Leveraging eDNA metabarcoding to characterize nearshore fish communities in Southeast Alaska: Do habitat and tide matter?

Nearshore marine habitats are critical for a variety of commercially important fish species, but assessing fish communities in these habitats is costly and time-intensive. Here, we leverage eDNA metabarcoding to characterize nearshore fish communities near Juneau, Alaska, USA, a high-latitude environment with large tidal swings, strong currents, and significant freshwater input. We investigated whether species richness and community composition differed across three habitat types (sand beaches, eelgrass beds, and rocky shorelines) and between high and low tides. Additionally, we tested whether replication of field samples and PCR reactions influenced either species richness or composition. We amplified a 12S mitochondrial locus in our samples and identified 188 fish amplicon sequence variants (ASVs), corresponding to 21 unique taxa, with approximately half of these resolved to single species. Species richness and composition inferred from eDNA differed substantially among habitats, with rock habitats containing fewer taxa and fewer overall detections than sand and eelgrass habitats. The effect of tide was more subtle and suggested a habitat-tide interaction, with differences in taxa between tides largely isolated to sand habitats. Power analyses indicated that additional field sampling is useful to detect subtle changes in species richness such as those due to tide. PCR replicates typically identified a small number of additional taxa. The most notable result from our study was that shore morphology appeared to substantially influence community structure. Rocky shorelines sloped quickly into deep water, while sand and eelgrass habitats descended much more gradually. We hypothesize that differences in taxa observed among habitats were largely due to lack of mixing between bottom and surface water, providing further evidence that eDNA transport is minimal and that many marine eDNA detections are derived from highly localized sampling locations. We suggest that future studies could explore the extent to which habitat and nearshore physical processes influence eDNA detections.

Blue mussels of the Mytilus edulis species complex from South America: The application of species delimitation models to DNA sequence variation

Smooth-shelled blue mussels, Mytilus spp., have a worldwide antitropical distribution and are ecologically and economically important. Mussels of the Mytilus edulis species complex have been the focus of numerous taxonomic and biogeographical studies, in particular in the Northern hemisphere, but the taxonomic classification of mussels from South America remains unclear. The present study analysed 348 mussels from 20 sites in Argentina, Chile, Uruguay and the Falkland Islands on the Atlantic and Pacific coasts of South America. We sequenced two mitochondrial locus, Cytochrome c Oxidase subunit I (625 bp) and 16S rDNA (443 bp), and one nuclear gene, ribosomal 18S rDNA (1770 bp). Mitochondrial and nuclear loci were analysed separately and in combination using maximum likelihood and Bayesian inference methods to identify the combination of the most informative dataset and model. Species delimitation using five different models (GMYC single, bGMYC, PTP, bPTP and BPP) revealed that the Mytilus edulis complex in South America is represented by three species: native M. chilensis, M. edulis, and introduced Northern Hemisphere M. galloprovincialis. However, all models failed to delimit the putative species Mytilus platensis. In contrast, however, broad spatial scale genetic structure in South America using Geneland software to analyse COI sequence variation revealed a group of native mussels (putatively M. platensis) in central Argentina and the Falkland Islands. We discuss the scope of species delimitation methods and the use of nuclear and mitochondrial genetic data to the recognition of species within the Mytilus edulis complex at regional and global scales.

Reevaluating claims of ecological speciation in Halichoeres bivittatus

Understanding the role of ecological processes in speciation has become one of the most active areas of research in marine population biology in recent decades. The traditional view was that allopatry was the primary driver of speciation in marine taxa, but the geography of the marine environment and the dispersal capabilities of many marine organisms render this view somewhat questionable. One of the earliest and most highly cited empirical examples of ecological speciation with gene flow in marine fishes is that of the slippery dick wrasse, Halichoeres bivittatus. Evidence for this cryptic or incipient speciation event was primarily in the form of a deep north-south divergence in a single mitochondrial locus, combined with a finding that these two haplotypes were associated with different habitat types in the Florida Keys and Bermuda, where they overlap. Here we examine habitat assortment in the Florida Keys using a broader sampling of populations and habitat types than were available for the original study, and find no evidence to support the claim that haplotype frequencies differ between habitat types, and little evidence to support any differences between populations. These results severely undermine claims of ecological speciation with gene flow in Halichoeres bivittatus. We argue that future claims of this type should be supported by multiple lines of evidence that illuminate potential mechanisms and allow researchers to rule out alternative explanations for spatial patterns of genetic differences.

