Population genomics in two cave-obligate invertebrates confirms extremely limited dispersal between caves.

Caves offer selective pressures that are distinct from the surface. Organisms that have evolved to exist under these pressures typically exhibit a suite of convergent characteristics, including a loss or reduction of eyes and pigmentation. As a result, cave-obligate taxa, termed troglobionts, are no longer viable on the surface. This circumstance has led to an understanding of highly constrained dispersal capabilities, and the prediction that, in the absence of subterranean connections, extreme genetic divergence between cave populations. An effective test of this model would involve (1) common troglobionts from (2) nearby caves in a cave-dense region, (3) good sample sizes per cave, (4) multiple taxa, and (5) genome-wide characterization. With these criteria in mind, we used RAD-seq to genotype an average of ten individuals of the troglobiotic spider Nesticus barri and the troglobiotic beetle Ptomaphagus hatchi, each from four closely located caves (ranging from 3 to 13 km apart) in the cave-rich southern Cumberland Plateau of Tennessee, USA. Consistent with the hypothesis of highly restricted dispersal, we find that populations from separate caves are indeed highly genetically isolated. Our results support the idea of caves as natural laboratories for the study of parallel evolutionary processes.

Predicting the Occurrence of Cave-Inhabiting Fauna Based on Features of the Earth Surface Environment.

One of the most challenging fauna to study in situ is the obligate cave fauna because of the difficulty of sampling. Cave-limited species display patchy and restricted distributions, but it is often unclear whether the observed distribution is a sampling artifact or a true restriction in range. Further, the drivers of the distribution could be local environmental conditions, such as cave humidity, or they could be associated with surface features that are surrogates for cave conditions. If surface features can be used to predict the distribution of important cave taxa, then conservation management is more easily obtained. We examined the hypothesis that the presence of major faunal groups of cave obligate species could be predicted based on features of the earth surface. Georeferenced records of cave obligate amphipods, crayfish, fish, isopods, beetles, millipedes, pseudoscorpions, spiders, and springtails within the area of Appalachian Landscape Conservation Cooperative in the eastern United States (Illinois to Virginia and New York to Alabama) were assigned to 20 x 20 km grid cells. Habitat suitability for these faunal groups was modeled using logistic regression with twenty predictor variables within each grid cell, such as percent karst, soil features, temperature, precipitation, and elevation. Models successfully predicted the presence of a group greater than 65% of the time (mean = 88%) for the presence of single grid cell endemics, and for all faunal groups except pseudoscorpions. The most common predictor variables were latitude, percent karst, and the standard deviation of the Topographic Position Index (TPI), a measure of landscape rugosity within each grid cell. The overall success of these models points to a number of important connections between the surface and cave environments, and some of these, especially soil features and topographic variability, suggest new research directions. These models should prove to be useful tools in predicting the presence of species in understudied areas.

Reproductive Seasonality in Nesticus (Araneae: Nesticidae) Cave Spiders.

Spiders of the family Nesticidae are members of cave communities around the world with cave-obligate (troglobiotic) species known from North America, Europe, Asia and the Indo-Pacific. A radiation of Nesticus (Araneae: Nesticidae) in the southern Appalachians includes ten troglobiotic species. Many of these species are of conservation interest due to their small ranges, with four species being single-cave endemics. Despite conservation concerns and their important role as predators in cave communities, we know little about reproduction and feeding in this group. We addressed this knowledge gap by examining populations of two species on a monthly basis for one year. We made further observations on several other species and populations, totaling 671 individual spider observations. This more than doubled the reported observations of reproduction and feeding in troglobiotic Nesticus. Female Nesticus carry egg sacs, facilitating the determination of the timing and frequency of reproduction. We found that Nesticus exhibit reproductive seasonality. Females carried egg sacs from May through October, with a peak in frequency in June. These spiders were rarely observed with prey; only 3.3% (22/671) of individuals were observed with prey items. The frequency at which prey items were observed did not vary by season. Common prey items were flies, beetles and millipedes. Troglobiotic species constituted approximately half of all prey items observed. This result represents a greater proportion of troglobiotic prey than has been reported for various troglophilic spiders. Although our findings shed light on the life history of troglobiotic Nesticus and on their role in cave ecosystems, further work is necessary to support effective conservation planning for many of these rare species.

