Sequence Divergence Estimates Research Articles

Evolution of the spinach sex-linked region within a rarely recombining pericentromeric region.

Sex chromosomes have evolved independently in many different plant lineages. Here, we describe reference genomes for spinach (Spinacia oleracea) X and Y haplotypes by sequencing homozygous XX females and YY males. The long arm of the 185 Mb chromosome 4 carries a 13 Mb X-linked region (XLR) and 24.1 Mb Y-linked region (YLR), of which 10 Mb is Y-specific. We describe evidence that this reflects insertions of autosomal sequences creating a "Y duplication region" or "YDR" whose presence probably directly reduces genetic recombination in the immediately flanking regions, although both the X and Y Sex-Linked Regions are within a large pericentromeric region of chromosome 4 that recombines rarely in meiosis of both sexes. Sequence divergence estimates using synonymous sites indicate that YDR genes started diverging from their likely autosomal progenitors about 3 MYA, around the time when the flanking YLR stopped recombining with the XLR. These flanking regions have a higher density of repetitive sequences in the YY than the XX assembly and include slightly more pseudogenes compared with the XLR, and the YLR has lost about 11% of the ancestral genes, suggesting some degeneration. Insertion of a male-determining factor would have caused Y-linkage across the entire pericentromeric region, creating physically small, highly recombining, terminal pseudo-autosomal regions. These findings provide a broader understanding of the origin of sex chromosomes in spinach.

The Rattlesnake W Chromosome: A GC-Rich Retroelement Refugium with Retained Gene Function Across Ancient Evolutionary Strata.

Sex chromosomes diverge after the establishment of recombination suppression, resulting in differential sex-linkage of genes involved in genetic sex determination and dimorphic traits. This process produces systems of male or female heterogamety wherein the Y and W chromosomes are only present in one sex and are often highly degenerated. Sex-limited Y and W chromosomes contain valuable information about the evolutionary transition from autosomes to sex chromosomes, yet detailed characterizations of the structure, composition, and gene content of sex-limited chromosomes are lacking for many species. In this study, we characterize the female-specific W chromosome of the prairie rattlesnake (Crotalus viridis) and evaluate how recombination suppression and other processes have shaped sex chromosome evolution in ZW snakes. Our analyses indicate that the rattlesnake W chromosome is over 80% repetitive and that an abundance of GC-rich mdg4 elements has driven an overall high degree of GC-richness despite a lack of recombination. The W chromosome is also highly enriched for repeat sequences derived from endogenous retroviruses and likely acts as a “refugium” for these and other retroelements. We annotated 219 putatively functional W-linked genes across at least two evolutionary strata identified based on estimates of sequence divergence between Z and W gametologs. The youngest of these strata is relatively gene-rich, however gene expression across strata suggests retained gene function amidst a greater degree of degeneration following ancient recombination suppression. Functional annotation of W-linked genes indicates a specialization of the W chromosome for reproductive and developmental function since recombination suppression from the Z chromosome.

Comparative Evidence of an Exceptional Impact of Gene Duplication on the Developmental Evolution of Drosophila and the Higher Diptera

The importance of gene duplication in developmental body plan evolution is well established, but for many megadiverse clades such as true flies (Diptera), a comprehensive understanding is still just emerging through comparative genomics. In a survey of 377 developmental gene families, we found that in addition to the pea aphid, which has been previously shown to be genome-wide enriched with gene duplicates and was included as positive control, more than twice as many expanded developmental gene families were observed in Drosophila (49) compared to mosquito (21), flour beetle (20), and honey bee (14). Synonymous sequence divergence estimates and ortholog conservation analyses in additional dipteran genomes revealed that most Drosophila gene duplicates are ancient and accumulated during a time window that reaches back to the origin of brachyceran flies, approximately 180 million years ago. Further, available genetic data suggest that more than half of the Drosophila developmental gene duplicates remained partially or even fully redundant despite their ancient separation. We therefore speculate that the exceptional accumulation of developmental gene duplicates in Drosophila and the higher Diptera was proximally driven by the evolution of fast development, benefiting from increased genetic robustness. At the same time, the concomitant increase of opportunities for gene duplicate diversification appears to have been a source for developmental and phenotypic innovation during the unparalleled diversification of brachyceran Diptera.

