Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty‐five accessions of the species‐rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species‐level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0–3 bp). Comparisons to previously published mitochondrial cox2–3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2–3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.     

Building the Coleoptera tree‐of‐life for >8000 species: composition of public DNA data and fit with Linnaean classification

The species representation of public databases is growing rapidly and permits increasingly detailed phylogenetic inferences. We present a supermatrix based on all gene sequences of Coleoptera available in Genbank for two nuclear (18S and 28S rRNA) and two mitochondrial (rrnL and cox1) genes. After filtering for unique species names and the addition of ?2000 unpublished sequences for cox1 and 18S rRNA, the resulting data matrix included 8441 species‐level terminals and 6600 aligned nucleotide positions. The concatenated matrix represents the equivalent of 2.17% of the 390 000 described species of Coleoptera and includes 152 beetle families. The remaining 29 families constitute small lineages with ?250 known species in total. Taxonomic coverage remains low for several major lineages, including Buprestidae (0.16% of described species), Staphylinidae (1.03%), Tenebrionidae (0.90%) and Cerambycidae (0.58%). The current taxon sampling was strongly biased towards the Northern Hemisphere. Phylogenetic trees obtained from the supermatrix were in very good agreement with the Linnaean classification, in particular at the family level, but lower for the subfamily and lowest for the genus level. The topology supports the basal split of Derodontidae and Scirtoidea from the remaining Polyphaga, and the broad paraphyly of Cucujoidea. The data extraction pipeline and detailed tree provide a framework for placement of any new sequences, including environmental samples, into a DNA‐based classification system of Coleoptera.     

Multilocus species delimitation in Mesoamerican Scaptotrigona stingless bees (Apidae: Meliponini) supports the existence of cryptic species

Accelerating taxonomic knowledge and making accurate species identifications are critically important given the current biodiversity crisis, particularly in biodiversity hotspots such as Mesoamerica. Objective species delimitation that reduces investigator‐driven bias is fundamental to the establishment of appropriate conservation strategies, above all in managed species. Previous morphological and molecular studies on three managed stingless bee species of the genus Scaptotrigona distributed in Mexico (S. mexicana, S. pectoralis and S. hellwegeri) suggested that both S. mexicana and S. hellwegeri are cryptic species complexes. Herein we tested species delimitation by analysing sequence information of five markers (two mitochondrial: cox1 and 16S, and three nuclear: ITS1, EF1‐α, ArgK) within a Bayesian coalescent framework to test the putative species. We obtained two different hypotheses using a Generalized Mixed Yule Coalescent (GMYC) model: four (cox1) and six (16S) species. After the species validation step with the Bayesian species‐delimitation analysis (BPP), we suggest that only S. mexicana is a complex of two species with different distribution (along the Pacific and the Atlantic coasts, respectively). We highly recommend avoiding colony exchange between geographical regions in order to conserve the genetic integrity of both taxa.     

Molecular Phylogeny and Evolutionary Relationships between the Ciliate Genera Peniculistoma and Mytilophilus (Peniculistomatidae,Pleuronematida)

Peniculistoma mytili and Mytilophilus pacificae are placed in the pleuronematid scuticociliate family Peniculistomatidae based on morphology and ecological preference for the mantle cavity of mytiloid bivalves. We tested this placement with sequences of the small subunit rRNA (SSUrRNA) and cytochrome c oxidase subunit 1 (cox1) genes. These species are very closely related sister taxa with no distinct genetic difference in the SSUrRNA sequence but about 21% genetic difference for cox1, supporting their placement together but separation as distinct taxa. Using infection frequencies, M. pacificae, like its sister species P. mytili, does not interact with Ancistrum spp., co‐inhabitants of the mantle cavity. On the basis of these ecological similarities, the fossil record of host mussels, and features of morphology and stomatogenesis of these two ciliates, we argue that M. pacificae derived from a Peniculistoma‐like ancestor after divergence of the two host mussels. Our phylogenetic analyses of pleuronematid ciliates includes the SSUrRNA gene sequence of Sulcigera comosa, a Histiobalantium‐like ciliate from Lake Baikal. We conclude: (i) that the pleuronematids are a monophyletic group; (ii) that the genus Pleuronema is paraphyletic; and (iii) that S. comosa is a Histiobalantium species. We transfer S. comosa to Histiobalantium and propose a new combination Histiobalantium comosa n. comb.     

