Michael W. Lloyd

Estimation of effective population size in continuously distributed populations: there goes the neighborhood

Use of genetic methods to estimate effective population size (Ne) is rapidly increasing, but all approaches make simplifying assumptions unlikely to be met in real populations. In particular, all assume a single, unstructured population, and none has been evaluated for use with continuously distributed species. We simulated continuous populations with local mating structure, as envisioned by Wright’s concept of neighborhood size (NS), and evaluated performance of a single-sample estimator based on linkage disequilibrium (LD), which provides an estimate of the effective number of parents that produced the sample (Nb). Results illustrate the interacting effects of two phenomena, drift and mixture, that contribute to LD. Samples from areas equal to or smaller than a breeding window produced estimates close to the NS. As the sampling window increased in size to encompass multiple genetic neighborhoods, mixture LD from a two-locus Wahlund effect overwhelmed the reduction in drift LD from incorporating offspring from more parents. As a consequence, never approached the global Ne, even when the geographic scale of sampling was large. Results indicate that caution is needed in applying standard methods for estimating effective size to continuously distributed populations.

Sequence Capture and Phylogenetic Utility of Genomic Ultraconserved Elements Obtained from Pinned Insect Specimens

Obtaining sequence data from historical museum specimens has been a growing research interest, invigorated by next-generation sequencing methods that allow inputs of highly degraded DNA. We applied a target enrichment and next-generation sequencing protocol to generate ultraconserved elements (UCEs) from 51 large carpenter bee specimens (genus Xylocopa), representing 25 species with specimen ages ranging from 2–121 years. We measured the correlation between specimen age and DNA yield (pre- and post-library preparation DNA concentration) and several UCE sequence capture statistics (raw read count, UCE reads on target, UCE mean contig length and UCE locus count) with linear regression models. We performed piecewise regression to test for specific breakpoints in the relationship of specimen age and DNA yield and sequence capture variables. Additionally, we compared UCE data from newer and older specimens of the same species and reconstructed their phylogeny in order to confirm the validity of our data. We recovered 6–972 UCE loci from samples with pre-library DNA concentrations ranging from 0.06–9.8 ng/μL. All investigated DNA yield and sequence capture variables were significantly but only moderately negatively correlated with specimen age. Specimens of age 20 years or less had significantly higher pre- and post-library concentrations, UCE contig lengths, and locus counts compared to specimens older than 20 years. We found breakpoints in our data indicating a decrease of the initial detrimental effect of specimen age on pre- and post-library DNA concentration and UCE contig length starting around 21–39 years after preservation. Our phylogenetic results confirmed the integrity of our data, giving preliminary insights into relationships within Xylocopa. We consider the effect of additional factors not measured in this study on our age-related sequence capture results, such as DNA fragmentation and preservation method, and discuss the promise of the UCE approach for large-scale projects in insect phylogenomics using museum specimens.

Dry habitats were crucibles of domestication in the evolution of agriculture in ants

The evolution of ant agriculture, as practised by the fungus-farming ‘attine’ ants, is thought to have arisen in the wet rainforests of South America about 55–65 Ma. Most subsequent attine agricultural evolution, including the domestication event that produced the ancestor of higher attine cultivars, is likewise hypothesized to have occurred in South American rainforests. The ‘out-of-the-rainforest’ hypothesis, while generally accepted, has never been tested in a phylogenetic context. It also presents a problem for explaining how fungal domestication might have occurred, given that isolation from free-living populations is required. Here, we use phylogenomic data from ultra-conserved element (UCE) loci to reconstruct the evolutionary history of fungus-farming ants, reduce topological uncertainty, and identify the closest non-fungus-growing ant relative. Using the phylogeny we infer the history of attine agricultural systems, habitat preference and biogeography. Our results show that the out-of-the-rainforest hypothesis is correct with regard to the origin of attine ant agriculture; however, contrary to expectation, we find that the transition from lower to higher agriculture is very likely to have occurred in a seasonally dry habitat, inhospitable to the growth of free-living populations of attine fungal cultivars. We suggest that dry habitats favoured the isolation of attine cultivars over the evolutionary time spans necessary for domestication to occur.

