Elizabeth L. Jockusch

The evolution of patterning of serially homologous appendages in insects.

Arthropod bodies are formed by a series of appendage-bearing segments, and appendages have diversified both along the body axis within species and between species. Understanding the developmental basis of this variation is essential for addressing questions about the evolutionary diversification of limbs. We examined the development of serially homologous appendages of two insect species, the beetle Tribolium castaneum and the grasshopper Schistocerca americana. Both species retain aspects of ancestral appendage morphology and development that have been lost in Drosophila, including branched mouthparts and direct development of appendages during embryogenesis. We characterized the expression of four genes important in proximodistal axis development of Drosophila appendages: the secreted signaling factors wingless and decapentaplegic, and the homeodomain transcription factors extradenticle and Distal-less. Our comparisons focus on two aspects of appendage morphology: differentiation of the main axis of serial homologues and the appearance of proximal branches (endites) in the mouthparts. Although Distal-less expression is similar in endites and palps of the mouthparts, the expression of other genes in the endites does not conform to their known roles in axial patterning, leading us to reject the hypothesis that branched insect mouthparts develop by reiteration of the limb patterning network. With the exception of decapentaplegic, patterning of the main appendage axis is generally more similar in direct homologues than in serial homologues. Interestingly, however, phylogenetic comparisons suggest that patterning of serial homologues was more similar in ancestral insects, and thus that the observed developmental differences did not cause the evolutionary divergence in morphology among serial homologues.

An evolutionary correlate of genome size change in plethodontid salamanders

Variation in the amount of nuclear DNA, the C–value, does not correlate with differences in morphological complexity. There are two classes of explanations for this observation, which is known as the ‘C–value paradox’. The quantity of DNA may serve a ‘nucleotypic’ function that is positively selected. Alternatively, large genomes may consist of junk DNA, which increases until it negatively affects fitness. Attempts to resolve the C–value paradox focus on the link between genome size and fitness. This link is usually sought in life history traits, particularly developmental rates. I examined the relationship among two life history traits, egg size and embryonic developmental time and genome size, in 15 species of plethodontid salamanders. Surprisingly, there is no correlation between egg size and developmental time, a relationship included in models of life history evolution. However, genome size is positively correlated with embryonic developmental time, a result that is robust with respect to many sources of variation in the data. Without information on the targets of natural selection it is not possible with these data to distinguish between nucleotypic and junk DNA explanations for the C–value paradox.

Extreme population subdivision throughout a continuous range: phylogeography of Batrachoseps attenuatus (Caudata: Plethodontidae) in western North America

Low‐vagility species with deep evolutionary histories are key to our understanding of the biogeographical history of geologically complex areas, such as the west coast of North America. We present a detailed study of the phylogeography of the salamander Batrachoseps attenuatus (Caudata: Plethodontidae) using sequences of the mitochondrial gene cob from 178 individuals sampled from throughout the species’ range. Sequences of three other mitochondrial genes (16S, cox1, nad4) and a nuclear gene (RAG‐1) were used to investigate the deeper evolutionary history of the species. We found high levels of genetic diversity and deep divergences within a mostly continuous distribution, with five genetically well‐differentiated and geographically structured mitochondrial DNA clades. Significant association between geographical and genetic distances within these clades suggests demographic stability, whereas Fus FS tests suggest demographic expansions in three of them. Mantel tests identify two biogeographical barriers, the San Andreas Fault and the Sacramento–San Joaquin Delta, as important in the diversification of lineages. The timing of the main splitting events between intraspecific lineages was estimated by applying relaxed molecular clock methods combining several mutation rates and a fossil calibration. The earliest splitting events are old (Pliocene/Miocene), with more recent (Pleistocene) subdivisions in some clades. Disjunct populations distributed along the western foothills of the Sierra Nevada colonized this area relatively recently from a single refugium east of San Francisco Bay. The combination of fine‐scale, comprehensive sampling with phylogenetic, historical demographic and hypothesis‐based tests allowed delineation of a complex biogeographical scenario with general implications for the study of codistributed taxa.

