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Abstract Background There are few genomic tools available in melon (Cucumis melo L.), a member of the Cucurbitaceae, despite its importance as a crop. Among these tools, genetic maps have been constructed mainly using marker types such as simple sequence repeats (SSR), restriction fragment length polymorphisms (RFLP) and amplified fragment length polymorphisms (AFLP) in different mapping populations. There is a growing need for saturating the genetic map with single nucleotide polymorphisms (SNP), more amenable for high throughput analysis, especially if these markers are located in gene coding regions, to provide functional markers. Expressed sequence tags (ESTs) from melon are available in public databases, and resequencing ESTs or validating SNPs detected in silico are excellent ways to discover SNPs. Results EST-based SNPs were discovered after resequencing ESTs between the parental lines of the PI 161375 (SC) × 'Piel de sapo' (PS) genetic map or using in silico SNP information from EST databases. In total 200 EST-based SNPs were mapped in the melon genetic map using a bin-mapping strategy, increasing the map density to 2.35 cM/marker. A subset of 45 SNPs was used to study variation in a panel of 48 melon accessions covering a wide range of the genetic diversity of the species. SNP analysis correctly reflected the genetic relationships compared with other marker systems, being able to distinguish all the accessions and cultivars. Conclusion This is the first example of a genetic map in a cucurbit species that includes a major set of SNP markers discovered using ESTs. The PI 161375 × 'Piel de sapo' melon genetic map has around 700 markers, of which more than 500 are gene-based markers (SNP, RFLP and SSR). This genetic map will be a central tool for the construction of the melon physical map, the step prior to sequencing the complete genome. Using the set of SNP markers, it was possible to define the genetic relationships within a collection of forty-eight melon accessions as efficiently as with SSR markers, and these markers may also be useful for cultivar identification in Occidental melon varieties.

Octocorals possess sclerites, small elements comprised of calcium carbonate (CaCO 3 ) that are important diagnostic characters in octocoral taxonomy. Among octocorals, sea pens comprise a unique order (Pennatulacea) that live in a wide range of depths. Habitat depth is considered to be important in the diversification of octocoral species, but a lack of information on sea pens has limited studies on their adaptation and evolution across depth. Here, we aimed to reveal trends of adaptation and evolution of sclerite shapes in sea pens with regards to habitat depth via phylogenetic analyses and ancestral reconstruction analyses. Colony form of sea pens is suggested to have undergone convergent evolution and the loss of axis has occurred independently across the evolution of sea pens. Divergences of sea pen taxa and of sclerite forms are suggested to depend on habitat depths. In addition, their sclerite forms may be related to evolutionary history of the sclerite and the surrounding chemical environment as well as water temperature. Three-flanged sclerites may possess the tolerance towards the environment of the deep sea, while plate sclerites are suggested to be adapted towards shallower waters, and have evolved independently multiple times. The common ancestor form of sea pens was predicted to be deep-sea and similar to family Pseudumbellulidae in form, possessing sclerites intermediate in form to those of alcyonaceans and modern sea pens such as spindles, rods with spines, and three-flanged sclerites with serrated edges sclerites, as well as having an axis and bilateral traits., Competing Interests: James Davis Reimer is an Academic Editor for PeerJ., (©2022 Kushida et al.)

A new species of Etelis is described based on 16 specimens collected from the Red Sea and Western Australia, with confirmed genetic records throughout the Indo-West Pacific. It is similar to and was often misidentified as Etelis carbunculus Cuvier, with both species sharing the diagnostic character of low number of developed gill rakers. Nonetheless, the two species are genetically divergent and differ morphologically in adult body length; proportions of eye, snout, cheek and caudal fin; shape of head, opercular spine and sagittal otolith; and coloration of the tip of the upper caudal fin. Etelis boweni has a wide Indo-west Pacific distribution that largely overlaps with E. carbunculus, and the two species are often caught on the same fishing line., (© 2021 Fisheries Society of the British Isles.)

