Cephalochordates, commonly known as amphioxus or lancelets, belong to the monophyletic group of chordates, which also includes tunicates and vertebrates (Figure 1A). Amphioxus are marine benthic animals that feed on phyto- and zooplankton by filtering the seawater, and they are distributed worldwide in sandy habitats of tropical and temperate seas (Bertrand and Escriva, 2011).

Figure 1

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A fascinating drawing by the Italian scientific illustrator Comingio Merculiano in the late 18^th century captures the lifestyle of amphioxus (Figure 2). Unlike the adult, the embryos and larvae are planktonic and, depending on the species, the larval phase can last up to several months in the open sea. Therefore, amphioxus have a high potential for offshore larval dispersion in new coastal areas until they undergo the process of metamorphosis and become juveniles. The juveniles already show the typical adult body plan and, at this stage, they adopt a benthic lifestyle, prevalently buried in the substrate.

Figure 2

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The name cephalochordate (i.e., cephalo- (head) and -chordate (notochord)), which was proposed by Ernst Haeckel in the 1860s (Haeckel, 1866), does a good job of describing the peculiarity of their anatomy, with the notochord extending to the front of the animal, beyond the cerebral vesicle (i.e. the most anterior structure of the central nervous system). The anatomy of cephalochordates is considered vertebrate-like, but simpler, having a prototypical chordate body plan. Chordate synapomorphies, present in amphioxus and vertebrates, include a dorsal hollow nerve chord and notochord, pharyngeal slits, segmented muscles and gonads, post anal tail, and homologs of pronephric kidney, pituitary and thyroid (Figure 1B). However, some typical vertebrate characteristics are not present in amphioxus such as paired sensory organs (image-forming eyes or ears), paired appendages and migrating neural crest cells. Their embryonic development includes 10 developmental periods, from the zygote to the adult (Carvalho et al., 2021; Bertrand et al., 2021), which are extremely well conserved among different amphioxus species.

An interesting anatomical feature of amphioxus development concerns their symmetry. In fact, larvae are completely asymmetrical, with the mouth and anus on the left side of the body. However, this asymmetry mostly disappears during metamorphosis, which produces an almost symmetrical adult animal (Paris et al., 2008).

The amphioxus genome also shows a high degree of conservation with vertebrate genomes, but with specific features. Amphioxus has orthologues for mostly all known vertebrate gene families, and the gene position and order in the genome, known as synteny, is also highly conserved which greatly benefits comparative analyses with vertebrates (Putnam et al., 2008; Marlétaz et al., 2018). However, the amphioxus genome has not undergone the two complete duplications that the vertebrate ancestor experienced (Figure 1A), although it has undergone numerous specific gene duplications (Brasó-Vives et al., 2022).

Moreover, the regulation of gene expression is much simpler than in vertebrates (Gil-Gálvez et al., 2022). And although it is still a point of debate, the amphioxus three-dimensional chromatin structure also seems to be less complicated than the vertebrate’s one (Acemel et al., 2016; Huang et al., 2023). Altogether, the crucial phylogenetic position, conserved morphological traits and genome organization make amphioxus a useful organism for answering fundamental questions in biology, particularly with respect to vertebrate evolution. Thus, over the last decades, cephalochordates have become an important animal model in the fields of evolutionary developmental biology (EvoDevo), immune system evolution, cell signalling, regeneration and genome evolution.

The proposed phylogenetic position of cephalochordates has, as with many other metazoan groups, undergone major changes in recent years. Cephalochordates used to be classified as the closest group to vertebrates within the chordates, one of the two deuterostome clades together with the Ambulacraria (Figure 1A). This position, as a sister group of vertebrates, was based above all on the conservation of numerous morphological characteristics, and also on some molecular studies based on rDNA (Winchell et al., 2002). Moreover, the other chordate subphylum, the tunicates, shows a great divergence at the morphological level, especially in adults, whose body plan is completely different from that of the prototypical chordate.

However, this classification has completely changed following studies using larger molecular data, which finally positioned the cephalochordate lineage as the earliest divergent group of chordates (Bourlat et al., 2006; Delsuc et al., 2006; Delsuc et al., 2008), and placing the tunicates as the sister group of the vertebrates. This new chordate phylogeny suggests an evolutionary explanation of why tunicates, despite their tremendous anatomical and genomic divergence, share some features with vertebrates that are absent in amphioxus such as migratory cells similar to those of the neural crest, or placode-like ectodermal regions (Abitua et al., 2015; Manni et al., 2004; Horie et al., 2018).

Another consequence of this newly proposed phylogenetic classification is a change in the hypothesis about the ancestral chordate lifestyle. In the past, it was hypothesised that vertebrates arose by neoteny from a sessile organism with free-living tadpole larvae (like ascidians) (Williams, 1996). Placing cephalochordates as the earliest divergent chordates suggests that instead the ancestral chordate could have been amphioxus-like with a free-living lifestyle even at the adult stage. This hypothesis is also reinforced by the fact that early vertebrate fossils, such as Haikouichthys or Haikouella, are similar to amphioxus in many aspects, such as their small size or their mobile and filter-feeding lifestyle (Mallatt and Chen, 2003; Shu et al., 2003a; Shu et al., 2003b).

Unfortunately, clear cephalochordate fossils have not yet been found. For many years, Pikaia, the Cambrian fossil from Burgess Shale in Canada, has been considered a basal chordate, but numerous features such as dorsal organ, posterior ventral area, posterior fusiform structure, anterior dorsal unit, and sigmoid rather than chevron‐shaped muscles, divide the paleontological community about the exact phylogenetic position of this controversial fossil, even if its unresolved position is most probably within chordates (Morris and Caron, 2012; Mallatt and Holland, 2013).

In 1774, the first scientific description of a specimen of amphioxus was made by Pallas from an animal from off the coast of Cornwall (United Kingdom) (Pallas et al., 1774). Years later, amphioxus were rediscovered in the Mediterranean Sea first by Costa, 1834, and, two years later, independently, by Yarrell, 1836. Apart from the difference in the nomenclature, the Cornish and Mediterranean specimens were considered the same species, Branchiostoma lanceolatum.

In 1847, Gray described a new specimen from the coast of Borneo, which he called Branchiostoma belcheri (Gray, 1847). Shortly afterwards, in 1852, a new species was described off Peru by Sundevall and named Branchiostoma elongatum (Sundevall, 1852). The same year, B. lanceolatum was also observed off northern Germany (Sundevall, 1852). It was then that a system capable of species classification became necessary, and the enumeration of myotomes anterior to the atriopore, between the atriopore and anus, and posterior to the anus was chosen, the global morphology being extremely similar between all described cephalochordates. Thus, the classification soon included four species (B. lanceolatum, B. belcheri, B. elongatum and Branchiostoma caribbaeum, described from the coasts of Rio de Janeiro in Brazil, Sundevall, 1853), distributed in a rather cosmopolitan way between the Mediterranean, the Atlantic and the Pacific oceans.

In 1876, based on differences in pharyngeal slits position and fin shape in animals from Torres Strait in Australia, Peters described a second cephalochordate genus, which he called Epigonichthys (Peters, 1876). Although a later study reclassified the same species as belonging to the genus Branchiostoma (Günther and Reptilia, 1882), Epigonichthys was later recognised as a new genus.

Finally, in 1893, Andrews described a third genus of cephalochordates, Asymmetron, for specimens from the Bahamas which he named Asymmetron lucayanum (Andrews, 1893). The main characteristics of this third genus were the presence of a single row of gonads and the asymmetrical metapleural folds, the left one ending at the level of the anus, while the right one was continuous behind with the ventral median fin. From this period on, many researchers began to describe new specimens from different locations and to define the classification of cephalochordates on the basis of the aforementioned meristic characteristics. This gave rise to discussions, with numerous synonyms for the same species or different species sharing the same name. Thus, new genera and subgenera were proposed, such as Amphioxus, Heteropleuron, Amphioxides, Dolichorhynchus, Paramphioxus, etc.

During this period, more than fifty species and ten genera of lancelets were described. Nevertheless, it was not until 1996 that Poss and Boschung made a compilation of all the described species and genera, and re-examined the different meristic data of each, to produce a list as correct as possible of the lancelet species described in the world (Poss and Boschung, 1996). This study reduced the total number of species to 29, and the number of genera to two, Branchiostoma and Epigonichthys. However, they defined Asymmetron as a synonym of Epigonichthys, since they failed to detect synapomorphies of the Epigonichthys group that would exclude Epigonichthys lucayanum (today called Asymmetron lucayanum) from this group. Thus, in the absence of arguments to support the fact that E. lucayanum (i.e. A. lucayanum) is the sister-taxon to all other Epigonichthys, they followed the classification proposed by Richardson and McKenzie, 1994 with only two genera (Epigonichthys and Branchiostoma) instead of three genera as proposed in Piyakarnchana and Vajropala, 1961.

Poss and Boschung described the challenge of classifying the different species of amphioxus through the use of meristic data as follows: “Multivariate analysis of meristic variation, using primarily American species, reveals considerable intraspecific variability in key taxonomic features. Some species exhibit wide variation in countable segments, whereas others are characterized by a narrow range” (Poss and Boschung, 1996). Furthermore, they clearly advocated the use of molecular techniques capable of distinguishing genetic differences and discriminating taxa with small morphological differences. In fact, it was through the use of modern molecular approaches, as well as through detailed morphological descriptions, that Nishikawa and Nohara confirmed the existence of three genera, Branchiostoma, Asymmetron and Epigonichthys (Nishikawa, 2004; Nohara et al., 2005). Two of them, Asymmetron and Epigonichthys, possess asymmetrical dextral gonads, and the third one, Branchiostoma, symmetrical gonads (Igawa et al., 2017).