Evidence for extreme sequence divergence between the male‐ and female‐transmitted mitochondrial genomes in the bivalve mollusc,Modiolus modiolus (Mytilidae)

Marine mussels of the family Mytilidae, as well as a number of other bivalves, have a unique system of mitochondrial DNA inheritance called doubly uniparental inheritance (DUI). DUI is characterized by the presence of an ‘F’ mitochondrial genome that is transmitted through mothers to daughters and sons, and an ‘M’ mitochondrial genome that is transmitted only from fathers to sons. In this paper, we demonstrate that DUI exists in the horse mussel, Modiolus modiolus (Linnaeus, 1758) and compare the pattern of molecular evolution of the M and F types in this species. Total DNA was isolated from M. modiolus male and female gonad tissues, as well as from spawned sperm cells. From these DNA samples, partial mitochondrial DNA fragments were amplified from both cytochrome c oxidase subunit I (cox1), and 16S ribosomal RNA (rrnL) genes. Based on cox1 and rrnL sequences, heteroplasmy was observed in M. modiolus and characterized by the resolution of two mitotypes: an F mitotype present in tissues of both males and females, and an M mitotype present in spawned sperm. Using standardized p‐distance and Tamura‐Nei values, M. modiolus is found to display the highest M/F conspecific sequence divergence for any member of the family Mytilidae (i.e. 38% M/F sequence divergence, which is 9% higher than any other intraspecific M/F comparison for the family Mytilidae when standardized using p‐distances across all taxa observed). Sequence analysis also indicated that the M. modiolus M mitotype evolves significantly faster than its conspecific F type. The findings discussed herein broaden the range of mytilid species known to exhibit DUI and they also establish a new threshold for the genetic divergence of male mytilid mitochondrial genomes.     

Identification of scuticociliates (Pseudocohnilembus persalinus,P. longisetus,Uronema marinum and Miamiensis avidus) based on the cox1 sequence

Scuticociliatosis is characterized as highly histophagous, causing systemic tissue destruction and high mortality in cultured marine fish. Some of the scuticociliates have been implicated as the causative agents of scuticociliatosis. Here, we describe our study to differentially identify various species in complex animal-sourced samples, namely olive flounder Paralichthys olivaceus and black rockfish Sebastes schlegelii suffering from scuticociliatosis. The mitochondrial cytochrome c oxidase 1 (cox1) gene from the scuticociliates was amplified and sequenced. The divergence percentage of small subunit ribosomal DNA sequence between average scuticociliate species was found to be low (8.3%) but the genetic divergence of cox1 sequence reached 23.5%, suggesting that a hyper-variable region of the cox1 gene could be used as a diagnostic DNA barcoding region. Thus, we developed species-specific primers for use in multiplex PCR of complex (pooled) samples. The primers yielded species-specific fragments (of distinct size) that allowed for simple, rapid, and effective identification and differentiation of multiple species present in a single sample.     

Genetic delineation between and within the widespread coccolithophore morpho‐species Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyta)

Emiliania huxleyi and Gephyrocapsa oceanica are abundant coccolithophore morpho‐species that play key roles in ocean carbon cycling due to their importance as both primary producers and cal‐cifiers. Global change processes such as ocean acidification impact these key calcifying species. The physiology of E. huxleyi, a developing model species, has been widely studied, but its genetic delineation from G. oceanica remains unclear due to a lack of resolution in classical genetic markers. Using nuclear (18S rDNA and 28S rDNA), mitochondrial (cox1, cox2, cox3, rpl16, and dam), and plastidial (16S rDNA, rbcL, tufA, and petA) DNA markers from 99 E. huxleyi and 44 G. oceanica strains, we conducted a multigene/multistrain survey to compare the suitability of different markers for resolving phylogenetic patterns within and between these two morpho‐species. The nuclear genes tested did not provide sufficient resolution to discriminate between the two morpho‐species that diverged only 291Kya. Typical patterns of incomplete lineage sorting were generated in phylogenetic analyses using plastidial genes. In contrast, full morpho‐species delineation was achieved with mitochondrial markers and common intra‐morpho‐species phylogenetic patterns were observed despite differing rates of DNA substitution. Mitochondrial genes are thus promising barcodes for distinguishing these coccolithophore morpho‐species, in particular in the context of environmental monitoring.     