Genetic variation of Prochilodus lineatus (Valenciennes, 1836) from Paraná, Baía, Miranda, and Corumbá rivers, Brazil.

The curimbatá (Prochilodus lineatus) is one of the migratory species in the Paraná River Basin impacted by the construction of dams. Mitochondrial DNA sequences and random amplified polymorphic DNA (RAPD) fragments were used to investigate genetic variability and geographic structure of five populations of curimbatá from the Paraná River Basin. A total of 1815 bp from seven polymerase chain reaction-amplified fragments representing five protein-coding mitochondrial genes were sequenced from 12 individuals. Estimates of nucleotide sequence divergence ranged from 0.00 to 0.95%. A total of 86 RAPD markers from 58 individuals were detected. Results from the Fisher exact test indicated that P. lineatus is not genetically subdivided, although significant differences in the frequencies of a few RAPD fragments were observed. This study provides useful information for stocking and management programs for resource planning of P. lineatus.

In-solution hybridization for mammalian mitogenome enrichment: pros, cons and challenges associated with multiplexing degraded DNA.

Here, we present a set of RNA-based probes for whole mitochondrial genome in-solution enrichment, targeting a diversity of mammalian mitogenomes. This probes set was designed from seven mammalian orders and tested to determine the utility for enriching degraded DNA. We generated 63 mitogenomes representing five orders and 22 genera of mammals that yielded varying coverage ranging from 0 to >5400X. Based on a threshold of 70% mitogenome recovery and at least 10× average coverage, 32 individuals or 51% of samples were considered successful. The estimated sequence divergence of samples from the probe sequences used to construct the array ranged up to nearly 20%. Sample type was more predictive of mitogenome recovery than sample age. The proportion of reads from each individual in multiplexed enrichments was highly skewed, with each pool having one sample that yielded a majority of the reads. Recovery across each mitochondrial gene varied with most samples exhibiting regions with gaps or ambiguous sites. We estimated the ability of the probes to capture mitogenomes from a diversity of mammalian taxa not included here by performing a clustering analysis of published sequences for 100 taxa representing most mammalian orders. Our study demonstrates that a general array can be cost and time effective when there is a need to screen a modest number of individuals from a variety of taxa. We also address the practical concerns for using such a tool, with regard to pooling samples, generating high quality mitogenomes and detail a pipeline to remove chimeric molecules.

DNA barcoding reveals species level divergence between populations of the microhylid frog genus Arcovomer (Anura: Microhylidae) in the Atlantic Rainforest of southeastern Brazil

The microhylid frogs belonging to the genus Arcovomer have been reported from lowland Atlantic Rainforest in the Brazilian states of Espírito Santo, Rio de Janeiro, and São Paulo. Here, we use DNA barcoding to assess levels of genetic divergence between apparently isolated populations in Espírito Santo and Rio de Janeiro. Our mtDNA data consisting of cytochrome oxidase subunit I (COI) nucleotide sequences reveals 13.2% uncorrected and 30.4% TIM2 + I + Γ corrected genetic divergences between these two populations. This level of divergence exceeds the suggested 10% uncorrected divergence threshold for elevating amphibian populations to candidate species using this marker, which implies that the Espírito Santo population is a species distinct from Arcovomer passarellii. Calibration of our model-corrected sequence divergence estimates suggests that the time of population divergence falls between 12 and 29 million years ago.