Octopus oliveri is a widespread and common rocky intertidal cephalopod that mates readily in the laboratory, but for which mating behavior has not been reported previously. Four sets of behavioral experiments were recorded wherein three males, small, medium & large in varying order, were introduced to each of six females, for a total of 24 individual females and 12 individual males utilized in the experiments. Video analysis shows that successful mating occurred in each of the mount, reach and beak-to-beak positions. Mating was observed for all males, regardless of size relative to the female, or order of introduction. Females showed preference for the first male to which they were introduced in experimental pairings rather than any specific male trait, and mating time increased significantly with increasing female size. Five novel microsatellite markers were developed and used to test paternity in the eleven broods resulting from these experimental pairings. We found skewed paternity in each brood, with early male precedence and male size being the best predictors of parentage. Multiple paternity was observed in every experimental cross but was estimated to be comparatively low in the field, suggesting that sperm limitation might be common in this species. We saw no evidence of direct sperm competition in Octopus oliveri , but larger males produced significantly more offspring. This study contributes to the growing research on cephalopod mating systems and indicates that octopus mating dynamics might be more variable and complex than thought previously., Competing Interests: Robert J. Toonen is an Academic Editor and the Section Editor for Aquatic Biology at PeerJ.

Millepora (Cnidaria: Hydrozoa: Milleporidae) spp. are distributed throughout shallow subtropical and tropical marine environments in the Indo-Pacific and Caribbean-Atlantic, and have traditionally been identified using pore characteristics and colony form. Until now, representatives of Millepora spp. on the island of Okinawa-jima, Japan, have been divided into five species; three branching species (Millepora intricata, M. tenera, M. dichotoma), one species with plate-like morphology (M. platyphylla), and one encrusting species (M. exaesa). There have been only a few reports from the Indo-Pacific that have studied the genetic diversity within Millepora spp., although phylogenetic analyses in the Caribbean-Atlantic have proven useful in delimiting closely-related species, while demonstrating that morphologically-based identification systems may have problems. In the present study, we sought to clarify taxonomic confusion of Millepora spp. in the Pacific by using sequence data of the ribosomal internal transcribed spacer (ITS-rDNA) of specimens from Okinawa, Japan and other localities (Johnston Atoll, Great Barrier Reef). Four separate clades were recovered from the ITS-rDNA analyses. Although we examined specimens of all three branching Millepora spp. previously reported from Okinawa-jima Island, in our phylogenetic analyses they were concentrated within a single clade, with only three specimens in other clades. Encrusting Millepora specimens were found within all clades, although it should be noted all species initially start as encrusting forms, and plate-like specimens were found within three clades. Our data also point to the existence of a previously unknown lineage within Millepora characterized by its ability to overgrow live scleractinian corals.

The diversity of DNA sequencing methods and algorithms for genome assemblies presents scientists with a bewildering array of choices. Here, we construct and compare eight candidate assemblies combining overlapping shotgun read data, mate-pair and Chicago libraries and four different genome assemblers to produce a high-quality draft genome of the iconic coral reef Pearlscale Pygmy Angelfish, Centropyge vrolikii (family Pomacanthidae). The best candidate assembly combined all four data types and had a scaffold N50 127.5 times higher than the candidate assembly obtained from shotgun data only. Our best candidate assembly had a scaffold N50 of 8.97 Mb, contig N50 of 189,827, and 97.4% complete for BUSCO v2 (Actinopterygii set) and 95.6% complete for CEGMA matches. These contiguity and accuracy scores are higher than those of any other fish assembly released to date that did not apply linkage map information, including those based on more expensive long-read sequencing data. Our analysis of how different data types improve assembly quality will help others choose the most appropriate de novo genome sequencing strategy based on resources and target applications. Furthermore, the draft genome of the Pearlscale Pygmy angelfish will play an important role in future studies of coral reef fish evolution, diversity and conservation.

The glasseye fish Heteropriacanthus, previously known as a monotypic genus, is now divided into three species based on morphological and genetic features. After examination on the type specimens and literature, herein we resurrect two junior binomens, H. carolinus (Cuvier, 1829) from the Indo-Pacific Ocean and H. fulgens (Lowe, 1838) from the northeastern Atlantic Ocean. Heteropriacanthus cruentatus (Lacepède, 1801) is now considered to be restricted to the Atlantic and southwestern Indian oceans. In light of these observations we discuss the evolutionary history of the genus.

