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DNA barcoding services

High-throughput processing of biological samples for DNA barcoding.

In 2010 Naturalis started a DNA barcoding program, aimed at producing 12,000 DNA barcodes annually. A single DNA barcode is defined as the section of the genome that is internationally accepted (IBOLECBOL) to be used for species recognition. For a general introduction on DNA barcoding, see the Barcode of Life website.

DNA barcoding team of Naturalis

To enable high-throughput we work with 96-well plates, of which one position is reserved for the negative control. This means that we require specimen input in multiples of 95. To get an indication of the variation of the DNA barcodes within a species and between species, a rule-of-thumb is that three specimens per species can be barcoded. Because of potential failure, more specimens per species can be collected. 

Flora and Fauna of the Netherlands

The Flora and Fauna of the Netherlands will receive special attention. The goal is to cover the entire flora and fauna of the Netherlands.

We are therefor looking for freshly collected material, identified by specialists in multiples of 95. If you fit this profile and see the value of the barcodes for your research, please do not hesitate to contact us.

All specimens collected so far have been registered online at Soortenregister.nl - search for a species and select the 'DNA barcode' tab.

Under 'Projects' you will find direct links to the list of collected specimen for each project at the species register website.

Projects

Overview of the current DNA barcoding projects. If you are interested in a certain project, see possibilities for cooperation or if you are able to obtain material that can be barcoded, please contact us through 

Marine invertebrates

Vertebrates

Fungi

The data produced within these projects will only be made publicly available through the international DNA barcode database BOLD after publication. If you are interested in using data before it has been made available then please contact us for cooperation.

Arachnida projects

BCP0033: Barcoding of Dutch spiders

Progress: 190 of 380 expected specimens, currently 87 barcodes

Constructing a library of the Dutch biota is a central goal of the NCB barcoding initiative. The current catalog of Dutch spiders exceeds 600 species. As such, they are one of the most diverse orders in the country. They are abundant predators in all terrestrial environments. While the taxonomic literature on Dutch species is generally of high quality, it is highly dependent on characteristics of the male and female genitalia, which are absent from juveniles. A complete DNA barcoding library would make the entire fauna at all ontological stages available for study.

Click here for an overview of the collected Dutch species.

A specimen

BCP0041: Rapid inventory of spiders from Vietnam
Progress: 190 of 1,140 specimens, currently no barcodes

Rapid structured inventories of spiders were conducted in three Vietnamese National Parks in 2009. Sampling from one-hectare plots yielded 300-400 adult specimens from each site representing a total of more than 250 species. Representatives of all morphospecies from two of three sites have already been sorted and photographed and work on the third site is in progress. The proposed research will yield DNA barcode sequences from one adult of each sex from each location, plus up to three additional specimens (as available), for each morphospecies. This will provide independent verification of our morphospecies concepts including matching sexes, and also verify shared species between sites.

BCP0069: Barcoding of Dutch Acarina

Progress: 200 of 250 expected specimens, currently no barcodes

TBD

 

Coleoptera projects

BCP0002: Barcoding of forensically relevant Cholevidae
Progress: 0 of 475 expected specimens, currently no barcodes

The family Cholevidae consists of around 1700 species world wide. They are generally found on cadavers, including human remains in forensic cases, making them potentially interesting for the field of forensic entomology. Due to the lack of a reference library, however, they have not been used in any cases. This project has been set up to create such a reference library. For further information, see the Cholevidae scratchpad.

BCP0047: Barcoding of Dutch Coleoptera

Progress: 1,844 of 5,000 expected specimens, currently 581 barcodes

This project is part of the barcoding of the complete Dutch flora and fauna. With almost 4,200 species, the Coleoptera are the richest order of insects in the Netherlands. The Dutch fauna is relatively well known, and a large number of species can be collected in a short time. Large taxonomic problems have already been sorted out in the past, enabling a more efficient process of collecting and identification.

Click here for an overview of the collected Dutch species.

