotropical species. For the first time, C. acutus, the American crocodile, was recovered as monophyletic when individuals from Antillean and continental populations were analyzed together. Our results also contradict previous inferences based on mitochondrial DNA data and a limited number of nuclear markers by robustly grouping Morelet's crocodile (C. moreletii) as the sister species to C. acutus, suggesting a novel phylogeographic hypothesis for the group. The present study punctuates the importance of using nuclear genome-wide information and representative sampling for resolving phylogenetic relationships, especially in broadly distributed species and those with complex evolutionary histories.

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Bemisia tabaci is a cryptic species complex that requires the use of molecular tools for identification. The most widely used approach for achieving this is the partial sequencing of the mitochondrial DNA cytochrome oxidase I gene (COI). A more reliable single nucleotide polymorphism (SNP)-based genotyping approach, using Nextera restriction-site-associated DNA (NextRAD) sequencing, has demonstrated the existence of six major haplogroups of B. tabaci on cassava in Africa. However, NextRAD sequencing is costly and time-consuming. We, therefore, developed a cheaper and more rapid diagnostic using the Kompetitive Allele-Specific PCR (KASP) approach. Seven sets of primers were designed to distinguish the six B. tabaci haplogroups based on the NextRAD data. Out of the 152 whitefly samples that were tested using these primer sets, 151 (99.3%) produced genotyping results consistent with NextRAD. The KASP assay was designed using NextRAD data on whiteflies from cassava in 18 countries across sub-Saharan Africa. This assay can, therefore, be routinely used to rapidly diagnose cassava B. tabaci by laboratories that are researching or monitoring this pest in Africa. This is the first study to develop an SNP-based assay to distinguish B. tabaci whiteflies on cassava in Africa, and the first application of the KASP technique for insect identification.

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Environmental variation along the geographical space can shape populations by natural selection. In the context of global warming and changing precipitation regimes, it is crucial to understand the role of environmental heterogeneity in tropical trees adaptation, given their disproportional contribution to water and carbon biogeochemical cycles. Here, we investigated how heterogeneity in freshwater availability along tropical wetlands has influenced molecular variations of the black mangrove (Avicennia germinans). A total of 57 trees were sampled at seven sites differing markedly in precipitation regime and riverine freshwater inputs. Using 2,297 genome-wide single nucleotide polymorphic markers, we found signatures of natural selection by the association between variations in allele frequencies and environmental variables, including the precipitation of the warmest quarter and the annual precipitation. Additionally, we found candidate loci for selection based on statistical deviations from neutral expectations of interpopulation differentiation. Most candidate loci within transcribed sequences were functionally associated with central aspects of drought tolerance or plant response to drought. Moreover, our results suggest the occurrence of the rapid evolution of a population, probably in response to sudden and persistent limitations in plant access to soil water, following a road construction in 1974. Observations supporting rapid evolution included the reduction in tree size and changes in allele frequencies and in transcript expression associated with increased drought tolerance through the accumulation of osmoprotectants and antioxidants, biosynthesis of cuticles, protection against protein degradation, stomatal closure, photorespiration and photosynthesis. We describe a major role of spatial heterogeneity in freshwater availability in the specialization of this typically tropical tree.

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BACKGROUND: In contrast to traditional models of purifying selection and a single aposematic signal in Müllerian complexes, some communities of unprofitable prey contain members with multiple aposematic patterns. Processes responsible for diversity in aposematic signaling are poorly understood and large multi-species communities are seldom considered. RESULTS: We analyzed the phylogeny and aposematic patterns of closely related Eniclases net-winged beetles in New Guinea using mtDNA and nextRAD data. We suggest three clades of closely related and incompletely reproductively isolated lineages, detail the extent of polymorphism among Eniclases, and categorize their low-contrast aposematic patterns. The warning signal of Eniclases consists of body shape and color, with ambiguous color perception under some circumstances, i.e., when resting on the undersides of leaves. Field observations suggest that perception of the aposematic signal is affected by beetle behavior and environmental conditions. Local communities containing Eniclases consisted of 7-85 metriorrhynchine species assigned to 3-10 colour patterns. CONCLUSION: As a result, we suggest that under certain light conditions the aposematic colour signal is less apparent than the body shape in net-winged beetle communities. We document variable environmental factors in our study area and highly diverse multi-species communities of other net-winged beetles. Which implies dynamically changing community structure in space and time. Variable environmental conditions and diverse community composition are suggested to be favorable for the persistence of multiple aposematic patterns, imperfect mimics, and intraspecific polymorphism. Further research should identify the relative effect of these factors on purifying selection and the alleles which are responsible for phenotypic differences.