Blooms of alien invasive marine algae have become common, greatly altering the health and stability of nearshore marine ecosystems. Concurrently, herbivorous fishes have been severely overfished in many locations worldwide, contributing to increases in macroalgal cover. We used a multi-pronged, interdisciplinary approach to test if higher biomass of herbivorous fishes inside a no-take marine reserve makes this area more resistant to invasive algal overgrowth. Over a two year time period, we (1) compared fish biomass and algal cover between two fished and one unfished patch reef in Hawai'i, (2) used acoustic telemetry to determine fidelity of herbivorous fishes to the unfished reef, and (3) used metabarcoding and next-generation sequencing to determine diet composition of herbivorous fishes. Herbivore fish biomass was significantly higher in the marine reserve compared to adjacent fished reefs, whereas invasive algal cover differed by species. Herbivorous fish movements were largely confined to the unfished patch reef where they were captured. Diet analysis indicated that the consumption of invasive algae varied among fish species, with a high prevalence of comparatively rare native algal species. Together these findings demonstrate that the contribution of herbivores to coral reef resilience, via resistance to invasive algae invasion, is complex and species-specific.

The greenfish sea cucumber Stichopus chloronotus is an economically and ecologically important sea cucumber species throughout its range. This species is widely distributed, inhabiting coral reefs of the Indo-Pacific Ocean. Our study evaluated population genetic structure and levels of genetic diversity in southern Japan. A total of 180 individuals were collected from eight locations from Okinawa and Okinoerabu Islands and sequenced using mitochondrial 16S ribosomal DNA (16S) and nuclear histone H3 (H3) gene. Only three 16S haplotypes were detected (518 bp) with haplotype diversity ranging from 0 to 0.56 and nucleotide diversity from 0 to 0.1%. H3 showed no variation among the studied locations. It is plausible that such results could be due to a shift to asexual reproduction . Additionally, the presence of the species on the east coast of Okinawa could only be detected in one location and all individuals consisted of a single haplotype. Genetic differences between the east and west coasts of Okinawa have been noticed in other coral reef organisms, and attributed to either ecological or biogeographical historical differences between the coasts due to differing levels of isolation during Pleistocene ice ages. Results from the present study should inform management and conservation policies of S. chloronotus in southern Japan., Competing Interests: James Reimer is an Academic Editor for PeerJ.

The number of goatfish species has increased recently, thanks in part to the application of molecular approaches to the taxonomy of a family with conservative morphology and widespread intraspecific color variation. A new subspecies Mulloidichthys flavolineatus flavicaudus Fernandez-Silva & Randall is described from the Red Sea and Arabian Sea, including Socotra and Gulf of Oman. It is characterized by a yellow caudal fin, 25-28 gill rakers, and 37-38 lateral-line scales and it is differentiated from nominal subspecies Mulloidichthys flavolineatus flavolineatus by 1.7% sequence divergence at the mitochondrial cytochrome b gene. The morphometric examination of specimens of Mulloidichthys flavolineatus flavolineatus revealed variation in head length, eye diameter, and barbel length, in western direction from the Hawaiian Islands, South Pacific, Micronesia, and the East Indies to the Indian Ocean. The population of Mulloidichthys flavolineatus flavicaudus subsp. n. in the Gulf of Aqaba differs from that of the remaining Red Sea by shorter barbels, smaller eyes, shorter head, and shorter pelvic fins. We present a list of 26 endemic fishes from the Gulf of Aqaba and discuss the probable basis for the endemism in the light of the geological history of this region.