Using phylogenetically-informed annotation (PIA) to search for light-interacting genes in transcriptomes from non-model organisms.

BACKGROUND: Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families. RESULTS: We generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository ( http://bitbucket.org/osiris_phylogenetics/pia/ ) and we demonstrate PIA on a publicly-accessible web server ( http://galaxy-dev.cnsi.ucsb.edu/pia/ ). CONCLUSIONS: Our new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.

A shift in germ layer allocation is correlated with large egg size and facultative planktotrophy in the echinoid Clypeaster rosaceus.

Egg size is a correlate of larval evolution in marine embryos. Comparing species with different egg sizes that develop via similar larvae reveals the flexibility and the constraints underlying larval forms. Clypeaster rosaceus is an echinoid that develops via a facultatively planktotrophic pluteus larva. Unlike most echinoids that develop via plutei, C. rosaceus (1) has a larger egg, with a correspondingly smaller ratio of surface area to volume, and (2) forms a large left coelom early in development. Given these characteristics, we predicted underlying changes in the allocation of embryonic tissues to germ layers. With a low surface-to-volume ratio, the C. rosaceus pluteus likely requires relatively less ectoderm than a typical pluteus, whereas the early formation of a large left coelom likely requires relatively more mesoderm than a typical pluteus. We tested this hypothesis by examining the cell lineage of C. rosaceus. We found that the boundary between ectoderm and endoderm in C. rosaceus has shifted relative to echinoids with more typical planktotrophic plutei and extends to or above the third cleavage plane at the equator of the embryo. This indicates a smaller proportional allocation to ectoderm and a larger proportional allocation to endomesoderm compared to echinoids with smaller egg sizes. On the basis of this observation, we develop a new model for the transition from obligate planktotrophy to lecithotrophy. We argue that species with larger eggs may allocate proportionally more tissue to structures selected for accelerated development. In the case of C. rosaceus, the larval cell lineage apportions more cells to endomesoderm and less to ectoderm due to the smaller surface-to-volume ratio of its larger eggs and the early formation of a large left coelom.

Phylogeography of the sand dollar genus Mellita: cryptic speciation along the coasts of the Americas.

Sand dollars of the genus Mellita are members of the sandy shallow-water fauna. The genus ranges in tropical and subtropical regions on the two coasts of the Americas. To reconstruct the phylogeography of the genus we sequenced parts of the mitochondrial cytochrome oxidase I and of 16S rRNA as well as part of the nuclear 28S rRNA gene from a total of 185 specimens of all ten described morphospecies from 31 localities. Our analyses revealed the presence of eleven species, including six cryptic species. Sequences of five morphospecies do not constitute monophyletic molecular units and thus probably represent ecophenotypic variants. The fossil-calibrated phylogeny showed that the ancestor of Mellita diverged into a Pacific lineage and an Atlantic+Pacific lineage close to the Miocene/Pliocene boundary. Atlantic M. tenuis, M. quinquiesperforata and two undescribed species of Mellita have non-overlapping distributions. Pacific Mellita consist of two highly divergent lineages that became established at different times, resulting in sympatric M. longifissa and M. notabilis. Judged by modern day ranges, not all divergence in this genus conforms to an allopatric speciation model. Only the separation of M. quinquiesperforata from M. notabilis is clearly due to vicariance as the result of the completion of the Isthmus of Panama. The molecular phylogeny calibrated on fossil evidence estimated this event as having occurred ~3 Ma, thus providing evidence that, contrary to a recent proposal, the central American Isthmus was not completed until this date.

Patterns of cave biodiversity and endemism in the Appalachians and Interior Plateau of Tennessee, USA.