Applicability of RAD‐tag genotyping for interfamilial comparisons: empirical data from two cetaceans

Restriction‐site‐associated DNA tag (RAD‐tag) sequencing has become a popular approach to generate thousands of SNPs used to address diverse questions in population genomics. Comparatively, the suitability of RAD‐tag genotyping to address evolutionary questions across divergent species has been the subject of only a few recent studies. Here, we evaluate the applicability of this approach to conduct genome‐wide scans for polymorphisms across two cetacean species belonging to distinct families: the short‐beaked common dolphin (Delphinus delphis; n = 5 individuals) and the harbour porpoise (Phocoena phocoena; n = 1 individual). Additionally, we explore the effects of varying two parameters in the Stacks analysis pipeline on the number of loci and level of divergence obtained. We observed a 34% drop in the total number of loci that were present in all individuals when analysing individuals from the distinct families compared with analyses restricted to intraspecific comparisons (i.e. within D. delphis). Despite relatively stringent quality filters, 3595 polymorphic loci were retrieved from our interfamilial comparison. Cetaceans have undergone rapid diversification, and the estimated divergence time between the two families is relatively recent (14–19 Ma). Thus, our results showed that, for this level of divergence, a large number of orthologous loci can still be genotyped using this approach, which is on par with two recent in silico studies. Our findings constitute one of the first empirical investigations using RAD‐tag sequencing at this level of divergence and highlights the great potential of this approach in comparative studies and to address evolutionary questions.     

Polymorphism of the <Emphasis Type="Italic">cox1</Emphasis> gene in cercariae isolates of bird schistosomes (Trematoda:Schistosomatidae) from ponds of Moscow and Moscow region

Polymorphism of a 810-bp fragment of mitochondrial cox1 gene was studied in 15 cercariae isolates of bird schistosomes (family Schistosomatidae), which were collected in water bodies of Moscow and Moscow oblast and represented three species: Trichobilharzia szidati, T. franki, and T. regenti. A substantial predominance of AT (65.4%) was characteristic of the cox1 sequences in all three species. Rare single nucleotide substitutions determined low (0.2–0.9%) intraspecific nucleotide and amino acid sequence diversity. Haplotype diversity h was high (80–100%) in all three species, suggesting a unique character for almost all cox1 sequences in the sample. Phylogenetic trees based on the nucleotide and amino acid sequence variations were constructed to study the relationships of the three schistosome species. A high support was observed for the main branching node that reflects differentiation of the monophyletic group Trichobilharzia and species of the genera Bilharziella (B. polonica), Dendritobilharzia (D. pulverulenta), and Gigantobilharzia (G. huronensis). Based on the nucleotide substitutions and amino acid polymorphisms, two groups of isolates, which parasitize Lymnaea stagnalis (T. szidati) and snails of the group Radix (T. franki and T. regenti) respectively, were isolated in the genus Trichobilharzia. The time of divergence between the two schistosome groups infecting snails of the genera Radix and Lymnaea was calculated from the cox1 nucleotide substitution rate, which is known for Asian and Indian blood flukes from the genus Schistosoma and is 2–3% per million years on average. Divergence of the three bird schistosome species under study and divergence of the Asian species of mammalian schistosomes were almost concurrent, dating back to 2.5–3.8 Myr ago. Factors responsible for the lack of intraspecific subdivision with respect to the cox1 in bird schistosomes and the lack of separation between two species (T. franki and T. regenti) are discussed.     

Phylogenetic Relationships Among Colletotrichum Pathogens of Strawberry and Design of PCR Primers for their Identification

Isolates of Colletotrichum acutatum, C. fragariae and C. gloeosporioides pathogenic to strawberry plants were examined by sequence analysis of the 5.8S‐ITS region. Phylogenetic relationships among isolates of Colletotrichum are, for the most part, congruent with the molecular groups established in earlier works. 5.8S‐ITS sequence analysis showed a high level of genetic divergence within C. acutatum. Isolates of this species clustered into two very distinct clusters with further subdivision. The divergences between C. fragariae and C. gloeosporioides were too low to distinguish them as separate species. On the basis of the sequence data, specific primers were designed both to identify isolates belonging to the genus Colletotrichum, and to distinguish isolates of the species C. acutatum. The specificity of these primers was validated by testing a wide range of strawberry isolates of Colletotrichum, non‐strawberry isolates of Colletotrichum and other fungi used as controls. Although the 5.8S‐ITS sequences were not polymorphic enough to allow the construction of C. gloeosporioides‐specific primers, specific PCR amplification followed by an MvnI digestion provides a tool to specifically identify strawberry isolates of C. gloeosporioides.     