Evolutionary genetic patterns in shallow coastal fishes are documented with dozens of studies, but corresponding surveys of deepwater fishes (>200m) are scarce. Here we investigate the evolutionary history of deepwater snappers (genus Etelis), comprised of three recognized Indo-Pacific species and one Atlantic congener, by constructing a phylogeny of the genus with two mtDNA loci and two nuclear introns. Further, we apply range-wide Indo-Pacific sampling to test for the presence and distribution of a putative cryptic species pair within E. carbunculus using morphological analyses and mtDNA cytochrome b sequences from 14 locations across the species range (N=1696). These analyses indicate that E. carbunculus is comprised of two distinct, non-interbreeding lineages separated by deep divergence (d=0.081 in cytochrome b). Although these species are morphologically similar, we identified qualitative differences in coloration of the upper-caudal fin tip and the shape of the opercular spine, as well as significant differences in adult body length, body depth, and head length. These two species have overlapping Indo-Pacific distributions, but one species is more widespread across the Indo-Pacific, whereas the other species is documented in the Indian Ocean and Western Central Pacific. The dated Etelis phylogeny places the cryptic species divergence in the Pliocene, indicating that the biogeographic barrier between the Indian and Pacific Oceans played a role in speciation. Based on historic taxonomy and nomenclature, the species more widespread in the Pacific Ocean is E. carbunculus, and the other species is previously undescribed (referred to here as E. sp.). The Atlantic congener E. oculatus has only recently (∼0.5Ma) diverged from E. coruscans in the Indo-Pacific, indicating colonization via southern Africa. The pattern of divergence at the Indo-Pacific barrier, and Pleistocene colonization from the Indian Ocean into the Atlantic, is concordant with patterns observed in shallow coastal fishes, indicating similar drivers of evolutionary processes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Microscopic mites of the genus Demodex live within the hair follicles of mammals and are ubiquitous symbionts of humans, but little molecular work has been done to understand their genetic diversity or transmission. Here we sampled mite DNA from 70 human hosts of diverse geographic ancestries and analyzed 241 sequences from the mitochondrial genome of the species Demodex folliculorum. Phylogenetic analyses recovered multiple deep lineages including a globally distributed lineage common among hosts of European ancestry and three lineages that primarily include hosts of Asian, African, and Latin American ancestry. To a great extent, the ancestral geography of hosts predicted the lineages of mites found on them; 27% of the total molecular variance segregated according to the regional ancestries of hosts. We found that D. folliculorum populations are stable on an individual over the course of years and that some Asian and African American hosts maintain specific mite lineages over the course of years or generations outside their geographic region of birth or ancestry. D. folliculorum haplotypes were much more likely to be shared within families and between spouses than between unrelated individuals, indicating that transmission requires close contact. Dating analyses indicated that D. folliculorum origins may predate modern humans. Overall, D. folliculorum evolution reflects ancient human population divergences, is consistent with an out-of-Africa dispersal hypothesis, and presents an excellent model system for further understanding the history of human movement.

Zooplanktonic taxa have a greater number of distinct populations and species than might be predicted based on their large population sizes and open-ocean habitat, which lacks obvious physical barriers to dispersal and gene flow. To gain insight into the evolutionary mechanisms driving genetic diversification in zooplankton, we developed eight microsatellite markers to examine the population structure of an abundant, globally distributed mesopelagic copepod, Haloptilus longicornis, at 18 sample sites across the Atlantic and Pacific Oceans (n = 761). When comparing our microsatellite results with those of a prior study that used a mtDNA marker (mtCOII, n = 1059, 43 sample sites), we unexpectedly found evidence for the presence of a cryptic species pair. These species were globally distributed and apparently sympatric, and were separated by relatively weak genetic divergence (reciprocally monophyletic mtCOII lineages 1.6% divergent; microsatellite FST ranging from 0.28 to 0.88 across loci, P < 0.00001). Using both mtDNA and microsatellite data for the most common of the two species (n = 669 for microsatellites, n = 572 for mtDNA), we also found evidence for allopatric barriers to gene flow within species, with distinct populations separated by continental landmasses and equatorial waters in both the Atlantic and Pacific Ocean basins. Our study shows that oceanic barriers to gene flow can act as a mechanism promoting allopatric diversification in holoplanktonic taxa, despite the high potential dispersal abilities and pelagic habitat for these species., (© 2014 John Wiley & Sons Ltd.)

