The ‘Tully Monster’ is not a vertebrate: characters,convergence and taphonomy in Palaeozoic problematic animals

The affinity of Tullimonstrum gregarium, a pincer‐mouthed, soft bodied bilaterian, has been subject to debate since its recovery from Carboniferous coal deposits at Mazon Creek, Illinois. After decades of impasse focused on mollusc, arthropod and annelid attributes, two recent, yet conflicting, high‐profile studies concluded that the ‘Tully Monster’ is a vertebrate, a relative of lampreys or jawed fishes. Here, we find that structures described as supporting vertebrate, and particularly crown vertebrate, affinity face significant challenges from biological, functional and taphonomic perspectives. Problems with comparator choice, interpretation of taphonomic processes at Mazon Creek and estimation of convergence within the bilaterian tree may have misled these recent studies, leading to conclusions which do not accommodate current understanding of the vertebrate record. For example, the absence of taphonomically‐expected synapomorphies in Tullimonstrum (e.g. otic capsules, body pigment) calls into question vertebrate identity and implies that convergence or deeper origins are responsible for vertebrate‐like traits. Further, phylogenetic placement within vertebrates is only made possible by the constraints of a chordate‐only dataset with limited outgroups and use of selective characters. Long‐discussed alternative placements among molluscs (e.g. heteropod gastropods), arthropods (e.g. anomalocarids) or elsewhere within non‐vertebrate deuterostomes are more congruent. Indeed, many of these lineages independently evolved vertebrate‐like traits, including complex eyes and ‘teeth’. Thus, given the totality of evidence, Tullimonstrum should be excluded from the vertebrate crown. Potential assignments for aberrant bilaterians, common throughout the Palaeozoic fossil record, need to be considered in light of the number and likelihood of required exceptions to established schemes.     

Hubrecht,Macfarlane, Jensen and Willmer: on the nature and testability of four versions of the nemertean theory of vertebrate origins

Four authors (A. A. W. Hubrecht, J. M. Macfarlane, D. D. Jensen and E. N. Willmer) have argued that nemerteans are the proximal invertebrate ancestors of vertebrates. While all these authors accept certain homologies (i.e. vertebrate pituitary and notochord with nemertean proboscis invagination and proboscis sheath or mesoderm), there is considerable variation among them in the derivation of the vertebrate nervous system, sense organs and foregut apertures. For example, Hubrecht suggests that the vertebrate nervous system developed from a solid dorsal nerve found in some nemerteans; Macfarlane suggests it developed by fusion of the left and right solid brains of nemerteans; Jensen suggests that the vertebrate tubular nervous system developed by the backward extension of the nemertean frontal organ and fusion with the nemertean dorsal and ventral brain ganglia. Modern EM, histochemical and biochemical methods can test empirically the four theories. Research testing these theories will also test the more popular acraniate theory, since that theory implies that vertebrate head structures evolved de novo in chordates and that no homologies should exist between vertebrate and nemertean head structures.     

Chemical signatures of soft tissues distinguish between vertebrates and invertebrates from the Carboniferous Mazon Creek Lagerstätte of Illinois

The chemical composition of fossil soft tissues is a potentially powerful and yet underutilized tool for elucidating the affinity of problematic fossil organisms. In some cases, it has proven difficult to assign a problematic fossil even to the invertebrates or vertebrates (more generally chordates) based on often incompletely preserved morphology alone, and chemical composition may help to resolve such questions. Here, we use in situ Raman microspectroscopy to investigate the chemistry of a diverse array of invertebrate and vertebrate fossils from the Pennsylvanian Mazon Creek Lagerstätte of Illinois, and we generate a ChemoSpace through principal component analysis (PCA) of the in situ Raman spectra. Invertebrate soft tissues characterized by chitin (polysaccharide) fossilization products and vertebrate soft tissues characterized by protein fossilization products plot in completely separate, non‐overlapping regions of the ChemoSpace, demonstrating the utility of certain soft tissue molecular signatures as biomarkers for the original soft tissue composition of fossil organisms. The controversial problematicum Tullimonstrum, known as the Tully Monster, groups with the vertebrates, providing strong evidence of a vertebrate rather than invertebrate affinity.     