A tansy beetle, Chrysolina graminis

Diptera projects

BCP0059: Forensic entomology

Progress: 0 of 190 expected specimens, currently no barcodes

TBD

BCP0061: Barcoding of Dutch Diptera

Progress: 285 of 1,100 expected specimens, currently 70 barcodes

TBD

Click here for an overview of the collected Dutch species.

BCP0065: Barcoding of Tephritidae

Progress: 190 of 285 expected specimens, currently no barcodes

TBD

Click here for an overview of the collected Dutch species.

BCP0067: Barcoding Dutch galls

Progress: 103 of 380 expected specimens, currently 86 barcodes

TBD

Hemiptera projects

BCP0053: Barcoding of Dutch Hemiptera

Progress: 665 of 950 expected specimens, currently 80 barcodes

Under the frame work of Biodiversity of the Netherlands, this project aims to barcode the Hemiptera of the Netherlands. The first two groups are taken into the consideration are Heteroptera and Auchenorrhyncha (Homoptera). The Psylloidea and Aphidoidea are considered as challenging tasks for later, due to the lack of identified and suitable material.

A crucifer shield bug, Eurydema Oleracea

Click here for an overview of the collected Dutch species.

BCP0054: Barcoding of Brazilian Hemiptera

Progress: 0 of 285 expected specimens, currently no barcodes

TBD

Hymenoptera projects

BCP0057: Barcoding of Dutch Formicidae

Progress: 190 of 190 expected specimens, currently no barcodes

TBD

Click here for an overview of the collected Dutch species.

BCP0059: Barcoding of Dutch Hymenoptera

Progress: 570 of 570 expected specimens, currently no barcodes

TBD

Click here for an overview of the collected Dutch species.

Odonata projects

BCP0046: All Odonate
Progress: 4,663 of 5,000 expected specimens, currently 828 barcodes

NBC Naturalis owns the larges and most diverse collection of Odonata in the world, to which fresh material from expeditions all over the world is added on a regular basis. Dragonflies have not been barcoded to a large extent, and this project aims to start off a global barcoding initiative by generating a considerable amount of barcodes. Pre-adult stages of dragonflies are difficult to identify with morphologic characters, but can provide good indication of water quality. This project therefore provides a valuable reference library for water researchers from organizations such as Rijkswaterstaat, the waterboards and ecology consultancies.

Four specimens of Odonata; Umma saphirina (top left), Nannophya pygmaea or Scarlet Dwarf (top right), Rhinocypha tincta (bottom left) and Coeliccia cyaneothorax (bottom right)

Click here for an overview of the collected Dutch species.

Lepidoptera projects

BCP0022: Barcoding of recent Lepidoptera collections

Progress: 2,375 of 2,660 expected specimens, currently 1,076 barcodes

Over the past 10 years , Lepidoptera expeditions of NCB Naturalis have focussed on Southeast Asia, mainly Vietnam. Much of this material is unique and in many cases even still unknown to science. The specimens are being sorted to morphospecies at first and efforts will then be made to identify and barcode these. The data will be used in biodiversity assessments and reciprocal identifications.

A member of the family Tortricidae

BCP0023: Barcoding of Nepticulidae and other leaf-miners
Progress: 1,500 of 2,500 expected specimens, currently 864 barcodes

Nepticulidae is one of the Lepidopteran families on which in NCB Naturalis is actively doing, taxonomic and phylogenetic research (see the  Nepticuloidea scratchpad). There are regular collecting trips, where the focus is on finding the larva to establish reliable links with hostplants. Where possible, the larva are reared to adults and studied further. As there are only a handful of specialists available worldwide to identify species of this family the DNA barcodes will prove invaluable, and simultaneously aid the ongoing research.

A larva of Stigmella hybnerella, mining in a leaf of the common hawthorn,Crataegus monogyna

BCP0025: Barcoding of Dutch Lepidoptera
Progress: 959 of 4,000 expected specimens, currently 187 barcodes

There are approximately 2800 species of moths and butterflies in the Netherlands. Lepidoptera are the group with worldwide the best barcode coverage. The Dutch will add to this with specialist knowledge of many of the smaller Lepidoptera in particular. Photographs taken for this project will also be used for the website Microlepidoptera.nl.