Here, we introduce ezRAD, a novel strategy for restriction site-associated DNA (RAD) that requires little technical expertise or investment in laboratory equipment, and demonstrate its utility for ten non-model organisms across a wide taxonomic range. ezRAD differs from other RAD methods primarily through its use of standard Illumina TruSeq library preparation kits, which makes it possible for any laboratory to send out to a commercial genomic core facility for library preparation and next-generation sequencing with virtually no additional investment beyond the cost of the service itself. This simplification opens RADseq to any lab with the ability to extract DNA and perform a restriction digest. ezRAD also differs from others in its flexibility to use any restriction enzyme (or combination of enzymes) that cuts frequently enough to generate fragments of the desired size range, without requiring the purchase of separate adapters for each enzyme or a sonication step, which can further decrease the cost involved in choosing optimal enzymes for particular species and research questions. We apply this method across a wide taxonomic diversity of non-model organisms to demonstrate the utility and flexibility of our approach. The simplicity of ezRAD makes it particularly useful for the discovery of single nucleotide polymorphisms and targeted amplicon sequencing in natural populations of non-model organisms that have been historically understudied because of lack of genomic information.

Ecological and societal disruptions by modern climate change are critically determined by the time frame over which climates shift beyond historical analogues. Here we present a new index of the year when the projected mean climate of a given location moves to a state continuously outside the bounds of historical variability under alternative greenhouse gas emissions scenarios. Using 1860 to 2005 as the historical period, this index has a global mean of 2069 (±18 years s.d.) for near-surface air temperature under an emissions stabilization scenario and 2047 (±14 years s.d.) under a 'business-as-usual' scenario. Unprecedented climates will occur earliest in the tropics and among low-income countries, highlighting the vulnerability of global biodiversity and the limited governmental capacity to respond to the impacts of climate change. Our findings shed light on the urgency of mitigating greenhouse gas emissions if climates potentially harmful to biodiversity and society are to be prevented.

The comparatively low cost of massive parallel sequencing technology, also known as next-generation sequencing (NGS), has transformed the isolation of microsatellite loci. The most common NGS approach consists of obtaining large amounts of sequence data from genomic DNA or enriched microsatellite libraries, which is then mined for the discovery of microsatellite repeats using bioinformatics analyses. Here, we describe a bioinformatics approach to isolate microsatellite loci, starting from the raw sequence data through a subset of microsatellite primer pairs. The primary difference to previously published approaches includes analyses to select the most accurate sequence data and to eliminate repetitive elements prior to the design of primers. These analyses aim to minimize the testing of primer pairs by identifying the most promising microsatellite loci.

Microsatellites are the markers of choice for a variety of population genetic studies. The recent advent of next-generation pyrosequencing has drastically accelerated microsatellite locus discovery by providing a greater amount of DNA sequencing reads at lower costs compared to other techniques. However, laboratory testing of PCR primers targeting potential microsatellite markers remains time consuming and costly. Here we show how to reduce this workload by screening microsatellite loci via bioinformatic analyses prior to primer design. Our method emphasizes the importance of sequence quality, and we avoid loci associated with repetitive elements by screening with repetitive sequence databases available for a growing number of taxa. Testing with the Yellowstripe Goatfish Mulloidichthys flavolineatus and the marine planktonic copepod Pleuromamma xiphias we show higher success rate of primers selected by our pipeline in comparison to previous in silico microsatellite detection methodologies. Following the same pipeline, we discover and select microsatellite loci in nine additional species including fishes, sea stars, copepods and octopuses.

The consistency of quantitative trait locus (QTL) effects among genetic backgrounds is a key factor for introgressing QTLs from initial mapping experiments into applied breeding programs. We have selected four QTLs (fs6.4, fw4.3, fw4.4 and fw8.1) involved in melon fruit morphology that had previously been detected in a collection of introgression lines derived from the cross between a Spanish cultivar, "Piel de Sapo," and the Korean accession PI161375 (Songwan Charmi). Introgression lines harboring these QTLs were crossed with an array of melon inbred lines representative of the most important cultivar types. Hybrids of the introgression and inbred lines, with the appropriate controls, were evaluated in replicated agronomic trials. The effects of the QTLs were consistent among the different genetic backgrounds, demonstrating the utility of these QTLs for applied breeding programs in modifying melon fruit morphology. Three QTLs, fw4.4, fs6.4 and fs12.1 were subjected to further study in order to map them more accurately by substitution mapping using a new set of introgression lines with recombination events within the QTL chromosome region. The position of the QTLs was narrowed down to 36-5 cM, depending on the QTL. The results presented in the current study set the basis for the use of these QTLs in applied breeding programs and for the molecular characterization of the genes underlying them.