Lasanius,an exceptionally preserved Silurian jawless fish from Scotland

The fossil record of non-biomineralizing, soft-bodied taxa is our only direct evidence of the early history of vertebrates. A robust reconstruction of the affinities of these taxa is critical to unlocking vertebrate origins and understanding the evolution of skeletal tissues, but these taxa invariably have unstable and poorly supported phylogenetic positions. At the cusp between mineralized bony vertebrates and entirely soft-bodied vertebrates is the enigmatic Lasanius, a purported anaspid from the Silurian of Scotland. Interpretations of its affinity and significance are conflicted, principally because of its poorly understood anatomy due to taphonomic distortion and loss of soft-tissues. Here we use an array of modern techniques to reassess the anatomy of Lasanius via a comprehensive study of 229 complete and partial specimens. A new reconstruction clarifies the identity and position of preserved features, including paired sensory organs, a notochord, and digestive tract, supporting the vertebrate affinities of this genus. SEM-EDS trace element mapping suggests a bone-like composition of mineralized parts, but finds no evidence for mineralized dermal armour. Phylogenetic analysis recovers Lasanius as an early stem-cyclostome, and subsequent analysis supports the rejection of alternative placements (such as stem-gnathostome). We highlight that while distinguishing between the early cyclostome and gnathostome condition is problematic, increasing confidence in the anatomy of key taxa, such as Lasanius, is vital for increased stability throughout the early vertebrate tree.     

DNA EVIDENCE FOR NONHYBRID ORIGINS OF PARTHENOGENESIS IN NATURAL POPULATIONS OF VERTEBRATES

Naturally occurring unisexual reproduction has been documented in less than 0.1% of all vertebrate species. Among vertebrates, true parthenogenesis is known only in squamate reptiles. In all vertebrate cases that have been carefully studied, the clonal or hemiclonal taxa have originated through hybridization between closely related sexual species. In contrast, parthenogenetic reproduction has arisen in invertebrates by a variety of mechanisms, including likely cases of “spontaneous” (nonhybrid) origin, a situation not currently documented in natural populations of vertebrates. Here, we present molecular data from the Neotropical night lizard genus Lepidophyma that provides evidence of independent nonhybrid origins for diploid unisexual populations of two species from Costa Rica and Panama. Our mitochondrial and nuclear phylogenies are congruent with respect to the unisexual taxa. Based on 14 microsatellite loci, heterozygosity (expected from a hybrid origin) is low in Lepidophyma reticulatum and completely absent in unisexual L. flavimaculatum. The unique value of this system will allow direct comparative studies between parthenogenetic and sexual lineages in vertebrates, with an enormous potential for this species to be a model system for understanding the mechanisms of nonhybrid parthenogenesis.     

AmphiD1/β, a dopamine D1/β-adrenergic receptor from the amphioxus <Emphasis Type="Italic">Branchiostoma floridae</Emphasis>: evolutionary aspects of the catecholaminergic system during development

Catecholamine receptors mediate wide-ranging functions in vertebrates and invertebrates but are largely unknown in invertebrate chordates such as amphioxus. Catecholaminergic cells have been described in amphioxus adults, but few data are known about the transmembrane signal transduction pathways and the expression pattern of related receptors during development. In Branchiostoma floridae, we cloned a full-length cDNA (AmphiD1/β) that corresponds to the dopamine D1/β receptor previously cloned from a related species of amphioxus, Branchiostoma lanceolatum, but no expression studies have been performed for such receptor in amphioxus. In B. floridae, AmphiD1/β encodes a polypeptide with typical G-protein-coupled receptor features, characterized by highest sequence similarity with D1 dopamine and β-adrenergic receptors. The expression of AmphiD1/β mRNA in different regions of the cerebral vesicle corresponds to that of D1-like receptors in vertebrate homologous structures. Furthermore, in situ experiments show that during development, the expression in the nervous system is restricted to cells located anteriorly. A further expression was found in larvae at the level of the endostyle, but it has no counterpart in the predominant expression domains of vertebrate dopamine and/or adrenergic receptor genes. At the same time, we compared the dopaminergic system, consisting of AmphiTH-expressing cells, with the AmphiD1/β expression. In conclusion, the identification of the AmphiD1/β receptor provides further basis for understanding the evolutionary history of the dopaminergic system at the transition from invertebrates and vertebrates.     

