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Mitochondrial pseudogenes in insect DNA barcoding: differing points of view on the same issue

Molecular tools have been used in taxonomy for the purpose of identification and classification of living organisms. Among these, a short sequence of the mitochondrial DNA, popularly known as DNA barcoding, has become very popular. However, the usefulness and dependability of DNA barcodes have been recently questioned because mitochondrial pseudogenes, non-functional copies of the mitochondrial DNA incorporated into the nuclear genome, have been found in various taxa. When these paralogous sequences are amplified together with the mitochondrial DNA, they may go unnoticed and end up being analyzed as if they were orthologous sequences. In this contribution the different points of view regarding the implications of mitochondrial pseudogenes for entomology are reviewed and discussed. A discussion of the problem from a historical and conceptual perspective is presented as well as a discussion of strategies to keep these nuclear mtDNA copies out of sequence analyzes.


Introduction
The classification and identification of living organisms, conducted by amateurs and professionals alike, has been classically based on the description and analysis of morphological features. While the general interest in documenting species diversity has grown exponentially over the years, the number of taxonomists and other professionals trained in species identification, such as parataxonomists (Jinbo et al. 2011), has steadily declined. Taking this scenario into account, several researchers have attempted to find different ways to accelerate and facilitate the process of species identification making it accessible to non-specialists.
Much of the recent taxonomic research has focused on the use of molecular tools in the classification and identification of living organisms. Among these efforts, the use of a short stretch of the mitochondrial cytochrome c oxidase subunit I, popularly known as DNA barcode, has received much attention (Hebert et al. 2003a, b, 2004, Janzen et al. 2005, Hajibabaei et al. 2006, Decaëns & Rougerie 2008, Janzen et al. 2009, Strutzenberger et al. 2010. Some authors are so partial to this technique that they have implied, or suggested, that DNA barcoding is superior to the classical, morphologically-based taxonomy, and that it should substitute morphology in species descriptions and identification as well as in studies trying to ascertain the relationships between them ).
Researchers who question the idea that DNA barcoding is a panacea that will solve all taxonomic problems have argued, among other things, that mitochondrial pseudogenes may lead to an overestimation of the actual species diversity, as well as to unreliable or misleading identifications based on barcoding sequences (Song et al. 2008, Buhay 2009, Hlaing et al. 2009, Hazkani-Covo et al. 2010.
In this contribution different views are compared and some of the problems mitochondrial pseudogenes may cause to insect DNA barcoding are discussed.

DNA Barcoding in Entomology
DNA barcoding, a taxonomic method that uses a short, standardized DNA sequence to identify species, has gained increased attention and acceptance from members of the scientific community interested in documenting the Earths' biodiversity (Hebert et al. 2003a, b, Savolainen et al. 2005, Ivanova et al. 2009, Janzen et al. 2009). Since its inauguration in 2004, the Consortium for the Barcode of Life -CBOL, managed primarily by the Canadian Centre for DNA Barcoding at the Biodiversity Institute of Ontario, University of Guelph, Ontario, Canada, has gathered partners from all over the world. Their objective is to build, in less than twenty years, a comprehensive database that will include barcode sequences of all extant eukaryotes , Ratnasingham & Hebert 2007, Jinbo et al. 2011.
One of the advantages of DNA barcoding with respect to traditional taxonomy is the speed and low costs involved in gathering and analyzing data , Strutzenberger et al. 2010. The creation of the CBOL's online database (The Barcode of Life Data System -BOLD: www.barcodinglife.org) has provided an incentive for numerous researchers to join the barcode initiative. The database is easy to access and provides free storage and retrieval of molecular, morphological and geographical data, besides built-in, integrated analysis tools such as tree reconstructions on the basis of genetic similarity (Ratnasingham & Hebert 2007, Frézal & Leblois 2008.
One of the premises on which DNA barcoding relies on is that the genetic variation among species is greater than the variation within other groups, not as a competitor against traditional taxonomy, but as a strong tool to assist in the discovery and description of new taxa. The projected growth of databases such as the BOLD system, which are capable to integrate morphological, physiological and ecological data, strengthen and give respectability to the method.

