Using SINEs to probe ancient explosive speciation: "hidden" radiation of African cichlids?

Cichlid fishes of the east African Great Lakes represent a paradigm of adaptive radiation. We conducted a phylogenetic analysis of cichlids including pan-African and west African species by using insertion patterns of short interspersed elements (SINEs) at orthologous loci. The monophyly of the east African cichlids was consistently supported by seven independent insertions of SINE sequences that are uniquely shared by these species. In addition, data from four other loci indicated that the genera Tilapia (pan-African) and Steatocranus (west African) are the closest relatives to east African cichlids. However, relationships among Tilapia, Steatocranus, and the east African clade were ambiguous because of incongruencies among topologies suggested by insertion patterns of SINEs at six other loci. One plausible explanation for this phenomenon is incomplete lineage sorting of alleles containing or missing a SINE insertion at these loci during ancestral speciation. Such incomplete sorting may have taken place earlier than 14 MYA, followed by random and stochastic fixation of the alleles in subsequent lineages. These observations prompted us to consider the possibility that cichlid speciation occurred at an accelerated rate during this period when the African Great Lakes did not exist. The SINE method could be useful for detecting ancient exclusive speciation events that tend to remain hidden during conventional sequence analyses because of accumulated point mutations.     

Phylogenetic relationships and ancient incomplete lineage sorting among cichlid fishes in Lake Tanganyika as revealed by analysis of the insertion of retroposons

Lake Tanganyika harbors numerous endemic species of extremely diverse cichlid fish that have been classified into 12 major taxonomic groups known as tribes. Analysis of short interspersed element (SINE) insertion data has been acknowledged to be a powerful tool for the elucidation of phylogenetic relationships, and we applied this method in an attempt to clarify such relationships among these cichlids. We studied insertion patterns of 38 SINEs in total, 24 of which supported the monophyly of three clades. The other 14 loci revealed extensive incongruence in terms of the patterns of SINE insertions. These incongruencies most likely stem from a period of adaptive radiation. One possible explanation for this phenomenon is the extensive incomplete lineage sorting of alleles for the presence or absence of a SINE during successive speciation events which took place about 5-10 MYA. The present study is the first to report the successful application of the SINE method in demonstrating the existence of such possible "ancient" incomplete lineage sorting. We discuss the possibility that it might potentially be very difficult to resolve the species phylogeny of a group that radiated explosively, even by resolving the genealogies of more than 10 nuclear loci, as a consequence of incomplete lineage sorting during speciation.     

Phylogenetic inconsistency caused by ancient sex-biased gene migration

Inferences of ancient sex-biased migration patterns using sex-linked genetic markers are usually difficult because of a stochastic process of allele fixation. Nevertheless, incongruent phylogenetic trees between different sex-linked markers and between sex-linked and autosomal markers are frequently interpreted as a signature of sex-biased migration without further statistical evaluation. I investigated the types of incongruent phylogenetic trees from which past sex-biased migration events can be statistically supported under the coalescent model. In the case of mammals, detecting a sex-biased migration pattern is not guaranteed by comparing the phylogenetic pattern of mitochondrial and Y-chromosomal loci. Likewise, evidence of introgression at a mitochondrial locus, but not at autosomal loci, does not support the hypothesis of an ancient female-biased migration pattern with statistical significance. In contrast, evidence of introgression at ≥ 5 unlinked autosomal loci, but not at a Y-chromosomal locus, would reject the null hypothesis of a sexually equal migration rate with statistical significance. A similar argument can be made to infer a male-biased migration pattern. Furthermore, the investigation of many recombining sex-biased markers such as X-chromosomal loci in mammals has the potential to efficiently detect ancient sex-biased demographic patterns.     

SINE insertions: powerful tools for molecular systematics

Short interspersed repetitive elements, or SINEs, are tRNA-derived retroposons that are dispersed throughout eukaryotic genomes and can be present in well over 10(4) total copies. The enormous volume of SINE amplifications per organism makes them important evolutionary agents for shaping the diversity of genomes, and the irreversible, independent nature of their insertion allows them to be used for diagnosing common ancestry among host taxa with extreme confidence. As such, they represent a powerful new tool for systematic biology that can be strategically integrated with other conventional phylogenetic characters, most notably morphology and DNA sequences. This review covers the basic aspects of SINE evolution that are especially relevant to their use as systematic characters and describes the practical methods of characterizing SINEs for cladogram construction. It also discusses the limits of their systematic utility, clarifies some recently published misunderstandings, and illustrates the effective application of SINEs for vertebrate phylogenetics with results from selected case studies. BioEssays 22:148-160, 2000.     

