Frequent Misclassification by mtDNA Barcoding as Revealed by nuDNA and/ or Testable Analysis of its Expression Products

The year 2003 was the starting point for a determined campaign that was to have a strong and long-lasting impact on alpha-taxonomy. Hebert [1] presented the term “DNA barcoding” and praised it as a ready-to-use silver bullet for reliable species identification. They claimed that all the 10–25 million animal species on earth could be quickly recognized by large-scale screening of a mitochondrial DNA reference gene with comparably low costs. This idea of a turbo-taxonomic approach received an enormous echo ranging from top-ranking science journals such as Nature [2] to popular media such as The Times [3]. Massive counter-evidence for disagreement of mtDNA classifications with other indicators of species identity was presented by a meta-analysis of 584 studies of 526 eumetazoan genera already in the same year by Funk & Omland [4] who detected mtDNA paraphyly or polyphyly in 23% of 2319 assayed species. This and numerous follow-up papers revealing mismatches between species identities and mtDNA indication in the years since then could not stop the development of the global Consortium for the Barcoding of Life (CBOL) and its some 50 national offshoots. The wide application of mtDNA barcoding continued even after Ross [5] presented another similarly broad meta-analysis confirming the conclusions of Funk & Omland [4]. Caused by much improvement in analysis of nuclear DNA (nuDNA), both regarding methodology and costs, we currently observe an avertion of several biodiversity students from mtDNA barcoding but the general tenacity to adhere to this method remains astonishing. A frequent answer in personal talks with convinced barcoders was that, though not denying occasional occurrence of paraphylies in many species, they deemed the frequency of misclassification on individual level to be negligibly low. Yet, are the frequencies really such low? Figuring out the true performance of Hebert’s barcoding requires to fulfill three preconditions. Precondition 1 is that the alternative methods checking the classification by mtDNA are testable. Testability, or falsification and verification of classification hypotheses is only possible when they are based, in one or the other way, on numeric analyses but not on subjective idiosyncratic decision. Finding real frequencies furthermore requires sufficiently high within-species numbers of samples (precondition 2) and not juxtaposition of alternative classification systems in singletons as seen in the trees frequently published. Most important, or essential, for realistic assessment of barcoding performance is the that the controlling classification systems have the highest likelihood to indicate “true” species identities (precondition 3). In a paper introducing the Gene and Gene Expression (GAGE) species concept, [6] wrote Species are separable clusters that have passed a threshold of evolutionary divergence and are exclusively defined by nuclear DNA sequences and/or their expression products. Nuclear DNA sequences and their expression products are different character systems but have a highly correlated indicative function. Character systems with the least risk of epigenetic or ontogenetic modification have superior indicative value when conflicts between character systems of integrative studies arise.

In other words, “true” species identities are best indicated when classification methods focus on the working points of evolution and this is nuDNA and those expression products of nuDNA least modified by environmental influence and thus most strongly correlating with nuDNA. These correlations are highest in protein sequences and morphology, considerable in behavioral traits and products of metabolism, and weak in ecological traits [7,8]. Another argument to favor morphology among the expression products is the leading role it played in the history of taxonomy since Linnaeus. Condition 1 (testability) and condition 3 (indication close to “true” species identities) are best fulfilled by an approach or working philosophy named by Seifert, Numeric Morphology- Based-Alpha-Taxonomy (NUMOBAT). Using the example of ants (Hymenoptera: Formicidae), I present here the results of 13 studies being in agreement with the three preconditions outlined above. Such hard checks, either nuDNA- or NUMOBAT-based (or both in combination) are still very rare and I have the impression that the advocates mtDNA barcoding mentally displace such studies. The account below lists up the percentage of disagreeing classifications by mtDNA barcoding with the checking systems given in square brackets. Note that “zoogeography” was added as indicator in a case of a strictly parapatric distribution. The error was
0% in Plagiolepis taurica and pyrenaica [9] [nuDNA and NUMOBAT],
1% in Temnothorax nylanderi and crassispinus [10] [NUMOBAT, zoogeography],
6% in 4 species of the Tapinoma nigerrimum group [11] [NUMOBAT],
7% in 5 species of the Cardiocondyla nuda group [12] [NUMOBAT],
15% in Formica pratensis and lugubris [13] [NUMOBAT],
16% in Cardiocondyla latifrons and micropila. (Heinze pers. comm) [NUMOBAT],
17% in 10 species of Tetramorium [14] [NUMOBAT, nuDNA],
19% in 17 species of Neotropical Linepithema [15] [nuDNA],
20% in 8 species of Serviformica (Purcell pers. comm) [nuDNA],
21% in 6 species of the Cataglyphis albicans group [16] [nuDNA],
23% in 3 species of African Cataglyphis [17] [nuDNA, NUMOBAT],
24% in 3 species of Tibetan Myrmica [8] [NUMOBAT, nuDNA],
32% in 2 species of Colobopsis [18] [NUMOBAT].

Averaging these data, the result is sobering: the mean estimated misclassification per individual within 13 studies over 10 genera and 66 species is 16.8%. Wrong indications of Hebert’s barcoding in the same range are supposed by subjective assessment of morphology for the genera Anochoetus and Odontomachus [19] and Solenopsis [20]. Furthermore, there is introgression of mtDNA into nuDNAdefined lineages in socially hybridogenetic ants [21]. In very broad study in Finland, Beresford [22] showed massive bidirectional introgression of heterospecific mtDNA into the populations of Formica polyctena and aquilonia. Looking at the data in the list above, there is clear trend that the lowest classification errors by mtDNA barcoding occur in species with parapatric zoogeography (the Plagiolepis and Temnothorax cases) or reduced frequency of outcrossing due to high frequency of intranidal mating (the Tapinoma nigerrimum and Cardiocondyla nuda group examples). In contrast, higher errors are more frequent in species groups performing extranidal mating or normal nuptial flights and having at least partially sympatric geographic ranges. These data support the idea that ancient hybridization with subsequent introgression of “wrong” matrilines is the most frequent source for mtDNA barcoding errors in ants whereas incomplete lineage sorting during species splitting is rarer and, in the cases reported here, probably responsible for the situation in the parapatric Colobopsis species.

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