Amphioxus Gli knockout disrupts the development of left-right asymmetry but has limited impact on neural patterning.

The Gli transcription factors are the primary mediators of Hedgehog (Hh) signaling. Vertebrate genomes contain multiple Gli paralogues with different functions downstream of Hh signal receipt, in part explaining the complexity of cellular responses to Hh that allow concentration-dependent target gene activation. Amphioxus is a chordate that split from the vertebrate lineage early in the evolution of chordates, before the genome duplications that occurred in early vertebrate evolution. It has a single Gli gene whose transcripts can be alternately spliced to yield two protein isoforms called GliS and GliL. We generated two knockout mutations in amphioxus Gli, one that affects the whole gene and a second that only affects GliL. Both knockouts showed major morphological and molecular defects in the development of left-right asymmetry, a phenotype that is similar but not identical to that previously found in Hh mutants. Hh signaling also patterns the amphioxus neural tube. Here, however, knockout of GliL showed no identifiable phenotype, while knockout of the full gene showed only small changes to the expression of one gene family, Olig. Other genes that were prominently affected by Hh knockout were not altered in expression in either knockout. Reasons for the differences between Hh and Gli knockouts in the pharynx and neural tube are discussed in the context of the likely different functions of amphioxus Gli isoforms. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00195-w.

Evolution of the expression and regulation of the nuclear hormone receptor ERR gene family in the chordate lineage.

The Estrogen Related Receptor (ERR) nuclear hormone receptor genes have a wide diversity of roles in vertebrate development. In embryos, ERR genes are expressed in several tissues, including the central and peripheral nervous systems. Here we seek to establish the evolutionary history of chordate ERR genes, their expression and their regulation. We examine ERR expression in mollusc, amphioxus and sea squirt embryos, finding the single ERR orthologue is expressed in the nervous system in all three, with muscle expression also found in the two chordates. We show that most jawed vertebrates and lampreys have four ERR paralogues, and that vertebrate ERR genes were ancestrally linked to Estrogen Receptor genes. One of the lamprey paralogues shares conserved expression domains with jawed vertebrate ERRγ in the embryonic vestibuloacoustic ganglion, eye, brain and spinal cord. Hypothesising that conserved expression derives from conserved regulation, we identify a suite of pan-vertebrate conserved non-coding sequences in ERR introns. We use transgenesis in lamprey and chicken embryos to show that these sequences are regulatory and drive reporter gene expression in the nervous system. Our data suggest an ancient association between ERR and the nervous system, including expression in cells associated with photosensation and mechanosensation. This includes the origin in the vertebrate common ancestor of a suite of regulatory elements in the 3' introns that drove nervous system expression and have been conserved from this point onwards.

Hmx gene conservation identifies the origin of vertebrate cranial ganglia.

The evolutionary origin of vertebrates included innovations in sensory processing associated with the acquisition of a predatory lifestyle1. Vertebrates perceive external stimuli through sensory systems serviced by cranial sensory ganglia, whose neurons arise predominantly from cranial placodes; however, the understanding of the evolutionary origin of placodes and cranial sensory ganglia is hampered by the anatomical differences between living lineages and the difficulty in assigning homology between cell types and structures. Here we show that the homeobox transcription factor Hmx is a constitutive component of vertebrate sensory ganglion development and that in the tunicate Ciona intestinalis, Hmx is necessary and sufficient to drive the differentiation programme of bipolar tail neurons, cells previously thought to be homologues of neural crest2,3. Using Ciona and lamprey transgenesis, we demonstrate that a unique, tandemly duplicated enhancer pair regulated Hmx expression in the stem-vertebrate lineage. We also show notably robust vertebrate Hmx enhancer function in Ciona, demonstrating that deep conservation of the upstream regulatory network spans the evolutionary origin of vertebrates. These experiments demonstrate regulatory and functional conservation between Ciona and vertebrate Hmx, and point to bipolar tail neurons as homologues of cranial sensory ganglia.

The evolutionary origins of the vertebrate olfactory system.