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.     

A mobile element based phylogeny of Old World monkeys

SINEs (Short INterspersed Elements) are a class of non-autonomous mobile elements that are <500 bp in length and have no open reading frames. Individual SINE elements are essentially homoplasy free with known ancestral states, making them useful genetic systems for phylogenetic studies. Alu elements are the most successful SINE in primate genomes and have been utilized for resolving primate phylogenetic relationships and human population genetics. However, no Alu based phylogenetic analysis has yet been performed to resolve relationships among Old World monkeys. Using both a computational approach and polymerase chain reaction display methodology, we identified 285 new Alu insertions from sixteen Old World monkey taxa that were informative at various levels of catarrhine phylogeny. We have utilized these elements along with 12 previously reported loci to construct a phylogenetic tree of the selected taxa. Relationships among all major clades are in general agreement with other molecular and morphological data sets but have stronger statistical support.     

Occurrence of Can-SINEs and intron sequence evolution supports robust phylogeny of pinniped carnivores and their terrestrial relatives

Investigating the dog genome we found 178 965 introns with a moderate length of 200–1000 bp. A screening of these sequences against 23 different repeat libraries to find insertions of short interspersed elements (SINEs) detected 45 276 SINEs. Virtually all of these SINEs (98%) belong to the tRNA-derived Can-SINE family. Can-SINEs arose about 55 million years ago before Carnivora split into two basal groups, the Caniformia (dog-like carnivores) and the Feliformia (cat-like carnivores). Genome comparisons of dog and cat recovered 506 putatively informative SINE loci for caniformian phylogeny. In this study we show how to use such genome information of model organisms to research the phylogeny of related non-model species of interest. Investigating a dataset including representatives of all major caniformian lineages, we analysed 24 randomly chosen loci for 22 taxa. All loci were amplifiable and revealed 17 parsimony-informative SINE insertions. The screening for informative SINE insertions yields a large amount of sequence information, in particular of introns, which contain reliable phylogenetic information as well. A phylogenetic analysis of intron- and SINE sequence data provided a statistically robust phylogeny which is congruent with the absence/presence pattern of our SINE markers. This phylogeny strongly supports a sistergroup relationship of Musteloidea and Pinnipedia. Within Pinnipedia, we see strong support from bootstrapping and the presence of a SINE insertion for a sistergroup relationship of the walrus with the Otariidae.     

Newly discovered young CORE-SINEs in marsupial genomes

Although recent mammalian genome projects have uncovered a large part of genomic component of various groups, several repetitive sequences still remain to be characterized and classified for particular groups. The short interspersed repetitive elements (SINEs) distributed among marsupial genomes are one example. We have identified and characterized two new SINEs from marsupial genomes that belong to the CORE-SINE family, characterized by a highly conserved "CORE" domain. PCR and genomic dot blot analyses revealed that the distribution of each SINE shows distinct patterns among the marsupial genomes, implying different timing of their retroposition during the evolution of marsupials. The members of Mar3 (Marsupialia 3) SINE are distributed throughout the genomes of all marsupials, whereas the Mac1 (Macropodoidea 1) SINE is distributed specifically in the genomes of kangaroos. Sequence alignment of the Mar3 SINEs revealed that they can be further divided into four subgroups, each of which has diagnostic nucleotides. The insertion patterns of each SINE at particular genomic loci, together with the distribution patterns of each SINE, suggest that the Mar3 SINEs have intensively amplified after the radiation of diprotodontians, whereas the Mac1 SINE has amplified only slightly after the divergence of hypsiprimnodons from other macropods. By compiling the information of CORE-SINEs characterized to date, we propose a comprehensive picture of how SINE evolution occurred in the genomes of marsupials.     

