SINE insertions in cladistic analyses and the phylogenetic affiliations of Tarsius bancanus to other primates

Transpositions of Alu sequences, representing the most abundant primate short interspersed elements (SINE), were evaluated as molecular cladistic markers to analyze the phylogenetic affiliations among the primate infraorders. Altogether 118 human loci, containing intronic Alu elements, were PCR analyzed for the presence of Alu sequences at orthologous sites in each of two strepsirhine, New World and Old World monkey species, Tarsius bancanus, and a nonprimate outgroup. Fourteen size-polymorphic amplification patterns exhibited longer fragments for the anthropoids (New World and Old World monkeys) and T. bancanus whereas shorter fragments were detected for the strepsirhines and the outgroup. From these, subsequent sequence analyses revealed three Alu transpositions, which can be regarded as shared derived molecular characters linking tarsiers and anthropoid primates. Concerning the other loci, scenarios are represented in which different SINE transpositions occurred independently in the same intron on the lineages leading both to the common ancestor of anthropoids and to T. bancanus, albeit at different nucleotide positions. Our results demonstrate the efficiency and possible pitfalls of SINE transpositions used as molecular cladistic markers in tracing back a divergence point in primate evolution over 40 million years old. The three Alu insertions characterized underpin the monophyly of haplorhine primates (Anthropoidea and Tarsioidea) from a novel perspective.     

Characterization of novel Alu- and tRNA-related SINEs from the tree shrew and evolutionary implications of their origins

We characterized two novel 7SL RNA-derived short interspersed nuclear element (SINE) families (Tu types I and II) and a novel tRNA-derived SINE family (Tu type III) from the tree shrew (Tupaia belangeri). Tu type I contains a monomer unit of a 7SL RNA-derived Alu-like sequence and a tRNA-derived region that includes internal RNA polymerase III promoters. Tu type II has a similar hybrid structure, although the monomer unit of the 7SL RNA-derived sequence is replaced by a dimer. Along with the primate Alu, the galago Alu type II, and the rodent B1, these two families represent the fourth and fifth 7SL RNA-derived SINE families to be identified. Furthermore, comparison of the Alu domains of Tu types I and II with those of other 7SL RNA-derived SINEs reveals that the nucleotides responsible for stabilization of the Alu domain have been conserved during evolution, providing the possibility that these conserved nucleotides play an indispensable role in retropositional activity. Evolutionary relationships among these 7SL RNA-derived SINE families, as well as phylogenetic relationships of their host species, are discussed.     

A novel family of tRNA-derived SINEs in the colugo and two new retrotransposable markers separating dermopterans from primates

Short interspersed nuclear elements (SINEs) provide a near homoplasy free and copious source of molecular evolutionary markers with precisely defined character polarity. Used as molecular cladistic markers in presence/absence analyses, they represent a powerful complement to phylogenetic reconstructions that are based on sequence comparisons on the level of nucleotide substitutions. Recent sequence comparisons of large data sets incorporating a broad eutherian taxonomic sample have led to considerations of the different primate infraorders to constitute a paraphyletic group. Statistically significant support against the monophyly of primates has been obtained by clustering the flying lemur-also termed colugo-(Cynocephalus, Dermoptera) amidst the primates as the sister group to anthropoid primates (New World monkeys, Old World monkeys, and hominoids). We discovered retrotransposed markers that clearly favor the monophyly of primates, with the markers specific to all extant primates but definitively absent at the orthologous loci in the flying lemur and other non-primates. By screening the colugo genome for phylogenetic informative SINEs, we also recovered a novel family of dermopteran specific SINE elements that we call CYN. This element is probably derived from the isoleucine tRNA and appears in monomeric, dimeric, and trimeric forms. It has no long tRNA unrelated region and no poly(A) linker between the monomeric subunits. The characteristics of the novel CYN-SINE family indicate a relatively recent history. Therefore, this SINE family is not suitable to solve the phylogenetic affiliation between dermopterans and primates. Nevertheless it is a valuable device to reconstruct the evolutionary steps from a functional tRNA to an interspersed SINE element.     