Evolution of the Integrin α and β Protein Families

A phylogenetic analysis of vertebrate and invertebrate α integrins supported the hypothesis that two major families of vertebrate α integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the αPS1 and αPS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including αPS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate αPS1 and αPS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of β integrins did not clearly resolve whether vertebrate β integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate β4 may have done so. In general, the phylogeny of neither α nor β integrins showed a close correspondence with patterns of α–β heterodimer formation or other functional characteristics. One major exception to this trend involved αL, αM, αX, and αD, a monophyletic group of immune cell-expressed α integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their β integrin partners. Received: 22 June 2000 / Accepted: 11 September 2000     

Opsin localization and chromophore retinoids identified within the basal brain of the lizard Anolis carolinensis

Since the beginning of this century evidence has accumulated which demonstrates that non-mammalian vertebrates possess photoreceptors situated deep within the brain. While many attempts have been made to localize these sensory cells, studies have either failed or been inconclusive. In this report we have used several experimental approaches to localize the deep brain photoreceptors of the lizard Anolis carolinensis. Using 3 antibodies that bind vertebrate cone opsins, we have immunolabelled cerebrospinal fluid (CSF)-contacting neurons located at the ventricular border within the nucleus ventromedialis of the septum. Western blot analysis indicates that these antibodies recognized a single 40 kD protein in ocular, anterior brain, and pineal extracts. Immunoblots of rodent brain did not show a similar protein band. We have also identified specific retinoids associated with phototransduction (11-cis and all-trans-3,4-didehydroretinaldehyde) within anterior brain extracts. This combined data provides the most detailed analysis of deep brain photoreceptors in any vertebrate. Consequently, we feel Anolis provides an excellent model to study this unexplored sensory system of the vertebrates.Abbreviations CSF contacting neurons cerebrospinal fluid-contacting neurons - HPLC high performance liquid chromatography - L:D light:dark cycle     

The evolution of the hedgehog gene family in chordates: insights from amphioxus hedgehog

 The hedgehog family of intercellular signalling molecules have essential functions in patterning both Drosophila and vertebrate embryos. Drosophila has a single hedgehog gene, while vertebrates have evolved at least three types of hedgehog genes (the Sonic, Desert and Indian types) by duplication and divergence of a single ancestral gene. Vertebrate Sonic-type genes typically show conserved expression in the notochord and floor plate, while Desert- and Indian-type genes have different patterns of expression in vertebrates from different classes. To determine the ancestral role of hedgehog in vertebrates, I have characterised the hedgehog gene family in amphioxus. Amphioxus is the closest living relative of the vertebrates and develops a similar body plan, including a dorsal neural tube and notochord. A single amphioxus hedgehog gene, AmphiHh, was identified and is probably the only hedgehog family member in amphioxus, showing the duplication of hedgehog genes to be specific to the vertebrate lineage. AmphiHh expression was detected in the notochord and ventral neural tube, tissues that express Sonic-type genes in vertebrates. This shows that amphioxus probably patterns its ventral neural tube using a molecular pathway conserved with vertebrates. AmphiHh was also expressed on the left side of the pharyngeal endoderm, reminiscent of the left-sided expression of Sonic hedgehog in chick embryos which forms part of a pathway controlling left/right asymmetric development. These data show that notochord, floor plate and possibly left/right asymmetric expression are ancestral sites of hedgehog expression in vertebrates and amphioxus. In vertebrates, all these features have been retained by Sonic-type genes. This may have freed Desert-type and Indian-type hedgehog genes from selective constraint, allowing them to diverge and take on new roles in different vertebrate taxa. Received: 20 July 1998 / Accepted: 23 September 1998     

Amphioxus expresses both vertebrate-type and invertebrate-type dopamine D1 receptors

