Land plants and DNA barcodes: short-term and long-term goals

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.     

The biogeography of mitochondrial and nuclear discordance in animals

Combining nuclear (nuDNA) and mitochondrial DNA (mtDNA) markers has improved the power of molecular data to test phylogenetic and phylogeographic hypotheses and has highlighted the limitations of studies using only mtDNA markers. In fact, in the past decade, many conflicting geographic patterns between mitochondrial and nuclear genetic markers have been identified (i.e. mito-nuclear discordance). Our goals in this synthesis are to: (i) review known cases of mito-nuclear discordance in animal systems, (ii) to summarize the biogeographic patterns in each instance and (iii) to identify common drivers of discordance in various groups. In total, we identified 126 cases in animal systems with strong evidence of discordance between the biogeographic patterns obtained from mitochondrial DNA and those observed in the nuclear genome. In most cases, these patterns are attributed to adaptive introgression of mtDNA, demographic disparities and sex-biased asymmetries, with some studies also implicating hybrid zone movement, human introductions and Wolbachia infection in insects. We also discuss situations where divergent mtDNA clades seem to have arisen in the absence of geographic isolation. For those cases where foreign mtDNA haplotypes are found deep within the range of a second taxon, data suggest that those mtDNA haplotypes are more likely to be at a high frequency and are commonly driven by sex-biased asymmetries and/or adaptive introgression. In addition, we discuss the problems with inferring the processes causing discordance from biogeographic patterns that are common in many studies. In many cases, authors presented more than one explanation for discordant patterns in a given system, which indicates that likely more data are required. Ideally, to resolve this issue, we see important future work shifting focus from documenting the prevalence of mito-nuclear discordance towards testing hypotheses regarding the drivers of discordance. Indeed, there is great potential for certain cases of mitochondrial introgression to become important natural systems within which to test the effect of different mitochondrial genotypes on whole-animal phenotypes.     

Influence of hydrogeographic history and hybridization on the distribution of genetic variation in the pupfishes Cyprinodon atrorus and C. bifasciatus

The evolutionary importance of hybridization in animals has been subject of much debate. In this study, we examined the influence of hydrogeographic history and hybridization on the present distribution of nuclear and mitochondrial DNA variation in two pupfish species, Cyprinodon atrorus and Cyprinodon bifasciatus. Results presented here indicate that there has been limited introgression of nuclear genes; however, mtDNA introgression has been substantial, with complete replacement of the C. bifasciatus mitochondrial genome by that of C. atrorus. Subsequent to this replacement, there has been diversification of mitochondrial haplotypes along major geographic regions in the basin. Evidence was also found that mitochondrial replacement follows a predictable, cyclical pattern in this system, with isolation and diversification followed by re-contact and replacement of C. bifasciatus mitochondrial haplotypes by those of C. atrorus. This pattern is best explained by a combination of a numeric bias towards C. atrorus and mating site selection rather than selection for C. atrorus mitochondrial genome. These results demonstrate the important role hybridization can play in evolution.     

Delineation of Two Sibling Red Algal Species,Gracilaria Gracilis and Gracilaria Dura (Gracilariales,Rhodophyta), Using Multiple DNA Markers: Resurrection of the Species G. Dura Previously Described in the Northern Atlantic 200 Years Ago1

Molecular markers belonging to the three different genomes, mitochondrial (cox2‐cox3 spacer), plastid (rbcL), and nuclear (internal transcribed spacer [ITS] 2 region), were used to compare samples of the two morphologically related species Gracilaria gracilis (Stackh.) Steentoft, L. M. Irvine et Farnham and G. dura (C. Agardh) J. Agardh collected along Atlantic coasts. In northern Europe, the distinction between these two species is ambiguous, and they are currently recognized under the single name of G. gracilis. The low but congruent patterns of genetic divergence observed for markers of the three genomic compartments highly suggest that these two taxa correspond effectively to two different genetic entities as previously described 200 years ago, based on morphological traits. However, thanks to the combination of different DNA markers, occurrence of “incongruent” cytotypes (i.e., mitotypes of G. dura associated with chlorotypes of G. gracilis) in individuals collected from Brittany, suggests interspecific hybridization between the two sibling species studied.     