SVA elements: a hominid-specific retroposon family

SVA is a composite repetitive element named after its main components, SINE, VNTR and Alu. We have identified 2762 SVA elements from the human genome draft sequence. Genomic distribution analysis indicates that the SVA elements are enriched in G+C-rich regions but have no preferences for inter- or intragenic regions. A phylogenetic analysis of the elements resulted in the recovery of six subfamilies that were named SVA_A to SVA_F. The composition, age and genomic distribution of the subfamilies have been examined. Subfamily age estimates based upon nucleotide divergence indicate that the expansion of four SVA subfamilies (SVA_A, SVA_B, SVA_C and SVA_D) began before the divergence of human, chimpanzee and gorilla, while subfamilies SVA_E and SVA_F are restricted to the human lineage. A survey of human genomic diversity associated with SVA_E and SVA_F subfamily members showed insertion polymorphism frequencies of 37.5% and 27.6%, respectively. In addition, we examined the amplification dynamics of SVA elements throughout the primate order and traced their origin back to the beginnings of hominid primate evolution, approximately 18 to 25 million years ago. This makes SVA elements the youngest family of retroposons in the primate order.     

Phylogeny of the macaques (Cercopithecidae: Macaca) based on Alu elements

Genus Macaca (Cercopithecidae: Papionini) is one of the most successful primate radiations. Despite previous studies on morphology and mitochondrial DNA analysis, a number of issues regarding the details of macaque evolution remain unsolved. Alu elements are a class of non-autonomous retroposons belonging to short interspersed elements that are specific to the primate lineage. Because retroposon insertions show very little homoplasy, and because the ancestral state (absence of the SINE) is known, Alu elements are useful genetic markers and have been utilized for analyzing primate phylogenentic relationships and human population genetic relationships. Using PCR display methodology, 298 new Alu insertions have been identified from ten species of macaques. Together with 60 loci reported previously, a total of 358 loci are used to infer the phylogenetic relationships of genus Macaca. With regard to earlier unresolved issues on the macaque evolution, the topology of our tree suggests that: 1) genus Macaca contains four monophyletic species groups; 2) within the Asian macaques, the silenus group diverged first, and members of the sinica and fascicularis groups share a common ancestor; 3) Macaca arctoides are classified in the sinica group. Our results provide a robust molecular phylogeny for genus Macaca with stronger statistical support than previous studies. The present study also illustrates that SINE-based approaches are a powerful tool in primate phylogenetic studies and can be used to successfully resolve evolutionary relationships between taxa at scales from the ordinal level to closely related species within one genus.     

SINEs of a nearly perfect character

Mobile elements have been recognized as powerful tools for phylogenetic and population-level analyses. However, issues regarding potential sources of homoplasy and other misleading events have been raised. We have collected available data for all phylogenetic and population level studies of primates utilizing Alu insertion data and examined them for potentially homoplasious and other misleading events. Very low levels of each potential confounding factor in a phylogenetic or population analysis (i.e., lineage sorting, parallel insertions, and precise excision) were found. Although taxa known to be subject to high levels of these types of events may indeed be subject to problems when using SINE analysis, we propose that most taxa will respond as the order Primates has--by the resolution of several long-standing problems observed using sequence-based methods.     

Chloroplast DNA restriction site variation in theVernonieae (Asteraceae), an initial appraisal of the relationship of New and Old World taxa and the monophyly ofVernonia

Chloroplast DNA restriction site variation was examined for 35 taxa in theVernonieae and four outgroup tribes, using 17 restriction enzymes mapped for ca. 900 restriction sites per species; 139 mutations were found to be phylogenetically informative. Phylogenetic trees were constructed using Wagner and weighted parsimony, and evaluated by bootstrap and decay analyses. Relationships of Old and New World taxa indicate complex geographical relationships; there was no clear geographic separation by hemisphere. The relationships between Old and New World Vernonias found here support prior morphological analyses. The sister group to all New and most Old World taxa was composed of a small group of Old World species including yellow-flowered, trinervate-leaved species previously postulated to be basal in the tribe. The majority of both New and Old World taxa are derived from a lineage beginning with the monotypic genusStokesia, an endemic of the southeastern United States. The genusVernonia was also found to be paraphyletic within both the New and Old World. Available data do not support either the separation ofVernonia or the tribeVernonieae into geographically distinct lineages. The pattern of relationships within theVernonieae for taxa from North America, Asia, Africa, Central and South America is most similar to that of several other groups of both plants and animals with a boreotropical origin, rather than an origin in Gondwanaland. Such a pattern of distribution suggests more ancient vicariant events than are routinely postulated for theAsteraceae.     