The appearance of Ulva laetevirens (Ulvophyceae, Chlorophyta) in the northeast coast of the United States of America

Introduced species may outcompete or hybridize with native species, resulting in the loss of native biodiversity or even alteration of ecosystem processes. In this study, we reported an alien distromatic Ulva species, which was found in an embayment (Holly Pond) connected with Long Island Sound, USA. The morphological and anatomical observations in combination with molecular data were used for its identification to species. Anatomy of collected specimens showed that the cell shape in rhizoidal and basal regions was round and the marginal teeth along the basal and median region were not found. These characteristics were primarily identical to the diagnostic characteristics of Ulva laetevirens Areschoug (Chlorophyta). The plastid-encoding tufA and nucleusencoding ITS1 were used for its molecular identification. Phylogenetic analysis for the tufA gene placed the specimens from Holly Pond in a well-supported clade along with published sequences of U. laetevirens identified early without any sequence divergence. In ITS tree, the sample also formed well-supported clades with the sequences of U. laetevirens with an estimated sequence divergence among the taxa in these clades as low as 1%. These findings confirmed the morpho-anatomical conclusion. Native to Australia, this species was reported in several countries along the Mediterranean coast after the late of 1990s. This is the first time that U. laetevirens is found in the northeast coast of United States and the second record for Atlantic North America.

Molecular phylogeny of hipposiderid bats (Chiroptera: Hipposideridae) from Solomon Islands and Cape York Peninsula, Australia

Throughout the Old World Tropics, examples of cryptic diversity have been demonstrated in leaf‐nosed bats from the Family Hipposideridae. In addition to cryptic diversity, the widespread distributions and graded morphologies of some species render the taxonomy of this family poorly resolved. We used sequences of the mitochondrial gene ND2 and the nuclear gene RAG1 to reconstruct molecular phylogenetic relationships for hipposiderid species. Specimens from Solomon Island and Cape York Peninsula, Australia, were sequenced and combined with published sequences from Southeast Asia. Our results suggest the presence of undescribed diversity in northern Australia. Sequences of ND2 and RAG1 for samples of Cape York Hipposideros ater were distinct from those of Southeast Asia, with corrected sequence divergence estimates for the mitochondrial gene exceeding 23%. In comparison, there was no cryptic diversity detected in Solomon Island specimens when compared to conspecifics from Southeast Asia. On the contrary, three species members of the diadema morphological group from the region exhibit variability in the mitochondrial gene ND2, far below that of other species included in the analyses.

Using Nuclear-encoded LSU and SSU rDNA Sequences to Identify the Eukaryotic Endosymbiont in Amphisolenia bidentata (Dinophyceae)

The marine dinoflagellate Amphisolenia bidentata possesses complete intracellular symbionts of prokaryotic and eukaryotic origin. This was confirmed ultrastructurally little over 20 years ago when it was showed that the eukaryotic endosymbiont had a nucleus, a chloroplast and mitochondria. We collected Amphisolenia bidentata cells in the Indian Ocean and the identity of the eukaryotic endosymbionts was investigated using both microscopical and molecular methods. Individual specimens of Amphisolenia bidentata were identified by light microscopy and selected for single-cell PCR. Host and endosymbiont nuclear-encoded LSU and SSU rDNA sequences were determined by PCR cloning. Blast searches showed the endosymbiont LSU sequence to have affinity to Pelagophyceae, an algal class within Chromalveolata that also includes dinoflagellates. Since more SSU rDNA sequences from pelagophytes are available we performed a SSU based phylogeny of chromalveolates. The eukaryotic endosymbiont clustered within a clade comprising flagellated and coccoid pelagophytes whereas Amphisolenia bidentata formed a sister taxon to other dinophysioids. Molecular data therefore resolved the endosymbiont in A. bidentata being a pelagophyte and thus identified the ninth novel chloroplast type in dinoflagellates and a new species association. Based on sequence divergence estimates and phylogenetic inference the endosymbiont in A. bidentata likely represents an undescribed genus of pelagophytes.