The use of molecular taxonomy to define species has brought some other surprises in the classification of amphioxus species, since it revealed the existence of cryptic species among animals that previously shared the same name. Thus, for example, specimens of B. belcheri from the Chinese and Japanese coasts were differentiated into two different species (B. belcheri and Branchiostoma tsingtauense) (Wang, 2004; Xu et al., 2005) even if later, according to the rule of priority, the name of B. tsingtauense was changed by B. japonicum (Wang, 2004; Zhang et al., 2006).

Another example of a cryptic species complex, revealed through the use of molecular approaches, concerns A. lucayanum, for which up to three genetically distinguished major groups of geographical populations have been discovered. For one of these groups, composed of animals collected in the Red Sea, the name Asymmetron rubrum has even been proposed (Subirana et al., 2020; Kon et al., 2006). Thus, all these studies suggest that the total number of amphioxus species in the world is probably underestimated, and that molecular characterisation is likely to increase the total number of extant species in the near future.

The different amphioxus species can be found in a cosmopolitan way in all tropical and temperate oceans of the world. Amphioxus have never been observed in freshwater, and although they are present worldwide, they have a preference for soils of more or less fine sand or shell deposits, and in most cases with little organic decay (see below). However, they are not always present in all suitable sandy sediments, which indicate that other factors, such as pollution or currents, may play an important role in the dynamic distribution of these benthic animals. Of course, this distribution refers to adult animals, which have a benthic lifestyle and are generally found at depths between very shallow water (i.e. 0.5 m deep) to 30–50 meters deep. There are also some exceptions since some specimens have been found at greater depths (i.e. about 180 m deep) (Wickstead, 1975) and in an anaerobic and sulfide-rich environment caused by the decomposing body of a whale at 229 m deep (Nishikawa, 2004), which does not exclude the possibility that deep-water species may be found in the future.

In contrast to adults, the embryonic and larval stages of amphioxus are planktonic. Thus, the amphioxus larvae can drift across oceans thanks to marine currents over a period of time that, depending on the species, can range from a couple of weeks to several months (Figure 3). The larvae are mainly found and distributed by coastal currents, although pelagic larvae have also been reported in places far away from the coast (Goldschmidt, 1905). The study of these larvae gave rise to the discussion of a different type of amphioxus, called Amphioxides, which were considered to be pelagic adults, although today there is a wide consensus on the larval nature of these individuals, which, however, have delayed their metamorphosis in a neotenic process (Bone, 1957).

Figure 3

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The distribution of each amphioxus species has been reviewed in Poss and Boschung, 1996. This kind of information can also be accessed in the Unesco database, OBIS (McEwan, 2001), where it can be observed that amphioxus are found in all tropical and temperate coasts of the world. An interesting aspect of the differences in distribution between species is that, while some species have been found at very distant locations around the globe, such as B. belcheri that can be found in practically all coasts of East Asia, Oceania and even the African coasts of the Indian Ocean, and similarly B. lanceolatum that is found along the entire Mediterranean coasts, the Atlantic coasts of Europe and North Africa (Caccavale et al., 2021b), and even in the Indian Ocean, other species are found in much more restricted areas. For example, Branchiostoma senegalensis or Branchiostoma gambiense were only described on the West Africa coasts. Several explanations can account for this, but the most widely accepted is that the amplitude of the species distribution depend upon the type of marine currents present in each area (Webb, 1975).

Adult amphioxus, as already mentioned, live on the seafloor, burrowed in well-ventilated substrates with a soft texture and without too much organic load. Different species have been described as living in different types of substrate, ranging from very fine sand, coarse sand and even shell deposits, with a clear preference of most of the species for coarse sand with low content of fine particles. This is the case of Branchiostoma nigeriense on the west coast of Africa (Webb and Hill, 1958; Webb, 1958), Branchiostoma caribaeum in Mississippi Sound and from South Carolina to Georgia (Boschung and Gunter, 1962; Cory and Pierce, 1967), B. senegalense in the off-shore shelf region off North West Africa (Gosselck and Spittler, 1979) and B. lanceolatum from the Mediterranean coast of southern France (Caccavale et al., 2021b; Desdevises et al., 2011). However, B. floridae from Tampa Bay in Florida seems to be an exception to this rule since they live in fine sand bottoms (Stokes and Holland, 1996a; Stokes, 1996).

All species of amphioxus are gonochoric, and only a few cases of hermaphroditism have been reported in both B. lanceolatum and B. belcheri (Yamaguchi and Henmi, 2003; Orton, 1914). In these cases, only a few female gonads (i.e., developing ovaries) were observed in a male (2–5 gonads out of a total of 45–50). A unique case of complete sex reversal has been described in B. belcheri, where a female amphioxus reared in the laboratory was sexually reversed into a male (Zhang et al., 2001).

Spawning, which consists in the release of thousands of oocytes and millions of spermatozoa in the water column, is concentrated, in most species, in one period of the year (i.e. the spawning season), which usually takes place during the warmer months (spring-summer). The spawning season duration varies between species, being shorter (between one and three months) in species living in temperate waters and longer (around six months) in tropical species. Spawning always occurs shortly after sunset, although the behaviour is different depending on the species. Thus, for example, in B. floridae, up to 90% of the animals spawn synchronously once every two or three weeks (Stokes and Holland, 1996b). On the other hand, in other species such as B. lanceolatum, spawning occurs gradually between the beginning and the end of the spawning season (Fuentes et al., 2004). An exception to this unique annual breeding season is the case of A. lucayanum, which spawn during two periods of the year (during the warm months of spring and summer, but also in autumn). Moreover, in this species, the moon cycle seems to play a major role since spawning is concentrated in the days preceding the new moon (Holland, 2011).

Concerning the feeding behaviour, the size of the particles filtered by different amphioxus species, as well as their diet, has been studied. Amphioxus are able to ingest sub-micron particles thanks to the mucus secreted by the endostyle. The size of these particles has been calculated in several species (i.e. B. lanceolatum, B. senegalense, B. floridae) and the results are quite similar regardless of the species. The size ranges from 0.062 to 100 µm, although in B. senegalense particles up to 300 µm were found (Gosselck et al., 1978; Ruppert et al., 2000; Riisgard and Svane, 1999). This particle size suggests that the amphioxus diet includes microbes as well as phytoplankton, even if, in addition to phytoplankton, crustaceans have also been found in the gut contents of B. senegalense and B. lanceolatum larvae (Gosselck and Kuehner, 1973; Webb, 1969). Moreover, much of the ingested material exits the anus undigested after 1–2 hours and most of the gut contents consist of detritus, suggesting that amphioxus are indiscriminate suspension feeders (Gosselck et al., 1978). A clear example of this indiscriminate filtering behaviour is the fact that several recent studies show how different species of amphioxus are capable of filtering microplastics present in the environment (Cheng et al., 2023; Xiang et al., 2022).

An interesting behaviour of adult amphioxus is that, as ciliary feeding progresses, the oral cirri, whose function is to prevent the entry of large particles, become blocked with these coarse detritus reducing the flow of water through the pharynx. When this occurs, the atrial floor is violently raised and lowered, and water is expelled from the atrium through the pharynx and oral hood, which unblocks the oral cirri (Dennell, 1950).

Several studies have focused on the lifespan of different amphioxus species, usually based on the size distribution of sampled individuals and taking into account that amphioxus grow continuously during their entire life (Stokes, 1996). These estimates include a lifespan of 2–3 years for B. floridae (Wells, 1926; Nelson, 1968; Futch and Dwinell, 1977), a maximum age of 2–3 years for B. belcheri (Chin, 1941; Chen et al., 2008), a lifespan of 4–5 years for B. senegalense in northwest Africa (Gosselck and Spittler, 1979), a lifespan of 5 years for B. lanceolatum in the Mediterranean Sea (Desdevises et al., 2011), which increases to 8 years in the relatively cold waters of Helgoland (Courtney, 1975).

The most likely causes of death in amphioxus, as in most wild animals, can be summarized as infections and predation. Thus, our own observations attest that amphioxus in the water column are attractive prey for fishes, and a description of amphioxus predators has been published. In this case, a stingray was observed to have a gut filled almost exclusively with amphioxus (B. floridae) in Tampa Bay (Stokes and Holland, 1992).

Concerning infections, in 1936 Ravitch-Stcherbo described the presence of a bacteria which produces a red pigment by putrid decomposition of tissues, and which is capable of infecting and killing amphioxus (B. lanceolatum) in captivity, but the strain of this bacterium was not described (Ravitch-Stcherbo, 1936). More recently, Zou and collaborators described the presence of a lethal bacteria and characterised it as Vibrio alginolyticus in B. belcheri (Zou et al., 2016). Finally, other causes of death like tumours, such as a chromaffinoma (Stolk, 1961), or the presence of parasites have also been described in amphioxus. Thus, in 1968 Azariah described the presence of a trypanorhynchan larvae, a cestode known to parasitize fishes, in several individuals of B. lanceolatum off the coast of Madras in India (Azariah, 1968), and Holland and collaborators also described the presence of parasitic larvae of the tapeworm Acanthobothrium brevissime in B. floridae (Holland et al., 2009).

In recent years, the worldwide growing interest in amphioxus as model organisms for different research studies has led various groups to develop new technical approaches to breed the animals in captivity. Thus, different tools and protocols have been developed for amphioxus maintenance and reproduction that allow obtaining large amounts of live embryos in the laboratory. These amphioxus aquaculture systems have been developed for the four most studied species, with slight differences concerning the day/night cycle, sea water recirculation, species-specific temperature regimes, natural or artificial seawater, the presence or not of sand in the tanks, and so forth (Fuentes et al., 2004; Fuentes et al., 2007; Holland and Yu, 2004; Yasui et al., 2007; Holland and Holland, 2010; Li et al., 2012; Li et al., 2013; Li et al., 2015; Theodosiou et al., 2011; Benito-Gutiérrez et al., 2013; Carvalho et al., 2017; Somorjai et al., 2008).