Vertebrates develop an olfactory system that detects odorants and pheromones through their interaction with specialized cell surface receptors on olfactory sensory neurons. During development, the olfactory system forms from the olfactory placodes, specialized areas of the anterior ectoderm that share cellular and molecular properties with placodes involved in the development of other cranial senses. The early-diverging chordate lineages amphioxus, tunicates, lampreys and hagfishes give insight into how this system evolved. Here, we review olfactory system development and cell types in these lineages alongside chemosensory receptor gene evolution, integrating these data into a description of how the vertebrate olfactory system evolved. Some olfactory system cell types predate the vertebrates, as do some of the mechanisms specifying placodes, and it is likely these two were already connected in the common ancestor of vertebrates and tunicates. In stem vertebrates, this evolved into an organ system integrating additional tissues and morphogenetic processes defining distinct olfactory and adenohypophyseal components, followed by splitting of the ancestral placode to produce the characteristic paired olfactory organs of most modern vertebrates.

Step-wise evolution of neural patterning by Hedgehog signalling in chordates.

In vertebrate embryos, Hedgehog (Hh) is expressed in some anterior basal plate domains and by notochord and floorplate cells, and ventral neural cells are patterned by the activities of Hh-regulated transcription factors. Hh signalling is antagonized by signals from the dorsal neural tube and loss of Hh leads to loss of ventral patterning as dorsal pattern expands. These mechanisms are critical for producing the neurons that implement motor responses to sensory inputs but understanding how they evolved has been hindered by lack of insight from commonly studied invertebrates where nervous system morphology and genetic mechanisms are non-conserved with vertebrates. The invertebrate chordate amphioxus, which expresses Hh in its notochord and floorplate, provides a window into the prevertebrate condition. We examined amphioxus neural development by manipulating Hh and downstream genes involved in neural pattern and cell identity. We show that Hh signalling regulates the differentiation of some neurons in amphioxus, including a subset of motor neurons. This demonstrates some conservation of mechanism between vertebrates and amphioxus. However, other aspects of neural patterning differ between the lineages. We suggest the complexity of Hh-dependent neural patterning in vertebrates evolved in a step-wise manner. Alongside other previously described regulatory changes, initial recruitment of Hh along the length of the axis occurred in an ancestor to the chordates to regulate the differentiation of a subset of neurons. This was followed, in the vertebrate lineage, by additional changes to the downstream gene regulatory network of transcription factors, giving Hh a broader role in dorsal-ventral neural patterning.

Evolution of vertebrate spinal cord patterning.

The vertebrate spinal cord is organized across three developmental axes, anterior-posterior (AP), dorsal-ventral (DV), and medial-lateral (ML). Patterning of these axes is regulated by canonical intercellular signaling pathways: the AP axis by Wnt, fibroblast growth factor, and retinoic acid (RA), the DV axis by Hedgehog, Tgfß, and Wnt, and the ML axis where proliferation is controlled by Notch. Developmental time plays an important role in which signal does what and when. Patterning across the three axes is not independent, but linked by interactions between signaling pathway components and their transcriptional targets. Combined this builds a sophisticated organ with many different types of cell in specific AP, DV, and ML positions. Two living lineages share phylum Chordata with vertebrates, amphioxus, and tunicates, while the jawless fish such as lampreys, survive as the most basally divergent vertebrate lineage. Genes and mechanisms shared between lampreys and other vertebrates tell us what predated vertebrates, while those also shared with other chordates tell us what evolved early in chordate evolution. Between these lie vertebrate innovations: genetic and developmental changes linked to evolution of new morphology. These include gene duplications, differences in how signals are received, and new regulatory connections between signaling pathways and their target genes.

Live Imaging of Cleavage Variability and Vesicle Flow Dynamics in Dextral and Sinistral Spiralian Embryos.

Spiral cleavage is a mode of embryonic cell division found in species from several Phyla, including molluscs, annelids and flatworms. It reflects a tilting in the direction of spindle orientation and cell division at the 4 to 8-cell stage, which may be dextral or sinistral, and propagates into later organismal asymmetry. Genetic analysis in a small number of gastropod molluscs shows the direction of spiral cleavage is determined by maternal genotype, though whether this is also the case more generally for spiralians, and whether spiral cleavage at the 4-8 cell stage is preceded by earlier internal chirality in any spiralian species, is unknown. Here we study the early cleavage stages of two equal-cleaving spiralians, the dextral annelid Spirobranchus lamarcki and the sinistral mollusc Biomphalaria glabrata, using light sheet microscopy to image subcellular vesicles in live embryos and asking if chirality of movement is identifiable. We observe variability in the early cleavage of S. lamarcki, including a viable 3-cell stage. Image data are analysed by both particle tracking and particle image velocimetry. Neither finds evidence for chiral movement in 1-, 2-, 3-, or 4-cell embryos, nor do we detect consistent differences between the embryos of the dextral and sinistrai species. The methodological and evolutionary implications of this are discussed.