Molecular phylogenetic analysis of slender salamanders genus Batrachoseps (Amphibia: Plethodontidae), from central coastal California with descriptions of four new species

Plethodontid salamanders of the genus Batrachoseps comprise a clade of morphologically similar, elongate species whose great genetic diversity is being revealed through molecular studies. We used allozymes and mtDNA sequences to study variation in 62 populations from central coastal California, treated most recently as members of the B. pacificus complex. Analyses of mtDNA data identify four lineages that are well differentiated from each other and do not form a monophyletic group. Instead, the central coastal lineages are multiply paraphyletic with respect to the southern California members of the pacificus group. Marked allozymic differences show that these four lineages are strongly differentiated, although some limited gene exchange may have occurred in the past. Each lineage is also morphologically distinctive, but the differences between them are subtle. Because these lineages appear to be evolving independently, we describe them as new species: B. luciae, B. incognitus and B. minor, distributed parapatrically from north to south in the Santa Lucia Mountains of coastal Monterey and San Luis Obispo Counties, and B. gavilanensis, occurring mainly inland from the range of B. luciae, centered on the Gabilan Mountains, but also extending to the Pacific coast at the north end of the range of the complex, along the northern border of Monterey Bay. Although no sympatry is known among any of the new species, B. luciae and B. gavilanensis are narrowly parapatric. Furthermore, all but B. luciae occur in sympatry with other members of the genus in at least a part of their geographic ranges. The new species may have arisen vicariantly with respect both to each other and to related forms in southern California, in part as a result of the dramatic tectonic movements that have characterized the last 15 million years of geological history in western North America.

Genetic patterning in the adult capitate antenna of the beetle Tribolium castaneum

Antenna structure varies widely among insects, in contrast to the well-conserved structure of legs. The adult capitate antenna of the red flour beetle, Tribolium castaneum, is composed of eleven articles, organized into four distinct morphological regions (scape, pedicel, funicle and club). Here, we report the use of RNA interference to examine the functions of 21 genes during antenna metamorphosis in T. castaneum. Genes with conserved functions relative to the developmental model species Drosophila melanogaster include Distal-less and EGF signaling (antennal growth), spineless (determination of antennal identity) and the Notch signaling pathway (antennal growth, joint formation, and sensory bristle development). However, the functions of many genes differed from those predicted from the Drosophila model. In addition to a conserved gap phenotype, depletion of dachshund transformed funicle articles toward club-like identity. Depletion of Distal-less or homothorax did not cause antenna-to-leg transformation. Lim1 was required only for development of the scape-pedicle joint. Depletion of odd-skipped-related genes led to the loss of the entire funicle, while spalt, rotund, spineless, and dachshund affected smaller regions. Growth and joint formation were linked developmentally in the funicle, but not in the club. Joint formation within the club required bric-a-brac, aristaless, apterous, and pdm. Gene functions are discussed in terms of a model of antenna development in T. castaneum. This model provides a contrast to knowledge of antenna development in D. melanogaster, insight into the likely ancestral mode of antenna development, and a framework for considering diverse antenna morphologies.

Body size evolution simultaneously creates and collapses species boundaries in a clade of scincid lizards

Speciation is generally viewed as an irreversible process, although habitat alterations can erase reproductive barriers if divergence between ecologically differentiated species is recent. Reversed speciation might also occur if geographical contact is established between species that have evolved the same reproductive isolating barrier in parallel. Here, we demonstrate a loss of intrinsic reproductive isolation in a clade of scincid lizards as a result of parallel body size evolution, which has allowed for gene flow where large-bodied lineages are in secondary contact. An mtDNA phylogeny confirms the monophyly of the Plestiodon skiltonianus species complex, but rejects that of two size-differentiated ecomorphs. Mate compatibility experiments show that the high degree of body size divergence imposes a strong reproductive barrier between the two morphs; however, the strength of the barrier is greatly diminished between parallel-evolved forms. Since two large-bodied lineages are in geographical contact in the Sierra Nevada Mountains of California, we were also able to test for postzygotic isolation under natural conditions. Analyses of amplified fragment length polymorphisms show that extensive gene exchange is occurring across the contact zone, resulting in an overall pattern consistent with isolation by distance. These results provide evidence of reversed speciation between clades that diverged from a common ancestor more than 12 Myr ago.

Why is limb regeneration possible in amphibians but not in reptiles, birds, and mammals?

