Thursday, October 30, 2014

Metabarcoding soil and leaf-litter

Today I came across a new very interesting paper published in Ecological Indicators. An international group of researchers used established metabarcoding protocols to compare soil and leaf-litter samples with malaise trap samples in southern China and to compare leaf-litter samples with canopy-fogging and morphologically identified spider samples in central Vietnam.

For those that are not so familiar with the term metabarcoding here a text fragment from my most successful blog post ever (sic!): Metabarcoding is a rapid method of biodiversity assessment that combines two technologies: DNA based identification and high-throughput DNA sequencing. It uses universal PCR primers to mass-amplify DNA Barcodes from mass collections of organisms or from environmental DNA. The PCR product is sent to a next generation sequencer and the result is a wealth of DNA sequences. Such sequence collections are auditable, because sites can be sampled by independent parties, or samples can be split, and analysed by certified entities following a standardized protocol. They can also be verified by fieldwork to confirm the presence or absence of particular species. These metabarcode data sets are taxonomically more comprehensive, many times quicker to produce, and less reliant on taxonomic expertise.

However, the leading question in this publication is focusing on the sampling technology rather than the actual analysis in laboratory: Is it possible to substitute ground-level (soil or leaf litter) samples for aboveground samples when conducting biodiversity surveys? The colleagues argue that a soil or leaf-litter sample can be collected in minutes, whereas an aboveground sample, such as from Malaise traps or canopy fogging, can require days to set up and run, during which time they are subject to theft, damage, and deliberate contamination.

It is no secret that we run a lot of malaise traps of the course of Canada's summer seasons and we had only very few instances of damage due to other animals (damage caused by a bear) or vandalism (one case during our school project). However, as reported in the paper there are examples were traps have been damaged by larger animals or uniformed people. New to me was the deliberate adulteration of environmental surveys mentioned. Another disillusioning fact to think about and frankly it makes me angry to think that brilliant minds have to waste their time designing experiments and monitoring tools to counteract potential fraud and vandalism. 

On the other hand it is great to learn that the world we didn't really dare to enter (soil and leaf litter samples) in our efforts to catalog the life on our planet one barcode at a time, can be explored just as effectively as the one literally just centimeters above, which we target with our hundreds of malaise traps:

Here we show that while the taxonomic compositions of soil and leaf-litter samples are very different from aboveground samples, both types of samples provide similar ecological information, in terms of ranking sites by species richness and differentiating sites by beta diversity. In fact, leaf-litter samples appear to be as or more powerful than Malaise-trap and canopy-fogging samples at detecting habitat differences. We propose that metabarcoded leaf-litter and soil samples be widely tested as a candidate method for rapid environmental monitoring in terrestrial ecosystems.

The authors are still cautious with respect to the prospects of metabarcoding but the technology has made substantial progress in the last few years. Some of the issues described already sound like mere technicalities that can be overcome with some creativity and smart testing. 

I think it is necessary to mention that they used different marker systems for the different samples. For the ground-level samples they took 18S and for anything above ground they used COI which could introduce a bias in the analysis but the researchers took this (at least partially) into account:

We caution that the higher prevalence of nematodes and annelids in the ground-level samples could reasonably be attributed to the different genetic markers used; the 18S primers were designed to amplify across the Metazoa, whereas our COI primers are only known to amplify successfully across the Arthropoda (Our COI primers cannot be used to amplify from soil and leaf-litter samples because >99% of returned OTUs are bacterial). Regardless, the taxonomic compositions of the metabarcode datasets are consistent with the microhabitats from which the samples were collected. Soil and leaf-litter microhabitats are indeed highly species-rich in spiders, mites, centipedes, millipedes, roundworms, and ringed worms, whereas canopy-fogging and Malaise-trap samples do capture mostly insects.

I have to admit that I disagree with the authors a little as it is my hope that efforts will be made to develop functional COI primers to amplify DNA Barcodes for all metazoan groups (only very few groups will remain to be a problem) which would allow us to match undetermined metabarcoding samples to reference specimens.