Click here for an overview of the collected Dutch species.

Terrestrial invertebrate projects

BCP0062: Barcoding of Dutch Neuroptera and Plecoptera

Progress: 0 of 380 expected specimens, currently no barcodes

TBD

BCP0064: Barcoding of Dutch soil invertebrates

Progress: 285 of 570 expected specimens, currently no barcodes

TBD

BCP0065: Barcoding of Dutch Cicada, Orthoptera and Ichneumonidae

Progress: 0 of 190 expected specimens, currently no barcodes

TBD

BCP0066: Barcoding of European Orthoptera

Progress: 350 of 700 expected specimens, currently no barcodes

 

Marine invertebrate projects

BCP0013: Barcoding of Crustacea

Progress: 475 of 1,805 expected specimens, currently 330 barcodes

This project has been created by combining two projects already in progress; the pontoniine shrimps and the gall crabs.

The subfamily Pontoniinae is one of the most radiated groups of shrimps in the world. More than 550 species in 108 genera have been recognized so far. These shrimps mainly inhabit shallow marine waters in tropical and subtropical regions, reaching highest diversity in reef areas. Almost all species are known to live in association with other marine organisms like sponges, coelenterates, echinoderms, bivalves and ascidians. Specimens associated with different hosts (but regarded as conspecific based on their morphology) might belong to separate cryptic species that could be identified on the basis of their barcodes.

In the most recent taxonomic revision of gall crabs, several new species were described (Kropp 1990). No molecular phylogenetic research on this group has been published yet. In this project, the collections of the Florida Museum of Natural History (350 specimens) and NCB Naturalis (500 specimens) are combined. Together these collections contain almost all described species for the Indo-Pacific region. With this project, a start can be made for the phylogenetic reconstruction of this relatively unknown group of crabs. Cryptic species are expected.

A specimen of pontoniine shrimp (photo: Charles Fransen)

BCP0035: Barcoding of Ovulidae
Progress: 95 of 190 expected specimens, currently no barcodes

The Ovulidae have always been considered a rich family. Back in 1994, Rosenberg argued that the variable characteristics in many Ovulidae and the interest of shell traders caused an overestimation of the number of species, and many species that have been described should be considered one and the same species. With the help of molecular techniques, we can determine where to draw boundaries between species and between variations within a species. This project can subsequently be used as a basis for phylogenetic research.

BCP0035: Barcoding of Dutch marine flora and fauna

Progress: 0 of 750 expected specimens, currently no barcodes

TBD

BCP0039: Sponge evolution

Progress: 600 of 600 expected specimens, currently 233 barcodes

Marine lakes are small bodies of seawater fully enclosed by land. They contain a unique fauna of poriferans, which has not been researched to larger extent before. Within this project, the communities of different lakes in Raja Ampat, West Papua and Indonesia are compared to eachother and to the outlying oceans to gain an idea of which species are endemic to the lakes. Are these lakes some sort of inside-out islands?

A specimen of Lissodendoryx fibrosa

Vertebrate projects

All fresh material that is brought into the museum will be subsampled for the DNA barcoding of Dutch birds (BCP0043), mammals (BCP0050), fish (BCP0051) and amphibians and reptiles (BCP0052).

BCP0043: Barcoding of Dutch Birds

Progress: 510 of 1000 expected specimens, currently 406 barcodes

Work on the DNA barcoding of birds collected in the Netherlands had already begun on a small scale at the Zoological Museum of Amsterdam (ZMA), allowing analytical protocols to be honed. This project will extend this work, analyzing available tissue material from the ZMA in 2011, and setting the stage for a subsequent effort to gather barcode records from all type material at the NCB (see also the 3D images of type specimen birds). Data will be used for calculating the molecular distances within populations and an accurate threshold of species level, necessary for delimitating cryptic species and hybrids.

Click here for an overview of the collected Dutch species.

