Kirk S. Zigler

Phylogeography and bindin evolution in Arbacia, a sea urchin genus with an unusual distribution

Among shallow water sea urchin genera, Arbacia is the only genus that contains species found in both high and low latitudes. In order to determine the geographical origin of the genus and its history of speciation events, we constructed phylogenies based on cytochrome oxidase I and sperm bindin from all its species. Both the mitochondrial and the nuclear gene genealogies show that Arbacia originated in the temperate zone of the Southern Hemisphere and gave rise to three species in the eastern Pacific, which were then isolated from the Atlantic by the Isthmus of Panama. The mid‐Atlantic barrier separated two additional species. The bindin data suggest that selection against hybridization is not important in the evolution of this molecule in this genus. Metz et al. in a previous publication found no evidence of selection on bindin of Arbacia and suggested that this might be due to allopatry between species, which obviated the need for species recognition. This suggestion formed the basis of the conclusion, widely spread in the literature, that the source of selection on sea urchin bindin (where it does occur) was reinforcement. However, the range of Arbacia spatuligera overlaps with that of two other species of Arbacia, and our data show that it is hybridizing with one of them. We found that even in the species that overlap geographically, there are no deviations from selective neutrality in the evolution of bindin.

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

BackgroundTools 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.ResultsWe 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/).ConclusionsOur 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.

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.

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 posses 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.

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.

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.

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.

A NEW HOST AND REPRODUCTION AT A SMALL SIZE FOR THE "SNAPPER-CHOKING ISOPOD" CYMOTHOA EXCISA (ISOPODA: CYMOTHOIDAE)

Cymothoa excisa Perty, 1833 (Isopoda: Cymothoidae), the “snapper-choking isopod,” is a fish parasite previously known from fifteen host species; it ranges along the Atlantic coast from Brazil to Massachusetts. However, no host information has previously been published for the species north of the Gulf of Mexico. We encountered this isopod in 3.0% (12/399) of weakfish (Cynoscion regalis) collected at St. Catherines Island, Georgia. This is the first report of weakfish as a host for C. excisa. We confirmed our morphological identifications by sequencing a fragment of the mitochondrial cytochrome oxidase I (COI) gene from our specimens and a previously identified C. excisa specimen from the Gulf of Mexico. Three C. excisa (body lengths 13-15 mm) were ovigerous with brood sizes ranging from 171 to 483 embryos. These represent some of the smallest ovigerous C. excisa known and the smallest brood sizes reported for the species. Due to morphological changes during development, the identification of immature cymothoid isopods is challenging. The COI sequences we report will facilitate identifying all life stages of this species in the future.

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.

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.