DnaJ chaperones contribute to canalization.

Canalization, an intrinsic robustness of development to external (environmental) or internal (genetic) perturbations, was first proposed over half a century ago. However, whether the robustness to environmental stress (environmental canalization [EC]) and to genetic variation (genetic canalization) are underpinned by the same molecular basis remains elusive. The recent discovery of the involvement of two endoplasmic reticulum (ER)-associated DnaJ genes in developmental buffering, orthologues of which are conserved across Metazoa, indicates that the role of ER-associated DnaJ genes might be conserved across the animal kingdom. To test this, we surveyed the ER-associated DnaJ chaperones in the nematode Caenorhabditis elegans. We then quantified the phenotype, in the form of variance and mean of seam cell counts, from RNA interference knockdown of DnaJs under three different temperatures. We find that seven out of eight ER-associated DnaJs are involved in either EC or microenvironmental canalization. Moreover, we also found two DnaJ genes not specifically associated with ER (DNAJC2/dnj-11 and DNAJA2/dnj-19) were involved in canalization. Protein expression pattern showed that these DnaJs are upregulated by heat stress, yet not all of them are expressed in the seam cells. Moreover, we found that most of the buffering DnaJs also control lifespan. We therefore concluded that a number of DnaJ chaperones, not limited to those associated with the ER, are involved in canalization as a part of the complex system that underlies development.

A Notch-regulated proliferative stem cell zone in the developing spinal cord is an ancestral vertebrate trait.

Vertebrates have evolved the most sophisticated nervous systems we know. These differ from the nervous systems of invertebrates in several ways, including the evolution of new cell types, and the emergence and elaboration of patterning mechanisms to organise cells in time and space. Vertebrates also generally have many more cells in their central nervous systems than invertebrates, and an increase in neural cell number may have contributed to the sophisticated anatomy of the brain and spinal cord. Here, we study how increased cell number evolved in the vertebrate central nervous system, investigating the regulation of cell proliferation in the lamprey spinal cord. Markers of proliferation show that a ventricular progenitor zone is found throughout the lamprey spinal cord. We show that inhibition of Notch signalling disrupts the maintenance of this zone. When Notch is blocked, progenitor cells differentiate precociously, the proliferative ventricular zone is lost and differentiation markers become expressed throughout the spinal cord. Comparison with other chordates suggests that the emergence of a persistent Notch-regulated proliferative progenitor zone was a crucial step for the evolution of vertebrate spinal cord complexity.

Developmental signature, synaptic connectivity and neurotransmission are conserved between vertebrate hair cells and tunicate coronal cells.

In tunicates, the coronal organ represents a sentinel checking particle entrance into the pharynx. The organ differentiates from an anterior embryonic area considered a proto-placode. For their embryonic origin, morphological features and function, coronal sensory cells have been hypothesized to be homologues to vertebrate hair cells. However, vertebrate hair cells derive from a posterior placode. This contradicts one of the principle historical criteria for homology, similarity of position, which could be taken as evidence against coronal cells/hair cells homology. In the tunicates Ciona intestinalis and C. robusta, we found that the coronal organ expresses genes (Atoh, Notch, Delta-like, Hairy-b, and Musashi) characterizing vertebrate neural and hair cell development. Moreover, coronal cells exhibit a complex synaptic connectivity pattern, and express neurotransmitters (Glu, ACh, GABA, 5-HT, and catecholamines), or enzymes for their synthetic machinery, involved in hair cell activity. Lastly, coronal cells express the Trpa gene, which encodes an ion channel expressed in hair cells. These data lead us to hypothesize a model in which competence to make secondary mechanoreceptors was initially broadly distributed through placode territories, but has become confined to different placodes during the evolution of the vertebrate and tunicate lineages.

The structure, splicing, synteny and expression of lamprey COE genes and the evolution of the COE gene family in chordates.