SINE Retrotransposition During the Evolution of the Pecoran Ruminants

SINE retrotransposition events have proven their value as phylogenetic markers in several eukaryotic taxa at different taxonomic levels. The genomes of ruminants contain three related SINE elements, Bov-tA, Bov-A2, and Bov-B. To estimate the time points of retrotransposition of individual copies of these SINEs, we designed PCR primers on database sequences containing SINE insertions in cattle, sheep, or goat genomes and tested for the presence of these copies in the genomes of other ruminants. It was checked by sequencing whether length variation of the PCR products reflected a SINE retrotransposition. One Bov-B and nine Bov-tA insertions were shared by cattle, sheep, goat, and giraffe, indicating an early retrotransposition event before the radiation of the Pecora, while three other Bov-tA and two Bov-B elements were apparently inserted later. The ruminant α-lactalbumine gene contains a hotspot of early and more recent Bov-tA insertions, a Bov-tA replacement as well as a recent Bov-B insertion. Three Bov-A2 insertions were found to be shared only by the Bovidae, the Bovini, and the Bos and Bison species, respectively, indicating that most Bov-A2 insertions are relatively recent. The time elapsed since the retrotransposition was also reflected in the degeneration of the direct repeats that flank SINE inserts. We suggest that retrotransposition of SINEs may serve as phylogenetic markers in the ruminant families, subfamilies, and even tribes. In addition, sequencing of SINE insertions revealed several other unique deletions/insertions that also may be informative for phylogenetic reconstructions of ruminants. Received: 19 January 2001 / Accepted: 6 June 2001     

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.     

A novel family of short interspersed repetitive elements (SINEs) from cichlids: the patterns of insertion of SINEs at orthologous loci support the proposed monophyly of four major groups of cichlid fishes in Lake Tanganyika

Short interspersed repetitive elements (SINEs) have been shown to be excellent markers of molecular phylogeny, since the integration of a SINE at a particular position in a genome can be considered an unambiguous derived homologous character. In the present study, we isolated a new family of SINEs from cichlids in Lake Tanganyika, whose speciation and diversification have been regarded as prime examples of explosive adaptive radiation. Members of this new SINE family, which we named the AFC family, are about 320 bp in length, and each has a tRNA- related region in its 5' region, as do most of the members of SINE families reported to date. A dot blot hybridization experiment showed that this family is distributed extensively in the genomes of cichlids in Africa, with estimated copy numbers of 2 x 10(3)-2 x 10(4) per haploid genome. Our investigations of the patterns of insertion of members of this family at six orthologous loci demonstrated clearly that four previously identified tribes, namely, the Lamprologini, Ectodini, Tropheini, and Perissodini, each form a monophyletic group. These results provide a basis for the elucidation of the phylogenetic framework of the cichlid fishes in Lake Tanganyika.      

SINEs, evolution and genome structure in the opossum

Short INterspersed Elements (SINEs) are non-autonomous retrotransposons, usually between 100 and 500 base pairs (bp) in length, which are ubiquitous components of eukaryotic genomes. Their activity, distribution, and evolution can be highly informative on genomic structure and evolutionary processes. To determine recent activity, we amplified more than one hundred SINE1 loci in a panel of 43 M. domestica individuals derived from five diverse geographic locations. The SINE1 family has expanded recently enough that many loci were polymorphic, and the SINE1 insertion-based genetic distances among populations reflected geographic distance. Genome-wide comparisons of SINE1 densities and GC content revealed that high SINE1 density is associated with high GC content in a few long and many short spans. Young SINE1s, whether fixed or polymorphic, showed an unbiased GC content preference for insertion, indicating that the GC preference accumulates over long time periods, possibly in periodic bursts. SINE1 evolution is thus broadly similar to human Alu evolution, although it has an independent origin. High GC content adjacent to SINE1s is strongly correlated with bias towards higher AT to GC substitutions and lower GC to AT substitutions. This is consistent with biased gene conversion, and also indicates that like chickens, but unlike eutherian mammals, GC content heterogeneity (isochore structure) is reinforced by substitution processes in the M. domestica genome. Nevertheless, both high and low GC content regions are apparently headed towards lower GC content equilibria, possibly due to a relative shift to lower recombination rates in the recent Monodelphis ancestral lineage. Like eutherians, metatherian (marsupial) mammals have evolved high CpG substitution rates, but this is apparently a convergence in process rather than a shared ancestral state.     