Species delimitation and digit number in a North African skink

Delimitation of species is an important and controversial area within evolutionary biology. Many species boundaries have been defined using morphological data. New genetic approaches now offer more objective evaluation and assessment of the reliability of morphological variation as an indicator that speciation has occurred. We examined geographic variation in morphology of the continuously distributed skink Chalcides mionecton from Morocco and used Bayesian analyses of nuclear and mitochondrial DNA (mtDNA) loci to examine: (i) their concordance with morphological patterns, (ii) support for species delimitation, (iii) timing of speciation, and (iv) levels of gene flow between species. Four digit individuals were found at sites between Cap Rhir (in the south) and the northern extreme of the range, whereas five-digit individuals were found in two disjunct areas: (i) south of Cap Rhir and (ii) the north of the range where they were often syntopic with four-digit individuals. The pattern of variation in generalized body dimensions was largely concordant with that in digit number, suggesting two general morphotypes. Bayesian analyses of population structure showed that individuals from sites south of Cap Rhir formed one genetic cluster, but that northern four- and five-digit individuals clustered together. Statistical support for delimitation of these genetic clusters into two species was provided by a recent Bayesian method. Phylogenetic–coalescent dating with external time calibrations indicates that speciation was relatively recent, with a 95% posterior interval of 0.46–2.66 mya. This postdates equivalent phylogenetic dating estimates of sequence divergence by approximately 1 Ma. Statistical analyses of a small number of independent loci provide important insights into the history of the speciation process in C. mionecton and support delimitation of populations into two species with distributions that are spatially discordant with patterns of morphological variation.

Identification of Lygus hesperus by DNA barcoding reveals insignificant levels of genetic structure among distant and habitat diverse populations.

BackgroundThe western tarnished plant bug Lygus hesperus is an economically important pest that belongs to a complex of morphologically similar species that makes identification problematic. The present study provides evidence for the use of DNA barcodes from populations of L. hesperus from the western United States of America for accurate identification.Methodology/Principal FindingsThis study reports DNA barcodes for 134 individuals of the western tarnished plant bug from alfalfa and strawberry agricultural fields in the western United States of America. Sequence divergence estimates of <3% reveal that morphologically variable individuals presumed to be L. hesperus were accurately identified. Paired estimates of Fst and subsequent estimates of gene flow show that geographically distinct populations of L. hesperus are genetically similar. Therefore, our results support and reinforce the relatively recent (<100 years) migration of the western tarnished plant bug into agricultural habitats across the western United States.Conclusions/SignificanceThis study reveals that despite wide host plant usage and phenotypically plastic morphological traits, the commonly recognized western tarnished plant bug belongs to a single species, Lygus hesperus. In addition, no significant genetic structure was found for the geographically diverse populations of western tarnished plant bug used in this study.

Correlated Genetic and Ecological Diversification in a Widespread Southern African Horseshoe Bat

The analysis of molecular data within a historical biogeographical framework, coupled with ecological characteristics can provide insight into the processes driving diversification. Here we assess the genetic and ecological diversity within a widespread horseshoe bat Rhinolophus clivosus sensu lato with specific emphasis on the southern African representatives which, although not currently recognized, were previously described as a separate species R. geoffroyi comprising four subspecies. Sequence divergence estimates of the mtDNA control region show that the southern African representatives of R. clivosus s.l. are as distinct from samples further north in Africa than they are from R. ferrumequinum, the sister-species to R. clivosus. Within South Africa, five genetically supported geographic groups exist and these groups are corroborated by echolocation and wing morphology data. The groups loosely correspond to the distributions of the previously defined subspecies and Maxent modelling shows a strong correlation between the detected groups and ecoregions. Based on molecular clock calibrations, it is evident that climatic cycling and related vegetation changes during the Quaternary may have facilitated diversification both genetically and ecologically.