Primate evolution of the alpha-globin gene cluster and its Alu-like repeats

The arrangement of alpha-globin genes in Old World and New World monkeys and a prosimian, galago, has been determined by restriction mapping. Recombinant DNAs containing galago and Old World monkey alpha-globin genes have been isolated and subjected to a partial sequence determination for comparison to alpha-globin genes in human, chimpanzee and non-primate mammals. The results of this extensive structural analysis are relevant to several topics concerning the evolution of primate alpha-globin genes and Alu family repeats. All orders of higher primates (i.e. Old and New World monkeys, chimpanzee and human) have the same arrangement of alpha-globin genes. In contrast, the arrangement and correction of galago alpha-globin genes differ from those of higher primates, but are similar to those of non-primate mammals. The 5' and 3'-flanking regions of the human alpha 1 gene are orthologous to the corresponding region in galago, identifying the human alpha 2 gene as the more recently duplicated gene. The human psi alpha 1 gene is found to be inactivated after divergence of the human and galago lineages but prior to the divergence of human and monkey. Orthologous Alu family members in human and monkey DNAs indicate that the dispersion of some Alu repeats occurred prior to the divergence of these lineages. However, the Alu-like repeats of prosimian and higher primates result from entirely independent events giving rise to different repeat elements inserted at distinct genomic positions.     

An alu-based phylogeny of lemurs (infraorder: lemuriformes)

LEMURS (INFRAORDER: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ～55-60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Short INterspersed Elements (SINEs), to construct a phylogeny of infraorder Lemuriformes. Alu elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (Microcebus murinus) was computationally assayed for synapomorphic Alu elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction) verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 Alu elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for Microcebus and Mirza as sister genera, with Cheirogaleus the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place Lemur and Hapalemur together to the exclusion of Eulemur and Varecia, with Varecia the sister lineage to the other three genera.     

HERV-K(OLD): ancestor sequences of the human endogenous retrovirus family HERV-K(HML-2)

Sequences homologous to the human endogenous retrovirus (HERV) family HERV-K(HML-2) are present in all Old World primate species. A previous study showed that a central region of the HERV-K(HML-2) gag genes in Hominoidea species displays a 96-bp deletion compared to the gag genes in lower Old World primates. The more ancient HERV-K(HML-2) sequences present in lower Old World primates were apparently not conserved during hominoid evolution, as opposed to the deletion variants. To further clarify the evolutionary origin of the HERV-K(HML-2) family, we screened GenBank with the 96-bp gag-sequence characteristic of lower Old World primates and identified, to date, 10 human sequence entries harboring either full-length or partially deleted proviral structures, probably representing remnants of a more ancient HERV-K(HML-2) variant. The high degree of mutations demonstrates the long-time presence of these HERV-K(OLD) proviruses in the genome. Nevertheless, they still belong to the HML-2 family as deduced from dot matrix and phylogenetic analyses. We estimate, based on the family ages of integrated Alu elements and on long terminal repeat (LTR) divergence data, that the average age of HERV-K(OLD) proviruses is ca. 28 million years, supporting an integration time before the evolutionary split of Hominoidea from lower Old World primates. Analysis of HERV-K(OLD) LTR sequences led to the distinction of two subgroups, both of which cluster with LTRs belonging to an evolutionarily older cluster. Taken together, our data give further insight into the evolutionary history of the HERV-K(HML-2) family during primate evolution.     

Consistency of SINE insertion topology and flanking sequence tree: quantifying relationships among cetartiodactyls

Short interspersed nuclear elements (SINEs) have been used to generate unambiguous phylogenetic topologies relating eukaryotic taxa. The irreversible nature of SINE retroposition is supported by a large body of comparative genome data and is a fundamental assumption inherent in the value of this qualitative method of inference. Here, we assess the key assumption of unidirectional SINE insertion by comparing the SINE insertion-derived topology and the phylogenetic tree based on seven independent loci of five taxa in the order Cetartiodactyla (Cetacea + Artiodactyla). The data sets and analyses were largely independent, but the loci were, by definition, linked, and thus their consistency supported an irreversible pattern of SINE retroposition. Moreover, our analyses of the flanking sequences provided estimates of divergence times among cetartiodactyl lineages unavailable from SINE insertion analysis alone. Unexpected rate heterogeneity among sites of SINE-flanking sequences and other noncoding DNA sequences were observed. Sequence simulations suggest that this rate heterogeneity may be an artifact resulting from the inaccuracies of the substitution model used.     