A new subspecies of giant sengi (Macroscelidea: Rhynchocyon) from coastal Kenya

A new subspecies of giant sengi or elephant-shrew, first documented in 2008, is described from northern coastal Kenya. All five currently described species and most known subspecies of Rhynchocyon are compared to this new lineage. Molecular analyses using mitochondrial and nuclear markers from the single DNA sample available for the new lineage show differences from other forms and reveal a close relationship with the allopatric golden-rumped sengi R. chrysopygus (0.43% divergence at the 12S mitochondrial locus). This level of 12S divergence is similar to that between other subspecies pairs within Rhynchocyon. Based on three voucher specimens and 843 images from camera traps, the new lineage is similar to R. chrysopygus in the rufous-maroon sides and shoulders but is distinguished by the lack of the golden rump, the presence of jet-black distal rump and thighs, dark dorsal line, and a pronounced nuchal crest of hairs. Though it also shows superficial pelage similarities to two Tanzania species, R. udzungwensis and the dark coastal form of R. cirnei macrurus, the new form has differences in pelage coloration that are clearly diagnosable from all other taxa. This new lineage has an allopatric distribution to all known Rhynchocyon taxa, with the closest congener being R. chrysopygus located 140 km apart. We estimate a potential range size for the new taxon of ~1980 km2 in the Boni and Dodori National Reserves with habitat consisting of mixed thickets and dry forests. Because of its close genetic relationship with R. chrysopygus, its allopatric distribution, and divergent coloration, the new subspecies is designated Rhynchocyon chrysopygus mandelai. The previously described populations of R. chrysopygus from southern coastal Kenya are now designated R. chrysopygus chrysopygus. As the current severe political insecurity in the area threatens the new taxon, we hope that its description will help establish immediate conservation priorities and action for the subspecies and its habitat.

Reevaluating claims of ecological speciation in Halichoeres bivittatus

Understanding the role of ecological processes in speciation has become one of the most active areas of research in marine population biology in recent decades. The traditional view was that allopatry was the primary driver of speciation in marine taxa, but the geography of the marine environment and the dispersal capabilities of many marine organisms render this view somewhat questionable. One of the earliest and most highly cited empirical examples of ecological speciation with gene flow in marine fishes is that of the slippery dick wrasse, Halichoeres bivittatus. Evidence for this cryptic or incipient speciation event was primarily in the form of a deep north-south divergence in a single mitochondrial locus, combined with a finding that these two haplotypes were associated with different habitat types in the Florida Keys and Bermuda, where they overlap. Here we examine habitat assortment in the Florida Keys using a broader sampling of populations and habitat types than were available for the original study, and find no evidence to support the claim that haplotype frequencies differ between habitat types, and little evidence to support any differences between populations. These results severely undermine claims of ecological speciation with gene flow in Halichoeres bivittatus. We argue that future claims of this type should be supported by multiple lines of evidence that illuminate potential mechanisms and allow researchers to rule out alternative explanations for spatial patterns of genetic differences.

Molecular Placement of an Outbreak-Causing Gall Wasp, Zapatella davisae (Hymenoptera: Cynipidae), with Comments on Phylogenetic Arrangements in the Tribe Cynipini

Gall wasps (Hymenoptera: Cynipidae) have fascinated researchers for centuries due to the elaborate diversity of charismatic galls they produce, the presence of unique reproductive systems (e.g., a form of cyclical parthenogenesis), the possible convergent evolution of semiparasitic gall wasp forms (i.e., “inquilines”), and their multitrophic interactions. While many classifications for gall wasps have been proposed, recent DNA sequence efforts combined with taxonomic revisions are beginning to clarify the evolutionary relationships of this group. To date, however, a well resolved phylogeny is lacking, complicating the study of outbreak-causing pest species. Outbreaks by one such species, the black oak gall wasp, Zapatella davisae Buffington & Melika (Hymenoptera: Cynipidae: Cynipini), have led to extensive damage and mortality of black oaks, Quercus velutina L. (Fagales: Fagaceae), in the northeastern United States. Here we sequenced fragments of the nuclear ribosomal gene 28S, and the nuclear protein coding gene long-wavelength opsin from samples of Z. davisae collected on Cape Cod, MA, and Long Island, NY. Using these sequences and sequences previously published from the mitochondrial locus cytochrome b, we performed Bayesian and maximum likelihood multilocus phylogenetic reconstructions based on a concatenated alignment including species of gall wasps in the tribe Cynipini from which all three loci were present in the GenBank database. Confirming morphological work, we find that Z. davisae is most closely related to species in the genera Callirhytis and Neuroterus, and appears to be a basal member of the “Quercus” section of the tribe Cynipini. We find that recent generic reclassifications within the Cynipini have made great progress towards clarifying the taxonomic relationships of species of gall-inducing wasps in this tribe, and we comment on several classifications that require additional research.

A Novel Disease of Mung Bean, Phytophthora Stem Rot Caused by a New Forma Specialis of Phytophthora vignae

