Molecular signatures that are distinctive characteristics of the vertebrates and chordates and supporting a grouping of vertebrates with the tunicates☆
Graphical abstract
Introduction
The phylum Chordata comprises Vertebrata as well as two other non-vertebrate taxa Urochordata and Cephalochordata (Nielsen, 1995, Gee, 1996, Jefferies, 1986). The chordates in turn are part of the superphylum Deuterostomes, which also includes the phyla Echinodermata, Hemichordata and a recently described phylum Xenoturbellida (Nielsen, 1995, Gee, 1996, Jefferies, 1986, Bourlat et al., 2006, Blair and Hedges, 2005). Because Vertebrata contains all known vertebrate species, an understanding of its evolutionary relationship to the other chordates and deuterostomes is of central importance to zoology (Blair and Hedges, 2005, Philippe and Telford, 2006, Edgecombe et al., 2011, Springer et al., 2004, Bourlat et al., 2006, Delsuc et al., 2006). Of the two non-vertebrate chordate taxa, cephalochordates are morphologically more similar to the vertebrates than to the adult urochordates (tunicates); thus, they are traditionally considered to be the closest relatives of vertebrates (Nielsen, 1995, Gee, 1996, Jefferies, 1986). A grouping of cephalochordates with vertebrates to the exclusion of urochordates is also observed in a number of phylogenetic studies based primarily on small subunit (SSU) and large subunit (LSU) rRNA gene sequences (Cameron et al., 2000, Mallatt and Winchell, 2007, Winchell et al., 2002). Additionally, the genomic organization of the Hox genes in cephalochordates also suggests that cephalochordates are more similar phylogenetically to the vertebrates than to the tunicates, whose genomes are highly divergent and not informative in this regard (Pascual-Anaya et al., 2013, Swalla and Smith, 2008). In contrast to these studies, the interrelationships among different deuterostomes and metazoan phyla have been examined in detail based on large datasets of sequences for nuclear proteins (Delsuc et al., 2006, Delsuc et al., 2008, Bourlat et al., 2006, Blair and Hedges, 2005). Surprisingly, the results of these studies strongly indicate that the urochordates and not cephalochordates are the sister taxon to the vertebrates. The distal branching of the tunicates from vertebrates in earlier studies was shown to be an artifact of long-branch attraction attributed to rapid evolution within the tunicates (Tsagkogeorga et al., 2010, Delsuc et al., 2006). In some of these studies, monophyly of the phylum Chordata was ambiguous using various phylogenetic methods (Delsuc et al., 2006, Winchell et al., 2002, Cameron et al., 2000, Glenner et al., 2004).
The inference from recent studies that tunicates are the closest relatives of vertebrates is of much importance for understanding the origin and evolution of vertebrates (Delsuc et al., 2006, Blair and Hedges, 2005, Bourlat et al., 2006, Swalla and Smith, 2008). Currently, the evidence that tunicates are more closely related to the vertebrates is entirely based on molecular phylogenetic studies (Delsuc et al., 2006, Blair and Hedges, 2005, Bourlat et al., 2006). However, several recent studies show that the inferences from molecular phylogenetic studies, even when they are based on large datasets involving multiple proteins, are sensitive to multiple confounding factors including differences in evolutionary rates among species, composition biases in sequences, on sampling of taxa, conflict in phylogenetic signal contained within the different amino acid sequences, and long-branch length attraction (Rokas et al., 2003, Jeffroy et al., 2006, Nosenko et al., 2013, Delsuc et al., 2008). Due to these factors, inferences from independent phylogenetic studies are often contradictory (Delsuc et al., 2006, Bourlat et al., 2006, Teeling and Hedges, 2013, Song et al., 2012, Philippe and Telford, 2006). Although in studies that group tunicates and vertebrates as a clade precautions were taken to guard against these artifacts (Delsuc et al., 2006, Blair and Hedges, 2005, Bourlat et al., 2006), it is important to confirm the relationship of vertebrates to the other chordate taxa by independent means.