Wednesday, October 29, 2014

Landscape genetics

Landscape genetics is a discipline that combines the fields of population genetics and landscape ecology to facilitate understanding of how geographical and environmental features structure genetic variation. Its analysis involves detection of genetic discontinuities and the correlation of these discontinuities with landscape features. The idea was developed about 11 years ago, so around the same time DNA Barcoding entered the stage. In a recently published review the leading author of the initial publication states that the main objective of modern landscape genetics is to improve our understanding of the effect of global change on genetic patterns to address these two key questions:
  • how has recent global change (i.e., land use and land cover as well as climate change) affected patterns of neutral and adaptive genetic variation; 
  • are species likely to adapt to ongoing global change on an ecological time scale?
The Asian tiger mosquito (Aedes albopictus), which is native to Southeast Asia, was spotted in Houston in 1985. By 1986 it had already reached states like Missouri or Florida, both not bordering Texas. Today it can be found in all of the southern states and as far north as Maine. The mosquito arrived in the U.S. in a shipment of used tires from Japan. Aedes albopictus lays eggs that can survive even if any water evaporates, so they're very easy to transport.

This little beast is a very potent vector as it is known be able to carry more than 50 different viruses among those Dengue or Chikungunya. It is an aggressive daytime biter with an affinity for humans. 

Looking at a map of the current range of Aedes albopictus in the U.S., it is impossible to know how the mosquito spread from its point of introduction, although it could hardly have been by wing power alone, since an adult mosquito flies less than a kilometer in its lifetime. In order to find out how the tiger mosquito spread from the point of introduction, a group of US researchers used landscape genetics as it provides a way to rigorously test competing hypotheses for dispersal.

As a first step they had to establish the genetic structure of the U.S. population. As a so called container mosquito, Aedes albopictus lays its eggs just above the waterline in old tires, flower pot saucers, water bowls, bird baths, and most importantly for this study in cemetery flower vases. To sample the mosquito population the colleagues collected larvae from abandoned flower vases in cemeteries both on the edge and within the core of the mosquitoes' U.S. range in both rural and urban areas.

The immature mosquitoes were raised to adults in the laboratory so the species could be accurately identified (well, DNA Barcoding might have helped to speed this up). Subsequently, DNA was extracted from clipped legs and genotyped at nine different microsatellite locations. The genetic structure retrieved by the microsatellite analysis was then compared to those predicted by 52 different models of mosquito dispersal that variously took into consideration habitat and highways.

It turned out that gene flow over long distances was correlated with highways and bodies of water. People had carried mosquitoes from the core of their range to its edge along highways, likely by semi-trailers or in cars. Wetlands and lakes were important, not because they are breeding sites, but because they tend to occur in areas where frequent rainfall refills artificial containers and supports mosquito growth.

The scientists also looked more closely at what was happening at the range edge. Because Aedes albopictus lays eggs in treeholes and is often found resting at forest edges, they expected forests at the northern edge of the mosquitoes' range to act as natural corridors for dispersal. However, it turned out forests were barriers rather than corridors, perhaps because Aedes albopictus had not been able to displace the native treehole mosquito, Aedes triseriatus.

Our results revealed a combined role of natural and human-aided dispersal throughout the range of Aedes albopictus only two decades after its initial introduction into the USA. Naturalized populations have become sufficiently dense that dispersal and recolonization are now naturally sustained, but long-distance dispersal, particularly between range-core and range-edge sites, is ongoing. A similar trend may be expected for introductions of other exotic species, particularly those that spread rapidly via human-aided transport and then establish dense, naturally connected sites. Our results affirm the importance of denying entry of exotic species as well as rapid responses to eradicate soon after introduction. In addition, multiple introductions can increase genetic diversity and adaptive potential for some established invasive species; potential ports of introduction should remain vigilant to continued importation of Aedes albopictus in used tires and other shipments with the potential to contain eggs and/or larvae from other continents where Aedes albopictus is now established. Finally, the spread of disease is often linked to human-aided transport, land-use change and climate-change ; recent advances in genetic and geographic techniques may improve the utility of landscape genetics as a viable assessment tool for mitigating disease risk, including disease vectors such as Aedes albopictus, at a global scale.