COE genes encode transcription factors that have been found in all metazoans examined to date. They possess a distinctive domain structure that includes a DNA-binding domain (DBD), an IPT/TIG domain and a helix-loop-helix (HLH) domain. An intriguing feature of the COE HLH domain is that in jawed vertebrates it is composed of three helices, compared to two in invertebrates. We report the isolation and expression of two COE genes from the brook lamprey Lampetra planeri and compare these to COE genes from the lampreys Lethenteron japonicum and Petromyzon marinus. Molecular phylogenetic analyses do not resolve the relationship of lamprey COE genes to jawed vertebrate paralogues, though synteny mapping shows that they all derive from duplication of a common ancestral genomic region. All lamprey genes encode conserved DBD, IPT/TIG and HLH domains; however, the HLH domain of lamprey COE-A genes encodes only two helices while COE-B encodes three helices. We also identified COE-B splice variants encoding either two or three helices in the HLH domain, along with other COE-A and COE-B splice variants affecting the DBD and C-terminal transactivation regions. In situ hybridisation revealed expression in the lamprey nervous system including the brain, spinal cord and cranial sensory ganglia. We also detected expression of both genes in mesenchyme in the pharyngeal arches and underlying the notochord. This allows us to establish the primitive vertebrate expression pattern for COE genes and compare this to that of invertebrate chordates and other animals to develop a model for COE gene evolution in chordates.

Cerberus-Nodal-Lefty-Pitx signaling cascade controls left-right asymmetry in amphioxus.

Many bilaterally symmetrical animals develop genetically programmed left-right asymmetries. In vertebrates, this process is under the control of Nodal signaling, which is restricted to the left side by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest diverging chordate lineage, has profound left-right asymmetry as a larva. We show that Cerberus, Nodal, Lefty, and their target transcription factor Pitx are sequentially activated in amphioxus embryos. We then address their function by transcription activator-like effector nucleases (TALEN)-based knockout and heat-shock promoter (HSP)-driven overexpression. Knockout of Cerberus leads to ectopic right-sided expression of Nodal, Lefty, and Pitx, whereas overexpression of Cerberus represses their left-sided expression. Overexpression of Nodal in turn represses Cerberus and activates Lefty and Pitx ectopically on the right side. We also show Lefty represses Nodal, whereas Pitx activates Nodal These data combine in a model in which Cerberus determines whether the left-sided gene expression cassette is activated or repressed. These regulatory steps are essential for normal left-right asymmetry to develop, as when they are disrupted embryos may instead form two phenotypic left sides or two phenotypic right sides. Our study shows the regulatory cassette controlling left-right asymmetry was in place in the ancestor of amphioxus and vertebrates. This includes the Nodal inhibitors Cerberus and Lefty, both of which operate in feedback loops with Nodal and combine to establish asymmetric Pitx expression. Cerberus and Lefty are missing from most invertebrate lineages, marking this mechanism as an innovation in the lineage leading to modern chordates.

Phylogenetics of Lophotrochozoan bHLH Genes and the Evolution of Lineage-Specific Gene Duplicates.

The gain and loss of genes encoding transcription factors is of importance to understanding the evolution of gene regulatory complexity. The basic helix-loop-helix (bHLH) genes encode a large superfamily of transcription factors. We systematically classify the bHLH genes from five mollusc, two annelid and one brachiopod genomes, tracing the pattern of bHLH gene evolution across these poorly studied Phyla. In total, 56-88 bHLH genes were identified in each genome, with most identifiable as members of previously described bilaterian families, or of new families we define. Of such families only one, Mesp, appears lost by all these species. Additional duplications have also played a role in the evolution of the bHLH gene repertoire, with many new lophotrochozoan-, mollusc-, bivalve-, or gastropod-specific genes defined. Using a combination of transcriptome mining, RT-PCR, and in situ hybridization we compared the expression of several of these novel genes in tissues and embryos of the molluscs Crassostrea gigas and Patella vulgata, finding both conserved expression and evidence for neofunctionalization. We also map the positions of the genes across these genomes, identifying numerous gene linkages. Some reflect recent paralog divergence by tandem duplication, others are remnants of ancient tandem duplications dating to the lophotrochozoan or bilaterian common ancestors. These data are built into a model of the evolution of bHLH genes in molluscs, showing formidable evolutionary stasis at the family level but considerable within-family diversification by tandem gene duplication.