The cephalochordate amphioxus (Branchiostoma floridae) has recently been placed as the most basal of all the chordates, which makes it an ideal organism for studying the molecular basis of the evolutionary transition from invertebrates to vertebrates. The biogenic amine, dopamine regulates many aspects of motor control in both vertebrates and invertebrates, and in both cases, its receptors can be divided into two main groups (D1 and D2) based on sequence similarity, ligand affinity and effector coupling. A bioinformatic study shows that amphioxus has at least three dopamine D1-like receptor sequences. We have recently characterized one of these receptors, AmphiD1/β, which was found to have high levels of sequence similarity to both vertebrate D1 receptors and to β-adrenergic receptors, but functionally appeared to be a vertebrate-type dopamine D₁ receptor. Here, we report on the cloning of two further dopamine D₁ receptors (AmphiAmR1 and AmphiAmR2) from adult amphioxus cDNA libraries and their pharmacological characterisation subsequent to their expression in cell lines. AmphiAmR1 shows closer structural similarities to vertebrate D₁-like receptors but shows some pharmacological similarities to invertebrate “DOP1” dopamine D₁-like receptors. In contrast, AmphiAmR2 shows closer structural and pharmacological similarities to invertebrate “INDR”-like dopamine D₁-like receptors.     

An amphioxus snail gene: Expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo

 Homologs of the Drosophila snail gene have been characterized in several vertebrates. In addition to being expressed in mesoderm during gastrulation, vertebrate snail genes are also expressed in presumptive neural crest and/or its derivatives. Given that neural crest is unique to vertebrates and is considered to be of fundamental importance in their evolution, we have cloned and characterized the expression of a snail gene from amphioxus, a cephalochordate widely accepted as the sister group of the vertebrates. We show that, at the amino acid sequence level, the amphioxus snail gene is a clear phylogenetic outgroup to all the characterized vertebrate snail genes. During embryogenesis snail expression initially becomes restricted to the paraxial or presomitic mesoderm of amphioxus. Later, snail is expressed at high levels in the lateral neural plate, where it persists during neurulation. Our results indicate that an ancestral function of snail genes in the lineage leading to vertebrates is to define the paraxial mesoderm. Furthermore, our results indicate that a cell population homologous to the vertebrate neural crest may be present in amphioxus, thus providing an important link in the evolution of this key vertebrate tissue. Received: 11 May 1998 / Accepted: 2 August 1998     

The origin of novel features by changes in developmental mechanisms: ontogeny and three‐dimensional microanatomy of polyodontode scales of two early osteichthyans

Recent advances in synchrotron imaging allow us to study the three‐dimensional (3D) histology of vertebrate fossils, including microfossils (e.g. teeth and scales) of early jawed vertebrates. These microfossils can often be scanned at submicron resolution (<1 µm) because of their small size. The resulting voxel (3D pixel) stacks can be processed into virtual thin sections revealing almost every internal detail of the samples, comparable to traditional thin sections. In addition, 3D models of the internal microanatomical structures, such as embedded odontodes and vasculature, can be assembled and examined in situ. Scales of two early osteichthyans, Psarolepis romeri from the Early Devonian of China and Andreolepis hedei from the Late Silurian of Sweden, were scanned using propagation phase‐contrast synchrotron X‐ray microtomography (PPC‐SRµCT), and 3D models of internal canal systems and buried odontodes were created from the scans. Based on these new data, we review the evolutionary origin of cosmine and its associated pore‐canal system, which has been long recognized as a synapomorphy of sarcopterygians. The first odontode that appeared during growth shows almost identical morphology in the two scales, but the second odontode of the Psarolepis scale shows a distinctive morphology with several pores on the surface. It is suggested that a shift from ridge‐like odontode to pore‐bearing odontode was the key step in the origin of cosmine, which was then elaborated further in more‐derived sarcopterygians. We perform a detailed comparison between the two scales and propose a primary homology framework to generate microanatomical characters, which can be used in the phylogenetic analysis of early osteichthyans when more 3D data become available. Our results highlight the importance of 3D data for the study of histology and ontogeny of the dermal skeleton of early jawed vertebrates, especially scales of the polyodontode type. The traditional microvertebrate collection is not only useful for biostratigraphic studies, but also preserves invaluable biological information about the growth of vertebrate hard tissues. Today, we are only beginning to understand the biological meaning of the new 3D data. The increasing availability of such data will enable, and indeed require, a complete revision of traditional palaeohistological studies on early vertebrates.     