Tuesday, October 28, 2014

Discoveries of the week

A new species of marine interstitial wormshrimp, Ingolfiella maldivensis, is described from coral sand on the inner and outer reef off Magoodhoo island, Faafu atoll, Maldives. Six females were found and compared to other species from the Maldives and those bordering the Indian Ocean and beyond. Morphological resemblance ties it to a species from the Caribbean island of Curaçao. Both species are found in shallow sublittoral interstitial spaces.

Obviously this new species was named after the group of islands where it was found, in the Republic of the Maldives. Wormshrimps are actually amphipods that live exclusively subterranean in most forms of aquatic habitats.
no DNA Barcodes

Tomopaguropsis ahkinpechensis

A new hermit crab species of the family Paguridae, Tomopaguropsis ahkinpechensis sp. n., is described from deep waters (780–827 m) of the Gulf of Mexico. This is the second species of Tomopaguropsis known from the western Atlantic, and the fifth worldwide. The new species is morphologically most similar to a species from Indonesia, T. crinita McLaughlin, 1997, the two having ocular peduncles that diminish in width distally, reduced corneas, dense cheliped setation, and males lacking paired pleopods 1. The calcified figs on the branchiostegite and anterodorsally on the posterior carapace, and the calcified first pleonal somite that is not fused to the last thoracic somite, are unusual paguroid characters. A discussion of the affinities and characters that define this new species is included, along with a key to all five species of Tomopaguropsis.

The species name is derived from the Mayan “Ah-Kin-Pech” (meaning “place of snakes and ticks”), a name given to a settlement where nowadays the Mexican city Campeche,, can be found. The new species was found near Campeche Bank.
no DNA Barcodes

Cynegetis chinensis
The first species of the genus Cynegetis Chevrolat is recorded from China. Cynegetis chinensis Wang & Ren, sp. n. is described from the Ningxia Province in North China. A key to the known species of Cynegetis is given. Diagnostic similarities and differences between Cynegetis and Subcoccinella Agassiz & Erichson are discussed and illustrated.

Cynegetis is a small genus, containing only two species occuring in the Palaearctic region. The group was not known to occur in China until some comprehensive investigations of Chinese ladybird collections revealed this new species, hence the name 'chinensis'.
no DNA Barcodes

Dorstenia luamensis
A new species of Dorstenia L. (Moraceae), D. luamensis M.E.Leal, is described from the Luama Wildlife Reserve, west of Lake Tanganyika and north of the town of Kalemie in the eastern part of the Democratic Republic of Congo (DRC). This species is endemic to the region and differs from any of the other species by its fernlike lithophytic habit and lack of latex. A description and illustration of this species is presented here. Dorstenia luamensis M.E.Leal inhabits moist and shady vertical rock faces close to small waterfalls in the forest; the species is distributed in small populations within the type locality, and merits the conservation status of endangered (EN).

A new member to the large fig family. The genus Dorstenia is the second largest in the family with 105 species. It is unique among all Moraceae due to extremely diverse growth habits and life forms. The species name of the 106th member of the genus refers the Luama Wildlife Reserve where the new species was collected.
no DNA Barcodes

Pilea matthewii, Pilea miguelii, Pilea nicholasii, Pilea nidiae
Pilea matthewii

Four new species of Pilea (Urticaceae) from the Andes of Venezuela are described and illustrated: Pilea matthewii sp. nov., P. miguelii sp. nov., P. nicholasii sp. nov., and P. nidiae sp. nov. The affinities of these species and their positions within the informal classifications of Pilea proposed by Weddell and Killip are discussed. Notes on other species of Pilea found in Venezuela also are presented.