叶绿体序列和核微卫星显示同域分布润楠属物种杂交严重

樟科润楠属(Machilus Nees)植物为常绿乔木,是亚热带常绿阔叶林常见的优势种,具有重要的经济价值,但属内分类和种间系统进化关系存在较大争议。为了解其可能的原因,该文选择12个同域分布的润楠属物种,采用72个叶绿体DNA序列片段和10个核微卫星位点进行分析。结果表明：(1) 72个叶绿体DNA序列片段共44 025 bp的序列上仅有48个变异位点,多态性极低。(2) 10个核微卫星标记在12个润楠属物种中通用性好、多态性高,不同物种间共享大量等位基因,独有等位基因少。(3)基于叶绿体DNA不能得到支持率高的一致系统进化树,微卫星标记支持建润楠(M.oreophila)、狭叶润楠(M.rehderi)和红楠(M.thunbergii)的分类地位,但其他物种间关系混乱且核-质系统进化关系冲突,其原因可能是种间频繁的杂交渐渗模糊了种间的界限。后续需综合形态学和多种分子标记,开发有效的分析工具,以理解润楠属内复杂的系统进化关系。     

Re-integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding

Species identification of earthworms is usually achieved by careful observation of morphological features, often sexual characters only present in adult specimens. Consequently, juveniles or cocoons are often impossible to identify, creating a possible bias in studies that aim to document species richness and abundance. DNA barcoding, the use of a short standardized DNA fragment for species identification, is a promising approach for species discrimination. When a reference library is available, DNA-based identification is possible for all life stages. In this study, we show that DNA barcoding is an unrivaled tool for high volume identification of juvenile earthworms. To illustrate this advance, we generated DNA barcodes for specimens of Lumbricus collected from three temperate grasslands in western France. The analysis of genetic distances between individuals shows that juvenile sequences unequivocally match DNA barcode clusters of previously identified adult specimens, demonstrating the potential of DNA barcoding to provide exhaustive specimen identification for soil ecological research.     

Phylogenetic network and physicochemical properties of nonsynonymous mutations in the protein-coding genes of human mitochondrial DNA

Theories on molecular evolution predict that phylogenetically recent nonsynonymous mutations should contain more non-neutral amino acid replacements than ancient mutations. We analyzed 840 complete coding-region human mitochondrial DNA (mtDNA) sequences for nonsynonymous mutations and evaluated the mutations in terms of the physicochemical properties of the amino acids involved. We identified 465 distinct missense and 6 nonsense mutations. 48% of the amino acid replacements changed polarity, 26% size, 8% charge, 32% aliphaticity, 13% aromaticity, and 44% hydropathy. The reduced-median networks of the amino acid changes revealed relatively few differences between the major continent-specific haplogroups, but a high variation and highly starlike phylogenies within the haplogroups. Some 56% of the mutations were private, and 25% were homoplasic. Nonconservative changes were more common than expected among the private mutations but less common among the homoplasic mutations. The asymptotic maximum of the number of nonsynonymous mutations in European mtDNA was estimated to be 1,081. The results suggested that amino acid replacements in the periphery of phylogenetic networks are more deleterious than those in the central parts, indicating that purifying selection prevents the fixation of some alleles.     

In defence of model‐based inference in phylogeography

Recent papers have promoted the view that model‐based methods in general, and those based on Approximate Bayesian Computation (ABC) in particular, are flawed in a number of ways, and are therefore inappropriate for the analysis of phylogeographic data. These papers further argue that Nested Clade Phylogeographic Analysis (NCPA) offers the best approach in statistical phylogeography. In order to remove the confusion and misconceptions introduced by these papers, we justify and explain the reasoning behind model‐based inference. We argue that ABC is a statistically valid approach, alongside other computational statistical techniques that have been successfully used to infer parameters and compare models in population genetics. We also examine the NCPA method and highlight numerous deficiencies, either when used with single or multiple loci. We further show that the ages of clades are carelessly used to infer ages of demographic events, that these ages are estimated under a simple model of panmixia and population stationarity but are then used under different and unspecified models to test hypotheses, a usage the invalidates these testing procedures. We conclude by encouraging researchers to study and use model‐based inference in population genetics.