SUMMARY The capacity to regenerate limbs is very high in amphibians and practically absent in other tetrapods despite the similarities in developmental pathways and ultimate morphology of tetrapod limbs. We propose that limb regeneration is only possible when the limb develops as a semiautonomous module and is not involved in interactions with transient structures. This hypothesis is based on the following two assumptions: To an important extent, limb development uses the same developmental mechanisms as normal limb development and developmental mechanisms that require interactions with transient structures cannot be recapitulated later. In amniotes limb development is early, shortly after neurulation, and requires inductive interactions with transient structures such as somites. In amphibians limb development is delayed relative to amniotes and has become decoupled from interactions with somites and other transient structures that are no longer present at this stage. The limb develops as a semi‐independent module. A comparison of the autonomy and timing of limb development in different vertebrate taxa supports our hypothesis and its assumptions. The data suggest a good correlation between self‐organizing and regenerative capacity. Furthermore, they suggest that whatever barriers amphibians overcame in the evolution of metamorphosis, they are the same barriers that need to be overcome to make limb regeneration possible in other taxa.

Extent With Modification: Leg Patterning in the Beetle Tribolium castaneum and the Evolution of Serial Homologs.

Serial homologs are similar structures that develop at different positions within a body plan. These structures share some, but not all, aspects of developmental patterning, and their evolution is thought to be constrained by shared, pleiotropic gene functions. Here we describe the functions of 17 developmental genes during metamorphic development of the legs in the red flour beetle, Tribolium castaneum. This study provides informative comparisons between appendage development in Drosophila melanogaster and T. castaneum, between embryonic and adult development in T. castaneum, and between the development of serially homologous appendages. The leg gap genes Distal-less and dachshund are conserved in function. Notch signaling, the zinc-finger transcription factors related to odd-skipped, and bric-à-brac have conserved functions in promoting joint development. homothorax knockdown alters the identity of proximal leg segments but does not reduce growth. Lim1 is required for intermediate leg development but not distal tarsus and pretarsus development as in D. melanogaster. Development of the tarsus requires decapentaplegic, rotund, spineless, abrupt, and bric-à-brac and the EGF ligand encoded by Keren. Metathoracic legs of T. castaneum have four tarsomeres, whereas other legs have five. Patterns of gene activity in the tarsus suggest that patterning in the middle of the tarsal region, not the proximal- or distal-most areas, is responsible for this difference in segment number. Through comparisons with other recent studies of T. castaneum appendage development, we test hypotheses for the modularity or interdependence of development during evolution of serial homologs.

Distal-less and dachshund pattern both plesiomorphic and apomorphic structures in chelicerates: RNA interference in the harvestman Phalangium opilio (Opiliones)

The discovery of genetic mechanisms that can transform a morphological structure from a plesiomorphic (=primitive) state to an apomorphic (=derived) one is a cardinal objective of evolutionary developmental biology. However, this objective is often impeded for many lineages of interest by limitations in taxonomic sampling, genomic resources, or functional genetic methods. In order to investigate the evolution of appendage morphology within Chelicerata, the putative sister group of the remaining arthropods, we developed an RNA interference (RNAi) protocol for the harvestman Phalangium opilio. We silenced the leg gap genes Distal‐less (Dll) and dachshund (dac) in the harvestman via zygotic injections of double‐stranded RNA (dsRNA), and used in situ hybridization to confirm RNAi efficacy. Consistent with the conserved roles of these genes in patterning the proximo‐distal axis of arthropod appendages, we observed that embryos injected with Dll dsRNA lacked distal parts of appendages and appendage‐like structures, such as the labrum, the chelicerae, the pedipalps, and the walking legs, whereas embryos injected with dac dsRNA lacked the medial podomeres femur and patella in the pedipalps and walking legs. In addition, we detected a role for these genes in patterning structures that do not occur in well‐established chelicerate models (spiders and mites). Dll RNAi additionally results in loss of the preoral chamber, which is formed from pedipalpal and leg coxapophyses, and the ocularium, a dorsal outgrowth bearing the eyes. In one case, we observed that an embryo injected with dac dsRNA lacked the proximal segment of the chelicera, a plesiomorphic podomere that expresses dac in wild‐type embryos. This may support the hypothesis that loss of the cheliceral dac domain underlies the transition to the two‐segmented chelicera of derived arachnids.

Detecting Species Borders Using Diverse Data Sets

Debates continue about the appropriate species-level taxonomy to use for plethodontid salamanders. Typically the debates center on what taxonomy is appropriate when geographically contiguous taxa meet and do not become sympatric (e.g., Highton, 1998; Petranka, 1998; Wake and Schneider, 1998), but also at issue is the status of allopatric populations. Differences of opinion are not new to the field of systematics, especially when knowledge is incomplete. What is new, and perhaps surprising, is that these disagreements persist despite the substantial data bases (morphology, allozymes, mtDNA sequences) now available.