The genus Pilea is another large genus with over 700 species. It belongs to the nettle family. Member species are found worldwide in tropical, subtropical, and temperate areas although the group is absent from Australia, New Zealand, and Europe.
All four species are named in honor of researchers that participated at various field expeditions in which the new species were collected.
no DNA Barcodes

Tynanthus densiflorus, Tynanthus espiritosantensis
Tynanthus densiflorus
Tynanthus is a genus of lianas that is broadly distributed through the Neotropics. Two new species of Tynanthus from Brazil are here described and illustrated: T. densiflorus, from Amazonas, and T. espiritosantensis, from Espírito Santo. T. densiflorus is recognized by the conspicuous interpetiolar glandular fields, a feature rarely found in Tynanthus, and the dense thyrses. Tynanthus espiritosantensis, on the other hand, is recognized by the bromeliad-like prophylls of the axillary buds and the lax thyrses. Information on the distribution, conservation status and morphologically similar species are provided.

Two new species of lianas. The term liana does not represent a taxonomic grouping, but is rather a description of the way the plant grows, much like the terms tree or shrub. Lianas may be found in many different plant families, here the family Bignoniaceae. The names refer to the density of flowers and the type locality respectively.
no DNA Barcodes

Monday, October 27, 2014

Barcoding Diatoms

Diatoms are microscopic algae living in both fresh and salt water.  They are unicellular organisms with silica impregnated cell walls. Living diatoms are among the most abundant forms of plankton and represent an essential part of the food chain in the ocean. Diatoms are responsible for at least 25% of global carbon dioxide fixation. Once dead, their shells accumulate on the seabed and eventually form siliceous sediment deposits.

Given that diatoms are photosynthetic algae, they are restricted to the sunlight zone, i.e. the depth of the water in a lake or ocean that is exposed to sufficient quantities of sunlight to allow for survival.  They are highly sensitive to any environmental changes such as light availability, temperature, salinity etc.  In general, diatoms prefer cold, nutrient rich waters. This is what makes them so valuable as indicators for water quality. The specific composition of diatom communities is a very sensitive instrument to measure changes in aquatic environments.

Diatoms have been regularly used as bioindicators to assess water quality of surface waters, especially in developed countries. Many of the widely used diatom indices have been developed from studies of European rivers and they are integrated in policies such as the European Water Framework Directive.

However, Diatom-based indices require unambiguous taxa identification to species level and that is challenging. Morphological approaches require expert taxonomic knowledge and often expensive infrastructure as many of the characters can only be detected by scanning electron microscopy or similar high-resolution technologies. 

It comes to no surprise that researchers working with diatoms are looking into the application of DNA Barcoding to overcome the difficulties of identification. The community is still discussing which marker to use but to me it seems they slowly gravitate towards a fragment of the 18S rDNA (V4 region) and thereby following the suggestions of the protist working group. A good example for this is a new study by a group of German researchers:

We here investigate how identification methods based on DNA (metabarcoding using NGS platforms) perform in comparison to morphological diatom identification and propose a workflow to optimize diatom fresh water quality assessments. 

Samples from seven different sites along the River Lusatian Neisse and the River Odra were taken and split into three subsamples. One of those was used for next generation sequencing of the 18S V4 region, the second for morphological analysis, and the third for the establishment of clone cultures from individual cells. The colleagues found that next generation sequencing almost always led to a higher number of identified taxa, which was subsequently verified by morphology. Taxa retrieval varies considerably but not necessarily because of natural variation but more as the result of varying taxonomic coverage in available reference databases. The authors conclude:

Next-generation sequencing based eDNA barcoding is not a swiss army knife, but provides a more comprehensive insight into diatom diversity or other protist communities and therefore could be the basis for the ecological projection of global diversity. If thoroughly conducted, the here presented approach not only bears the potential to supplement and improve the old identification system, but beyond that opens up many new opportunities and challenges: diversity data from NGS eDNA barcoding of environmental samples can easily be compared and combined on different spatial (α-, β-, γ-diversity), temporal and taxonomical levels. Therefore, it is applicable for large scale biomonitoring and the quality management of water bodies, for example under governmental frameworks. 