Using species distribution data, we developed a georeferenced database of troglobionts (cave-obligate species) in Tennessee to examine spatial patterns of species richness and endemism, including >2000 records for 200 described species. Forty aquatic troglobionts (stygobionts) and 160 terrestrial troglobionts are known from caves in Tennessee, the latter having the greatest diversity of any state in the United States. Endemism was high, with 25% of terrestrial troglobionts (40 species) and 20% of stygobionts (eight species) known from just a single cave and nearly two-thirds of all troglobionts (130 species) known from five or fewer caves. Species richness and endemism were greatest in the Interior Plateau (IP) and Southwestern Appalachians (SWA) ecoregions, which were twice as diverse as the Ridge and Valley (RV). Troglobiont species assemblages were most similar between the IP and SWA, which shared 59 species, whereas the RV cave fauna was largely distinct. We identified a hotspot of cave biodiversity with a center along the escarpment of the Cumberland Plateau in south-central Tennessee defined by both species richness and endemism that is contiguous with a previously defined hotspot in northeastern Alabama. Nearly half (91 species) of Tennessee's troglobiont diversity occurs in this region where several cave systems contain ten or more troglobionts, including one with 23 species. In addition, we identified distinct troglobiont communities across the state. These communities corresponded to hydrological boundaries and likely reflect past or current connectivity between subterranean habitats within and barriers between hydrological basins. Although diverse, Tennessee's subterranean fauna remains poorly studied and many additional species await discovery and description. We identified several undersampled regions and outlined conservation and management priorities to improve our knowledge and aid in protection of the subterranean biodiversity in Tennessee.

Natural hybridization in the sea urchin genus Pseudoboletia between species without apparent barriers to gamete recognition.

Marine species with high dispersal potential often have huge ranges and minimal population structure. Combined with the paucity of geographic barriers in the oceans, this pattern raises the question as to how speciation occurs in the sea. Over the past 20 years, evidence has accumulated that marine speciation is often linked to the evolution of gamete recognition proteins. Rapid evolution of gamete recognition proteins in gastropods, bivalves, and sea urchins is correlated with gamete incompatibility and contributes to the maintenance of species boundaries between sympatric congeners. Here, we present a counterexample to this general pattern. The sea urchins Pseudoboletia indiana and P. maculata have broad ranges that overlap in the Indian and Pacific oceans. Cytochrome oxidase I sequences indicated that these species are distinct, and their 7.3% divergence suggests that they diverged at least 2 mya. Despite this, we suspected hybridization between them based on the presence of morphologically intermediate individuals in sympatric populations at Sydney, Australia. We assessed the opportunity for hybridization between the two species and found that (1) individuals of the two species occur within a meter of each other in nature, (2) they have overlapping annual reproductive cycles, and (3) their gametes cross-fertilize readily in the laboratory and in the field. We genotyped individuals with intermediate morphology and confirmed that many were hybrids. Hybrids were fertile, and some female hybrids had egg sizes intermediate between the two parental species. Consistent with their high level of gamete compatibility, there is minimal divergence between P. indiana and P. maculata in the gamete recognition protein bindin, with a single fixed amino acid difference between the two species. Pseudoboletia thus provides a well-characterized exception to the idea that broadcast spawning marine species living in sympatry develop and maintain species boundaries through the divergence of gamete recognition proteins and the associated evolution of gamete incompatibility.

Egg energetics, fertilization kinetics, and population structure in echinoids with facultatively feeding larvae.

Larvae of marine invertebrates either arise from small eggs and feed during their development or arise from large eggs that proceed to metamorphosis sustained only from maternal provisioning. Only a few species are known to possess facultatively feeding larvae. Of about 250 echinoid species with known mode of development, only two, Brisaster latifrons and Clypeaster rosaceus, are known to develop through facultatively planktotrophic larvae. To obtain more information on this form of development and its consequences, we determined egg size and egg energetic and protein content of these two species. We found that eggs of B. latifrons resemble those of species with nonfeeding larvae in these characteristics more than those of C. rosaceus. We also compared DNA sequences of the cytochrome oxidase (COI) gene from the Caribbean C. rosaceus to those of the sympatric planktotrophic developer C. subdepressus and also to those of the eastern Pacific species C. europacificus to estimate the degree of divergence between species with different developmental modes. Comparison of COI sequences of C. rosaceus from Panama and Florida revealed that there is no geographic differentiation in this species. Cross-fertilization experiments between C. rosaceus and C. subdepressus indicated that bidirectional gametic incompatibility has evolved between the two species.