Mitochondrial Pseudogenes
Pseudogenes, also known as nuclear mitochondrial DNA (NUMTs), are non-functional copies of mitochondrial sequences that have become incorporated into the nuclear genome. The transfer of mitochondrial genes to the nuclear DNA may happen through direct transfer, or may be mediated by RNAs, in which case viral elements are believed to participate (Williams & Knowlton 2001, D'Errico et al. 2004, Frézal & Leblois 2008, Song et al. 2008). According to Strugnell & Lindgren (2007), when transferred to the nucleus, the mitochondrial gene loses its original function, and is free to accumulate mutations, even though the mutation rate of the nuclear DNA is slower than that of the mitochondrial genome.
Pseudogenes have been long known to occur in prokaryotes, where they usually originate when errors in the transcription process cause a gene to "die". A pseudogene is structurally similar to the stretch of DNA it originates from, but may lack a start codon, have duplicated termination codons, and/or abnormal regulatory sequences on either end. For this reason, unlike functional genes, pseudogenes cannot be translated into functional proteins (D'Errico et al. 2004, Gerstein & Zheng 2006. Pseudogenes have been detected in several eukaryotes; they vary in number, size and abundance (Bensasson et al. 2001a, Richly & Leister 2004, Timmis et al. 2004, Arthofer et al. 2010. In humans, for instance, NUMTs are very common, and five of them are known to cause diseases (Hazkani-Covo et al. 2010).
Besides mitochondrial pseudogenes, two other types of pseudogenes exist: processed and unprocessed. The former are copied from RNA and are not found in the same chromosome they originated from. They lack introns and regulatory sequences. Unprocessed pseudogenes, by contrast, can be found in the same chromosome where they originated, and may have introns and regulatory sequences, just as a functional gene (D'Errico et al. 2004).
NUMTs may originate anywhere in the mitochondrial DNA, and may occur as a unique copy in different parts of the genome. These fragments are usually less than 1kb long, but longer fragments seem to be common in mammals (Bensasson et al. 2001a, Richly & Leister 2004, Arthofer et al. 2010).
According to Sorenson & Quinn (1998), even though NUMTs are very similar to their source DNA sequences, they have various degrees of functionality; because they are located in different parts of the cell and away from their origin, they are subject to different evolutionary pressures. This is why misleading conclusions can be reached when pseudogenes are unknowingly included in analyzes of mitochondrial sequences. According to Hazkani-Covo et al. (2010), the generation of NUMTs is an important evolutionary process in continuous development.