The chordate as well as vertebrate clades are presently distinguished from other animals only on the basis of a limited number of morphological characteristics (Nielsen, 1995, Gee, 1996, Jefferies, 1986, Swalla and Smith, 2008). Besides the morphological characteristics, no other reliable molecular or biochemical property is known that is specifically shared by either all chordates or all vertebrates and can be used to distinguish these important groups of animals from all others. The availability of genome sequences from large numbers of animal species covering the diversity of metazoan taxa now provides a valuable resource for identifying novel molecular markers that are diagnostic for different animal taxa. Conserved signature indels (CSIs) in protein sequences constitute one type of rare genetic changes (RGCs) that provide very useful markers for this purpose, and they have been used extensively for evolutionary and systematic studies (Rokas and Holland, 2000, Springer et al., 2004, Baldauf and Palmer, 1993, Rivera and Lake, 1992, Gupta et al., 1994, Gupta, 1998, Gupta, 2014, Bhandari et al., 2012). Although the shared presence of CSIs in protein sequences in some cases can represent homoplasy or lateral gene transfers (Bapteste and Philippe, 2002, Gupta, 2012), in general, when a conserved indel of a definite length is found uniquely in a phylogenetically related group of organisms, its most parsimonious explanation is inheritance from the most recent common ancestor (Nielsen, 1995, Gee, 1996, Jefferies, 1986). Thus, conserved signature indel(s) provide powerful means to support or refute a given phylogenetic hypothesis.
In the present work, I have examined sequence alignments of >3000 proteins from different metazoan species to identify conserved signature indels that are specific for either the Vertebrata or groupings of vertebrates with other animal taxa (particularly the other chordate lineages). These studies have identified seven CSIs in 6 widely distributed proteins that are uniquely found in either all sequenced vertebrate species, or all sequenced chordate species, but which are not present in any other animal groups/phyla. The unique shared presence of these CSIs (synapomorphies) in these animal groups provides evidence that these groups are monophyletic, and the identified characteristics provide novel molecular means for distinguishing members of these groups from other animal taxa. Additionally, the present study has identified 2 other CSIs in widely distributed proteins that are uniquely shared by all sequenced vertebrate species and the urochordate species (Ciona intestinalis and Oikopleura dioica), but which are not found in Branchiostoma (cephalochordate) or other deuterostome species. The specific presence of these CSIs in these two chordate lineages provides strong and independent confirmation that the vertebrate species are more closely related to the tunicates (or Urochordata subphylum) than to the cephalochordates.
Section snippets
Materials and methods
Identification of conserved signature indels that are specific for the chordates or vertebrates was performed as described in earlier work (Gupta, 2014, Gupta, 1998, Gupta and Golding, 1996). Briefly, for these studies, Blastp searches were performed on >2000 proteins from the genome of Ciona intestinalis (Satou et al., 2008). For each protein for which high-scoring homologs were found in assorted vertebrates as well as non-vertebrate species, sequences for 20–25 homologs from divergent
Results
The aim of this study is to identify novel molecular markers in the form of conserved signature indels in protein sequences specific for either the Vertebrata or other chordate subphyla. The presence or absence of conserved indels in gene/protein sequences is generally not affected by factors such as differences in the evolutionary rates among species, composition biases, long-branch attraction, etc., which significantly affect phylogenetic analyses of base substitutions (Rokas and Holland, 2000
Discussion
The phylum Chordata, which includes vertebrates and two non-vertebrate subphyla (Urochordata and Cephalochordata), is presently recognized only on the basis of certain morphological characteristics viz. the notochord, a hollow dorsal nerve cord, pharyngeal gills and a muscular post-anal tail (Nielsen, 1995, Gee, 1996, Jefferies, 1986). Besides these characteristics, some of which are observed only at certain developmental stages in some members, no other reliable molecular or biochemical
Acknowledgments
This work was supported by a research grant (No. 249924) from the Natural Science and Engineering Research Council of Canada. I thank Rajni Gupta, Fariq Aziz and Reena Fabros for assistance in the creation of sequence alignments and in the formatting of the signature files. The assistance of Mobolaji Adeolu and Jeffery Chan in creating the Graphical Abstract of the present work is also much appreciated.
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This paper was edited by the Associate Editor A. Larson.