Identification of genes for engineering the male germline of Aedes aegypti and Ceratitis capitata.

BACKGROUND: Synthetic biology approaches are promising new strategies for control of pest insects that transmit disease and cause agricultural damage. These strategies require characterised modular components that can direct appropriate expression of effector sequences, with components conserved across species being particularly useful. The goal of this study was to identify genes from which new potential components could be derived for manipulation of the male germline in two major pest species, the mosquito Aedes aegypti and the tephritid fruit fly Ceratitis capitata. RESULTS: Using RNA-seq data from staged testis samples, we identified several candidate genes with testis-specific expression and suitable expression timing for use of their regulatory regions in synthetic control constructs. We also developed a novel computational pipeline to identify candidate genes with testis-specific splicing from this data; use of alternative splicing is another method for restricting expression in synthetic systems. Some of the genes identified display testis-specific expression or splicing that is conserved across species; these are particularly promising candidates for construct development. CONCLUSIONS: In this study we have identified a set of genes with testis-specific expression or splicing. In addition to their interest from a basic biology perspective, these findings provide a basis from which to develop synthetic systems to control important pest insects via manipulation of the male germline.

Identification of molecular signatures specific for distinct cranial sensory ganglia in the developing chick.

BACKGROUND: The cranial sensory ganglia represent populations of neurons with distinct functions, or sensory modalities. The production of individual ganglia from distinct neurogenic placodes with different developmental pathways provides a powerful model to investigate the acquisition of specific sensory modalities. To date there is a limited range of gene markers available to examine the molecular pathways underlying this process. RESULTS: Transcriptional profiles were generated for populations of differentiated neurons purified from distinct cranial sensory ganglia using microdissection in embryonic chicken followed by FAC-sorting and RNAseq. Whole transcriptome analysis confirmed the division into somato- versus viscerosensory neurons, with additional evidence for subdivision of the somatic class into general and special somatosensory neurons. Cross-comparison of distinct ganglia transcriptomes identified a total of 134 markers, 113 of which are novel, which can be used to distinguish trigeminal, vestibulo-acoustic and epibranchial neuronal populations. In situ hybridisation analysis provided validation for 20/26 tested markers, and showed related expression in the target region of the hindbrain in many cases. CONCLUSIONS: One hundred thirty-four high-confidence markers have been identified for placode-derived cranial sensory ganglia which can now be used to address the acquisition of specific cranial sensory modalities.

Molecular basis of canalization in an ascidian species complex adapted to different thermal conditions.

Canalization is a result of intrinsic developmental buffering that ensures phenotypic robustness under genetic variation and environmental perturbation. As a consequence, animal phenotypes are remarkably consistent within a species under a wide range of conditions, a property that seems contradictory to evolutionary change. Study of laboratory model species has uncovered several possible canalization mechanisms, however, we still do not understand how the level of buffering is controlled in natural populations. We exploit wild populations of the marine chordate Ciona intestinalis to show that levels of buffering are maternally inherited. Comparative transcriptomics show expression levels of genes encoding canonical chaperones such as Hsp70 and Hsp90 do not correlate with buffering. However the expression of genes encoding endoplasmic reticulum (ER) chaperones does correlate. We also show that ER chaperone genes are widely conserved amongst animals. Contrary to previous beliefs that expression level of Heat Shock Proteins (HSPs) can be used as a measurement of buffering levels, we propose that ER associated chaperones comprise a cellular basis for canalization. ER chaperones have been neglected by the fields of development, evolution and ecology, but their study will enhance understanding of both our evolutionary past and the impact of global environmental change.

Draft genome assemblies and predicted microRNA complements of the intertidal lophotrochozoans Patella vulgata (Mollusca, Patellogastropoda) and Spirobranchus (Pomatoceros) lamarcki (Annelida, Serpulida).

MicroRNAs (miRNA) are small non-coding RNAs that act post-transcriptionally to regulate gene expression levels. Some studies have indicated that microRNAs may have low homoplasy, and as a consequence the phylogenetic distribution of microRNA families has been used to study animal evolutionary relationships. Limited levels of lineage sampling, however, may distort such analyses. Lophotrochozoa is an under-sampled taxon that includes molluscs, annelids and nemerteans, among other phyla. Here, we present two novel draft genomes, those of the limpet Patella vulgata and polychaete Spirobranchus (Pomatoceros) lamarcki. Surveying these genomes for known microRNAs identifies numerous potential orthologues, including a number that have been considered to be confined to other lineages. RT-PCR demonstrates that some of these (miR-1285, miR-1287, miR-1957, miR-1983 and miR-3533), previously thought to be found only in vertebrates, are expressed. This study provides genomic resources for two lophotrochozoans and reveals patterns of microRNA evolution that could be hidden by more restricted sampling.