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The specific mechanisms that result in the success of any species invasion case are difficult to document. Reproductive strategies are often cited as a primary driver of invasive success, with human activities further facilitating invasions by, for example, acting as seed vectors for dispersal via road, train, air, and marine traffic, and by producing efficient corridors for movement including canals, drainages, and roadways. Sahara mustard (Brassica tournefortii) is a facultative autogamous annual native to Eurasia that has rapidly invaded the southwestern United States within the past century, displacing natives, and altering water-limited landscapes in the southwest. We used a genotyping-by-sequencing approach to study the population structure and spatial geography of Sahara mustard from 744 individuals from 52 sites across the range of the species' invasion. We also used herbaria records to model range expansion since its initial introduction in the 1920s. We found that Sahara mustard occurs as three populations in the United States unstructured by geography, identified three introduction sites, and combined herbaria records with genomic analyses to map the spread of the species. Low genetic diversity and linkage disequilibrium are consistent with self-fertilization, which likely promoted rapid invasive spread. Overall, we found that Sahara mustard experienced atypical expansion patterns, with a relatively constant rate of expansion and without the lag phase that is typical of many invasive species.

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The migration of anadromous fish in heterogenic environments unceasingly imposes a selective pressure that results in genetic variation for local adaptation. However, discrimination of anadromous fish populations by fine-scale local adaptation is challenging because of their high rate of gene flow, highly connected divergent population, and large population size. Recent advances in next-generation sequencing (NGS) have expanded the prospects of defining the weakly structured population of anadromous fish. Therefore, we used NGS-based restriction site-associated DNA (NextRAD) techniques on 300 individuals of an anadromous Hilsa shad (Tenualosa ilisha) species, collected from nine strategic habitats, across their diverse migratory habitats, which include sea, estuary, and different freshwater rivers. The NextRAD technique successfully identified 15,453 single nucleotide polymorphism (SNP) loci. Outlier tests using the FST OutFLANK and pcadapt approaches identified 74 and 449 SNPs (49 SNPs being common), respectively, as putative adaptive loci under a divergent selection process. Our results, based on the different cluster analyses of these putatively adaptive loci, suggested that local adaptation has divided the Hilsa shad population into two genetically structured clusters, in which marine and estuarine collection sites were dominated by individuals of one genetic cluster and different riverine collection sites were dominated by individuals of another genetic cluster. The phylogenetic analysis revealed that all the riverine populations of Hilsa shad were further subdivided into the north-western riverine (turbid freshwater) and the north-eastern riverine (clear freshwater) ecotypes. Among all of the putatively adaptive loci, only 36 loci were observed to be in the coding region, and the encoded genes might be associated with important biological functions related to the local adaptation of Hilsa shad. In summary, our study provides both neutral and adaptive contexts for the observed genetic divergence of Hilsa shad and, consequently, resolves the previous inconclusive findings on their population genetic structure across their diverse migratory habitats. Moreover, the study has clearly demonstrated that NextRAD sequencing is an innovative approach to explore how dispersal and local adaptation can shape genetic divergence of non-model anadromous fish that intersect diverse migratory habitats during their life-history stages.

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Recent theoretical advances have hypothesized a central role of habitat persistence on population genetic structure and resulting biodiversity patterns of freshwater organisms. Here, we address the hypothesis that lotic species, or lineages adapted to comparably geologically stable running water habitats (streams and their marginal habitats), have high levels of endemicity and phylogeographic structure due to the persistent nature of their habitat. We use a nextRAD DNA sequencing approach to investigate the population structure and phylogeography of a putatively widespread New Guinean species of diving beetle, Philaccolilus ameliae (Dytiscidae). We find that P. ameliae is a complex of morphologically cryptic, but geographically and genetically well-differentiated clades. The pattern of population connectivity is consistent with theoretical predictions associated with stable lotic habitats. However, in two clades, we find a more complex pattern of low population differentiation, revealing dispersal across rugged mountains and watersheds of New Guinea up to 430 km apart. These results, while surprising, were also consistent with the original formulation of the habitat template concept by Southwood, involving lineage-idiosyncratic evolution in response to abiotic factors. In our system, low population differentiation might reflect a young species in a phase of range expansion utilizing vast available habitat. We suggest that predictions of life history variation resulting from the dichotomy between lotic and lentic organisms require more attention to habitat characterization and microhabitat choice. Our results also underpin the necessity to study fine-scale processes but at a larger geographical scale, as compared to solely documenting macroecological patterns, to understand ecological drivers of regional biodiversity. Comprehensive sampling especially of tropical lineages in complex and threatened environments such as New Guinea remains a critical challenge.