Moestrupia oblonga gen. & comb. nov. (syn.: Gyrodinium oblongum), a new marine dinoflagellate genus characterized by light and electron microscopy, photosynthetic pigments and LSU rDNA sequence

Hansen G. and Daugbjerg N. 2011. Moestrupia oblonga gen. et comb. nov. (syn.: Gyrodinium oblongum), a new marine dinoflagellate genus characterized by light and electron microscopy, photosynthetic pigments and LSU rDNA sequence. Phycologia 50: 583–599. DOI: 10.2216/11-11.1A small-sized, peridinin-containing, athecate dinoflagellate (13–17 µm long) was isolated into clonal culture from a water sample collected at a nearshore location in Tenerife, Spain (October 2004). Based on phenotypic characters (size, shape, pyrenoid and nucleus position), the culture was identified as Gyrodinium oblongum. However, a detailed ultrastructural examination revealed a number of features that did not fit into the current delineation of the genus Gyrodinium or any other dinoflagellate. Likewise, the molecular phylogeny and sequence divergence estimates based on partial LSU rDNA sequences indicated that this isolate has a taxonomically isolated position, and we therefore propose the new genus Moestrupia to accommodate this species within the Dinophyceae. The ultrastructural study uncovered a number of unique features for Moestrupia, for example, a microtubule supported ventral flange situated along the right cingular border and onto the episome. Also, the exit point of the peduncle, through a lip-like protrusion situated in a cavity on the episome, is new in dinoflagellates. The transverse flagellum appeared less coiled compared to other dinoflagellates, and its distal end terminated some distance within the wide sulcus. The upper cingulum border had a distinct rim supported by microtubules; whereas, the lower cingulum border was indistinct. The apical groove was short and curved and bordered on each side by a delicate rim, which may represent amphiesmal vesicles. It ran from the anterior part of the ventral flange to the middorsal side of the episome. Thus, it differs markedly from that in species of Gyrodinium and emphasizes it being distantly related to this genus.

Evolutionary genetics of an S-like polymorphism in Papaveraceae with putative function in self-incompatibility.

Background Papaver rhoeas possesses a gametophytic self-incompatibility (SI) system not homologous to any other SI mechanism characterized at the molecular level. Four previously published full length stigmatic S-alleles from the genus Papaver exhibited remarkable sequence divergence, but these studies failed to amplify additional S-alleles despite crossing evidence for more than 60 S-alleles in Papaver rhoeas alone.Methodology/Principal FindingsUsing RT-PCR we identified 87 unique putative stigmatic S-allele sequences from the Papaveraceae Argemone munita, Papaver mcconnellii, P. nudicuale, Platystemon californicus and Romneya coulteri. Hand pollinations among two full-sib families of both A. munita and P. californicus indicate a strong correlation between the putative S-genotype and observed incompatibility phenotype. However, we also found more than two S-like sequences in some individuals of A. munita and P. californicus, with two products co-segregating in both full-sib families of P. californicus. Pairwise sequence divergence estimates within and among taxa show Papaver stigmatic S-alleles to be the most variable with lower divergence among putative S-alleles from other Papaveraceae. Genealogical analysis indicates little shared ancestral polymorphism among S-like sequences from different genera. Lack of shared ancestral polymorphism could be due to long divergence times among genera studied, reduced levels of balancing selection if some or all S-like sequences do not function in incompatibility, population bottlenecks, or different levels of recombination among taxa. Preliminary estimates of positive selection find many sites under selective constraint with a few undergoing positive selection, suggesting that self-recognition may depend on amino acid substitutions at only a few sites.Conclusions/SignificanceBecause of the strong correlation between genotype and SI phenotype, sequences reported here represent either functional stylar S-alleles, tightly linked paralogs of the S-locus or a combination of both. The considerable complexity revealed in this study shows we have much to learn about the evolutionary dynamics of self-incompatibility systems.