An emerging soil-borne disease resembling Phytophthora stem rot was observed on mung bean plants grown in Anhui, China. To identify the causal agent, diseased plants and soil samples from 13 fields were collected to isolate the pathogen. Twenty-two Phytophthora isolates were recovered from the samples and conducted for detailed identification. Based on morphological and molecular characterizations, all the isolates were consistently identified as Phytophthora vignae. Phylogenetic analysis using eight nuclear loci sequences of the internal transcribed spacer (ITS) region, rRNA gene large subunit (LSU), a partial sequence of the beta-tubulin (β-tubulin) gene, the translation elongation factor 1 alpha (EF1-α), the 60S ribosomal protein L10 (60SL10), enolase (Enl) gene, the heat shock protein 90 (HSP90), and the triose phosphate isomerase/glyceraldehyde-3-phosphate dehydrogenase (TigA) and a mitochondrial locus cytochrome c oxidase subunit I (cox1) revealed that the mung bean isolates group in the same clade as P. vignae and its two formae speciales, P. vignae f. sp. adzukicola and P. vignae f. sp. vignae. A host specificity test showed that the mung bean isolates of P. vignae are pathogenic towards mung bean with a much stronger virulence and towards adzuki bean with a relatively weak virulence, but non-pathogenic to the other tested legume crops, soybean, cowpea, pea, common bean, faba bean, and chickpea. The host range of mung bean isolates significantly differs from that of the P. vignae f. sp. adzukicola and f. sp. vignae, based on our results and the previous studies. Thus, the pathogen causing Phytophthora stem rot of mung bean is proposed as a new forma specialis of P. vignae, designated as P. vignae f. sp. mungcola.

Comprehensive evidence for subspecies designations in Cook’s Petrel Pterodroma cookii with implications for conservation management

Summary Cook’s Petrel Pterodroma cookii is an endemic New Zealand seabird that has experienced a large range decline since the arrival of humans and now only breeds on two offshore islands (Te Hauturu-o-Toi/Little Barrier Island and Whenua Hou/Codfish Island) at the extreme ends of its former distribution. Morphological, behavioural, and mitochondrial cytochrome oxidase 1 (CO1) sequence data led a previous study to recognise the two extant populations as distinct conservation management units. Here, we further examine the genetic relationship between the extant populations using two nuclear introns (β-fibint7 and PAX). Using one mitochondrial locus (CO1), we also investigate the past distribution of a single nucleotide polymorphism (SNP) that differentiates the modern populations using bone and museum skins sourced from within its former range across New Zealand’s North and South Islands. We found significant population genetic structure between the two extant Cook’s Petrel populations for one of the two nuclear introns (β-fibint7). The mitochondrial DNA CO1 analysis indicated that the SNP variant found in the Codfish Island population was formerly widely distributed across both the North and South Islands, whereas the Little Barrier Island variant was detected only in North Island samples. We argue that these combined data support the recognition of the extant populations as different subspecies. Previous names for these taxa exist, thus Cook’s Petrel from Little Barrier Island becomes Pterodroma cookii cookii and Cook’s Petrel from Codfish Island becomes P. c. orientalis. Furthermore, we suggest that both genetic and non-genetic data should be taken into consideration when planning future mainland translocations. Namely, any translocations on the South Island should be sourced from Codfish Island and future translocations on the North Island should continue to be sourced from Little Barrier Island only.

A qPCR Assay for the Detection of Phytophthora cinnamomi Including an mRNA Protocol Designed to Establish Propagule Viability in Environmental Samples

Phytophthora cinnamomi causes root and collar rot in many plant species in natural ecosystems and horticulture. A species-specific primer and probe PCIN5 were designed based on a mitochondrial locus encoding subunit 2 of cytochrome c oxidase (cox2). Eight PCR primers, including three forward and five reverse, were designed and tested in all possible combinations. Annealing temperatures were optimized for each primer pair set to maximize both specificity and sensitivity. Each set was tested against P. cinnamomi and two closely related clade 7 species, P. parvispora and P. niederhauseri. From these tests, five primer pairs were selected based on specificity and, with a species-specific P. cinnamomi probe, used to develop quantitative real-time PCR (qPCR) assays. The specificity of the two most sensitive qPCR assays was confirmed using the genomic DNA of 29 Phytophthora isolates, including 17 isolates of 11 species from clade 7, and representative species from nine other clades (all except clade 3). The assay was able to detect as little as 150 ag of P. cinnamomi DNA and showed no cross-reaction with other Phytophthora species, except for P. parvispora, a very closely related species to P. cinnamomi, which showed late amplification at high DNA concentrations. The efficiency of the qPCR protocol was evaluated with environmental samples including roots and associated soil from plants artificially infected with P. cinnamomi. Different RNA isolation kits were tested and evaluated for their performance in the isolation of RNA from environmental samples, followed by cDNA synthesis, and qPCR assay. Finally, a protocol was recommended for determining the presence of P. cinnamomi in recalcitrant environmental samples.

The species of Uroleucon (Hemiptera: Aphididae) living on Adesmia (Fabaceae) in Argentina, with the description of a new species

Uroleucon Mordvilko is the largest genus in the Macrosiphini (Hemiptera, Aphididae) and most of its species live on species of Asteraceae. Uroleucon nahuelhuapense Nieto Nafría & von Dohlen sp. n. is described from apterous viviparous females collected on Adesmia boronioides (Fabaceae) in a locality of Neuquén province (Argentina). The morphological characters and the sequence of the tRNAleu-COII mitochondrial locus support the inclusion of the new species in the genus Uroleucon. Apterous viviparous females of Uroleucon adesmiae Mier Durante & Ortego collected in two Argentinean localities have also been studied and ranges of several quantitative features have been modified. The identification key for the Uroleucon species known in South America by Nieto Nafría et al. (2007) has been modified to include the new species.