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BACKGROUND: In Loreto Department, Peru, a successful 2005-2010 malaria control programme (known as PAMAFRO) included massive distribution of long-lasting insecticidal nets (LLINs). Additional local distribution of LLINs occurred in individual villages, but not between 2012 and 2015. A 2011-2012 study of the primary regional malaria vector Anopheles darlingi detected a trend of increased exophagy compared with pre-PAMAFRO behaviour. For the present study, An. darlingi were collected in three villages in Loreto in 2013-2015 to test two hypotheses: (1) that between LLIN distributions, An. darlingi reverted to pre-intervention biting behaviour; and, (2) that there are separate sub-populations of An. darlingi in Loreto with distinct biting behaviour. RESULTS: In 2013-2015 An. darlingi were collected by human landing catch during the rainy and dry seasons in the villages of Lupuna and Cahuide. The abundance of An. darlingi varied substantially across years, villages and time periods, and there was a twofold decrease in the ratio of exophagic:endophagic An. darlingi over the study period. Unexpectedly, there was evidence of a rainy season population decline in An. darlingi. Plasmodium-infected An. darlingi were detected indoors and outdoors throughout the night, and the monthly An. darlingi human biting rate was correlated with the number of malaria cases. Using nextRAD genotyping-by-sequencing, 162 exophagic and endophagic An. darlingi collected at different times during the night were genotyped at 1021 loci. Based on model-based and non-model-based analyses, all genotyped An. darlingi belonged to a homogeneous population, with no evidence for genetic differentiation by biting location or time. CONCLUSIONS: This study identified a decreasing proportion of exophagic An. darlingi in two villages in the years between LLIN distributions. As there was no evidence for genetic differentiation between endophagic and exophagic An. darlingi, this shift in biting behaviour may be the result of behavioural plasticity in An. darlingi, which shifted towards increased exophagy due to repellence by insecticides used to impregnate LLINs and subsequently reverted to increased endophagy as the nets aged. This study highlights the need to target vector control interventions to the biting behaviour of local vectors, which, like malaria risk, shows high temporal and spatial heterogeneity.

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PREMISE OF THE STUDY: The Andean highlands are a hotspot of domestication, yet our understanding of the origins of early Andean agriculture remains fragmentary. Key questions of where, when, how many times, and from what progenitors many Andean crops were domesticated remain unanswered. The Andean lupine crop tarwi (Lupinus mutabilis) is a regionally important pulse crop with exceptionally high seed protein and oil content and is the focus of modern breeding efforts, but its origins remain obscure. METHODS: A large genome-wide DNA polymorphism data set was generated using nextRADseq to infer relationships among more than 200 accessions of Andean Lupinus species, including 24 accessions of L. mutabilis and close relatives. Phylogenetic and demographic analyses were used to identify the likely progenitor of tarwi and elucidate the area and timing of domestication in combination with archaeological evidence. KEY RESULTS: We infer that tarwi was domesticated once in northern Peru, most likely in the Cajamarca region within, or adjacent to the extant distribution of L. piurensis, which is the most likely wild progenitor. Demographic analyses suggest that tarwi split from L. piurensis around 2600 BP and suffered a classical domestication bottleneck. The earliest unequivocal archaeological evidence of domesticated tarwi seeds is from the Mantaro Valley, central Peru ca. 1800 BP. CONCLUSIONS: A single origin of tarwi from L. piurensis in northern Peru provides a robust working hypothesis for the domestication of this regionally important crop and is one of the first clear-cut examples of a crop originating in the highlands of northern Peru.

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Conservation genomics has become an increasingly popular term, yet it remains unclear whether the non-invasive sampling that is essential for many conservation-related studies is compatible with the minimum requirements for harnessing next-generation sequencing technologies. Here, we evaluated the feasibility of using genotyping-by-sequencing of non-invasively collected hair samples to simultaneously identify and genotype single nucleotide polymorphisms (SNPs) in a climate-sensitive mammal, the American pika (Ochotona princeps). We identified and genotyped 3,803 high-confidence SNPs across eight sites distributed along two elevational transects using starting DNA amounts as low as 1 ng. Fifty-five outlier loci were detected as candidate gene regions under divergent selection, constituting potential targets for future validation. Genome-wide estimates of gene diversity significantly and positively correlated with elevation across both transects, with all low elevation sites exhibiting significant heterozygote deficit likely due to inbreeding. More broadly, our results highlight a range of issues that must be considered when pairing genomic data collection with non-invasive sampling, particularly related to field sampling protocols for minimizing exogenous DNA, data collection strategies and quality control steps for enhancing target organism yield, and analytical approaches for maximizing cost-effectiveness and information content of recovered genomic data.