Mesozoic fossils (&gt;145 Mya) suggest the antiquity of the subgenera of Daphniaand their coevolution with chaoborid predators

BackgroundThe timescale of the origins of Daphnia O. F. Mueller (Crustacea: Cladocera) remains controversial. The origin of the two main subgenera has been associated with the breakup of the supercontinent Pangaea. This vicariance hypothesis is supported by reciprocal monophyly, present day associations with the former Gondwanaland and Laurasia regions, and mitochondrial DNA divergence estimates. However, previous multilocus nuclear DNA sequence divergence estimates at < 10 Million years are inconsistent with the breakup of Pangaea. We examined new and existing cladoceran fossils from a Mesozoic Mongolian site, in hopes of gaining insights into the timescale of the evolution of Daphnia.ResultsWe describe new fossils of ephippia from the Khotont site in Mongolia associated with the Jurassic-Cretaceous boundary (about 145 MYA) that are morphologically similar to several modern genera of the family Daphniidae, including the two major subgenera of Daphnia, i.e., Daphnia s. str. and Ctenodaphnia. The daphniid fossils co-occurred with fossils of the predaceous phantom midge (Chaoboridae).ConclusionsOur findings indicate that the main subgenera of Daphnia are likely much older than previously known from fossils (at least 100 MY older) or from nuclear DNA estimates of divergence. The results showing co-occurrence of the main subgenera far from the presumed Laurasia/Gondwanaland dispersal barrier shortly after formation suggests that vicariance from the breakup of Pangaea is an unlikely explanation for the origin of the main subgenera. The fossil impressions also reveal that the coevolution of a dipteran predator (Chaoboridae) with the subgenus Daphnia is much older than previously known -- since the Mesozoic.

Sexual Isolation in Acinetobacter baylyi Is Locus-Specific and Varies 10,000-Fold Over the Genome

Naturally transformable bacteria acquire chromosomal DNA from related species at lower frequencies than from cognate DNA sources. To determine how genome location affects heterogamic transformation in bacteria, we inserted an nptI marker into random chromosome locations in 19 different strains of the Acinetobacter genus (>24% divergent at the mutS/trpE loci). DNA from a total of 95 nptI-tagged isolates was used to transform the recipient Acinetobacter baylyi strain ADP1. A total of >1300 transformation assays revealed that at least one nptI-tagged isolate for each of the strains/species tested resulted in detectable integration of the nptI marker into the ADP1 genome. Transformation frequencies varied up to approximately 10,000-fold among independent nptI insertions within a strain. The location and local sequence divergence of the nptI flanking regions were determined in the transformants. Heterogamic transformation depended on RecA and was hampered by DNA mismatch repair. Our studies suggest that single-locus-based studies, and inference of transfer frequencies from general estimates of genomic sequence divergence, is insufficient to predict the recombination potential of chromosomal DNA fragments between more divergent genomes. Interspecies differences in overall gene content, and conflicts in local gene organization and synteny are likely important determinants of the genomewide variation in recombination rates between bacterial species.

Molecular phylogenetics and taxonomy of leaf-toed geckos (Phyllodactylidae: Phyllodactylus) inhabiting the peninsula of Baja California

Herein we assess the phylogenetic relationships and taxonomy of geckos of the genus Phyllodactylus inhabiting the peninsula of Baja California, Mexico using five mitochondrial and two nuclear genes. Phylogenetic analysis using maximum parsimony (MP) and Bayesian inference (BI) recovered three distinct peninsular clades with high statistical support. Sequence divergence estimates between peninsular taxa approached 13%. Two of the species, P. unctus and P. xanti are Cape Region endemics, whereas P. nocticolus is widespread throughout much of the peninsula and extreme southern California. Monophyly of the peninsular taxa was strongly supported. In the MP analysis, P. unctus rooted at the base of the peninsular clade, resolving P. xanti and P. nocticolus as sister taxa. Conversely, BI placed P. nocticolus and P. unctus as sister taxa. These data provide further evidence for a trans-peninsular seaway near the Isthmus of La Paz, severing the Cape Region from the rest of the peninsula. The analysis also supports the validity of P. nocticolus as a distinct species and suggests a single invasion to the peninsula from mainland Mexico, presumably during tectonic activity during the Miocene.

SYMBIODINIUM NATANS SP. NOV.: A "FREE-LIVING" DINOFLAGELLATE FROM TENERIFE (NORTHEAST-ATLANTIC OCEAN)(1).