Adult amphioxus with mature gonads can be artificially induced to spawn in the laboratory during the breeding season under controlled conditions. This is a prerequisite for the in vitro fertilization of eggs and the achievement of synchronized embryo’s cultures. Different methodologies have proven effective for successful spawning induction depending on the species: the first of these was an electric shock in B. floridae (Holland and Holland, 1989), but this approach also induced unfertilised egg activation in other species, such as B. lanceolatum, so a different approach was required. A water temperature change 36 hours prior spawning is employed for species like B. lanceolatum (Fuentes et al., 2004; Fuentes et al., 2007). Gonad maturation is a prerequisite for spawning induction, but it is seasonally restricted to the breeding season and is often quite difficult to obtain in captive animals. Nevertheless, excellent results have been obtained using tropical species (B. floridae and B. belcheri) for which it has been possible to significantly increase the reproductive period artificially in the laboratory, beyond the limited breading season (Li et al., 2013; Holland and Li, 2021).

Animal husbandry, therefore, allows obtaining large amounts of eggs and embryos on demand, opening the door to modern functional approaches to study developmental gene function and the molecular mechanisms of gene and genomic regulation. The first studies focusing on gene expression using amphioxus embryos were based on classical analyses through in situ hybridization in the 1990s (Holland et al., 1992). The first functional studies were carried out through the use of pharmacological treatments capable of activating, inhibiting or modifying certain signalling pathways (Bertrand et al., 2017). Other methods to manipulate gene expression through gene overexpression or gene knockdown by microinjection in the unfertilized eggs of mRNAs or morpholinos have also been developed in different amphioxus species (Aldea et al., 2019; Onai et al., 2010; Schubert et al., 2005).

Classical embryo micromanipulation techniques, including grafting, have also been developed (Le Petillon et al., 2020). Importantly, through the use of the TALEN and CRISPR-Cas9 gene-editing approaches, and Tol2-based transgenesis, it has been possible to obtain knock out and transgenic lines in B. floridae and B. belcheri for different genes, which has lifted an important brake on functional studies using amphioxus and has boosted the research in the evolutionary developmental biology field (EvoDevo) (Holland and Li, 2021; Li et al., 2014; Li et al., 2017; Hu et al., 2017; Zhong et al., 2020; Ren et al., 2020; Zhu et al., 2020; Zou et al., 2021; Su et al., 2020; Kozmikova and Kozmik, 2015).

Finally, high-throughput sequencing techniques have made it possible to obtain the complete chromosome-level genome assembly of four amphioxus species, B. floridae, B. lanceolatum, B. belcheri and B. japonicum (Putnam et al., 2008; Marlétaz et al., 2018; Brasó-Vives et al., 2022; Huang et al., 2023; Huang et al., 2014), thus opening the door to functional and comparative genomics studies. The use of new sequencing techniques at the level of single cells has been producing significant amount of information in recent years in various animal models. Amphioxus has not been left behind and this technique has also started to generate interesting results in several species (Lin et al., 2020; Satoh et al., 2021; Ma et al., 2022).

In this article we have focused mainly on known data on the biology and natural history of amphioxus. However, most of the recent scientific work published on amphioxus focuses on the evolution of developmental mechanisms and genomes. As we have presented in this review, because of their phylogenetic position among chordates, their prototypical characteristics, and the possibility of obtaining a large amount of externally developing and transparent embryos, amphioxus were mainly used to try to understand how the evolution of genomes and of the control of developmental processes led to the morphological complexity found in extant vertebrates.

Concerning genomics, obtaining whole genome sequences for amphioxus (Putnam et al., 2008; Marlétaz et al., 2018; Huang et al., 2014), and also for tunicates (Dehal et al., 2002; Dehal and Boore, 2005), allowed the 2R hypothesis proposed by Ohno, 1970 to be confirmed. According to this hypothesis, two rounds of whole genome duplications took place during the early evolutionary history of vertebrates, although data in lamprey suggest that only one of these duplications might be shared by gnathostomes (jawed vertebrates) and cyclostomes (jawless vertebrates including lampreys and hagfish) (Simakov et al., 2020). Amphioxus genomic data also helped reconstructing the chordate ancestral karyotype, and the evolution of gene families in this clade. Finally, recent epigenomic analyses showed that chromatin conformation evolution (Acemel et al., 2016; Huang et al., 2023) and complexification of developmental gene regulation (Marlétaz et al., 2018) and of the interconnectivity between signalling pathways (Gil-Gálvez et al., 2022) might have participated to the emergence of vertebrate specific traits.

Studies of amphioxus development, through the analysis of gene expression or function, and of the role of different intercellular communication pathways, led to several key advances in our understanding of morphological evolution within the chordate group. First, conservation of the expression of orthologous genes in homologous structures between amphioxus and vertebrates allowed highlighting the key actors controlling the formation of chordate synapomorphic traits. Hence it has been shown, for example, that both amphioxus and vertebrates possess an embryonic territory at the gastrula stage called the dorsal organizer, which is responsible for early axial patterning and for neural induction (Le Petillon et al., 2017; Yu et al., 2007).

On the other hand, studies in amphioxus also pointed out differences with vertebrate developmental modalities that could be linked to the emergence of vertebrate traits such as an unsegmented head musculature (Aldea et al., 2019; Bertrand et al., 2011; Meister et al., 2022). Lastly, embryological studies on amphioxus may also shed light on unsuspected roles of certain signals in the control of chordate development. For example, a recent work showed the role of the nitric oxide pathway in normal pharyngeal development through an interaction with the retinoic acid signaling pathway in amphioxus (Caccavale et al., 2021a), which calls for a more detailed examination of embryonic function of nitric oxide in vertebrates.

The interest in the study of cephalochordate biology and ecology has experienced alternating periods of great popularity and long periods of stagnation. Today, however, the number of research groups using amphioxus as a model organism is growing and they are located all over the world. Moreover, the interest in amphioxus covers a wide spectrum of research fields, ranging from classical embryology, through EvoDevo, to functional and comparative genomics (see Box 1 for a list of outstanding questions about the natural history of amphioxus).

Unlike many other animal models, there is not a specific meeting for researchers interested in amphioxus, although the European Society for Evolutionary Developmental Biology (https://evodevo.eu) has sponsored a satellite meeting dedicated to amphioxus for the past decade. This meeting, which takes places every two years, is typically attended by almost one hundred participants.

Modern technological approaches are driving amphioxus research, and undoubtedly the technical developments we have discussed, and in particular the obtaining of the transcriptomic profile of each cell type, will help resolving old questions in the future. Examples of such questions include the evolutionary appearance of the neural crest cells typical of vertebrates, or the evolution and complexification of the vertebrate brain from that of the chordate ancestor. The multiplication of data produced by high-throughput sequencing techniques will also certainly raise new and interesting questions, as much on the evolutionary level as on that of embryonic development or physiology of amphioxus.

Whatever the future of research using different species of amphioxus as model organisms, one point on which the entire scientific community agrees is that this is an extremely exciting time to be working with this fascinating little animal.

No new data was generated for this article.

1. 1. Abitua PB
   2. Gainous TB
   3. Kaczmarczyk AN
   4. Winchell CJ
   5. Hudson C
   6. Kamata K
   7. Nakagawa M
   8. Tsuda M
   9. Kusakabe TG
   10. Levine M

   (2015) The pre-vertebrate origins of neurogenic placodes

   Nature 524:462–465.

   https://doi.org/10.1038/nature14657

   • PubMed
   • Google Scholar
2. 1. Acemel RD
   2. Tena JJ
   3. Irastorza-Azcarate I
   4. Marlétaz F
   5. Gómez-Marín C
   6. de la Calle-Mustienes E
   7. Bertrand S
   8. Diaz SG
   9. Aldea D
   10. Aury J-M
   11. Mangenot S
   12. Holland PWH
   13. Devos DP
   14. Maeso I
   15. Escrivá H
   16. Gómez-Skarmeta JL

   (2016) A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation

   Nature Genetics 48:336–341.

   https://doi.org/10.1038/ng.3497

   • Google Scholar
3. 1. Aldea D
   2. Subirana L
   3. Keime C
   4. Meister L
   5. Maeso I
   6. Marcellini S
   7. Gomez-Skarmeta JL
   8. Bertrand S
   9. Escriva H

   (2019) Genetic regulation of amphioxus somitogenesis informs the evolution of the vertebrate head mesoderm

   Nature Ecology & Evolution 3:1233–1240.

   https://doi.org/10.1038/s41559-019-0933-z

   • PubMed
   • Google Scholar
4. Book

   1. Andrews EA

   (1893)

   An undescribed acraniate: Asymmetron Lucayanum

   In: Martin HN, Brooks WK, editors. Studies from the Biological Laboratory. Baltimore: The Johns Hopkins Press. pp. 213–247.

   • Google Scholar
5. 1. Azariah J

   (1968)

   Occurrence of a trypanorhynchan larva in amphioxus (Branchiostoma lanceolatum)

   Current Science 37:439–440.