Friday, October 24, 2014

The Sugarcane Borer

The sugarcane borer moth, Diatraea saccharalis, is widespread throughout the Western Hemisphere, and is considered an introduced species in the southern United States. Although this moth has a wide distribution and is a pest of many crop plants including sugarcane, corn, sorghum and rice, it is considered one species.

The larvae bore into the sugarcane stalks. In mature plants the tops tend to weaken or die, sometimes breaking off. In young plants the inner whorl of leaves is killed, resulting in a condition known as "dead heart." The amount and purity of juice that can be extracted from cane is reduced when borers are present, and sucrose yield may be decreased 10 to 20%. Lastly, when seed cane is attacked, the tunneling by borers makes the seed piece susceptible to fungal infection. Sugarcane borer attacks plants in the family Poaceae (true grasses). Though principally a pest of sugarcane, this insect also will feed on other crops such as corn, rice, sorghum, and sudangrass. However, the damage to those is usually fairly modest.

Despite the damage caused by this pest species only few studies have investigated the existence of cryptic species or the population structure of this moth. Especially for species with a widespread distribution it should be determined whether they are truly one species, or rather a complex of sibling species. In addition, it is unknown if this insect may have been introduced into the southern United States once or on several occasions which would be reflected in genetically distinct populations which can vary in their susceptibility to natural enemies and control measures. 

New research, just published in PLoSONE, focused on the population structure of  sugarcane borers in the southern United States with the hopes to contribute to its management as well as to help identify future introductions and their likely region of origin. A group of US researchers investigated this question by collecting D. saccharalis in Texas, Louisiana and Florida and by examining their population structure using amplified fragment length polymorphisms (AFLPs). In addition, a 658 base pair region of the mitochondrial DNA COI gene was sequenced from several individuals from each southern United States population. The mitochondrial COI sequences were compared to publicly available COI sequences for D. saccharalis, to investigate potential source populations for those established in the southern US, as well as to estimate the number of potential cryptic species which may exist within this species.

The DNA Barcode data of the study indicated the existence of at least three distinct lineages: A Florida lineage, a lineage including Texas, Louisiana and Mexico, and a third lineage from South America that includes Brazil, Argentina, and Bolivia. In the literature a fourth lineage from Colombia is also discussed. Both AFLP and COI analysis show that the Florida lineage represents a genetically distinct cluster. This degree of genetic divergence suggests that Florida D. saccharalis could represent a distinct species.

The authors make clear that it would be premature to speak of several new cryptic species:
To be robust, defining species limits should include multiple lines of evidence. Such an approach is referred to as integrative taxonomy and should include morphological, behavioral, molecular and geographic data. Thus, although our data strongly suggests the existence of a D. saccharalis cryptic species complex, further lines of evidence would provide additional support of this assertion.

However, even if we don't know if we are looking at several distinct species or not, the study represents a big step forward:  Genetically distinct lineages may differ in their damage potential and/or in their vulnerability to pest control strategies such as biological control. The ability to characterize and identify genotypes of D. saccharalis and related species or as of yet undiscovered species will improve pest management efforts against this pest and improve area-wide control efforts across its geographic distribution.

Thursday, October 23, 2014

Milkfish fry fishery

According to the Food and Agriculture Organization of the United Nations (FAO) Milkfish (Chanos chanos) is one of the most important food fish species in the world. In Indonesia, Taiwan and the Philippines, more than a quarter of a million tonnes of milkfish are harvested annually in brackish ponds, which represents about 60% of the total fish production from aquaculture in Southeast Asia. This huge amount sourced from a single fish commodity is projected to further increase in the coming years to meet the dietary protein needs of the ever-growing population in Southeast Asia. 

Milkfish farming in Southeast Asia started about six centuries ago. Culture methods in a variety of enclosures are constantly being improved upon. The traditional milkfish industry depended totally on an annual restocking of farm ponds with juvenile fish reared from wild-caught fry. Seasonal and annual variations in fry availability made the industry vulnerable. During the past decade, research focused on the mass production of fry in hatcheries to become independent from wild-caught fry. However, it seems this hasn't been taken up completely. Large quantities of fry are still taken from the Ocean and that causes another problem:

Milkfish fry fishery, an important industry in the Philippines, uses non-selective fishing gears and push nets in coastal areas which lead to the capture of other non-targeted juvenile aquatic species. Unfortunately, information on the amount and the identity of by-catch species is lacking thus the extent of impact of the fry fishery is not known.