The evolution of teleost pigmentation and the fish‐specific genome duplication

Teleost fishes have evolved a unique complexity and diversity of pigmentation and colour patterning that is unmatched among vertebrates. Teleost colouration is mediated by five different major types of neural‐crest derived pigment cells, while tetrapods have a smaller repertoire of such chromatophores. The genetic basis of teleost colouration has been mainly uncovered by the cloning of pigmentation genes in mutants of zebrafish Danio rerio and medaka Oryzias latipes. Many of these teleost pigmentation genes were already known as key players in mammalian pigmentation, suggesting partial conservation of the corresponding developmental programme among vertebrates. Strikingly, teleost fishes have additional copies of many pigmentation genes compared with tetrapods, mainly as a result of a whole‐genome duplication that occurred 320–350 million years ago at the base of the teleost lineage, the so‐called fish‐specific genome duplication. Furthermore, teleosts have retained several duplicated pigmentation genes from earlier rounds of genome duplication in the vertebrate lineage, which were lost in other vertebrate groups. It was hypothesized that divergent evolution of such duplicated genes may have played an important role in pigmentation diversity and complexity in teleost fishes, which therefore not only provide important insights into the evolution of the vertebrate pigmentary system but also allow us to study the significance of genome duplications for vertebrate biodiversity.     

入侵红火蚁对脊椎动物的影响研究进展

红火蚁(Solenopsis invicta)具有扩散迅速、攻击性强等特点,常对入侵地区造成巨大的生态危害。虽然很多文献报道了红火蚁对无脊椎动物的影响,但红火蚁对脊椎动物影响的研究较少,也缺乏系统的总结。综述了红火蚁对不同脊椎动物类群的影响和主要的研究方法(野外调查、案例分析、实验室评估、比较分析和人工移除红火蚁),以及脊椎动物对红火蚁的行为和生理防御机制。一般而言,红火蚁常以腹部末端的螯针攻击其他动物并释放毒液,可直接捕食、伤害各种脊椎动物的卵、幼体和成体,并影响动物的行为。此外,红火蚁可降低生境内无脊椎动物的丰度,间接影响捕食者的种群数量。尽管红火蚁是陆生生物,但鱼类可能取食掉落在水面的有翅繁殖蚁而中毒,且漂浮于水面的红火蚁蚁筏也可能对鱼类造成威胁。由于两栖类动物通常体型较小,运动能力较弱,且皮肤缺乏角质层与鳞片毛发等结构的保护,极易受到红火蚁的攻击和伤害。对于鸟类和爬行类,红火蚁主要捕食卵和幼体,并干扰成体的筑巢和育幼行为,降低其繁殖成功率。此外,红火蚁对小型哺乳动物的觅食行为和种群数量均可造成负面影响,并可蛰刺伤害牛羊等家畜,对畜牧业造成巨大的经济损失。一些动物对红火蚁具有行为防御,如用附肢蹬掉体表的红火蚁,或避免取食或接触红火蚁。此外,一些动物能对红火蚁的毒素产生抗体,或调整体内荷尔蒙水平以应对红火蚁的攻击。然而,仍不清楚这些行为和生理反应是否普遍存在于其他脊椎动物,以及能在多大程度上保护其免受红火蚁伤害。值得注意的是,几乎所有的相关研究都是由国外研究者完成的,建议我国学者更多的关注红火蚁对我国经济物种、珍稀动物和食蚁物种的影响。     

Comparative genomics of Lbx loci reveals conservation of identical Lbx ohnologs in bony vertebrates

Background  

Lbx/ladybird genes originated as part of the metazoan cluster of Nk homeobox genes. In all animals investigated so far, both the protostome genes and the vertebrate Lbx1 genes were found to play crucial roles in neural and muscle development. Recently however, additional Lbx genes with divergent expression patterns were discovered in amniotes. Early in the evolution of vertebrates, two rounds of whole genome duplication are thought to have occurred, during which 4 Lbx genes were generated. Which of these genes were maintained in extant vertebrates, and how these genes and their functions evolved, is not known.     