Gene regulation by SINES and inosines: biological consequences of A-to-I editing of Alu element inverted repeats

The Alu elements are conserved ~300 nucleotide long repeat sequences that belong to the SINE family of retrotransposons found abundantly in primate genomes. Although the vast majority of Alu elements appear to be genetically inert, it has been tempting to consider the great majority of them as â€?junk DNA. However, a growing line of evidence suggests that transcribed Alu RNAs are in fact functionally involved in a number of diverse biological processes. Pairs of inverted Alu repeats in RNA can form duplex structures that lead to A-to-I editing by the ADAR enzymes. In this review we discuss the possible biological effects of Alu editing, with particular focus on the regulation of gene expression by inverted Alu repeats in the 3a€?-UTR regions of mRNAs.     

A molecular phylogeny of the Old World cypresses (<Emphasis Type="Italic">Cupressus</Emphasis>: Cupressaceae): evidence from nuclear and chloroplast DNA sequences

Studies of phylogenetic relationships among cypresses of the Old World (Cupressus; Cupressaceae) have been plagued by unresolved relationships, poor branch support, and conflict between data sets and methods of analysis. In this study, we combined 5.4 kb of aligned DNA sequence and 157 binary characters with previously published data in examining phylogenetic relationships among Cupressus species. Bayesian and parsimony analysis of the combined data or of the nuclear data alone always recovered three principal clades of Cupressus; however, tests of phylogenetic incongruence could not distinguish between competing relationships among the three principal Cupressus lineages. In contrast, incongruence tests often found statistically significant conflict between the nuclear and plastid data, particularly with respect to the placement of C. chengiana. Consistent with previous studies and prevailing taxonomic opinion, we find C. darjeelingensis more closely related to cypresses of the New World (Hesperocyparis). In contrast, we placed accessions of C. assamica and C. tonkinensis, two putatively Old World species suggested to be misidentified New World taxa by some authors, within well-supported Old World clades. Statistical analysis of genetic distances suggests instances in which taxa recognized as distinct species by some authors are identical or nearly so and may best be considered a single taxon. Conversely, we identify instances in which infraspecific taxa are more distantly related to one another than those traditionally recognized as distinct species. Factors confounding cypress taxonomies, including poor morphological differentiation, misidentification, and the use of accessions of questionable provenance, are discussed.     

Biogeography of Old World emballonurine bats (Chiroptera: Emballonuridae) inferred with mitochondrial and nuclear DNA

Extant bats of the genus Emballonura have a trans-Indian Ocean distribution, with two endemic species restricted to Madagascar, and eight species occurring in mainland southeast Asia and islands in the western Pacific Ocean. Ancestral Emballonura may have been more widespread on continental areas, but no fossil identified to this genus is known from the Old World. Emballonura belongs to the subfamily Emballonurinae, which occurs in the New and Old World. Relationships of all Old World genera of this subfamily, including Emballonura and members of the genera Coleura from Africa and western Indian Ocean islands and Mosia nigrescens from the western Pacific region, are previously unresolved. Using 1833 bp of nuclear and mitochondrial genes, we reconstructed the phylogenetic history of Old World emballonurine bats. We estimated that these lineages diverged around 30 million years ago into two monophyletic sister groups, one represented by the two taxa of Malagasy Emballonura, Coleura and possibly Mosia, and the other by a radiation of Indo-Pacific Emballonura, hence, rendering the genus Emballonura paraphyletic. The fossil record combined with these phylogenetic relationships suggest at least one long-distance dispersal event across the Indian Ocean, presumably of African origin, giving rise to all Indo-Pacific Emballonura species (and possibly Mosia). Cladogenesis of the extant Malagasy taxa took place during the Quaternary giving rise to two vicariant species, E. atrata in the humid east and E. tiavato in the dry west.     

Gene conversion as a secondary mechanism of short interspersed element (SINE) evolution.

The Alu repetitive family of short interspersed elements (SINEs) in primates can be subdivided into distinct subfamilies by specific diagnostic nucleotide changes. The older subfamilies are generally very abundant, while the younger subfamilies have fewer copies. Some of the youngest Alu elements are absent in the orthologous loci of nonhuman primates, indicative of recent retroposition events, the primary mode of SINE evolution. PCR analysis of one young Alu subfamily (Sb2) member found in the low-density lipoprotein receptor gene apparently revealed the presence of this element in the green monkey, orangutan, gorilla, and chimpanzee genomes, as well as the human genome. However, sequence analysis of these genomes revealed a highly mutated, older, primate-specific Alu element was present at this position in the nonhuman primates. Comparison of the flanking DNA sequences upstream of this Alu insertion corresponded to evolution expected for standard primate phylogeny, but comparison of the Alu repeat sequences revealed that the human element departed from this phylogeny. The change in the human sequence apparently occurred by a gene conversion event only within the Alu element itself, converting it from one of the oldest to one of the youngest Alu subfamilies. Although gene conversions of Alu elements are clearly very rare, this finding shows that such events can occur and contribute to specific cases of SINE subfamily evolution.     