We examined a free-living Symbiodinium species by light and electron microscopy and nuclear-encoded partial LSU rDNA sequence data. The strain was isolated from a net plankton sample collected in near-shore waters at Tenerife, the Canary Islands. Comparing the thecal plate tabulation of the free-living Symbiodinium to that of S. microadriaticum Freud., it became clear that a few but significant differences could be noted. The isolate possessed two rather than three antapical plates, six rather than seven to eight postcingular plates, and finally four rather than five apical plates. The electron microscopic study also revealed the presence of an eyespot with brick-shaped contents in the sulcal region and a narrow anterior plate with small knob-like structures. Bayesian analysis revealed the free-living Symbiodinium to be a member of the earliest diverging clade A. However, it did not group within subclade AI (=temperate A) or any other subclades within clade A. Rather, it occupied an isolated position, and this was also supported by sequence divergence estimates. On the basis of comparative analysis of the thecal plate tabulation and the inferred phylogeny, we propose that the Symbiodinium isolate from Tenerife is a new species (viz. S. natans). To elucidate further the species diversity of Symbiodinium, particularly those inhabiting coral reefs, we suggest combining morphological features of the thecal plate pattern with gene sequence data. Indeed, future examination of motile stages originating from symbiont isolates will demonstrate if this proves a feasible way to identify and characterize additional species of Symbiodinium and thus match ribotypes or clusters of ribotypes to species.

Timing and evolution of the most recent common ancestor of the Korean clade HIV subtype B based on Nef and Vif sequences

Molecular phylogenetic studies of the HIV-1 isolated from Koreans have suggested the presence of the so-called "Korean clade", which can be defined as a cluster free of foreign isolates. The Korean clade accounts for more than 60% of Korean isolates and exerts characteristic amino acid sequences. Thus, it is merited to estimate when this Korean clade first emerged in order to understand the evolutionary pattern of the Korean clade. We analyzed and reconstructed the most recent common ancestor (MRCA) sequences from nef (n=229) and vif (n=179) Korean clade sequences. Linear regression analyses of sequence divergence estimates were plotted against sampling years to infer the year in which there was zero divergence from the MRCA sequences. MRCA sequences suggested the Korean clade was first emerged around 1984, before the first detection of HIV-1 in Korea in 1985. Further studies on synonymous and nonsynonymous substitution rates suggested positive selection event for the Korean clade, while other subtype B had undergone negative to neutral evolution.

Comparison of the genetic relationships and osteological aspects in six branchiostegid fish species (perciformes)

We analyzed partial sequences of cytochrome b (cyt‐b), a mitochondrial DNA (mtDNA) gene, to determine the genetic relationships between six horsehead fish species: Branchiostegus japonicus, Branchiostegus albus, Branchiostegus auratus, Branchiostegus argentatus, Branchiostegus wardi, and an unidentified Branchiostegus species. The specimens were collected in Korea, China, Japan, and Vietnam. We compared their molecular phylogenetic relationships inferred from mtDNA cyt‐b sequences with an osteological analysis. The unidentified species, B. sp., was similar to B. albus in terms of the lack of triangular silver‐white dot at the posterior region of eyes (vs. large one present in B. japonicus), but was also similar to B. japonicus in terms of the presence of a straight‐shaped first hemal spine (vs. a curve‐shaped hemal spine in B. albus). Analysis of the mtDNA cyt‐b sequences indicated that the smallest estimated sequence divergence was between the B. japonicus and B. sp. (0.70–0.94%), whereas the largest difference was between B. auratus and B. argentatus (23.06–23.36%). Both the maximum parsimony and maximum likelihood trees showed that the B. sp. was closely clustered with B. japonicus, and that B. auratus was most distant from the other species. When comparing the osteological characters, UPGMA tree showed that the B. japonicus and B. sp. were the most closely clustered species, and B. auratus was the most distantly clustered fish relative to the other species. The shape of the nasal, otolith and first hemal spine was informative for distinguishing B. auratus from the other species. These osteological differences were consistent with the differences in mtDNA.