Hilary A. Smith

Extensive introgression in a malaria vector species complex revealed by phylogenomics

Introduction The notion that species boundaries can be porous to introgression is increasingly accepted. Yet the broader role of introgression in evolution remains contentious and poorly documented, partly because of the challenges involved in accurately identifying introgression in the very groups where it is most likely to occur. Recently diverged species often have incomplete reproductive barriers and may hybridize where they overlap. However, because of retention and stochastic sorting of ancestral polymorphisms, inference of the correct species branching order is notoriously challenging for recent speciation events, especially those closely spaced in time. Without knowledge of species relationships, it is impossible to identify instances of introgression. Rationale Since the discovery that the single mosquito taxon described in 1902 as Anopheles gambiae was actually a complex of several closely related and morphologically indistinguishable sibling species, the correct species branching order has remained controversial and unresolved. This Afrotropical complex contains the world’s most important vectors of human malaria, owing to their close association with humans, as well as minor vectors and species that do not bite humans. On the basis of ecology and behavior, one might predict phylogenetic clustering of the three highly anthropophilic vector species. However, previous phylogenetic analyses of the complex based on a limited number of markers strongly disagree about relationships between the major vectors, potentially because of historical introgression between them. To investigate the history of the species complex, we used whole-genome reference assemblies, as well as dozens of resequenced individuals from the field. Results We observed a large amount of phylogenetic discordance between trees generated from the autosomes and X chromosome. The autosomes, which make up the majority of the genome, overwhelmingly supported the grouping of the three major vectors of malaria, An. gambiae, An. coluzzii, and An. arabiensis. In stark contrast, the X chromosome strongly supported the grouping of An. arabiensis with a species that plays no role in malaria transmission, An. quadriannulatus. Although the whole-genome consensus phylogeny unequivocally agrees with the autosomal topology, we found that the topology most often located on the X chromosome follows the historical species branching order, with pervasive introgression on the autosomes producing relationships that group the three highly anthropophilic species together. With knowledge of the correct species branching order, we are further able to uncover introgression between another species pair, as well as a complex history of balancing selection, introgression, and local adaptation of a large autosomal inversion that confers aridity tolerance. Conclusion We identify the correct species branching order of the An. gambiae species complex, resolving a contentious phylogeny. Notably, lineages leading to the principal vectors of human malaria were among the first in the complex to radiate and are not most closely related to each other. Pervasive autosomal introgression between these human malaria vectors, including nonsister vector species, suggests that traits enhancing vectorial capacity can be acquired not only through de novo mutation but also through a more rapid process of interspecific genetic exchange. Time-lapse photographs of an adult anopheline mosquito emerging from its pupal case. RELATED ITEMS IN ScienceD. E. Neafsey et al., Science 347, 1258522 (2015) Introgressive hybridization is now recognized as a widespread phenomenon, but its role in evolution remains contested. Here, we use newly available reference genome assemblies to investigate phylogenetic relationships and introgression in a medically important group of Afrotropical mosquito sibling species. We have identified the correct species branching order to resolve a contentious phylogeny and show that lineages leading to the principal vectors of human malaria were among the first to split. Pervasive autosomal introgression between these malaria vectors means that only a small fraction of the genome, mainly on the X chromosome, has not crossed species boundaries. Our results suggest that traits enhancing vectorial capacity may be gained through interspecific gene flow, including between nonsister species. Mosquito adaptability across genomes Virtually everyone has first-hand experience with mosquitoes. Few recognize the subtle biological distinctions among these bloodsucking flies that render some bites mere nuisances and others the initiation of a potentially life-threatening infection. By sequencing the genomes of several mosquitoes in depth, Neafsey et al. and Fontaine et al. reveal clues that explain the mystery of why only some species of one genus of mosquitoes are capable of transmitting human malaria (see the Perspective by Clark and Messer). Science, this issue 10.1126/science.1258524 and 10.1126/science.1258522; see also p. 27 Comparison of several genomes reveals the genetic history of mosquitoes’ ability to vector malaria among humans. [Also see Perspective by Clark and Messer]