A new study from the Philippines shows that by-catch fish species of the milkfish fry industry included various marketable food fish, culture species and aquarium trade species. The researchers used DNA Barcoding to  identify postlarval and juvenile fish samples that were collected from the catch of local fishers using traditional fishing gears and push nets, with milkfish fry as target species.

By-catch in fisheries is a well known global problem. In 2003 it was estimated that approximately 20 million metric tonnes, representing about a quarter of the total world catch are actually by-catch. This represents a serious threat to biodiversity and coastal ecosystem integrity. The present study shows the utility of DNA Barcoding in identifying the juvenile fish species threatened as by-catch in fry fishery. It proves to be very valuable in aiding management efforts for the sustainability of these natural resources.

Wednesday, October 22, 2014

Biological Control of Hemlock Woolly Adelgid

hemlock woolly adelgid
The hemlock woolly adelgid (Adelges tsugae), a native of Asia, is a <1 mm long reddish purple insect that lives within its own protective coating. White, woolly masses that shelter these sap-feeding insects can be found at the bases of hemlock needles along infested branches. The presence of these white sacs, which resemble tiny cotton balls, indicate that a tree is infested. The hemlock woolly adelgid is a threat to North American hemlock forests. As of 2007, 50% of the geographic range of eastern hemlock (Tsuga canadensis) had been impacted. The feeding activities of these hemipterans reduces new shoot growth, premature needle drop, thinned crowns, branch tip dieback, and eventual tree death. 

Laricobius osakensis
Aside from recommending the use of insecticides researchers focused on biological control measures for this pest. In 2006 the derodontid Laricobius osakensis was imported from Japan to the United States for study in quarantine facilities as a potential biological control agent. Four years later it was granted release from quarantine by the U.S. Department of Agriculture. However, it seems that this was premature. A new study published in the Southeastern Naturalist describes what happened not much later:

However, after sequencing DNA barcodes for members of the L. osakensis colony in the fall of 2011, it was discovered that the colony was contaminated by another Japanese species, Laricobius naganoensis. 

The problem is that regulations clearly state that insects shipped from abroad must not contain unauthorized species; therefore the presence of L. naganoensis within the L. osakensis colony resulted in the placement of the L. osakensis colony back into quarantine before beetles were released in the field.

Laricobius naganoensis is a species that lives in sympatry with Laricobius osakensis. Both species are morphologically very similar which makes it difficult to differentiate them. Females cannot be reliably differentiated using morphology and males only by their genitalia but this identification requires dissection of dead specimens which is something one would like to avoid if the goal is to release the beetles as biological control agent.

The researchers were looking for  a quick and inexpensive assay to identify both species and evaluated non-lethal DNA extraction methods. They designed a restriction fragment length polymorphism (RFLP) assay based on two enzymes (AluI and MboII ) using DNA Barcodes. In addition they found out that a single antenna from a specimen is sufficient to retrieve a DNA Barcode sequence. Further research will have to show if the removal of an antenna will have an impact on beetle survival and reproduction but this is a very promising result:

Without the proper permits, L. naganoensis cannot be released legally in the US. Therefore, distinguishing between L. osakensis and L. naganoensis is currently necessary for universities and state or federal agencies that will be importing L. osakensis from Japan for biological control of Hemlock Woolly Adelgid. The RFLP assay developed here is less expensive and less time consuming than DNA sequencing, and the equipment needed for this assay is available in most basic molecular labs. The enzymes AluI and MboII were each sufficient for distinguishing the species. However, since there is likely to be more natural diversity than we have sampled to date, possibly resulting in additional banding patterns, we recommend using both enzymes independently and sequencing any individuals for which the assay results do not match or which produce new gel patterns not reported here.