   • Google Scholar
6. 1. Benito-Gutiérrez È
   2. Weber H
   3. Bryant DV
   4. Arendt D
   5. Escriva H

   (2013) Methods for generating year-round access to amphioxus in the laboratory

   PLOS ONE 8:e71599.

   https://doi.org/10.1371/journal.pone.0071599

   • Google Scholar
7. 1. Bertrand S
   2. Camasses A
   3. Somorjai I
   4. Belgacem MR
   5. Chabrol O
   6. Escande ML
   7. Pontarotti P
   8. Escriva H

   (2011) Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits

   PNAS 108:9160–9165.

   https://doi.org/10.1073/pnas.1014235108

   • PubMed
   • Google Scholar
8. 1. Bertrand S
   2. Escriva H

   (2011) Evolutionary crossroads in developmental biology: amphioxus

   Development 138:4819–4830.

   https://doi.org/10.1242/dev.066720

   • PubMed
   • Google Scholar
9. 1. Bertrand S
   2. Le Petillon Y
   3. Somorjai IML
   4. Escriva H

   (2017) Developmental cell-cell communication pathways in the cephalochordate amphioxus: actors and functions

   The International Journal of Developmental Biology 61:697–722.

   https://doi.org/10.1387/ijdb.170202sb

   • PubMed
   • Google Scholar
10. 1. Bertrand S
    2. Carvalho JE
    3. Dauga D
    4. Matentzoglu N
    5. Daric V
    6. Yu JK
    7. Schubert M
    8. Escrivá H

    (2021) The ontology of the amphioxus anatomy and life cycle

    Frontiers in Cell and Developmental Biology 9:668025.

    https://doi.org/10.3389/fcell.2021.668025

    • PubMed
    • Google Scholar
11. 1. Bone Q

    (1957) The problem of the ‘amphioxides’ larva

    Nature 180:1462–1464.

    https://doi.org/10.1038/1801462a0

    • PubMed
    • Google Scholar
12. 1. Boschung H
    2. Gunter G

    (1962)

    Distribution and variation of Branchiostoma caribaeum in Mississippi Sound

    Tulane Studies of Zoology 9:245–247.

    • Google Scholar
13. 1. Bourlat SJ
    2. Juliusdottir T
    3. Lowe CJ
    4. Freeman R
    5. Aronowicz J
    6. Kirschner M
    7. Lander ES
    8. Thorndyke M
    9. Nakano H
    10. Kohn AB
    11. Heyland A
    12. Moroz LL
    13. Copley RR
    14. Telford MJ

    (2006) Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida

    Nature 444:85–88.

    https://doi.org/10.1038/nature05241

    • PubMed
    • Google Scholar
14. 1. Brasó-Vives M
    2. Marlétaz F
    3. Echchiki A
    4. Mantica F
    5. Acemel RD
    6. Gómez-Skarmeta JL
    7. Hartasánchez DA
    8. Le Targa L
    9. Pontarotti P
    10. Tena JJ
    11. Maeso I
    12. Escriva H
    13. Irimia M
    14. Robinson-Rechavi M

    (2022) Parallel evolution of amphioxus and vertebrate small-scale gene duplications

    Genome Biology 23:243.

    https://doi.org/10.1186/s13059-022-02808-6

    • Google Scholar
15. 1. Caccavale F
    2. Annona G
    3. Subirana L
    4. Escriva H
    5. Bertrand S
    6. D’Aniello S

    (2021a) Crosstalk between nitric oxide and retinoic acid pathways is essential for amphioxus pharynx development

    eLife 10:e58295.

    https://doi.org/10.7554/eLife.58295

    • PubMed
    • Google Scholar
16. 1. Caccavale F
    2. Osca D
    3. D’Aniello S
    4. Crocetta F

    (2021b) Molecular taxonomy confirms that the northeastern Atlantic and Mediterranean Sea harbor a single lancelet, Branchiostoma lanceolatum (Pallas, 1774) (Cephalochordata: Leptocardii: Branchiostomatidae)

    PLOS ONE 16:e0251358.

    https://doi.org/10.1371/journal.pone.0251358

    • PubMed
    • Google Scholar
17. 1. Carvalho JE
    2. Lahaye F
    3. Schubert M

    (2017) Keeping amphioxus in the laboratory: an update on available husbandry methods

    The International Journal of Developmental Biology 61:773–783.

    https://doi.org/10.1387/ijdb.170192ms

    • Google Scholar
18. 1. Carvalho JE
    2. Lahaye F
    3. Yong LW
    4. Croce JC
    5. Escrivá H
    6. Yu J-K
    7. Schubert M

    (2021) An updated staging system for cephalochordate development: One table suits them all

    Frontiers in Cell and Developmental Biology 9:668006.

    https://doi.org/10.3389/fcell.2021.668006

    • PubMed
    • Google Scholar
19. 1. Chen Y
    2. Shin PKS
    3. Cheung SG

    (2008) Growth, secondary production and gonad development of two co-existing amphioxus species (Branchiostoma belcheri and B. malayanum) in subtropical Hong Kong

    Journal of Experimental Marine Biology and Ecology 357:64–74.

    https://doi.org/10.1016/j.jembe.2007.12.028

    • Google Scholar
20. 1. Cheng J
    2. Meistertzheim A-L
    3. Leistenschneider D
    4. Philip L
    5. Jacquin J
    6. Escande M-L
    7. Barbe V
    8. Ter Halle A
    9. Chapron L
    10. Lartaud F
    11. Bertrand S
    12. Escriva H
    13. Ghiglione J-F

    (2023) Impacts of microplastics and the associated plastisphere on physiological, biochemical, genetic expression and gut microbiota of the filter-feeder amphioxus

    Environment International 172:107750.

    https://doi.org/10.1016/j.envint.2023.107750

    • PubMed
    • Google Scholar
21. 1. Chin T

    (1941)

    Studies on the biology of the Amoy amphioxus Branchiostoma belcheri Gray

    The Philip J Sci 75:369–424.

    • Google Scholar
22. 1. Cory RL
    2. Pierce EL

    (1967) Distribution and ecology of lancelets (order amphioxi) over the continental shelf of the southeastern United States

    Limnology and Oceanography 12:650–656.

    https://doi.org/10.4319/lo.1967.12.4.0650

    • Google Scholar
23. Book

    1. Costa OG

    (1834)

    Cenni Zoologici, Ossia Descrizione Somaria delle specie Nuove Di Animali Discoperti in diverse Contrade del Regno Nell'Anno 1834

    In: Costa OG, editors. Annuario Zoologico. Napoli: Tipografia di Azzolino e Comp. pp. 49–50.

    • Google Scholar
24. 1. Courtney WAM

    (1975)

    The temperature relationships and age-structure of North Sea and Mediterranean populations of Branchiostoma lanceolatum

    Symp. Zool. Soc. London 36:213–233.

    • Google Scholar
25. 1. Dehal P
    2. Satou Y
    3. Campbell RK
    4. Chapman J
    5. Degnan B
    6. De Tomaso A
    7. Davidson B
    8. Di Gregorio A
    9. Gelpke M
    10. Goodstein DM
    11. Harafuji N
    12. Hastings KEM
    13. Ho I
    14. Hotta K
    15. Huang W
    16. Kawashima T
    17. Lemaire P
    18. Martinez D
    19. Meinertzhagen IA
    20. Necula S
    21. Nonaka M
    22. Putnam N
    23. Rash S
    24. Saiga H
    25. Satake M
    26. Terry A
    27. Yamada L
    28. Wang HG
    29. Awazu S
    30. Azumi K
    31. Boore J
    32. Branno M
    33. Chin-Bow S
    34. DeSantis R
    35. Doyle S
    36. Francino P
    37. Keys DN
    38. Haga S
    39. Hayashi H
    40. Hino K
    41. Imai KS
    42. Inaba K
    43. Kano S
    44. Kobayashi K
    45. Kobayashi M
    46. Lee BI
    47. Makabe KW
    48. Manohar C
    49. Matassi G
    50. Medina M
    51. Mochizuki Y
    52. Mount S
    53. Morishita T
    54. Miura S
    55. Nakayama A
    56. Nishizaka S
    57. Nomoto H
    58. Ohta F
    59. Oishi K
    60. Rigoutsos I
    61. Sano M
    62. Sasaki A
    63. Sasakura Y
    64. Shoguchi E
    65. Shin-i T
    66. Spagnuolo A
    67. Stainier D
    68. Suzuki MM
    69. Tassy O
    70. Takatori N
    71. Tokuoka M
    72. Yagi K
    73. Yoshizaki F
    74. Wada S
    75. Zhang C
    76. Hyatt PD
    77. Larimer F
    78. Detter C
    79. Doggett N
    80. Glavina T
    81. Hawkins T
    82. Richardson P
    83. Lucas S
    84. Kohara Y
    85. Levine M
    86. Satoh N
    87. Rokhsar DS

    (2002) The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins

    Science 298:2157–2167.

    https://doi.org/10.1126/science.1080049

    • PubMed
    • Google Scholar
26. 1. Dehal P.
    2. Boore JL

    (2005) Two rounds of whole genome duplication in the ancestral vertebrate

    PLOS Biology 3:e314.

    https://doi.org/10.1371/journal.pbio.0030314

    • PubMed
    • Google Scholar
27. 1. Delsuc F
    2. Brinkmann H
    3. Chourrout D
    4. Philippe H

    (2006) Tunicates and not cephalochordates are the closest living relatives of vertebrates

    Nature 439:965–968.

    https://doi.org/10.1038/nature04336

    • PubMed
    • Google Scholar
28. 1. Delsuc F
    2. Tsagkogeorga G
    3. Lartillot N
    4. Philippe H

    (2008) Additional molecular support for the new chordate phylogeny

    Genesis 46:592–604.

    https://doi.org/10.1002/dvg.20450

    • PubMed
    • Google Scholar
29. 1. Dennell R

    (1950) Note on the feeding of Amphioxus (Branchiostoma bermudae)

    Proc. Roy. Soc. Edinb 64:229–234.

    https://doi.org/10.1017/S0080455X00000345

    • Google Scholar
30. 1. Desdevises Y
    2. Maillet V
    3. Fuentes M
    4. Escriva H

    (2011) A snapshot of the population structure of Branchiostoma lanceolatum in the Racou Beach, France, during its spawning season

    PLOS ONE 6:e18520.

    https://doi.org/10.1371/journal.pone.0018520

    • PubMed
    • Google Scholar
31. 1. Fuentes M
    2. Schubert M
    3. Dalfo D
    4. Candiani S
    5. Benito E
    6. Gardenyes J
    7. Godoy L
    8. Moret F
    9. Illas M
    10. Patten I
    11. Permanyer J
    12. Oliveri D
    13. Boeuf G
    14. Falcon J
    15. Pestarino M
    16. Fernandez JG
    17. Albalat R
    18. Laudet V
    19. Vernier P
    20. Escriva H

    (2004) Preliminary observations on the spawning conditions of the European amphioxus (Branchiostoma lanceolatum) in captivity

    Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 302:384–391.

    https://doi.org/10.1002/jez.b.20025

    • PubMed
    • Google Scholar
32. 1. Fuentes M
    2. Benito E
    3. Bertrand S
    4. Paris M
    5. Mignardot A
    6. Godoy L
    7. Jimenez-Delgado S
    8. Oliveri D
    9. Candiani S
    10. Hirsinger E
    11. D’Aniello S
    12. Pascual-Anaya J
    13. Maeso I
    14. Pestarino M
    15. Vernier P
    16. Nicolas J-F
    17. Schubert M
    18. Laudet V
    19. Geneviere AM
    20. Albalat R
    21. Garcia Fernandez J
    22. Holland ND
    23. Escriva H

    (2007) Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum)

    Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 308:484–493.

    https://doi.org/10.1002/jez.b.21179

    • PubMed
    • Google Scholar
33. 1. Futch CR
    2. Dwinell SE

    (1977)

    Nearshore marine ecology at Hutchinson Island, Florida: 1971–1974: IV. Lancelets and fishes

    Florida Marine Research Publications 24:1–23.

