Ministry of higher and secondary special education of the republic of uzbekistan urgench state university

2.3. Details of the evolutionary history from invertebrates to vertebrates

The evolutionary pathway from advanced invertebrates through primitive chordates to vertebrates has been a subject of extensive investigation and vigorous discussion for more than a century.

Chordates are categorized as deuterostomes, which are characterized by several features that include, for example, radial cleavage, the fate of the blastopore that does not form a mouth, an enterocoelic coelom, and a tripartite body plan.
Traditionally, the deuterostomes include pogonophorans, chaetognaths, echinoderms, hemichordates, and chordates (urochordates, cephalochordates, and vertebrates), but recent studies have forced the categorization of pogonophorans near the annelids.
The chaetognath (arrowworm) remains a mystery in terms of its ancestry. Recent advances in molecular biology have made it possible to answer some of the questions posed by evolutionary biologists. Comparisons based on molecular data, such as the amino acid sequences of certain proteins and the nucleotide sequences of certain RNAs and DNAs, provide powerful tools with which to examine phylogenetic relationships among animal groups since these molecular characteristics can be interpreted more objectively than others.
Unfortunately, pioneer studies with sequences of 5S rRNA and partial sequences of 18S rRNA failed to affirm the monophyly ofdeuterostomes, probably because the radiation of bilaterally symmetrical animals occurred over a very short period of time. However, the monophyly of the deuterostomes was suggested by Lake after application of his original method for the construction ofphylogenetic trees. A recent study by Stock and Whitt provided evidence from sequences of 18S rRNAs that lampreys and hagfishes form a natural group, but conclusive support from molecularphylogenetic analysis for the monophyly of the deuterostomes has not yet been obtained.
To further our knowledge of the phylogeny of deuterostomes, we determined almost the entire sequences of 18S rDNAs from two chaetognaths, five echinoderms, a hemichordate, and two urochordates (a larvacean and a salp) and, together with the sequences of other deuterostomes (an ascidian, a cephalochordate, and vertebrates) and protostomes (an arthropod and a mollusc), we reexamined the evolutionary history of these animals.⁶
Biological Materials. The animals examined in the present study were the chaetognaths (arrowworms) Paraspadella gotoi and Sagitta crassaforma naikaiensis; the echinoderms, which included Antedon serrata (comatulid), Asterias amurensis (starfish), Ophioplocus japonicus (brittle star), Strongylocentrotus intermedius (sea urchin), and Stichopus japonicus (sea cucumber); the hemichordate (acorn worm) Balanoglossus carnosus as well as the urochordates Oikopleura sp. (larvacean) and Thalia democratica (salp).
Isolation of DNA, Amplification of 18S rDNA, and Sequencing of Amplified DNA. High molecular weight genomic DNA was extracted from gonads or tissues as described.
Almost the entire length of the 18S rDNA was amplified by the PCR using the primers 5'-CTGGTTGATCCTGCCAG-3' and 5'-CACCTACGGA(AT)ACCTTG-3'. One ofthe primers was phosphorylated prior to PCR at the 5' terminus. Amplified DNA was digested with A exonuclease to obtain singlestranded DNA.
With the single-stranded DNA as a template, the nucleotide sequence was determined by dideoxynucleotide chain termination (16). In addition to the two primers described above, primers
5'-CCGGAGAGGGAGCCTGA-3',
5'-CAGCAGCCGCGGTAATT-3',
5'-GCGAAAGCATTTGCCAA-3',
5'-GAAACT(TC)AAAGGAAT-3',
5'-ACGGGCGGTGTGT(AG)C-3',
and antisense analogues of these primers were used for sequencing. Comparison of Sequences and Inferences About Phylogeny. Sequences were aligned on the basis of maximum nucleotide similarity. Using the aligned sequences, we calculated evolutionary distance in a pairwise manner.
The phylogenetic tree was constructed by the neighbor-joining method. The degree of support for internal branches of the tree was further assessed by bootstrapping. The phylogenetic tree was also constructed by the maximum-likelihood method using the DNAML program from the PHYLIP package, and the fastDNAml program.
Sequences of >1700 nucleotides of 18S rDNA were determined for two chaetognaths, five echinoderms, a hemichordate, and two urochordates (a larvacean and a salp). The phylogenetic relationships among deuterostomes, arthropods, and molluscs were analyzed by comparing 1369 unambiguously aligned sites (gaps are not included). We chose platyhelminthes as an outgroup with which to examine the monophyly of deuterostomes since platyhelminthes diverged at the deepest node in the metazoan branch.
Structural similarities and evolutionary distance values were calculated pairwise as described by Jukes and Cantor between the aligned sequences; the results are summarized. Using this matrix, we first constructed the phylogenetic tree by the neighbor-joining method. The tree shown in Fig. 2.3.1 indicates that the echinoderms, the hemichordate, the urochordates, the cephalochordate, and the vertebrates form a discrete group. The grouping of deuterostomes was supported by a high value obtained by bootstrapping (71.2%). Next, using the same alignment, we constructed another phylogenetic tree by the maximum-likelihood method using the fastDNAml program .
The tree topology was identical to that of the neighbor-joining method, except for the grouping of urochordates and cephalochordate vertebrates (data not shown). Therefore, it is highly probable that these animals had a common ancestry and that the features that characterize deuterostomes are homologous but not convergent. In contrast to the general interpretation of the relationship between deuterostomes and other groups of animals, as mentioned above, L0vtrup (25) claimed that molluscs and vertebrates have common ancestors that differed from those ofechinoderms. It is evident, however, that L0vtrup's theory is not supported by the present phylogenetic tree derived from 18S rDNAs.