Identification and tissue-specific expression of a promethin-like homolog in amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

Promethins have been shown to be present in the vertebrates examined so far, yet little is known to date about them in invertebrates. Here we isolated a cDNA encoding a promethin-like homolog from the gut cDNA library of the amphioxus Branchiostoma belcheri, a cephalochordate occupying a nodal position transient from invertebrates to vertebrates. It contained a 504 bp open reading frame corresponding to a protein of 167 amino acids. Primary structural examination showed that the deduced promethin-like homolog was a transmembrane protein with three potential transmembrane helices, resembling the vertebrate promethins. Phylogenetic analysis showed that B. belcheri promethin-like homolog was located at the base of the vertebrate counterparts, suggesting that it represents the archetype of vertebrate promethins. Both Northern blotting and in situ hybridization histochemistry revealed a tissue-specific expression pattern of promethin-like gene, like that of mammalian promethins. This is the first report on invertebrate promethin-like homolog, paving the way for further insights into the evolution and function of promethins.     

Induction by alkaloids and phenobarbital of Family 4 Cytochrome P450s in Drosophila : evidence for involvement in host plant utilization

In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds. A degenerate 3^′ RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships. Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs. Received: 14 August 1997 / Accepted: 24 March 1998     

Phylogenies of Developmentally Important Proteins Do Not Support the Hypothesis of Two Rounds of Genome Duplication Early in Vertebrate History

It has been proposed that two rounds of duplication of the entire genome (polyploidization) occurred early in vertebrate history (the 2R hypothesis); and the observation that certain gene families important in regulating development have four members in vertebrates, as opposed to one in Drosophila, has been adduced as evidence in support of this hypothesis. However, such a pattern of relationship can be taken as support of the 2R hypothesis only if (1) the four vertebrate genes can be shown to have diverged after the origin of vertebrates, and (2) the phylogeny of the four vertebrate genes (A–D) exhibits a topology of the form (AB) (CD), rather than (A) (BCD). In order to test the 2R hypothesis, I constructed phylogenies for nine protein families important in development. Only one showed a topology of the form (AB) (CD), and that received weak statistical support. In contrast, four phylogenies showed topologies of the form (A) (BCD) with statistically significant support. Furthermore, in two cases there was significant support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes: in one case there was significant support for duplication of the vertebrate genes at least prior to the divergence of vertebrates and urochordates, and in one case there was weak support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes. Taken together with other recently published phylogenies of developmentally important genes, these results provide strong evidence against the 2R hypothesis. Received: 22 December 1997 / Accepted: 5 October 1998     

Characterization of approximately 50 kb of the immunoglobulin VH locus of the Japanese pufferfish, Fugu rubripes

The variable region of the immunoglobulin heavy chain is created by a somatic rearrangement of a limited number of germline genes. This mechanism of gene assembly [V(D)J recombination] has been found to take place only in jawed vertebrates (gnathostomes). To understand how this mechanism evolved and diversified it is necessary to study the genomic organization of the heavy-chain gene in different vertebrate lineages. Since there is scant sequence information on the VH locus in fish, shotgun sequencing of a cosmid clone containing part of the VH genomic region of the Japanese pufferfish, Fugu rubripes, was undertaken. Eight full-length VH genes were isolated and characterized. They have higher homology to trout genes, but show the same structural features as VHs found in other vertebrates. Two VH subgroups have been identified whose members are interspersed. The frequency of synonymous and nonsynonymous substitution for VH comparisons between family members was found to be higher in the complementarity-determining regions than in the framework regions. Finally, there are four other genes interspersed with the VH genes, one of which is the first full-length retrotransposon element characterized in vertebrates. Received: 27 October 1999 / Revised: 15 December 1999     

Identification and expression of an elastase homologue in <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis> with implications to the origin of vertebrate pancreas

Elastases have been identified in a variety of organisms ranging from bacteria to insects to mammals, yet little is known to date about them in amphioxus, a model animal for insights into the origin and evolution of vertebrates. In this study we demonstrate the presence of an elastase homologue, named BbElas, in Branchiostoma belcheri. The recombinant BbElas hydrolyses the elastase specific substrate N-succinyl-Ala-Ala-Ala p-nitroanilide, which can be inhibited by the serine proteinase inhibitor PMSF, the elastase-specific inhibitor elastatinal and the cysteine proteinase inhibitor PCMB. Phylogenetic analysis shows that BbElas represents the archetype of vertebrate elastases, hinting at the clues that the different isoforms of vertebrate elastases are originated from an ancestral gene like BbElas. Our results also suggest that the mid-gut in amphioxus is to homologous vertebrate pancreas, a novel proposal which deserves further study.