Phylogeny,divergence times and biogeography of window flies (Scenopinidae) and the therevoid clade (Diptera: Asiloidea)

The evolution of the ‘therevoid’ clade, with an emphasis on window flies (Scenopinidae), is presented by combining DNA sequence data with morphological characters for living and fossil species. The therevoid clade represents a group of four families (Apsilocephalidae, Evocoidae, Scenopinidae and Therevidae) of lower brachyceran Diptera in the superfamily Asiloidea. A comprehensive phylogenetic analysis using parsimony and likelihood methods was undertaken using extensive taxon sampling from all families and subfamilies, and compared with outgroup taxa sampled from the related families Asilidae, Mydidae, Apioceridae and Empididae. Fifty‐nine morphological characters (adult, larval and pupal) were combined with 6.4 kb of DNA sequences for two ribosomal genes (16S and 18S ribosomal DNA) and three protein‐encoding genes [cytochrome oxidase I (COI), triose phosphate isomerase (TPI) and the CPSase region of carbamoyl‐phosphate synthase‐aspartate transcarbamoylase‐dihydroorotase (CAD)]. Results from combined analyses of morphological and molecular data for 78 taxa representing all families of the therevoid clade are presented. Specific hypotheses of the relationship between respective families and subfamilies were tested statistically using four‐cluster likelihood mapping. The therevoid clade is a well‐supported monophyletic group within Asiloidea, with Evocoidae sister to Apsilocephalidae and Therevidae sister to Scenopinidae. Temporal and zoogeographical aspects of therevoid clade evolution were investigated using Bayesian divergence time estimates and Lagrange ancestral range scenarios. The effect of inclusion of fossils as terminal taxa on phylogenetic and divergence time estimation was investigated, with morphological scoring for fossil representatives included in the analyses rather than used simply as minimum age constraints. In each analysis there was either improvement in estimation, or only marginal and localized loss in tree resolution, and with younger estimates of divergence time across the tree. The historical biogeography of the therevoid clade was examined with multiple trans‐Antarctic vicariance events between Australasia and South America evident during the Late Cretaceous to early Palaeogene. Scenopininae is newly subdivided into two tribes, Metatrichini trib.n. and Scenopinini Fallén stat.r. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:4974EBF8‐3117‐4189‐B6DE‐7D5BF9B23E53 .     

Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats

In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free‐ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post‐mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already‐described taeniid species. Indeed, we detected three well‐defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.     

DNA sequences repaired at pachytene exhibit strong homology among distantly related higher plants

Moderately repetitive DNA sequences in Lilium (cv Enchantment) which undergo a meiotic-specific repair synthesis during pachytene (P-DNA) were previously shown to exist as families of very low internal sequence divergence. The present study concerns P-DNA sequence preservation among higher plants. The relative abundance of these sequences in a variety of plant species and their divergence relative to Enchantment P-DNA was determined through C₀t analysis and thermal denaturation of hybrid duplexes. Nearly all of the P-DNA sequence families of Enchantment were found to be present in the genomes of a number of monocot species and the dicot Vicia faba. Sequence content is highly conserved, with less than 6% divergence between Lilium and distantly related species such as Zea mays and Secale cereale. However, the number of repeats per P-DNA family varies considerably in different species, being particularly low among the Poales. P-DNA differs from most high thermal stability (HTS) sequence families of Enchantment which, although exhibiting a high degree of internal homology, are not present as repetitive DNA in the genomes of the other species examined. For most HTS families, the lack of internal divergence probably reflects their fairly recent introduction into the moderately repetitive DNA class, while P-DNA sequences represent evolutionarily ancient families which are the products of strong selective pressure for an indispensable meiotic function.     

Genetic diversity of two mitochondrial DNA genes in Spirometra erinaceieuropaei (Cestoda: Diphyllobothridae) from Poland

The tapeworm species Spirometra erinaceieuropaei was documented mainly in Asia and Europe. In recent years, plerocercoid larvae (spargana) of this parasite have been found in different hosts in north‐eastern Poland. The evolutionary history and way of S. erinaceieuropaei spreading across Eurasia have been not described yet. However, this phenomenon could be closely related to the evolutionary history and migration routes of studied tapeworm host species. We investigated the genetic variability and divergence pattern among S. erinaceieuropaei populations in intermediate and paratenic hosts from north‐eastern Poland based on complete mitochondrial sequences of cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) genes. Analysis of 319 consolidated sequences of these two genes showed no genetic structure across study area. Comparison of sequences from Poland and China showed distinct separation of S. erinaceieuropaei populations from these two regions. They split from their common ancestor approximately 28.6 million years ago. Demographic expansion of Polish population of S. erinaceieuropaei started from glacial refugia approximately 12.5 thousand years ago, and recent population expansion has been observed in the tapeworm population from north‐eastern Poland.     

Tetrahymena australis (Protozoa,Ciliophora): A Well‐Known But “Non‐Existing” Taxon – Consideration of Its Identification,Definition and Systematic Position

A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.     