Phylogenomic species delimitation and host-symbiont coevolution in the fungus-farming ant genus Sericomyrmex Mayr (Hymenoptera: Formicidae): ultraconserved elements (UCEs) resolve a recent radiation

Ants in the Neotropical genus Sericomyrmex Mayr cultivate fungi for food. Both ants and fungi are obligate, coevolved symbionts. The taxonomy of Sericomyrmex is problematic because the morphology of the worker caste is generally homogeneous across all of the species within the genus, species limits are vague, and the relationships between them are unknown. We used ultraconserved elements (UCEs) as genome‐scale markers to reconstruct evolutionary history and to infer species boundaries in Sericomyrmex. We recovered an average of ∼990 UCE loci for 88 Sericomyrmex samples from across the geographical range of the genus as well as for five outgroup taxa. Using maximum likelihood and species‐tree approaches, we recovered nearly identical topologies across datasets with 50–95% matrix completeness. We identify nine species‐level lineages in Sericomyrmex, including two new species. This is less than the previously described 19 species, even accounting for two species for which we had no UCE samples, which brings the total number of Sericomyrmex species to 11. Divergence‐dating analyses recovered 4.3 Ma as the crown‐group age estimates for Sericomyrmex, indicating a recent, rapid radiation. We also sequenced mitochondrial cytochrome oxidase subunit I (COI) for 125 specimens. Resolution and support for clades in our COI phylogeny are weak, indicating that COI is not an appropriate species‐delimitation tool. However, taxa within species consistently cluster together, suggesting that COI is useful as a species identification (‘DNA barcoding’) tool. We also sequenced internal transcribed spacer (ITS) and large subunit (LSU) for 32 Sericomyrmex fungal cultivars. The fungal phylogeny confirms that Sericomyrmex fungi are generalized higher‐attine cultivars, interspersed with Trachymyrmex‐associated fungal species, indicating cultivar sharing and horizontal transfer between these two genera. Our results indicate that UCEs offer immense potential for delimiting and resolving relationships of problematic, recently diverged species.

The power to detect recent fragmentation events using genetic differentiation methods.

Habitat loss and fragmentation are imminent threats to biological diversity worldwide and thus are fundamental issues in conservation biology. Increased isolation alone has been implicated as a driver of negative impacts in populations associated with fragmented landscapes. Genetic monitoring and the use of measures of genetic divergence have been proposed as means to detect changes in landscape connectivity. Our goal was to evaluate the sensitivity of Wright’s F st, Hedrick’ G’st, Sherwin’s MI, and Jost’s D to recent fragmentation events across a range of population sizes and sampling regimes. We constructed an individual-based model, which used a factorial design to compare effects of varying population size, presence or absence of overlapping generations, and presence or absence of population sub-structuring. Increases in population size, overlapping generations, and population sub-structuring each reduced F st, G’st, MI, and D. The signal of fragmentation was detected within two generations for all metrics. However, the magnitude of the change in each was small in all cases, and when N e was >100 individuals it was extremely small. Multi-generational sampling and population estimates are required to differentiate the signal of background divergence from changes in Fst, G’st, MI, and D associated with fragmentation. Finally, the window during which rapid change in Fst, G’st, MI, and D between generations occurs can be small, and if missed would lead to inconclusive results. For these reasons, use of F st, G’st, MI, or D for detecting and monitoring changes in connectivity is likely to prove difficult in real-world scenarios. We advocate use of genetic monitoring only in conjunction with estimates of actual movement among patches such that one could compare current movement with the genetic signature of past movement to determine there has been a change.

Phylogenomics, biogeography and diversification of obligate mealybug-tending ants in the genus Acropyga.

Acropyga ants are a widespread clade of small subterranean formicines that live in obligate symbiotic associations with root mealybugs. We generated a data set of 944 loci of ultraconserved elements (UCEs) to reconstruct the phylogeny of 41 representatives of 23 Acropyga species using both concatenation and species-tree approaches. We investigated the biogeographic history of the genus through divergence dating analyses and ancestral range reconstructions. We also explored the evolution of the Acropyga-mealybug mutualism using ancestral state reconstruction methods. We recovered a highly supported species phylogeny for Acropyga with both concatenation and species-tree analyses. The age for crown-group Acropyga is estimated to be around 30Ma. The geographic origin of the genus remains uncertain, although phylogenetic affinities within the subfamily Formicinae point to a Paleotropical ancestor. Two main Acropyga lineages are recovered with mutually exclusive distributions in the Old World and New World. Within the Old World clade, a Palearctic and African lineage is suggested as sister to the remaining species. Ancestral state reconstructions indicate that Old World species have diversified mainly in close association with xenococcines from the genus Eumyrmococcus, although present-day associations also involve other mealybug genera. In contrast, New World Acropyga predominantly evolved with Neochavesia until a recent (10-15Ma) switch to rhizoecid mealybug partners (genus Rhizoecus). The striking mandibular variation in Acropyga evolved most likely from a 5-toothed ancestor. Our results provide an initial evolutionary framework for extended investigations of potential co-evolutionary interactions between these ants and their mealybug partners.