    • Google Scholar
34. 1. Gil-Gálvez A
    2. Jiménez-Gancedo S
    3. Pérez-Posada A
    4. Franke M
    5. Acemel RD
    6. Lin CY
    7. Chou C
    8. Su YH
    9. Yu JK
    10. Bertrand S
    11. Schubert M
    12. Escrivá H
    13. Tena JJ
    14. Gómez-Skarmeta JL

    (2022) Gain of gene regulatory network interconnectivity at the origin of vertebrates

    PNAS 119:e2114802119.

    https://doi.org/10.1073/pnas.2114802119

    • PubMed
    • Google Scholar
35. Book

    1. Goldschmidt R

    (1905)

    Amphioxides. Wiss. Ergebn. Der Deutschen Tiefsee-Expedition,’Valdivia’

    Biodiversity Library.

    • Google Scholar
36. 1. Gosselck F
    2. Kuehner E

    (1973) Investigations on the biology of Branchiostoma senegalense larvae off the northwest African coast

    Marine Biology 22:67–73.

    https://doi.org/10.1007/BF00388911

    • Google Scholar
37. 1. Gosselck F
    2. Kell V
    3. Spittler P

    (1978) On the feeding of Branchiostoma senegalense (Acrania: Branchiostomidae)

    Marine Biology 46:175–179.

    https://doi.org/10.1007/BF00391534

    • Google Scholar
38. 1. Gosselck F
    2. Spittler P

    (1979) Age structure, growth, and weight of Branchiostoma senegalense (acrania, branchiostomidae) off north-west africa

    Internationale Revue Der Gesamten Hydrobiologie Und Hydrographie 64:541–550.

    https://doi.org/10.1002/iroh.19790640418

    • Google Scholar
39. 1. Gray JE

    (1847) Description of a new species of amphioxus from Borneo

    Annals and Magazine of Natural History 19:463–464.

    https://doi.org/10.1080/037454809495995

    • Google Scholar
40. Report

    1. Günther A
    2. Reptilia B

    (1882)

    Pisces in Report on the Zoological Collections Made in the Indo-Pacific Ocean during the Voyage of HMS Alert 1881–1882

    London: British Museum.

    • Google Scholar
41. Book

    1. Haeckel E

    (1866) Generelle Morphologie Der Organismen

    Berlin: Reimer.

    https://doi.org/10.1515/9783110848281

    • Google Scholar
42. 1. Holland ND
    2. Holland LZ

    (1989) Fine Structural Study of the Cortical Reaction and Formation of the Egg Coats in a Lancelet (= Amphioxus), Branchiostoma floridae (Phylum Chordata: Subphylum Cephalochordata = Acrania)

    The Biological Bulletin 176:111–122.

    https://doi.org/10.2307/1541578

    • Google Scholar
43. 1. Holland PW
    2. Holland LZ
    3. Williams NA
    4. Holland ND

    (1992) An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution

    Development 116:653–661.

    https://doi.org/10.1242/dev.116.3.653

    • PubMed
    • Google Scholar
44. 1. Holland LZ
    2. Yu JK

    (2004) Cephalochordate (amphioxus) embryos: procurement, culture, and basic methods

    Methods in Cell Biology 74:195–215.

    https://doi.org/10.1016/s0091-679x(04)74009-1

    • PubMed
    • Google Scholar
45. 1. Holland ND
    2. Campbell TG
    3. Garey JR
    4. Holland LZ
    5. Wilson NG

    (2009) The Florida amphioxus (Cephalochordata) hosts larvae of the tapeworm Acanthobothrium brevissime: natural history, anatomy and taxonomic identification of the parasite

    Acta Zoologica 90:75–86.

    https://doi.org/10.1111/j.1463-6395.2008.00343.x

    • Google Scholar
46. 1. Holland ND
    2. Holland LZ

    (2010) Laboratory spawning and development of the Bahama lancelet, Asymmetron lucayanum (cephalochordata): fertilization through feeding larvae

    The Biological Bulletin 219:132–141.

    https://doi.org/10.1086/BBLv219n2p132

    • PubMed
    • Google Scholar
47. 1. Holland ND

    (2011) Spawning periodicity of the lancelet, Asymmetron lucayanum (Cephalochordata), in Bimini, Bahamas

    Italian Journal of Zoology 78:478–486.

    https://doi.org/10.1080/11250003.2011.594097

    • Google Scholar
48. Book

    1. Holland LZ
    2. Li G

    (2021) Laboratory culture and mutagenesis of Amphioxus (Branchiostoma Floridae)

    In: Carroll DJ, Stricker SA, editors. Developmental Biology of the Sea Urchin and Other Marine Invertebrates: Methods and Protocols. New York, NY: Humana Press. pp. 1–29.

    https://doi.org/10.1007/978-1-0716-0974-3

    • Google Scholar
49. 1. Horie R
    2. Hazbun A
    3. Chen K
    4. Cao C
    5. Levine M
    6. Horie T

    (2018) Shared evolutionary origin of vertebrate neural crest and cranial placodes

    Nature 560:228–232.

    https://doi.org/10.1038/s41586-018-0385-7

    • PubMed
    • Google Scholar
50. 1. Hu G
    2. Li G
    3. Wang H
    4. Wang Y

    (2017) Hedgehog participates in the establishment of left-right asymmetry during amphioxus development by controlling Cerberus expression

    Development 144:4694–4703.

    https://doi.org/10.1242/dev.157172

    • PubMed
    • Google Scholar
51. 1. Huang S
    2. Chen Z
    3. Yan X
    4. Yu T
    5. Huang G
    6. Yan Q
    7. Pontarotti PA
    8. Zhao H
    9. Li J
    10. Yang P
    11. Wang R
    12. Li R
    13. Tao X
    14. Deng T
    15. Wang Y
    16. Li G
    17. Zhang Q
    18. Zhou S
    19. You L
    20. Yuan S
    21. Fu Y
    22. Wu F
    23. Dong M
    24. Chen S
    25. Xu A

    (2014) Decelerated genome evolution in modern vertebrates revealed by analysis of multiple lancelet genomes

    Nature Communications 5:5896.

    https://doi.org/10.1038/ncomms6896

    • Google Scholar
52. 1. Huang Z
    2. Xu L
    3. Cai C
    4. Zhou Y
    5. Liu J
    6. Xu Z
    7. Zhu Z
    8. Kang W
    9. Cen W
    10. Pei S
    11. Chen D
    12. Shi C
    13. Wu X
    14. Huang Y
    15. Xu C
    16. Yan Y
    17. Yang Y
    18. Xue T
    19. He W
    20. Hu X
    21. Zhang Y
    22. Chen Y
    23. Bi C
    24. He C
    25. Xue L
    26. Xiao S
    27. Yue Z
    28. Jiang Y
    29. Yu JK
    30. Jarvis ED
    31. Li G
    32. Lin G
    33. Zhang Q
    34. Zhou Q

    (2023) Three amphioxus reference genomes reveal gene and chromosome evolution of chordates

    PNAS 120:e2201504120.

    https://doi.org/10.1073/pnas.2201504120

    • PubMed
    • Google Scholar
53. 1. Igawa T
    2. Nozawa M
    3. Suzuki DG
    4. Reimer JD
    5. Morov AR
    6. Wang Y
    7. Henmi Y
    8. Yasui K

    (2017) Evolutionary history of the extant amphioxus lineage with shallow-branching diversification

    Scientific Reports 7:1157.

    https://doi.org/10.1038/s41598-017-00786-5

    • PubMed
    • Google Scholar
54. 1. Kon T
    2. Nohara M
    3. Nishida M
    4. Sterrer W
    5. Nishikawa T

    (2006) Hidden ancient diversification in the circumtropical lancelet Asymmetron lucayanum complex

    Marine Biology 149:875–883.

    https://doi.org/10.1007/s00227-006-0271-y

    • Google Scholar
55. 1. Kozmikova I
    2. Kozmik Z

    (2015) Gene regulation in amphioxus: An insight from transgenic studies in amphioxus and vertebrates

    Marine Genomics 24 Pt 2:159–166.

    https://doi.org/10.1016/j.margen.2015.06.003

    • PubMed
    • Google Scholar
56. 1. Le Petillon Y
    2. Luxardi G
    3. Scerbo P
    4. Cibois M
    5. Leon A
    6. Subirana L
    7. Irimia M
    8. Kodjabachian L
    9. Escriva H
    10. Bertrand S