The evolution of sea urchin sperm bindin.

Sea urchins have been model organisms for the study of fertilization for more than a century. Fertilization in sea urchins happens externally, which facilitates the study of sperm-egg attachment and fusion, and means that all of the molecules involved in gamete recognition and fusion are associated with the gametes. Sea urchin sperm bindin was the first "gamete recognition protein" to be isolated and characterized (Vacquier and Moy 1977), and bindin has since been studied by developmental biologists interested in fertilization, by biochemists interested in membrane fusion and by evolutionary biologists interested in reproductive isolation and speciation. Research on bindin was last reviewed thirteen years ago by Vacquier et al. (1995) in an article titled "What have we learned about sea urchin sperm bindin?" in which the authors reviewed the identification, isolation and early molecular examinations of bindin. Research since then has focused on bindin's potential role in fusing egg and sperm membranes, comparisons of bindin between distantly related species, studies within genera linking bindin evolution to reproductive isolation, and studies within species looking at fertilization effects of individual bindin alleles. In addition, the egg receptor for bindin has been cloned and sequenced. I review this recent research here.

Evolution of direct-developing larvae: selection vs loss.

Observations of a sea urchin larvae show that most species adopt one of two life history strategies. One strategy is to make numerous small eggs, which develop into a larva with a required feeding period in the water column before metamorphosis. In contrast, the second strategy is to make fewer large eggs with a larva that does not feed, which reduces the time to metamorphosis and thus the time spent in the water column. The larvae associated with each strategy have distinct morphologies and developmental processes that reflect their feeding requirements, so that those that feed exhibit indirect development with a complex larva, and those that do not feed form a morphologically simplified larva and exhibit direct development. Phylogenetic studies show that, in sea urchins, a feeding larva, the pluteus, is the ancestral form and the morphologically simplified direct-developing larva is derived. The current hypothesis for evolution of the direct-developing larval form in sea urchins suggests that major developmental changes occur by neutral loss of larval features after the crucial transition to a nonfeeding life history strategy. We present evidence from Clypeaster rosaceus, a sea urchin with a life history intermediate to the two strategies, which indicates that major developmental changes for accelerated development have been selected for in a larva that can still feed and maintains an outward, pluteus morphology. We suggest that transformation of larval form has resulted from strong selection on early initiation and acceleration of adult development.

Sea urchin bindin divergence predicts gamete compatibility.

Studies on the evolution of reproductive proteins have shown that they tend to evolve more rapidly than other proteins, frequently under positive selection. Progress on understanding the implications of these patterns is possible for marine invertebrates, where molecular evolution can be linked to gamete compatibility. In this study, we surveyed data from the literature from five genera of sea urchins for which there was information on gamete compatibility, divergence of the sperm-egg recognition protein bindin, and mitochondrial divergence. We draw three conclusions: (1) bindin divergence at nonsynonymous sites predicts gamete compatibility, whereas (2) bindin divergence at synonymous sites and mitochondrial DNA divergence do not, and (3) as few as 10 amino acid changes in bindin can lead to complete gamete incompatibility between species. Using mitochondrial divergence as a proxy for time, we find that complete gamete incompatibility can evolve in approximately one and a half million years, whereas sister species can maintain complete gamete compatibility for as long as five million years.

Speciation on the coasts of the new world: phylogeography and the evolution of bindin in the sea urchin genus Lytechinus.