Three specimens of the Bearded Reedling, Panurus biarmicus

BCP0050: Barcoding of Dutch mammals
Progress: 15 of 95 expected specimens, currently no barcodes

TBD

BCP0051: Barcoding of Dutch fish

Progress: 13 of 190 expected specimens, currently no barcodes

TBD

BCP0052: Barcoding of Dutch amphibians and reptiles

Progress: 0 of 95 expected specimens, currently no barcodes

TBD

BCP0058: Bird-Strike

Progress: 23 of 100 expected cases

Birds are regular victims of collisions with air traffic, sometimes with considerable damage to planes. The remains may be difficult to identify, while identifications are important to prevent these “bird strikes”. Requests for identifications of material are sent in to the expert centrum, which sends all tissue material that cannot be used for morphologic identification to the DNA barcoding facility, usually preserved on Whatmann FTA cards. At this moment, NCB Naturalis has a contract with the Royal Netherlands Air Force. The BOLD ID engine (which includes data generated by project BCP0043) is be used to make identifications from generated DNA sequences.

Tracheophyta projects

BCP0008: Barcoding of winter-hardy ferns of Hortus Botanicus

Progress: 0 of 380 expected specimens, currently no barcodes

TBD

BCP0008: Barcoding of tropical ferns of Hortus Botanicus

Progress: 0 of 380 expected specimens, currently no barcodes

TBD

BCP0008: Barcoding of Cycadales of Hortus Botanicus

Progress: 95 of 380 expected specimens, currently no barcodes

TBD

BCP0008: Barcoding of orchid species in living collection of Hortus Botanicus

Progress: 1,045 of 1,045 expected specimens

Over the past 25 years, many tropical orchids have been collected during the Flora Malesiana expeditions and added to the living collection of the Hortus Botanicus in Leiden. This project will generate DNA barcodes from that collection, with a focus on all the vouchered Bulbophyllinae, Coelogyninae and Cypripedioideae.

A specimen of Coelogyne miniata (photo: Rogier van Vugt)

BCP0015: Barcoding of Dutch vascular plants
Progress: 1045 of 5000 expected specimens

This is one of the larger projects of the DNA barcoding program. It will focus on all vascular plants included in Heukels' Flora van Nederland from 2005. Material will be collected by various groups and individuals, including FLORON.

Click here for an overview of the collected Dutch species.

BCP0018: A barcode reference library for Pelargonium

Progress: 87 of 87 expected specimens, currently no barcodes

Pelargonium is a predominantly South African genus well-known for its horticultural importance, but also as a metabolomics model system. The genus has been subjected to several phylogenetic studies and is well-represented in living collections of botanical gardens. All evidence indicates there to be two main Pelargonium clades, one of them comprising 80% of all 280known species. This clade has seen extensive evolution in floral and vegetative morphology, probably in response to the establishment of Mediterranean type climate in the South African Cape.

Bryophyta projects

BCP0001: Barcoding of Dutch mosses

Progress: 685 of 1500 expected specimens, currently 612 barcodes (ITS-2 and trnL-F)

This is a project in cooperation with the Dutch Bryological and Lichenological Society. Mosses are important for the functioning of terrestrial ecosystems and are often used in biomonitoring. Due to the simple morphology and large phenotypical variation, the mosses are often difficult to identify. DNA barcoding can assist in the molecular identification of species in ecological research and various other applications.

A specimen of pale bristle-moss, Orthotrichum pallens

Click here for an overview of the collected Dutch species.

Fungi projects

BCP0011: Barcoding of Dutch mushrooms

Progress: 190 of 760 expected specimens, currently no barcodes

The multi-volume Flora Agaricina Neerlandica provides identification keys, extensive descriptions, and illustrations of all agarics and boleti occurring in the Netherlands, with data on their ecology and distribution, based on vouchered specimens deposited at NHN. Out of the vast number of species, barcoding ectomycorrhizal (ECM) agaric species will be prioritize, because progress in fungal ecology is severely hampered by the lack of reliable reference sequences for identification of root- and soil samples. The proposed barcoding of ECM fungi will greatly enhance ecological and biodiversity studies worldwide, resulting in a greater recognition of the NCB Naturalis collection abroad. Many ECM genera are species rich and are taxonomically challenging. Generating sequences for types and other well-documented collections will create a reliable backbone for systematic studies.