    (2017) Nodal/activin pathway is a conserved neural induction signal in chordates

    Nature Ecology & Evolution 1:1192–1200.

    https://doi.org/10.1038/s41559-017-0226-3

    • PubMed
    • Google Scholar
57. 1. Le Petillon Y
    2. Bertrand S
    3. Escrivà H

    (2020) Spawning induction and embryo micromanipulation protocols in the Amphioxus Branchiostoma lanceolatum

    Methods in Molecular Biology 2047:347–359.

    https://doi.org/10.1007/978-1-4939-9732-9_19

    • PubMed
    • Google Scholar
58. 1. Li G
    2. Yang X
    3. Shu Z
    4. Chen X
    5. Wang Y

    (2012) Consecutive spawnings of Chinese amphioxus, Branchiostoma belcheri, in captivity

    PLOS ONE 7:e50838.

    https://doi.org/10.1371/journal.pone.0050838

    • PubMed
    • Google Scholar
59. 1. Li G.
    2. Shu Z
    3. Wang Y

    (2013) Year-round reproduction and induced spawning of Chinese amphioxus, Branchiostoma belcheri, in laboratory

    PLOS ONE 8:e75461.

    https://doi.org/10.1371/journal.pone.0075461

    • PubMed
    • Google Scholar
60. 1. Li G
    2. Feng J
    3. Lei Y
    4. Wang J
    5. Wang H
    6. Shang L-K
    7. Liu D-T
    8. Zhao H
    9. Zhu Y
    10. Wang Y-Q

    (2014) Mutagenesis at specific genomic loci of amphioxus Branchiostoma belcheri using TALEN method

    Journal of Genetics and Genomics 41:215–219.

    https://doi.org/10.1016/j.jgg.2014.02.003

    • PubMed
    • Google Scholar
61. 1. Li G
    2. Wang J
    3. Yuan L
    4. Wang H
    5. Wang Y-Q

    (2015) A simple method for selecting spawning-ready individuals out from laboratorial cultured amphioxus population

    Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 324:629–635.

    https://doi.org/10.1002/jez.b.22640

    • PubMed
    • Google Scholar
62. 1. Li G
    2. Liu X
    3. Xing C
    4. Zhang H
    5. Shimeld SM
    6. Wang Y

    (2017) Cerberus-Nodal-Lefty-Pitx signaling cascade controls left-right asymmetry in amphioxus

    PNAS 114:3684–3689.

    https://doi.org/10.1073/pnas.1620519114

    • PubMed
    • Google Scholar
63. 1. Lin C-Y
    2. Lu M-YJ
    3. Yue J-X
    4. Li K-L
    5. Le Pétillon Y
    6. Yong LW
    7. Chen Y-H
    8. Tsai F-Y
    9. Lyu Y-F
    10. Chen C-Y
    11. Hwang S-PL
    12. Su Y-H
    13. Yu J-K
    14. Mullins MC

    (2020) Molecular asymmetry in the cephalochordate embryo revealed by single-blastomere transcriptome profiling

    PLOS Genetics 16:e1009294.

    https://doi.org/10.1371/journal.pgen.1009294

    • Google Scholar
64. 1. Ma P
    2. Liu X
    3. Xu Z
    4. Liu H
    5. Ding X
    6. Huang Z
    7. Shi C
    8. Liang L
    9. Xu L
    10. Li X
    11. Li G
    12. He Y
    13. Ding Z
    14. Chai C
    15. Wang H
    16. Qiu J
    17. Zhu J
    18. Wang X
    19. Ding P
    20. Zhou S
    21. Yuan Y
    22. Wu W
    23. Wan C
    24. Yan Y
    25. Zhou Y
    26. Zhou Q-J
    27. Wang G-D
    28. Zhang Q
    29. Xu X
    30. Li G
    31. Zhang S
    32. Mao B
    33. Chen D

    (2022) Joint profiling of gene expression and chromatin accessibility during amphioxus development at single-cell resolution

    Cell Reports 39:110979.

    https://doi.org/10.1016/j.celrep.2022.110979

    • Google Scholar
65. 1. Mallatt J
    2. Chen J

    (2003) Fossil sister group of craniates: predicted and found

    Journal of Morphology 258:1–31.

    https://doi.org/10.1002/jmor.10081

    • PubMed
    • Google Scholar
66. 1. Mallatt J
    2. Holland N

    (2013) Pikaia gracilens Walcott: stem chordate, or already specialized in the Cambrian?

    Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 320:247–271.

    https://doi.org/10.1002/jez.b.22500

    • PubMed
    • Google Scholar
67. 1. Manni L
    2. Lane NJ
    3. Joly JS
    4. Gasparini F
    5. Tiozzo S
    6. Caicci F
    7. Zaniolo G
    8. Burighel P

    (2004) Neurogenic and non-neurogenic placodes in ascidians

    Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 302:483–504.

    https://doi.org/10.1002/jez.b.21013

    • PubMed
    • Google Scholar
68. 1. Marlétaz F
    2. Firbas PN
    3. Maeso I
    4. Tena JJ
    5. Bogdanovic O
    6. Perry M
    7. Wyatt CDR
    8. de la Calle-Mustienes E
    9. Bertrand S
    10. Burguera D
    11. Acemel RD
    12. van Heeringen SJ
    13. Naranjo S
    14. Herrera-Ubeda C
    15. Skvortsova K
    16. Jimenez-Gancedo S
    17. Aldea D
    18. Marquez Y
    19. Buono L
    20. Kozmikova I
    21. Permanyer J
    22. Louis A
    23. Albuixech-Crespo B
    24. Le Petillon Y
    25. Leon A
    26. Subirana L
    27. Balwierz PJ
    28. Duckett PE
    29. Farahani E
    30. Aury J-M
    31. Mangenot S
    32. Wincker P
    33. Albalat R
    34. Benito-Gutiérrez È
    35. Cañestro C
    36. Castro F
    37. D’Aniello S
    38. Ferrier DEK
    39. Huang S
    40. Laudet V
    41. Marais GAB
    42. Pontarotti P
    43. Schubert M
    44. Seitz H
    45. Somorjai I
    46. Takahashi T
    47. Mirabeau O
    48. Xu A
    49. Yu J-K
    50. Carninci P
    51. Martinez-Morales JR
    52. Crollius HR
    53. Kozmik Z
    54. Weirauch MT
    55. Garcia-Fernàndez J
    56. Lister R
    57. Lenhard B
    58. Holland PWH
    59. Escriva H
    60. Gómez-Skarmeta JL
    61. Irimia M

    (2018) Amphioxus functional genomics and the origins of vertebrate gene regulation

    Nature 564:64–70.

    https://doi.org/10.1038/s41586-018-0734-6

    • PubMed
    • Google Scholar
69. 1. McEwan IJ

    (2001) Bakers yeast rises to the challenge: reconstitution of mammalian steroid receptor signalling in S. cerevisiae

    Trends in Genetics 17:239–243.

    https://doi.org/10.1016/s0168-9525(01)02273-9

    • PubMed
    • Google Scholar
70. 1. Meister L
    2. Escriva H
    3. Bertrand S

    (2022) Functions of the FGF signalling pathway in cephalochordates provide insight into the evolution of the prechordal plate

    Development 149:dev200252.

    https://doi.org/10.1242/dev.200252

    • PubMed
    • Google Scholar
71. 1. Morris SC
    2. Caron JB

    (2012) Pikaia gracilens Walcott, a stem-group chordate from the Middle Cambrian of British Columbia

    Biological Reviews of the Cambridge Philosophical Society 87:480–512.

    https://doi.org/10.1111/j.1469-185X.2012.00220.x

    • PubMed
    • Google Scholar
72. 1. Nelson GE

    (1968)

    Amphioxus in old Tampa Bay, Florida

    Quarterly Journal of the Florida Academy of Sciences 31:93–100.

    • Google Scholar
73. 1. Nishikawa T

    (2004) A new deep-water lancelet (Cephalochordata) from off Cape Nomamisaki, SW Japan, with a proposal of the revised system recovering the genus Asymmetron

    Zoological Science 21:1131–1136.

    https://doi.org/10.2108/zsj.21.1131

    • PubMed
    • Google Scholar
74. 1. Nohara M
    2. Nishida M
    3. Miya M
    4. Nishikawa T

    (2005) Evolution of the mitochondrial genome in cephalochordata as inferred from complete nucleotide sequences from two Epigonichthys species

    Journal of Molecular Evolution 60:526–537.

    https://doi.org/10.1007/s00239-004-0238-x

    • PubMed
    • Google Scholar
75. Book

    1. Ohno S

    (1970) Evolution by Gene Duplication

    Berlin, Heidelberg: Springer-Verlag.

    https://doi.org/10.1007/978-3-642-86659-3

    • Google Scholar
76. 1. Onai T
    2. Yu J-K
    3. Blitz IL
    4. Cho KWY
    5. Holland LZ

    (2010) Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus

    Developmental Biology 344:377–389.

    https://doi.org/10.1016/j.ydbio.2010.05.016

    • PubMed
    • Google Scholar
77. 1. Orton JH

    (1914) On a hermaphrodite specimen of Amphioxus with notes on experiments in rearing amphioxus

    Journal of the Marine Biological Association of the United Kingdom 10:506–512.

    https://doi.org/10.1017/S0025315400008262

    • Google Scholar
78. Book

    1. Pallas PS
    2. Richmond CW
    3. Tucker MB

    (1774) Spicilegia Zoologica: Quibus Novae Imprimis et Obscurae Animalium Species Iconibus, Descriptionibus Atque Commentariis Illustrantur. Fasciculus Decimus

    Lange: Berolini.

    https://doi.org/10.5962/bhl.title.39832

    • Google Scholar
79. 1. Paris M
    2. Escriva H
    3. Schubert M
    4. Brunet F
    5. Brtko J
    6. Ciesielski F
    7. Roecklin D
    8. Vivat-Hannah V
    9. Jamin EL
    10. Cravedi J-P
    11. Scanlan TS
    12. Renaud J-P
    13. Holland ND
    14. Laudet V

    (2008) Amphioxus postembryonic development reveals the homology of chordate metamorphosis

    Current Biology 18:825–830.

    https://doi.org/10.1016/j.cub.2008.04.078

    • PubMed
    • Google Scholar
80. 1. Peters WCH

    (1876)

    Über Epigonichthys cultellus eine neue Gattung und Art der Leptocardii

    Ber. Akad. Wiss. Berlin 1875:322–326.