History of the Relationship Between NUMTs and Insects
The first record of a mitochondrial pseudogene in Metazoa was for Locusta migratoria (Linnaeus, 1758) (Orthoptera : Acrididae) (Gellissen et al. 1983), when sequences homologous to stretches of the mitochondrial DNA were found in the nuclear genome.
After the first discovery of NUMTs in Orthoptera, thirteen years passed until an important contribution involving pseudogenes was published in insect molecular research. In 1996, Sunnucks & Hales reported on numerous transpositions of mitochondrial sequences similar to the cytochrome oxidase I and II in Sitobion Mordvilko, 1914 (Hemiptera : Aphididae). According to the authors, the non-mitochondrial copies of at least three species seemed to have originated even before transposition. In the same year, Zhang & Hewitt (1996) detected highly conserved pseudogenes in the nucleus of Schistocerca gregaria (Forskal, 1775) (Orthoptera : Acrididae) that had been amplified along with authentic mitochondrial sequences. They observed that pseudogene amplification seemed more common when the source specimen had been preserved dry for longer periods of time, and without drawing further conclusions on that comment, suggested that researchers should seek for NUMTs when conducting population biology research using the mitochondrial DNA as a marker in order to avoid potentially misleading evidence.
Five years after the works mentioned above, Bensasson et al. (2001a) found pseudogenes in all 10 species of Acrididae (Orthoptera) studied, distributed in four subfamilies (Podisminae, Calliptaminae, Cyrtacanthacridinae and Gomphocerinae). Until then, grasshoppers were among the groups believed to harbor a great number of pseudogenes. According to Bensasson et al. (2001a), at least in  Neotrop.,vol. 12,no. 3 Orthoptera, the evolution of NUMTs seems to involve two steps. First, horizontal transfer, which is the simple transposition of mitochondrial DNA to the nucleus; second, post-transfer replication in the nucleus, which allows for the continuation of the pseudogene. According to the summary compiled by Bensasson et al. (2001b), pseudogenes have been found in a total of 82 species of eukaryotes, corresponding to approximately 21 species of insects, particularly in Orhtoptera and Hemiptera. The great majority of reports on NUMTs in animals have been for vertebrates (Blanchard & Schmidt 1996, Sorenson & Quinn 1998, Bensasson et al. 2001b). Other important publications on insect molecular studies appeared in the beginning of the twenty-first century, reporting on the discovery of mitochondrial pseudogenes. For instance, Harrison et al. (2003) located about 100 pseudogenes in Drosophila melanogaster Meigen, 1830 (Diptera: Drosophilidae). Richly & Leister (2004) found pseudogenes in 13 eukaryote species, including Drosophila melanogaster, but failed to find any in Anopheles gambiae Giles, 1926 (Diptera : Culicidae). The variation in the abundance of NUMTs in closely related species in that study, when compared with the variation found for other eukaryotes, was explained as a function of two factors. First, among-species differences in the rate of sequence transfer from the mitochondrial to the nuclear DNA; and second, among-species differences in the rate of loss of NUMTs in the nucleus. These conclusions were corroborated by a similar study by D'Errico et al. (2004), who utilized only D. melanogaster as a representative of Hexapoda.
In 2006, Brower re-evaluated data on Astraptes fulgerator (Walch, 1775) (Lepidoptera: Hesperiidae) and found NUMTs among the barcode sequences published by Hebert et al. (2004). Later, Pamilo et al. (2007) searched for pseudogenes in four insect species, D. melanogaster; A. gambiae; Apis mellifera Linnaeus, 1758 (Hymenoptera : Apidae) and Tribolium castaneum (Herbst, 1797) (Coleoptera : Tenebrionidae), and suggested that the rate of transfer of mitochondrial genes to the nuclear DNA in Apis mellifera and Tribolium castaneum is high with respect to the dipterans sampled. After analyzing the number of NUMTs (>2000) and the relationship between the number of base-pairs transferred per 1 Kb of nuclear sequence (>1.0) in their samples, they also concluded that A. mellifera has the greatest number of NUMTs in the animal kingdom. Hlaing et al. (2009) searched for NUMTs in the genome of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae), D. melanogaster and A. gambiae, concluding that the first species has more NUMTs than the other two. They also concluded that many of the NUMTs detected had originated more recently and for that reason they were difficult to distinguish from their functional counterparts. The authors suggested that similar cases might pose a great problem for DNA barcoding.
Hazkani-Covo et al. (2010) studied sequences of 85 eukaryotes in search for pseudogenes, which they referred to as "molecular Poltergeists". They found NUMTs in 72 species, absent from the study of Richly & Leister (2004). One of their new records was Bombyx mori (Linnaeus, 1758) (Lepidoptera: Bombycidae) with 0.0016% of the nuclear genome composed of NUMTs (the record for Metazoa, Apis mellifera, is 0.081%). Magnacca & Brown (2010) found barcode-like pseudogenes in Hylaeus Fabricius, 1793 (Hymenoptera : Colletidae), which were easily distinguished from their functional counterparts in their nucleotide sequences and translated amino-acids.

Implications of NUMTs for Entomological Studies using DNA Barcoding
Using the subunit I of the cytochrome c oxidase, Hebert et al. (2004) divided Astraptes fulgerator (Lepidoptera : Hesperiidae) into ten different species, most of which (six to seven species) were corroborated by morphological, ethological and ecological evidence. The remaining (cryptic) species were defined based solely on their barcode sequences. Even though the authors did not rule out the possibility that pseudogenes were a problem, they stressed that only 2.8% of their sequences were likely to have been amplified from NUMTs.
The contribution by Hebert et al. (2004) was criticized by Brower (2006) and Song et al. (2008), who suggested that the number of cryptic species had been overestimated because mitochondrial pseudogenes of Astraptes fulgerator had been amplified by the universal primers used by Herbert and collaborators. The critique is based on the fact that when paralogous genes are used in the place of orthologous ones, the assumptions of phylogenetic reconstructions are violated, leading to erroneous reconstructions.
In a study involving DNA barcode sequences as well as pseudogenes of various orthopterans, Song et al. (2008) concluded that the presence of NUMTs in their analysis led to an overestimation of the number of species. Even though they expressed some pessimism that NUMTs can be completely eliminated, they suggested some strategies to help identify these alien sequences: search for ambiguity among sequences, noise, or double peaks in the electropherogram or chromatogram ( Figure 1); sequence translation in search for additional termination codons and the comparison of the amplified sequences with other, published sequences from closely related species. Hebert et al. (2004) suggested sequencing freshly collected specimens (preserved for less than 10 years) and using reverse transcriptase followed by PCR (RT-PCR) to prevent pseudogene amplification, particularly for taxa known to carry NUMTs. Even though they excluded 13 sequences from their analysis because their electropherogram revealed double peaks, they failed to mention other strategies they might have used to look for NUMTs, for instance, searching for additional termination codons. Later, Ratnasingham & Hebert (2007) declared that all sequences submitted to the BOLD system are scrutinized with various tools in search for abnormalities typical of pseudogenes, including the search for termination codons and translation into protein for comparison with the cytochrome c oxidase I product. Zhang & Hewitt (1996) compared the nuclear copies of the mtDNA to mitochondrial heteroplasmy (the presence of more than one type of mtDNA within cells). The latter, a common cause of degenerative diseases, also causes trouble in sequence analysis. The authors mentioned some strategies that can be used to avoid pseudogene contamination in molecular data, such as the use of specific primers, search for well delimited peaks in the chromatograms and termination codons, comparison with other sequences, and re-sampling when radically divergent sequences are found, or contradictory topologies are recovered in an analysis. An additional strategy, mentioned by Calvignac et al. (2011) to avoid NUMTs is to evaluate the rate of evolution of potential pseudogenes as they tend to evolve faster with respect to their paralogous counterparts. Moulton et al. (2010) tested the strategy of using specifically designed primers to amplify the barcode region as a means to avoid co-amplification of NUMTs in 11 species of Orthoptera. Their results casted more doubts on the barcode method, because the use of specific primers only eliminated NUMTs from sequences of one species, and merely reduced the amount of amplified pseudogenes from the others. Several of the pseudogenes found lacked termination codons, a determining factor that makes their identification difficult. Moulton and collaborators regarded the presence of NUMTs as a challenge to insect DNA barcoding, and suggested that a lot more control on sequence quality needs to be exerted, and further studies http://www.biotaneotropica.org.br/v12n3/en/abstract?thematic-review+bn02412032012 http://www.biotaneotropica.org.br Neotrop.,vol. 12,no. 3 on the ubiquity of NUMTs need to be conducted, if the COI is to be used as a universal marker.