Transmission and Scanning Electron Microscopy of the Accessory Cells and Chorion During Development of Ciona intestinalis Type B Embryos and the Impact of Their Removal on Cell Morphology.

Spawned ascidian oocytes are surrounded by a membrane called the chorion (or vitelline coat) and associated with two populations of maternally-supplied cells. Outside the chorion are follicle cells, which may affect the buoyancy of eggs. Inside the chorion are test cells, which during oogenesis provision the egg and which after fertilisation contribute to the larval tunic. The structure of maternal cells may vary between species. The model ascidian Ciona intestinalis has been recently split into two species, currently named type A and type B. The ultrastructure of extraembryonic cells and structures from type A embryos has been reported. Here we describe the ultrastructure of follicle and test cells from C. intestinalis type B embryos. Test cells are about 5 µm in diameter and line the inside of the chorion of developing embryos in a dense sheet. Follicle cells are large (> 100 µm long) and spike-shaped, with many large vesicles. Terminal electron dense granules are found towards the tips of spikes, adjacent to cytoplasm containing numerous small electron dense bodies connected by filaments. These are probably vesicles containing material for the terminal granules. Removal of maternal structures and cells just after fertilisation, as commonly used in many experiments manipulating C. intestinalis development, has been reported to affect embryonic patterning. We examined the impact of this on embryonic ectoderm cells by scanning electron microscopy. Cells of embryos that developed without maternal structures still developed cilia, but had indistinct cell boundaries and a more flattened appearance than those that developed within the chorion.

Characterization of two neurogenin genes from the brook lamprey lampetra planeri and their expression in the lamprey nervous system.

CONCLUSIONS: Neurogenins are required for the specification of neuronal precursors and regulate the expression of basic Helix-Loop-Helix genes involved in neuronal differentiation. Jawed vertebrates possess three Neurogenin paralogy groups and their combined expression covers the entire nervous system, apart from the autonomic nervous system. RESULTS: Here we report the isolation of two Neurogenin genes, LpNgnA and LpNgnB, from the lamprey Lampetra planeri. Phylogenetic analyses show both genes have orthologues in other lamprey species and in a hagfish. Neither gene shows evidence of orthology to specific jawed vertebrate Neurogenin paralogues. LpNgnA is expressed in the ventricular zone of regions of the brain and spinal cord, with expression in the brain demarcating brain sub-compartments including the pallium, tegmentum, tectum, and dorsal thalamus. In the peripheral nervous system, LpNgnA is expressed in cranial sensory placodes and their derivatives, and in the dorsal root ganglia. LpNgnB is expressed transiently in placodal head ectoderm and throughout the central nervous system in early development, and in a small population cells that form part of the macula. CONCLUSIONS: Combined, LpNgnA and LpNgnB were detected in most cell populations marked by Neurogenin gene expression in jawed vertebrates, with the exception of the cerebellum, retina and the non-neural expression sites. Developmental Dynamics 244:1096-1108, 2015. © 2015 Wiley Periodicals, Inc.

Symmetrical reproductive compatibility of two species in the Ciona intestinalis (Ascidiacea) species complex, a model for marine genomics and developmental biology.

The sea squirt Ciona intestinalis species complex is a widely used model system for genomics and developmental biology, as well as ecology. Contrary to previous reports, here we show no difference in the success of development and hatching between hybrid and conspecific crosses between the two species within this complex known as types A and B, from a region in the English Channel where they are sympatric. We grew laboratory hybrids in the field for three months, and successfully obtained reproductive adults. In back-crosses of F1 laboratory hybrids to parental types, normal larvae were obtained. We conclude that hybrid crosses generate viable offspring and the resulting hybrids are interfertile with types A and B. However we also show that introgression in the natural sympatric population remains low. We discuss possible pre-zygotic and post-zygotic mechanisms which reproductively isolate these species.