    • Google Scholar
81. 1. Piyakarnchana T
    2. Vajropala K

    (1961)

    Some ecological factors that limit the distribution of three species of lancelets in the Gulf of Thailand

    J. Nat. Res. Council Thailand 2:1–6.

    • Google Scholar
82. 1. Poss SG
    2. Boschung HT

    (1996)

    Lancelets (Cephalochordata: Branchiostomatidae): How many species are valid

    Israel J Zool 42:S13–S66.

    • Google Scholar
83. 1. Putnam NH
    2. Butts T
    3. Ferrier DEK
    4. Furlong RF
    5. Hellsten U
    6. Kawashima T
    7. Robinson-Rechavi M
    8. Shoguchi E
    9. Terry A
    10. Yu J-K
    11. Benito-Gutiérrez E
    12. Dubchak I
    13. Garcia-Fernàndez J
    14. Gibson-Brown JJ
    15. Grigoriev IV
    16. Horton AC
    17. de Jong PJ
    18. Jurka J
    19. Kapitonov VV
    20. Kohara Y
    21. Kuroki Y
    22. Lindquist E
    23. Lucas S
    24. Osoegawa K
    25. Pennacchio LA
    26. Salamov AA
    27. Satou Y
    28. Sauka-Spengler T
    29. Schmutz J
    30. Shin-I T
    31. Toyoda A
    32. Bronner-Fraser M
    33. Fujiyama A
    34. Holland LZ
    35. Holland PWH
    36. Satoh N
    37. Rokhsar DS

    (2008) The amphioxus genome and the evolution of the chordate karyotype

    Nature 453:1064–1071.

    https://doi.org/10.1038/nature06967

    • Google Scholar
84. Conference

    1. Ravitch-Stcherbo J

    (1936)

    De l’origine bactérienne du pigment rouge de l’Amphioxus lanceolatum, cause de sa mort et destruction Trudy Sevastopol Biol Stan Akad Nauk SSSR

    SSSR. pp. 287–296.

    • Google Scholar
85. 1. Ren Q
    2. Zhong Y
    3. Huang X
    4. Leung B
    5. Xing C
    6. Wang H
    7. Hu G
    8. Wang Y
    9. Shimeld SM
    10. Li G

    (2020) Step-wise evolution of neural patterning by Hedgehog signalling in chordates

    Nature Ecology & Evolution 4:1247–1255.

    https://doi.org/10.1038/s41559-020-1248-9

    • PubMed
    • Google Scholar
86. 1. Richardson BJ
    2. McKenzie AM

    (1994) Taxonomy and distribution of Australian Cephalochordates (Chordata : Cephalochordata)

    Invertebrate Systematics 8:1443.

    https://doi.org/10.1071/IT9941443

    • Google Scholar
87. 1. Riisgard HU
    2. Svane I

    (1999) Filter feeding in lancelets (amphioxus), Branchiostoma lanceolatum

    Invertebrate Biology 118:423.

    https://doi.org/10.2307/3227011

    • Google Scholar
88. 1. Ruppert EE
    2. Nash TR
    3. Smith AJ

    (2000) The size range of suspended particles trapped and ingested by the filter-feeding lancelet Branchiostoma floridae (Cephalochordata: Acrania)

    Journal of the Marine Biological Association of the United Kingdom 80:329–332.

    https://doi.org/10.1017/S0025315499001903

    • Google Scholar
89. 1. Satoh N
    2. Tominaga H
    3. Kiyomoto M
    4. Hisata K
    5. Inoue J
    6. Nishitsuji K

    (2021) A preliminary single-cell RNA-seq analysis of embryonic cells that express Brachyury in the Amphioxus, Branchiostoma japonicum

    Frontiers in Cell and Developmental Biology 9:696875.

    https://doi.org/10.3389/fcell.2021.696875

    • PubMed
    • Google Scholar
90. 1. Schubert M
    2. Yu J-K
    3. Holland ND
    4. Escriva H
    5. Laudet V
    6. Holland LZ

    (2005) Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus

    Development 132:61–73.

    https://doi.org/10.1242/dev.01554

    • PubMed
    • Google Scholar
91. 1. Shu D-G
    2. Morris SC
    3. Han J
    4. Zhang Z-F
    5. Yasui K
    6. Janvier P
    7. Chen L
    8. Zhang X-L
    9. Liu J-N
    10. Li Y
    11. Liu H-Q

    (2003a) Head and backbone of the Early Cambrian vertebrate Haikouichthys

    Nature 421:526–529.

    https://doi.org/10.1038/nature01264

    • PubMed
    • Google Scholar
92. 1. Shu D
    2. Morris SC
    3. Zhang ZF
    4. Liu JN
    5. Han J
    6. Chen L
    7. Zhang XL
    8. Yasui K
    9. Li Y

    (2003b) A new species of yunnanozoan with implications for deuterostome evolution

    Science 299:1380–1384.

    https://doi.org/10.1126/science.1079846

    • PubMed
    • Google Scholar
93. 1. Simakov O
    2. Marlétaz F
    3. Yue J-X
    4. O’Connell B
    5. Jenkins J
    6. Brandt A
    7. Calef R
    8. Tung C-H
    9. Huang T-K
    10. Schmutz J
    11. Satoh N
    12. Yu J-K
    13. Putnam NH
    14. Green RE
    15. Rokhsar DS

    (2020) Deeply conserved synteny resolves early events in vertebrate evolution

    Nature Ecology & Evolution 4:820–830.

    https://doi.org/10.1038/s41559-020-1156-z

    • Google Scholar
94. 1. Somorjai IML
    2. Camasses A
    3. Rivière B
    4. Escrivà H

    (2008) Development of a semi-closed aquaculture system for monitoring of individual amphioxus (Branchiostoma lanceolatum), with high survivorship

    Aquaculture 281:145–150.

    https://doi.org/10.1016/j.aquaculture.2008.05.023

    • Google Scholar
95. 1. Stokes MD
    2. Holland ND

    (1992) Southern stingray (Dasyatis americana) feeding on lancelets (Branchiostoma floridae)

    Journal of Fish Biology 41:1043–1044.

    https://doi.org/10.1111/j.1095-8649.1992.tb02732.x

    • Google Scholar
96. 1. Stokes MD

    (1996) Larval settlement, post-settlement growth and secondary production of the Florida lancelet (= amphioxus) Branchiostoma floridae

    Marine Ecology Progress Series 130:71–84.

    https://doi.org/10.3354/meps130071

    • Google Scholar
97. 1. Stokes MD
    2. Holland ND

    (1996a)

    Life-history characteristics of the Florida lancelet, Branchiostoma floridae: some factors affecting population dynamics in Tampa Bay

    Israel Journal of Zoology 42:67–86.

    • Google Scholar
98. 1. Stokes MD
    2. Holland ND

    (1996b) Reproduction of the Florida lancelet (Branchiostoma floridae): Spawning patterns and fluctuations in gonad indexes and nutritional reserves

    Invertebrate Biology 115:349.

    https://doi.org/10.2307/3227024

    • Google Scholar
99. 1. Stolk A

    (1961) Two types of ribonucleoprotein in the nucleolus of intestinal carcinoma of the newt following injection of herring-sperm deoxyribonucleic acid

    Nature 192:1215–1216.

    https://doi.org/10.1038/1921215a0

    • PubMed
    • Google Scholar
100. 1. Su L
     2. Shi C
     3. Huang X
     4. Wang Y
     5. Li G

     (2020) Application of CRISPR/Cas9 nuclease in Amphioxus genome editing

     Genes 11:1311.

     https://doi.org/10.3390/genes11111311

     • PubMed
     • Google Scholar
101. 1. Subirana L
     2. Farstey V
     3. Bertrand S
     4. Escriva H

     (2020) Asymmetron lucayanum: How many species are valid?

     PLOS ONE 15:e0229119.

     https://doi.org/10.1371/journal.pone.0229119

     • PubMed
     • Google Scholar
102. 1. Sundevall C

     (1852)

     Ny art af Amphioxus

     Öfversigt Af Kongl. Vetenskaps-Akademiens Forhandlinger 9:147–148.

     • Google Scholar
103. 1. Sundevall CJ

     (1853)

     Ny art af Branchiostoma (Amphioxus caribaeum)

     Öfversigt Af Kongl. Vetenskaps-Akademiens Forhandlinger 10:11–13.

     • Google Scholar
104. 1. Theodosiou M
     2. Colin A
     3. Schulz J
     4. Laudet V
     5. Peyrieras N
     6. Nicolas J-F
     7. Schubert M
     8. Hirsinger E

     (2011) Amphioxus spawning behavior in an artificial seawater facility

     Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 316:263–275.

     https://doi.org/10.1002/jez.b.21397

     • PubMed
     • Google Scholar
105. 1. Wang Y

     (2004)

     Taxonomic status of amphioxus Branchiostoma belcheri in Xiamen Beach estimated by homologous sequence of Cyt b gene

     Acta Zool Sinica 50:60–66.