Conclusions
Based on all previously published data and discussions about mitochondrial pseudogenes in DNA barcoding, we conclude that, if ignored, NUMTs pose a major problem for taxonomic and phylogenetic studies based exclusively on barcode sequences. Increased control on submission sequences, amplification from fresh material, use of specific primers, careful analysis of chromatograms, and comparison with other sequences should be mandatory to reduce the risk of contamination with NUMTs. In other words, the barcode protocol needs to be adjusted to accommodate for the new information regarding the prevalence of pseudogenes.  http://www.biotaneotropica.org.br http://www.biotaneotropica.org.br/v12n3/en/abstract?thematic-review+bn02412032012 Biota Neotrop.,vol. 12,no. 3 Different steps must be prioritized in future studies using DNA barcoding (Figure 2), as previously suggested by others (Zhang & Hewitt 1996, Song et al. 2008, Calvignac et al. 2011, and methodologies must be specified in such a manner as to allow other researchers to make inferences on the reliability of each dataset. Sequences that resemble a pseudogene should be removed as early as possible, beginning with chromatogram analysis in search for suspicious peaks.
Since the barcode sequence was proposed as a universal marker by Hebert et al. (2003a, b), the number of entomological studies using it have grown exponentially. However, despite the problems discussed in the present study, the majority of them fail to mention the possibility of contamination with pseudogenes in their data, or have neglected to use methodologies aimed to mitigate the problem (Janzen et al. 2005, Hajibabaei et al. 2006, Craft et al. 2010, Dasmahapatra et al. 2010. There are two possible explanations for this behavior: ignorance regarding the prevalence of pseudogenes and/or hurry to publish. Fewer studies using COI in insect taxonomy, however, are more reliable despite the fact that they fail to mention NUMTs, because they also use morphological and/or ecological information (Burns et al. 2007, Decaëns & Rougerie 2008 as corroborating evidence. The DNA Barcoding revolution has introduced a strong tool to aid in the taxonomy and phylogenetic systematics, being particularly useful in pairing individuals of different sexes and uncovering cryptic species, which are very important to understand our biodiversity. However, it should not be treated as a substitute for any technique, nor should it be used, along with their programs and system (BOLD), as the only source of evidence in the place of morphological, ecological and natural history evidence, as it has been the case in some studies. The COI is simply a tool that provides additional evidence, and therefore should be treated as such.
More studies should be conducted in order to understand the prevalence of mitochondrial pseudogenes in the various insect orders. Existing data to date are not very informative and only report on the presence and quantity of NUMTs in some species within a few orders. The pseudogenes are definitely important contaminants in molecular studies using DNA barcoding, and should be searched for, analyzed, and disposed of when detected.