The Salmon Smai Family of Short Interspersed Repetitive Elements (Sines): Interspecific and Intraspecific Variation of the Insertion of Sines in the Genomes of Chum and Pink Salmon

The genomes of chum salmon and pink salmon contain a family of short interspersed repetitive elements (SINEs), designated the salmon SmaI family. It is restricted to these two species, a distribution that suggests that this SINE family might have been generated in their common ancestor. When insertions of the SmaI SINEs at 10 orthologous loci of these species were analyzed, however, it was found that there were no shared insertion sites between chum and pink salmon. Furthermore, at six loci where SmaI SINEs have been species-specifically inserted in chum salmon, insertions of SINEs were polymorphic among populations of chum salmon. By contrast, at four loci where SmaI SINEs had been species-specifically inserted in pink salmon, the SINEs were fixed among all populations of pink salmon. The interspecific and intraspecific variation of the SmaI SINEs cannot be explained by the assumption that the SmaI family was amplified in a common ancestor of these two species. To interpret these observations, we propose several possible models, including introgression and the horizontal transfer of SINEs from pink salmon to chum salmon during evolution.     

Details of Retropositional Genome Dynamics That Provide a Rationale for a Generic Division: The Distinct Branching of All the Pacific Salmon and Trout (Oncorhynchus) from the Atlantic Salmon and Trout (Salmo)

Salmonid species contain numerous short interspersed repetitive elements (SINEs), known collectively as the HpaI family, in their genomes. Amplification and successive integration of individual SINEs into the genomes have occurred during the evolution of salmonids. We reported previously a strategy for determining the phylogenetic relationships among the Pacific salmonids in which these SINEs were used as temporal landmarks of evolution. Here, we provide evidence for extensive genomic rearrangements that involved retropositions and deletions in a common ancestor of all the Pacific salmon and trout. Our results provide genetic support for the recent phylogenetic reassignment of steelhead and related species from the genus Salmo to the genus Oncorhynchus. Several other informative loci identified by insertions of HpaI SINEs have been isolated, and previously proposed branching orders of the Oncorhynchus species have been confirmed. The authenticity of our phylogenetic tree is supported both by the isolation of more than two informative loci per branching point and by the congruence of all our data, which suggest that the period between succesive speciations was sufficiently long for each SINE that had been amplified in the original species to become fixed in all individuals of that species.     

Short interspersed elements (SINEs) in plants: origin, classification, and use as phylogenetic markers

Short interspersed elements (SINEs) are a class of dispersed mobile sequences that use RNA as an intermediate in an amplification process called retroposition. The presence-absence of a SINE at a given locus has been used as a meaningful classification criterion to evaluate phylogenetic relations among species. We review here recent developments in the characterisation of plant SINEs and their use as molecular makers to retrace phylogenetic relations among wild and cultivated Oryza and Brassica species. In Brassicaceae, further use of SINE markers is limited by our partial knowledge of endogenous SINE families (their origin and evolution histories) and by the absence of a clear classification. To solve this problem, phylogenetic relations among all known Brassicaceae SINEs were analyzed and a new classification, grouping SINEs in 15 different families, is proposed. The relative age and size of each Brassicaceae SINE family was evaluated and new phylogenetically supported subfamilies were described. We also present evidence suggesting that new potentially active SINEs recently emerged in Brassica oleracea from the shuffling of preexisting SINE portions. Finally, the comparative evolution history of SINE families present in Arabidopsis thaliana and Brassica oleracea revealed that SINEs were in general more active in the Brassica lineage. The importance of these new data for the use of Brassicaceae SINEs as molecular markers in future applications is discussed.     

Population structure of Coilia nasus in the Yangtze River revealed by insertion of short interspersed elements

Coilia nasus is found in the Yangtze River and the coastal waters of China, Korea, and Japan. Two ecotypes (anadromous and freshwater-resident populations) are distributed throughout the Yangtze River basin based on their ecology and behavior, but relatively little is known about the population structure of this species. Analysis of short interspersed element (SINE) insertions, which vary among individuals, has been acknowledged to provide a unique way to study population divergence. SINEs isolated from C. nasus were characterized, and this enabled analysis of the SINE insertion pattern in six populations distributed throughout the Yangtze River basin. In all populations, four SINE loci displayed individual polymorphism, and two SINE loci showed a stochastic loss in all individuals of two resident populations. The correlation between genetic and geographic populations indicated a degree of genetic isolation in this species. In contrast with Coilia grayii and Coilia mystus, two SINE loci appeared only in C. nasus. Sequencing analysis indicated that the high insertion variability of SINEs was attributed mainly to the tails, which contained various repeat copies. The results in this study will be useful for sustainable management of fishery resources and conservation of this species.     