Beginning with E. Mayr's study in 1954, tropical sea urchins have played an important role in studies of speciation in the sea, but what are the processes of cladogenesis and divergence that give rise to new species in this group? We attempt to answer this question in the genus Lytechinus. Unlike the majority of other tropical sea urchin genera, which have circumtropical distributions, Lytechinus is mostly confined to the tropics and subtropics of the New World. We sequenced a region of mitochondrial cytochrome oxidase I and the entire molecule of nuclear bindin (a sperm gamete recognition protein) of nearly all species in the genus, and we assayed isozymes of three partially sympatric closely related species and subspecies. We found that in both mitochondrial DNA (mtDNA) and in bindin the genus Lytechinus is paraphyletic, encompassing Sphaerechinus granularis as the sister species of L. euerces. The rest of the species are arranged in an Atlantic clade composed of L. williamsi and L. variegatus, and a Pacific clade containing L. anamesus, L. pictus, L. semituberculatus, and L. panamensis. Divergence between these clades suggests that they were separated no later than the closure of the Isthmus of Panama, and possibly before this time. Our data confirm that L. anamesus and L. pictus from California are a single species, and provide no evidence of differentiation between L. variegatus variegatus from the Caribbean and L. variegatus atlanticus from Bermuda. Lytechinus variegatus variegatus mtDNA is distinct from that of L. variegatus carolinus from the North American seaboard and the Gulf of Mexico, whereas their bindins are very similar. However, there is clear evidence of introgression of mtDNA between the two subspecies and they share alleles in all sampled isozyme loci. Lytechinus williamsi from the Caribbean shares mtDNA haplotypes with L. variegatus variegatus, and they also share isozymes in all assayed loci. Their bindin, however, is distinct and coalesces within each morphospecies. A private clade of mtDNA in L. williamsi may be indicative of former differentiation in the process of being swamped by introgression, or of recent speciation. Recent sudden expansions in effective population size may explain the predominance of a few mitochondrial haplotypes common to the two species. Despite the high divergence of bindin (relative to differentiation of mtDNA) between L. variegatus and L. williamsi, comparison of amino acid replacement to silent substitutions by various methods uncovered no evidence for positive selection on the bindin of any clade of Lytechinus. With the possible exception of L. williamsi and L. variegatus, our results are consistent with a history of allopatric speciation in Lytechinus. The molecular results from Lytechinus, along with those of other similar studies of sea urchins, suggest that the general speciation patterns deduced in the middle of last century by Mayr from morphology and geography have held up, but also have uncovered peculiarities in the evolution of each genus.

Adaptive evolution of bindin in the genus Heliocidaris is correlated with the shift to direct development.

Sea urchins are widely used to study both fertilization and development. In this study we combine the two fields to examine the evolution of reproductive isolation in the genus Heliocidaris. Heliocidaris tuberculata develops indirectly via a feeding larva, whereas the only other species in the genus, H. erythrogramma, has evolved direct development through a nonfeeding larva. We estimated the time of divergence between H. erythrogramma and H. tuberculata from mitochondrial DNA divergence, quantified levels of gametic compatibility between the two species in cross-fertilization assays, and examined the mode of evolution of the sperm protein bindin by sequencing multiple alleles of the two species. Bindin is the major component of the sea urchin sperm acrosomal vesicle, and is involved in sperm-egg attachment and fusion. Based on our analyses, we conclude that: the two species of Heliocidaris diverged less than five million years ago, indicating that direct development can evolve rapidly in sea urchins; since their divergence, the two species have become gametically incompatible; Heliocidaris bindin has evolved under positive selection; and this positive selection is concentrated on the branch leading to H. erythrogramma. Three hypotheses can explain the observed pattern of selection on bindin: (1) it is a correlated response to the evolution of direct development in H. erythrogramma; (2) it is the result of an intraspecific process acting in H. erythrogramma but not in H. tuberculata; or (3) it is the product of reinforcement on the species that invests more energy into each egg to avoid hybridization.

250 million years of bindin evolution.

Bindin plays a central role in sperm-egg attachment and fusion in sea urchins (echinoids). Previous studies determined the DNA sequence of bindin in two orders of the class Echinoidea, representing 10% of all echinoid species. We report sequences of mature bindin from five additional genera, representing four new orders, including the distantly related sand dollars, heart urchins, and pencil urchins. The six orders in which bindin is now known include 70% of all echinoids, and indicate that bindin was present in the common ancestor of all extant sea urchins more than 250 million years ago. Over this span of evolutionary time there has been (1). remarkable conservation in the core region of bindin, particularly in a stretch of 29 amino acids that has not changed at all; (2). conservation of a motif of basic amino acids at the cleavage site between preprobindin and mature bindin; (3). more than a twofold change in length of mature bindin; and (4). emergence of high variation in the sequences outside the core, including the insertion of glycine-rich repeats in the bindins of some orders, but not others.