Using the guild ratio to characterize pelagic rotifer communities

Ecological research is moving from a species-based to a functional-based approach to better understand the underlying principles that govern community dynamics. Studies of functional-based ecology, however, have been limited for zooplankton and particularly for rotifers. While rotifers show a variety of trophi types and coronal shapes, suggesting the importance of niche differentiation in their feeding strategy, relatively little is known of how this relates to rotifer dynamics. We used the guild ratio (GR′, a ratio of raptorial to microphagous species), an index based on a functional trait (i.e. feeding strategy), as a novel approach to rotifer dynamics. We extracted the seasonal GR′ by using seasonal trend decomposition and investigated similarities between study sites (Lake Washington, USA and Lake Caldonazzo, Italy) and its relation to cladocerans by cross-correlation analysis. Our study indicated that (i) raptorial and microphagous rotifers showed alternating dominance, and that raptorial rotifers and cladocerans had a synchronous pattern, (ii) the seasonal pattern of the GR′ was consistent across different sampling frequencies, and (iii) the GR′ was similar in both lakes. We interpreted these patterns as the general strength of the GR′: discernment of species–environment relationships and robustness across sampling regimes. The limitations of the GR′ (i.e. species identity is neglected, simplification of food preferences) can also be seen as its strong point: synthesis of multi-species patterns. In addition, the independence of GR′ from species-level identification and its potential to make use of datasets with infrequent sampling intervals and low taxon resolution could further support its innovative aspect.

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera)

BackgroundMate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating.ResultsA 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons.ConclusionOwing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation.

Exposure to dsRNA Elicits RNA Interference in Brachionus manjavacas (Rotifera)

RNA interference (RNAi) is a powerful technique for functional genomics, yet no studies have reported its successful application to zooplankton. Many zooplankton, particularly microscopic metazoans of phylum Rotifera, have unique life history traits for which genetic investigation has been limited. In this paper, we report the development of RNAi methods for rotifers, with the exogenous introduction of double-stranded RNA (dsRNA) through the use of a lipofection reagent. Transfection with dsRNA for heat shock protein 90, the membrane-associated progesterone receptor, and mitogen-activated protein kinase significantly increased the proportion of non-reproductive females. Additionally, a fluorescence-based lectin binding assay confirmed the significant suppression of four of six glycosylation enzymes that were targeted with dsRNA. Suppression of mRNA transcripts was confirmed with quantitative PCR. Development of RNAi for rotifers promises to enhance the ability for assessing genetic regulation of features critical to their life history and represents a key step toward functional genomics research in zooplankton.

Analysing threshold effects in the sexual dynamics of cyclically parthenogenetic rotifer populations

Sex in some cyclically parthenogenetic rotifers is triggered when a threshold population density is achieved. In Brachionus, accumulation of a mixis-inducing protein (MIP) that the rotifers excrete into the medium is the proximal signal. Models explicitly accounting for MIP dynamics suggest that either (1) a positive feedback of MIP concentration on MIP production or (2) a switching relationship between the proportion of sexual females and MIP concentration is needed to produce a threshold response of mixis to population density. In order to examine both possibilities, experiments were conducted to estimate the sexual response to a dilution series of medium containing the MIP. The resulting sexual response patterns are consistent with the existence of a threshold response of mixis, but are insufficient to reject one hypothesized mechanism for the threshold response in favor of the other. However, the observed smooth increase of sexual offspring with MIP concentration is consistent with the hypothesis of positive feedback on MIP production, and deserves further investigation.

Molecular evolution of the membrane associated progesterone receptor in the Brachionus plicatilis (Rotifera, Monogononta) species complex

Many studies have investigated physiological roles of the membrane associated progesterone receptor (MAPR), but little is known of its evolution. Marked variations in response to exogenous progesterone have been reported for four brachionid rotifer species, suggesting differences in progesterone signaling and reception. Here we report sequence variation for the MAPR gene in the Brachionus plicatilis species complex. Phylogenetic analysis of this receptor is compared with relatedness based on cytochrome c oxidase subunit 1 sequences. Nonsynonymous to synonymous site substitution rate ratios, amino acid divergence, and variations in predicted phosphorylation sites are examined to assess evolution of the MAPR among brachionid clades.

Genome-wide QTL mapping of saltwater tolerance in sibling species of Anopheles (malaria vector) mosquitoes

Although freshwater (FW) is the ancestral habitat for larval mosquitoes, multiple species independently evolved the ability to survive in saltwater (SW). Here, we use quantitative trait locus (QTL) mapping to investigate the genetic architecture of osmoregulation in Anopheles mosquitoes, vectors of human malaria. We analyzed 1134 backcross progeny from a cross between the obligate FW species An. coluzzii, and its closely related euryhaline sibling species An. merus. Tests of 2387 markers with Bayesian interval mapping and machine learning (random forests) yielded six genomic regions associated with SW tolerance. Overlap in QTL regions from both approaches enhances confidence in QTL identification. Evidence exists for synergistic as well as disruptive epistasis among loci. Intriguingly, one QTL region containing ion transporters spans the 2Rop chromosomal inversion that distinguishes these species. Rather than a simple trait controlled by one or a few loci, our data are most consistent with a complex, polygenic mode of inheritance.