An ancient retrovirus-like element contains hot spots for SINE insertion

Vertebrate retrotransposons have been used extensively for phylogenetic analyses and studies of molecular evolution. Information can be obtained from specific inserts either by comparing sequence differences that have accumulated over time in orthologous copies of that insert or by determining the presence or absence of that specific element at a particular site. The presence of specific copies has been deemed to be an essentially homoplasy-free phylogenetic character because the probability of multiple independent insertions into any one site has been believed to be nil. Mys elements are a type of LTR-containing retrotransposon present in Sigmodontine rodents. In this study we have shown that one particular insert, mys-9, is an extremely old insert present in multiple species of the genus Peromyscus. We have found that different copies of this insert show a surprising range of sizes, due primarily to a continuing series of SINE (short interspersed element) insertions into this locus. We have identified two hot spots for SINE insertion within mys-9 and at each hot spot have found that two independent SINE insertions have occurred at identical sites. These results have major repercussions for phylogenetic analyses based on SINE insertions, indicating the need for caution when one concludes that the existence of a SINE at a specific locus in multiple individuals is indicative of common ancestry. Although independent insertions at the same locus may be rare, SINE insertions are not homoplasy-free phylogenetic markers.     

Inheritance of nuclear DNA markers in gynogenetic haploid pink salmon

We describe the inheritance of 460 PCR-based loci in the polyploid-derived pink salmon (Oncorhynchus gorbuscha) genome using gynogenetic haploid embryos. We detected a length polymorphism in a growth hormone gene (GH-2) intron that is caused by an 81 bp insertion homologous to the 3' end of the salmonid short interspersed repetitive element (SINE) SmaI. Such insertion polymorphisms within species bring into question the use of SINEs as phylogenetic markers. We confirmed that a microsatellite locus encodes a PCR-null allele that is responsible for an apparent deficit of heterozygotes in a population sample from Prince William Sound. Another set of microsatellite primers amplified alleles of the same molecular weight from both loci of a duplicated pair. In our analysis of several PCR-based multilocus techniques, we failed to detect evidence of comigrating fragments produced by duplicated loci. Segregation analysis of PCR-based markers using gynogenetic haploid embryos ensures that the interpretation of molecular variation is not complicated by heterozygosity, diploidy, or gene duplication. We urge investigators to test the inheritance of polymorphisms in salmonids prior to using them to measure genetic variation.     

Retroposition of the AFC Family of SINEs (Short Interspersed Repetitive Elements) Before and During the Adaptive Radiation of Cichlid Fishes in Lake Malawi and Related Inferences About Phylogeny

Lake Malawi is home to more than 450 species of endemic cichlids, which provide a spectacular example of adaptive radiation. To clarify the phylogenetic relationships among these fish, we examined the presence and absence of SINEs (short interspersed repetitive elements) at orthologous loci. We identified six loci at which a SINE sequence had apparently been specifically inserted by retroposition in the common ancestor of all the investigated species of endemic cichlids in Lake Malawi. At another locus, unique sharing of a SINE sequence was evident among all the investigated species of endemic non-Mbuna cichlids with the exception of Rhamphochromis sp. The relationships were in good agreement with those deduced in previous studies with various different markers, demonstrating that the SINE method is useful for the elucidation of phylogenetic relationships among cichlids in Lake Malawi. We also characterized a locus that exhibited transspecies polymorphism with respect to the presence or absence of the SINE sequence among non-Mbuna species. This result suggests that incomplete lineage sorting and/or interspecific hybridization might have occurred or be occurring among the species in this group, which might potentially cause misinterpretation of phylogenetic data, in particular when a single-locus marker, such as a sequence in the mitochondrial DNA, is used for analysis. Received: 15 December 2000 / Accepted: 30 January 2001