No evidence for natural selection on endogenous borna-like nucleoprotein elements after the divergence of Old World and New World monkeys

Endogenous Borna-like nucleoprotein (EBLNs) elements were recently discovered as non-retroviral RNA virus elements derived from bornavirus in the genomes of various animals. Most of EBLNs appeared to be defective, but some of primate EBLN-1 to -4, which appeared to be originated from four independent integrations of bornavirus nucleoprotein (N) gene, have retained an open reading frame (ORF) for more than 40 million years. It was therefore possible that primate EBLNs have encoded functional proteins during evolution. To examine this possibility, natural selection operating on all ORFs of primate EBLN-1 to -4 was examined by comparing the rates of synonymous and nonsynonymous substitutions. The expected number of premature termination codons in EBLN-1 generated after the divergence of Old World and New World monkeys under the selective neutrality was also examined by the Monte Carlo simulation. As a result, natural selection was not identified for the entire region as well as parts of ORFs in the pairwise analysis of primate EBLN-1 to -4 and for any branch of the phylogenetic trees for EBLN-1 to -4 after the divergence of Old World and New World monkeys. Computer simulation also indicated that the absence of premature termination codon in the present-day EBLN-1 does not necessarily support the maintenance of function after the divergence of Old World and New World monkeys. These results suggest that EBLNs have not generally encoded functional proteins after the divergence of Old World and New World monkeys.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Baleen whale phylogeny and a past extensive radiation event revealed by SINE insertion analysis

Baleen whales (suborder Mysticeti) comprise 11 extant species that are classified into four families. Although several phylogenetic hypotheses about these taxa have been proposed, their phylogenetic relationships remain confused. We addressed this problem using short interspersed repetitive element (SINE) insertion data, which now are regarded as almost ideal shared, derived characters at the molecular level. We reconstructed the phylogenetic relationships of baleen whales by characterizing 36 informative SINE loci. One of the intriguing conclusions is that balaenopterids and eschrichtiids radiated very rapidly during a very short evolutionary period. During this period, speciation occurred in balaenopterids and eschrichtiids while newly inserted SINE loci remains polymorphic. Later on, these SINEs were sorted incompletely into each lineage. Thus, there are now inconsistencies among species regarding the presence or absence of a given SINE. This is in sharp contrast to the phylogeny of toothed whales, for which no SINE inconsistencies have been found. Furthermore, we found monophyletic groupings between humpback and fin whales as well as between (sei+Bryde's) whales and blue whales, both of which have not previously been recognized. The comprehensive SINE insertion data, together with the mitochondrial DNA phylogeny that was recently completed (Sasaki, T., M. Nikaido, H. Healy et al. 2005. Mitochondrial phylogenetics and evolution of mysticete whales. Syst. Biol. 56:77-90; Rychel, A. L., T. W. Reeder, and A. Berta. 2004. Phylogeny of mysticete whales based on mitochondrial and nuclear data. Mol. Phylogenet. Evol. 32:892-901), provide a nearly complete picture of the evolutionary history of baleen whales.     

Phylogenetic roots of Alu-mediated rearrangements leading to cancer.

There are over a million Alu repetitive elements dispersed throughout the human genome, and a high level of Alu-sequence similarity ensures a strong propensity for unequal crossover events, some of which have lead to deleterious oncogenic rearrangements. Furthermore, Alu insertions introduce consensus 3' splice sites, which potentially facilitate alternative splicing. Not surprisingly, Alu-mediated defective splicing has also been associated with cancer. To investigate a possible correlation between the expansion of Alu repeats associated with primate divergence and predisposition to cancer, 4 Alu-mediated rearrangements--known to be the basis of cancer--were selected for phylogenetic analysis of the necessary genotype. In these 4 cases, it was determined that the different phylogenetic age of the oncogenic recombination-prone genotype reflected the evolutionary history of Alu repeats spreading to new genomic sites. Our data implies that the evolutionary expansion of Alu repeats to new genomic locations establishes new predispositions to cancer in various primate species.