Fungi projects

BCP0011: Barcoding of Dutch mushrooms

Progress: 190 of 760 expected specimens, currently no barcodes

The multi-volume Flora Agaricina Neerlandica provides identification keys, extensive descriptions, and illustrations of all agarics and boleti occurring in the Netherlands, with data on their ecology and distribution, based on vouchered specimens deposited at NHN. Out of the vast number of species, barcoding ectomycorrhizal (ECM) agaric species will be prioritized for the following, because progress in fungal ecology is severely halted by the shortage of reliable reference sequences for identification of root- and soil samples. The proposed barcoding of ECM fungi will greatly enhance ecological and biodiversity studies globally, resulting in a greater recognition of the NCB Naturalis collection abroad. Many ECM genera are species rich and are taxonomically challenging. Generating sequences for types and other well-documented collections will create a reliable backbone for systematic studies.

 

DNA barcoding lab

Facilities & Methods

What is DNA barcoding?

Since December 2011, a video of the DNA barcoding facility of Naturalis has been shown in the LiveScience exhibit of the museum.

Facilities

Two laboratory rooms are equipped for DNA barcoding. The largest (90m2) functions as a biologic lab, the smaller room (50m2) is used for the molecular steps.

In the biology lab specimens are photographed and a tissue sample is taken for DNA extraction, if this has not been done already. In the molecular room the DNA is extracted, PCR is prepared and then this is run in a separate small room that is shared with the DNA markerpoint.

The PCR results are checked on an agarose gel and sent to the outsourced sequencing company. The returned raw data is then processed into DNA barcodes and uploaded to BOLD.

In the future more of our methods will be published on this website, meanwhile please contact us through e-mail if you are interested in more details.

Barcoding labflow

Protocollen (Dutch)

Hier zijn de meest recente versies van alle protocollen specifiek voor de DNA barcoding faciliteit te vinden. Het is aan te raden deze site te gebruiken als bron voor bestanden.

Verzamelprotocollen

Vanwege de grote doorvoer kunnen wij alleen materiaal accepteren dat op juiste wijze wordt aangeleverd. De juiste wijze van verzamelen wordt in onderstaande protocollen uitgelegd, kies het protocol dat voor het materiaal van toepassing is. Uitgangspunt bij het opstellen van de protocollen was in eerste plaats conservering van het DNA, gevolgd door gemak voor de verzamelaar en mogelijkheid tot snelle doorvoer op het lab.

Verzamelprotocol Entomologie

Verzamelprotocol Vaatplanten

Registratie bestanden

Er dienen altijd twee bestanden te worden ingevuld. Het eerste is het SampleSheet bestand en geeft aan wat in welk buisje zit. Dit bestand wordt gebruikt in het lab. Het tweede bestand is voor de taxonomie- en verzamelgegevens. Er is keuze uit een zoölogie en een botanie versie. Deze gegevens worden opgenomen in het collectieregistratiesysteem, komen terecht op de labels en uiteraard in BOLD. Zowel het SampleSheet als het SampleData bestand moeten, correct ingevuld, aanwezig zijn op de barcoding faciliteit voordat we met het materiaal gaan beginnen.

SampleSheet
Registratie welk exemplaar in welk rekje/buisje zit.
Versie 1.3, nieuw per 05/02/2013

SampleData Zoologie
Registratie taxonomie- en verzamelgegevens zoölogische samples. 
Versie 1.2, nieuw per 30/11/2011

SampleData Botanie
Registratie taxonomie- en verzamelgegevens botanische samples. 
Versie 1.2, nieuw per 30/11/2011

SampleData Mycologie
Registratie taxonomie- en verzamelgegevens mycologische samples. 
Versie 2.1, nieuw per 08/08/2013

Het collectieregistratiesysteem voor botanische objecten in gebruik bij Naturalis is BRAHMS. Hoewel de SampleData botanie gemaakt is om compatibel te zijn met BRAHMS, hebben sommige velden een dergelijk lastige uitleg dat wij aanraden om de BRAHMS handleiding bij het invullen van de SampleData Botanie er bij te houden.

