Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II

Hughes, Karen W.; Morris, S. D.; Reboredo-Segovia, Ana L.

doi:10.3897/mycokeys.10.5126

Cited by 15 publications

(13 citation statements)

References 34 publications

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“…In analogy, for sequence types found only in a single study, some sound skepticism is perhaps in place given the sequence quality-related issues involved in studies based on cloning as well as next-generation sequencing (Hyde et al 2013;Lindahl et al 2013;Hughes et al 2015). However, there are examples to the contrary for both of these situations: sequence types found only in one particular study have proved to be authentic, and "species" found in several different studies have proved to be chimeras (Brown et al 2015;Nilsson et al 2015).…”

Section: Sorting Of the List Of Taxa (Sequence Or Study Count)mentioning

confidence: 92%

Top 50 most wanted fungi

Nilsson¹,

Wurzbacher²,

Bahram³

et al. 2016

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Environmental sequencing regularly recovers fungi that cannot be classified to any meaningful taxonomic level beyond "Fungi". There are several examples where evidence of such lineages has been sitting in public sequence databases for up to ten years before receiving scientific attention and formal recognition. In order to highlight these unidentified lineages for taxonomic scrutiny, a search function is presented that produces updated lists of approximately genus-level clusters of fungal ITS sequences that remain unidentified at the phylum, class, and order levels, respectively. The search function (https://unite.ut.ee/top50.php) is implemented in the UNITE database for molecular identification of fungi, such that the underlying sequences and fungal lineages are open to third-party annotation. We invite researchers to examine these enigmatic fungal lineages in the hope that their taxonomic resolution will not have to wait another ten years or more. Key words

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Section: Sorting Of the List Of Taxa (Sequence Or Study Count)mentioning

confidence: 92%

Top 50 most wanted fungi

Nilsson¹,

Wurzbacher²,

Bahram³

et al. 2016

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“…These usually do not focus on taxonomy, but just on a very rough classification, and consequently, quality control is not necessarily focussed on the DNA sequences generated due to the large amount of data. In addition, PCR can produce chimeric sequences ( Hughes et al . 2015 ), especially when DNA derived from multiple species is used (e.g.…”

Section: Ten Reasonsmentioning

confidence: 99%

Ten reasons why a sequence-based nomenclature is not useful for fungi anytime soon

et al. 2018

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Abstract:The large number of species still to be discovered in fungi, together with an exponentially growing number of environmental sequences that cannot be linked to known taxa, has fuelled the idea that it might be necessary to formally name fungi on the basis of sequence data only. Here we object to this idea due to several shortcomings of the approach, ranging from concerns regarding reproducibility and the violation of general scientific principles to ethical issues. We come to the conclusion that sequence-based nomenclature is potentially harmful for mycology as a discipline. Additionally, a classification based on sequences as types is not within reach anytime soon, because there is a lack of consensus regarding common standards due to the fast pace at which sequencing technologies develop.

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“…Therefore, it is very important to detect and filter out such sequences to avoid false diversity estimates (Wintzingerode et al 1997), increasing the number of OTUs and novel discovery of ''species'' from erroneous sequence data (Smith et al 2010;Hoshino 2012). However, it is difficult to detect chimeras as normally they have a short length, and occur near the end of a template (Hughes et al 2015). Most fungal sequence data from high-throughput sequencing available in GenBank were obtained from amplicon sequencing, with ITS as the barcoding locus (Schoch et al 2012).…”

Section: Formation Of Chimerasmentioning

confidence: 99%

Can we use environmental DNA as holotypes?

et al. 2018

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The advantages and disadvantages of giving a valid name to a sequence of DNA detected from environmental specimens is presently a hot debate amongst the mycological community. The idea of using intracellular DNA (''mgDNA'') from environmental samples as holotypes seems at face value, to be a good idea, considering the expansion of knowledge among these 'dark taxa' or 'dark matter fungi' that it could provide (i.e. sequence based taxa without physical specimens and formal nomenclature). However, the limitations of using mgDNA as holotypes needs careful thought, i.e. can we use a short mgDNA fragment, which may contain a small amount of genetic information, to allow discrimination between species? What is the point and are the potential problems of giving valid scientific names to mgDNA? Numerous mycologists and taxonomists, who have many years of experience working on the taxonomy and phylogeny of different groups of fungi, are concerned about the consequences of providing valid names to mgDNA. There has been much debate, through several publications on the considerable problems of using mgDNA as holotypes. The proponents have tried to debate the virtues of using mgDNA as holotypes. Those against have shown that identification to species using mgDNA does not work in many fungal groups, while those for have shown cases where species can be identified with mgDNA. Different disciplines have different reasons and opinions for using mgDNA as holotypes, however even groups of the same disciplines have dissimilar ideas. In this paper we explore the use of mgDNA as holotypes. We provide evidences and opinions as to the use of mgDNA as holotypes from our own experiences. In no way do we attempt to degrade the study of DNA from environmental samples and the expansion of knowledge in to the dark taxa, but relate the issues to fungal taxonomy. In fact we show the value of using sequence data from these approaches, in dealing with the discovery of already named taxa, taxa numbers and ecological roles. We discuss the advantages and the pitfalls of using mgDNA from environmental samples as holotypes. The impacts of expanding the nomenclatural concept to allow using mgDNA from environmental samples as holotypes are also discussed. We provide evidence from case studies on Botryosphaeria, Colletotrichum, Penicillium and Xylaria. The case studies show that we cannot use mgDNA due to their short fragments and the fact that most ITS sequence data presently result from environmental sequencing. We conclude from the evidence that it is highly undesirable to use mgDNA as holotypes in naming fungal species. If this approach adopted, it would result in numerous problems where species identification cannot be confirmed due to limited sequence data available for the holotypes. We also propose an alternative DNA-based system for naming DNA based species which would provide considerably less problems and should be adopted.

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Cloning of ribosomal ITS PCR products creates frequent, non-random chimeric sequences – a test involving heterozygotes between Gymnopus dichrous taxa I and II

Cited by 15 publications

References 34 publications

Top 50 most wanted fungi

Top 50 most wanted fungi

Ten reasons why a sequence-based nomenclature is not useful for fungi anytime soon

Can we use environmental DNA as holotypes?

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