FIG. 2.3.1. Phylogenetic relationships among deuterostomes, as deduced from comparisons of 18S rDNA sequences. Sequences were aligned in terms ofmaximum-likelihood (gaps were excluded from phylogenetic analysis), and the phylogenetic tree was constructed by neighbor-joining. Scale bar indicates an evolutionary distance of 0.02 nucleotide substitution per position in the sequence. The degree of support for internal branches of the tree was assessed by bootstrapping; numbers at each branch indicate percentage of times that a node was supported in 500 bootstrap pseudoreplications.

The Phylogenetic Posion of Chaoaths. The phylogenetic status of chaetognaths is rather mysterious. Chaetognaths (arrowworms) are marine, largely planktonic animals. They share some common characteristics with deuterostomes during their ontogeny: radial cleavage, ablastopore at the rear end of the body, and a postanal tail.

However, the morphology ofadults-namely, a coelom without a peritoneum, chitin in their teeth and grasping spines, and a thick cuticle-suggests their similarity to pseudocoelomate groups. Their nervous system is more like that of protostomes.
Moreover, even during ontogeny, they do not pass through the dipleurula stage that is seen in every deuterostome phylum. As is evident from Table 1 and Fig. 1, the chaetognaths cannot be included in the deuterostomes by analysis of neighbor-joining. The best tree obtained by the maximumlikelihood analysis also did not support the sister-grouping of chaetognaths with deuterostomes (data not shown).
The high rate of substitutions found in chaetognath sequences is reflected in the branch length, which might affect the topology of the tree. Nonetheless, we failed to find any evidence that the chaetognaths should be included in the deuterostome group. This result is consistent with that of a very recent study by Telford and Holland; the affinity of chaetognaths with deuterostomes was not supported by analysis of 18S rDNA of another species, Sagitta elegans.
It is likely that chaetognaths evolved independently during an early stage of metazoan evolution and that the similarities in terms of embryology to deuterostomes may be the result of convergence. The Origin and Evolution of Chordates.
Next, we examined the phylogenetic relationships among deuterostomes. As shown in Fig. 2.3.1, the 18S rDNA tree indicates that deuterostomes can be divided into five groups: echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates.
Consistent with the results of a previous study (10), the monophyly of the five classes of echinoderms is supported by the high value obtained by bootstrapping (86.4%). The phylogenetic relationships among the extant five classes of echinoderms will be discussed elsewhere (30). Urochordates also form a discrete group of monophyletic origin in the tree (bootstrapping, 100%1).
With respect to the relationships among the five groups of deuterostomes, the 18S rDNA tree showed branching of a group that consisted of echinoderms, hemichordates, and urochordates from a group that consisted of cephalochordates and vertebrates. However, the value after bootstrapping for each of these branchings was low (44.4% and 45.0o%, respectively). Therefore, we examined the deuterostome phylogeny further by both neighbor-joining and maximumlikelihood, using one representative species from each group.
The evolutionary pathways toward the vertebrates have been discussed for more than a century, with emphasis on determining whether the ancestors of chordates were freeliving animals or sessile and immotile. The hypothesis proposed by Garstang, which has been widely accepted, states that cephalochordates and vertebrates emerged from a sessile, ascidian-like ancestry as a result of neotenic evolution. In the urochordate lineage, it is proposed that the ascidian-like ancestry gave rise to pelagic salps and then to larvaceans, the emergence of larvaceans being also the result of neotenic evolution.
Berrill proposed that neotenic evolution from the ascidian-like ancestors gave rise only to the larvaceans, from which the cephalochordate-vertebrate lineage evolved. By contrast, Tokioka proposed that the ancestors of chordates were free-living animals from which cephalochordates and vertebrates evolved directly and that, in the urochordate lineage, larvaceans with a lower tendency toward pelagic life emerged first, followed by the emergence of sessile-ascidians and pelagic salps. Jollie also regarded the ancestors of chordates as free-living animals, although he united cephalochordates and urochordates in a monophyletic group.⁷