Klasse

Voor Nederlands materiaal raden wij het gebruik van het softwarepakket KLASSE aan om taxonomie- en verzamelgegevens bij te houden. Het is in dat geval niet nodig om een SampleData bestand in te vullen, een export file vanuit KLASSE is genoeg. Hiervoor is een handleiding beschikbaar specifiek voor DNA barcoding materiaal.

Fotografie

Voor de fotografie van exemplaren die als voucher dienen bij DNA barcodes zijn door BOLD fotografie richtlijnen opgesteld. In sommige gevallen wijken wij hier echter van af, vanwege taxonomische relevantie of als de foto's ook voor andere doeleinden gebruikt gaan worden. Wij proberen zo veel mogelijk aan te sluiten bij het tevens lopende digitalisatie project van Naturalis, en alle foto's komen in de nog op te zetten beeldbank terecht. Neem voor vragen over fotografie even contact met ons op.

Protocols (English)

Collecting / Preservation

Many different chemicals are used for collecting specimen for natural history collections. Some of these may have negative effects on DNA. A short section in the collection protocol is devoted to this, but we have also gathered all our knowledge in these extensive guidelines. If any chemical that you use is still missing, please contact us.

Extensive guidelines for preserving specimen or tissue for later DNA work
Version 1.0 by Camiel Doorenweerd & Kevin Beentjes, January 2012

KingFisher DNA Extraction

At the barcoding facility we use a Thermo Fisher KingFisher Flex for the DNA extractions, with slightly modified protocols from those of the manufacturers kits. We modified all steps to be on the safe side, as we commonly work with low concentrations of DNA.

To save the robot protocols use right click > save as...

Macherey Nagel (NucleoMag)

Qiagen (BioSprint)

Thermo Fisher (KingFisher DNA Kit)

DNA barcoding lab

Contact

The DNA barcoding facility is located in the Sylvius building of Leiden University, on the sixth floor, wing 4. On the third and sixth floor of this building the DNA markerpoint laboratory can be found; the joint molecular lab of the Biology Section of Leiden University and Naturalis.

Phone: +31 71 527 1912
E-mail: 

Address:
Naturalis Biodiversity Center
DNA barcoding
Sylviusweg 72*
2333 BE Leiden

*Not all GPS/satnav systems recognize Sylviusweg 72; in that case Wassenaarseweg 72 can be programmed.

Map of the Bio-Science Park

Publications

These publications have been created using data produced in the DNA Barcoding facility.

2011

Schilthuizen, M., Scholte, C., van Wijk, R.E.J., Dommershuijzen, J., van der Horst, D., Schlochtern, M.M.Z., Lievers, R., Groenenberg, D.S.J., 2011. Using DNA-barcoding to make the necrobiont beetle family Cholevidae accessible for forensic entomology. Forensic Science International 210, 91-95. Link

2012

van Nieukerken E. J., Doorenweerd C., Stokvis F. R., Groenenberg Dick S. J., 2012. DNA barcoding of the leaf-mining moth subgenus Ectoedemia s. str. (Lepidoptera: Nepticulidae) with COI and EF1-α: two are better than one in recognising cryptic species.Contributions to Zoology 81, 1-24. Link

van Nieukerken Erik, Wagner David, Baldessari Mario, Mazzon Luca, Angeli Gino, Girolami Vincenzo, Duso Carlo, Doorenweerd Camiel, 2012. Antispila oinophylla new species (Lepidoptera, Heliozelidae), a new North American grapevine leafminer invading Italian vineyards: taxonomy, DNA barcodes and life cycle. Zookeys 170, 29-77. Link

van Nieukerken E. J., Mutanen M., Doorenweerd C., 2012. DNA barcoding resolves species complexes in Stigmella salicis and S. aurella species groups and shows additional cryptic speciation in S. salicis (Lepidoptera: Nepticulidae). Entomologisk Tidskrift 132, 235-255.



For more information about our DNA barcoding services, please contact Arjen Speksnijder, Head of Laboratories

arjen.speksnijder@naturalis.nl