Does genetic diversity of restored sites differ from natural sites? A comparison of Vallisneria americana (Hydrocharitaceae) populations within the Chesapeake Bay

The goal of ecological restoration is to re-establish self-sustaining ecosystems that will resist future perturbation without additional human input. We focus here on the re-establishment of submersed aquatic macrophyte beds in the restoration of the Chesapeake Bay estuary. Degraded environmental conditions are often to blame for poor bed establishment, but genetic factors could also be contributing to low survival. We quantified the effect of restoration practices on genetic diversity in the submersed aquatic plant species Vallisneria americana Michx. (Hydrocharitaceae) in the Chesapeake Bay. In 2007, we collected 440 shoots from 8 restored/natural site pairs and 4 restoration stock repositories, and genotyped those individuals at 10 microsatellite loci. Restoration practices do not appear to negatively impact genetic diversity, and basic measures of genetic diversity within restored sites overlap with natural sites. However, small population size of restored sites, significant inbreeding coefficients within 3 sites, and low overlap of allele composition among sites provide cause for concern. These problems are relatively minor, and we propose several corrections that would alleviate them altogether. Managers should be encouraged by our findings as well as the current state of the genetic diversity within V. americana restoration efforts.

The structure of population genetic diversity in Vallisneria americana in the Chesapeake Bay: implications for restoration

Submersed aquatic macrophyte beds provide important ecosystem services, yet their distribution and extent has declined worldwide in aquatic ecosystems. Effective restoration of these habitats will require, among other factors, reintroduction of genetically diverse source material that can withstand short- and long-term environmental fluctuations in environmental conditions. We examined patterns of genetic diversity in Vallisneriaamericana because it is a cosmopolitan freshwater submersed aquatic macrophyte and is commonly used for restoring freshwater habitats. We sampled 26 naturally occurring populations of V. americana in the Chesapeake Bay estuary and its tributaries and found that the majority of populations have high genotypic diversity and are not highly inbred. Fourteen of the populations had high allelic and genotypic diversity and could serve as source sites for restoration material. However, substantial geographic structuring of genetic diversity suggests that caution should be used in moving propagules to locations distant from their source. In particular, we suggest that propagules at least be limited within four primary geographic areas that correspond to freshwater tidal and non-tidal, oligohaline, and seasonally mesohaline areas of the Chesapeake Bay.

Development of 11 polymorphic microsatellite markers in a macrophyte of conservation concern, Vallisneria americana Michaux (Hydrocharitaceae)

Vallisneria americana Michaux (wild celery) is currently a target of submersed aquatic vegetation restoration efforts in the Chesapeake Bay watershed. To aid these efforts, we have developed 11 polymorphic microsatellite markers to assess the distribution and degree of genetic diversity in both restored and naturally occurring populations in the Chesapeake Bay. In 59 individuals from two populations, we detected two to 10 total alleles per locus. Observed heterozygosity ranged from 0.125 to 0.929, and two loci exhibited significant deviations from Hardy–Weinberg equilibrium in at least one of the populations assayed.

Next-generation museum genomics: Phylogenetic relationships among palpimanoid spiders using sequence capture techniques (Araneae: Palpimanoidea)

Historical museum specimens are invaluable for morphological and taxonomic research, but typically the DNA is degraded making traditional sequencing techniques difficult to impossible for many specimens. Recent advances in Next-Generation Sequencing, specifically target capture, makes use of short fragment sizes typical of degraded DNA, opening up the possibilities for gathering genomic data from museum specimens. This study uses museum specimens and recent target capture sequencing techniques to sequence both Ultra-Conserved Elements (UCE) and exonic regions for lineages that span the modern spiders, Araneomorphae, with a focus on Palpimanoidea. While many previous studies have used target capture techniques on dried museum specimens (for example, skins, pinned insects), this study includes specimens that were collected over the last two decades and stored in 70% ethanol at room temperature. Our findings support the utility of target capture methods for examining deep relationships within Araneomorphae: sequences from both UCE and exonic loci were important for resolving relationships; a monophyletic Palpimanoidea was recovered in many analyses and there was strong support for family and generic-level palpimanoid relationships. Ancestral character state reconstructions reveal that the highly modified carapace observed in mecysmaucheniids and archaeids has evolved independently.