Evolutionary conservation,diversity and specificity of LTR‐retrotransposons in flowering plants: insights from genome‐wide analysis and multi‐specific comparison

The availability of complete or nearly complete genome sequences from several plant species permits detailed discovery and cross‐species comparison of transposable elements (TEs) at the whole genome level. We initially investigated 510 long terminal repeat‐retrotransposon (LTR‐RT) families comprising 32 370 elements in soybean (Glycine max (L.) Merr.). Approximately 87% of these elements were located in recombination‐suppressed pericentromeric regions, where the ratio (1.26) of solo LTRs to intact elements (S/I) is significantly lower than that of chromosome arms (1.62). Further analysis revealed a significant positive correlation between S/I and LTR sizes, indicating that larger LTRs facilitate solo LTR formation. Phylogenetic analysis revealed seven Copia and five Gypsy evolutionary lineages that were present before the divergence of eudicot and monocot species, but the scales and timeframes within which they proliferated vary dramatically across families, lineages and species, and notably, a Copia lineage has been lost in soybean. Analysis of the physical association of LTR‐RTs with centromere satellite repeats identified two putative centromere retrotransposon (CR) families of soybean, which were grouped into the CR (e.g. CRR and CRM) lineage found in grasses, indicating that the ‘functional specification’ of CR pre‐dates the bifurcation of eudicots and monocots. However, a number of families of the CR lineage are not concentrated in centromeres, suggesting that their CR roles may now be defunct. Our data also suggest that the envelope‐like genes in the putative Copia retrovirus‐like family are probably derived from the Gypsy retrovirus‐like lineage, and thus we propose the hypothesis of a single ancient origin of envelope‐like genes in flowering plants.     

Uncovering Cryptic Diversity in Two Amoebozoan Species Using Complete Mitochondrial Genome Sequences

The Amoebozoa are a major eukaryotic lineage that encompasses a wide range of amoeboid organisms. The group is understudied from a systematic perspective: molecular tools have only been applied in the last 15 yr. Hence, there is an undersampling of both genes and taxa in the group especially compared to plants, animals, and fungi. Here, we present the complete mitochondrial genomes of two ubiquitous and abundant morpho‐species (Acanthamoeba castellanii and Vermamoeba vermiformis). Both have mitochondrial genomes of close relatives previously available, enabling insights into recent divergences at a genomic scale, while simultaneously offering comparisons with divergence estimates obtained from traditionally used single genes, SSU rDNA and cox1. The newly sequenced mt genomes are significantly divergent from their previously sequenced conspecifics (A. castellannii 16.4% divergence at nucleotide level and 10.4% amino acid; V. vermiformis 21.6% and 13.1%, respectively), while divergence at the small subunit ribosomal DNA is below 1% within both species. Morphological analyses determined that these lineages are indistinguishable from their previously sequenced counterparts. Phylogenetic reconstructions using 26 mt genes also indicate a level of divergence that is comparable to divergence among species, while reconstructions using the small subunit ribosomal DNA (SSU rDNA) do not. In addition, we demonstrate that between closely related taxa, there are high levels of synteny, which can be explored for primer design to obtain larger fragments than the traditional barcoding genes. We conclude that, although most systematic work has relied on SSU, this gene alone can severely underestimate diversity. Thus, we suggest that the mt genome emerges as an alternative for unraveling the lower level phylogenetic relationships of Amoebozoa.     

An analysis of nucleotide and amino acid variability in the barcode region of cytochrome c oxidase I (cox1) in fishes

The 655 bp cytochrome c oxidase subunit I barcode region of single specimens of 388 species of fishes (four Holocephali, 61 Elasmobranchii and 323 Actinopterygii) was examined. All but two (Urolophus cruciatus and Urolophus sufflavus) showed different cox1 nucleotide sequences (99.5% species discrimination); the two that could not be resolved are suspected to hybridize. Most of the power of cox1 nucleotide sequence analysis for species identification comes from the degenerate nature of the genetic code and the highly variable nature of the third codon position of amino acids. Variation at the third codon position is bimodally distributed, and the more variable mode is dominated by amino acids with four or six codons, while the less variable mode is dominated by amino acids with two codons. The ratio of nonsynonymous to synomymous changes is much less than one, indicating that this gene is subject to strong purifying selection. Consequently, cox1 amino acid sequence diversity is much less than nucleotide sequence diversity and has very poor species resolution power. Fourteen of the 16 amino acid residues recognized as having important functions in the region of cox1 sequenced were completely conserved over all 388 species (and the bovine cox1 sequence), with one fish species varying at one of these sites, and three fish at another site. No significant differences in amino acid conservation were observed between residues in helices, strands and turns. Patterns of nucleotide and amino acid variability were very similar between elasmobranchs and actinopterygians.