     • Google Scholar
106. 1. Webb JE

     (1958) The ecology of Lagos Lagoon V. Some physical properties of lagoon deposits

     Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 241:393–419.

     https://doi.org/10.1098/rstb.1958.0009

     • Google Scholar
107. 1. Webb JE
     2. Hill MB

     (1958) The ecology of Lagos lagoon. IV. On the reactions of Branchiostoma nigeriense Webb to its environment

     Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 241:355–391.

     https://doi.org/10.1098/rstb.1958.0008

     • Google Scholar
108. 1. Webb JE

     (1969) On the feeding and behaviour of the larva of Branchiostoma lanceolatum

     Marine Biology 3:58–72.

     https://doi.org/10.1007/BF00355593

     • Google Scholar
109. 1. Webb JE

     (1975)

     The distribution of amphioxus

     Symp. Zool. Soc. Lond 36:179–212.

     • Google Scholar
110. 1. Wells MM

     (1926) Collecting amphioxus

     Science 64:187–188.

     https://doi.org/10.1126/science.64.1651.187

     • PubMed
     • Google Scholar
111. Book

     1. Wickstead J

     (1975)

     Chordata: Acrania (Cephalochordata)

     In: Giese AC, Pearse JS, editors. Reproduction of Marine Invetebrates. Vol. II. Entroprocta and Lesser Coelomates. New York: Academic Press. pp. 283–319.

     • Google Scholar
112. 1. Williams JB

     (1996) Sessile lifestyle and origin of chordates

     New Zealand Journal of Zoology 23:111–133.

     https://doi.org/10.1080/03014223.1996.9518072

     • Google Scholar
113. 1. Winchell CJ
     2. Sullivan J
     3. Cameron CB
     4. Swalla BJ
     5. Mallatt J

     (2002) Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data

     Molecular Biology and Evolution 19:762–776.

     https://doi.org/10.1093/oxfordjournals.molbev.a004134

     • PubMed
     • Google Scholar
114. 1. Xiang K
     2. He Z
     3. Fu J
     4. Wang G
     5. Li H
     6. Zhang Y
     7. Zhang S
     8. Chen L

     (2022) Microplastics exposure as an emerging threat to ancient lineage: A contaminant of concern for abnormal bending of amphioxus via neurotoxicity

     Journal of Hazardous Materials 438:129454.

     https://doi.org/10.1016/j.jhazmat.2022.129454

     • PubMed
     • Google Scholar
115. 1. Xu QS
     2. Ma F
     3. Wang YQ

     (2005) Morphological and 12S rRNA gene comparison of two Branchiostoma species in Xiamen waters

     Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 304:259–267.

     https://doi.org/10.1002/jez.b.21036

     • PubMed
     • Google Scholar
116. 1. Yamaguchi T
     2. Henmi Y

     (2003) Biology of the amphioxus, Branchiostoma belcheri in the Ariake Sea, Japan. II. Reproduction

     Zoological Science 20:907–918.

     https://doi.org/10.2108/zsj.20.907

     • PubMed
     • Google Scholar
117. Book

     1. Yarrell W

     (1836)

     A History of British Fishes

     London: John van Voors.

     • Google Scholar
118. 1. Yasui K
     2. Urata M
     3. Yamaguchi N
     4. Ueda H
     5. Henmi Y

     (2007) Laboratory culture of the Oriental lancelet Branchiostoma belcheri

     Zoological Science 24:514–520.

     https://doi.org/10.2108/zsj.24.514

     • PubMed
     • Google Scholar
119. 1. Yu J-K
     2. Satou Y
     3. Holland ND
     4. Shin-I T
     5. Kohara Y
     6. Satoh N
     7. Bronner-Fraser M
     8. Holland LZ

     (2007) Axial patterning in cephalochordates and the evolution of the organizer

     Nature 445:613–617.

     https://doi.org/10.1038/nature05472

     • PubMed
     • Google Scholar
120. 1. Zhang S
     2. Li G
     3. Zhu J
     4. Su F

     (2001) Sex reversal of the female amphioxus Branchiostoma belcheri tsingtauense reared in the laboratory

     Journal of the Marine Biological Association of the United Kingdom 81:181–182.

     https://doi.org/10.1017/S0025315401003599

     • Google Scholar
121. 1. Zhang Q-J
     2. Zhong J
     3. Fang S-H
     4. Wang Y-Q

     (2006) Branchiostoma japonicum and B. belcheri are distinct lancelets (Cephalochordata) in Xiamen waters in China

     Zoological Science 23:573–579.

     https://doi.org/10.2108/zsj.23.573

     • PubMed
     • Google Scholar
122. 1. Zhong Y
     2. Herrera-Úbeda C
     3. Garcia-Fernàndez J
     4. Li G
     5. Holland PWH

     (2020) Mutation of amphioxus Pdx and Cdx demonstrates conserved roles for ParaHox genes in gut, anus and tail patterning

     BMC Biology 18:68.

     https://doi.org/10.1186/s12915-020-00796-2

     • PubMed
     • Google Scholar
123. 1. Zhu X
     2. Shi C
     3. Zhong Y
     4. Liu X
     5. Yan Q
     6. Wu X
     7. Wang Y
     8. Li G

     (2020) Cilia-driven asymmetric Hedgehog signalling determines the amphioxus left-right axis by controlling Dand5 expression

     Development 147:dev182469.

     https://doi.org/10.1242/dev.182469

     • PubMed
     • Google Scholar
124. 1. Zou Y
     2. Ma C
     3. Zhang Y
     4. Du Z
     5. You F
     6. Tan X
     7. Zhang P-J

     (2016) Isolation and characterization of Vibrio alginolyticus from cultured amphioxus Branchiostoma belcheri tsingtauense

     Biologia 71:757–762.

     https://doi.org/10.1515/biolog-2016-0102

     • Google Scholar
125. 1. Zou J
     2. Wu X
     3. Shi C
     4. Zhong Y
     5. Zhang L
     6. Yan Q
     7. Su L
     8. Li G

     (2021) A potential method for rapid screening of amphioxus founder harboring germline mutation and transgene

     Frontiers in Cell and Developmental Biology 9:702290.

     https://doi.org/10.3389/fcell.2021.702290

     • PubMed
     • Google Scholar

Decision letter after peer review:

Thank you for submitting your article "The cephalochordate amphioxus as a model to study vertebrate development and evolution" to eLife for consideration as a Feature Article. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a member of the eLife Features Team (Peter Rodgers). The following individuals involved in review of your submission have agreed to reveal their identity: JK Sky Yu, Tokiharu Takahashi, and Filipe Castro.

The reviewers and editors have discussed the reviews and we have drafted this decision letter to help you prepare a revised submission.

Summary:

This manuscript provides a valuable review of the scientific history, basic biology, and recent research on cephalochordates, focusing on the contributions of amphioxus as a model for comparative genomics and evolutionary developmental biology. Recent technological advances have taken amphioxus research to the next level, and this review will undoubtedly be valuable to those seeking background knowledge on amphioxus. The key outstanding questions regarding amphioxus biology are also well organized and provide an excellent research framework for coming years. However, there are a number of points that need to be addressed to make the article suitable for publication.

Essential revisions:

1) Line 83-84; a recent paper (Huang et al. 2023 PNAS 120 e2201504120) has shown that the amphioxus Hox cluster is also divided into two TAD domains from gastrula stage, similar to that in vertebrates. Thus, the 3-D chromatin architecture of amphioxus genome may not be much simpler than that of vertebrates.

2) Line 98-100. Here the authors only cite Delsuc et al. 2006 Nature paper in this sentence; however, in this paper the phylogenetic analysis did not recover the monophyletic chordate clade and grouped cephalochordates with echinoderms. A later paper by Bourlat et al., (Nature 444:85-88, 2006) corrected this problem by including more data from invertebrate deuterostome animals for phylogenetic analyses, and further support the Olfactores (tunicates + vertebrates) hypothesis. Subsequently Dulsuc et al. also published another paper (genesis, 46:592-604, 2008) with broader sampling to correct their previous analysis, and they also recovered the monophyletic chordate group and further support cephalochordates as the most basal lineage while tunicates and vertebrates are sister groups. I would suggest the authors to also cite these later publications for completeness of information.

3) Line 102-104; I would suggest the authors to include some appropriate references here about those traits that are shared between tunicates and vertebrates but absent in cephalochordates. About MHB evolution, I think it is still a contentious issue. There are expression data suggesting a MHB-like region exists in amphioxus CNS (Castro 2006 Dev Biol 295:40-51; Albuixech-Crespo 2017 PLoS Biol 15: e2001573), but whether it has secondary organizer function remains unknown. In addition, a MHB-like gene regulatory program appears to be present in hemichordate S. kowalevskii (Pani 2012 Nature 483:289-2924), suggesting a conserved gene module for AP patterning of the embryonic ectoderm was already evolved in the deuterostome common ancestor.

4) Line 163-164; the last part of this sentence is incorrect. Both Asymmetron and Epigonichthys have gonads only on the right side of the animals, whereas Branchiostoma has one row of gonads on each side of the body (see references such as: Igawa 2017 Sci Rep 7: 1157; Holland and Holland 2022 Curr Topics Dev Bio 147:563-594).

5) Line 318-320; The paper in PNAS by Huang et al. mentioned above has further increased the number of chromosome-level genome assemblies to four amphioxus species, as well as Hi-C data from B. floridae, B. japonicum, and B. belcheri. The authors should include these new results into this review.

In addition, there are quite a few valuable bulk RNA-seq transcriptome datasets from several Branchiostoma and Asymmetron species, which have contributed extensively to many comparative analyses and phylogenomics studies. Again, the authors should consider mention this work.

6) Lines 286-323 ("Technical advances in amphioxus research")

In this section, the authors first present a chronological overview of amphioxus husbandry and spawning induction techniques. They then summarise the technical advances in functional experiments, most of which have been made in recent years. However, they overlook the descriptive studies, which, although they may appear old-fashioned, have undoubtedly contributed to and still play an important role in amphioxus research. In order to give the readers a context for understanding the whole picture of modern amphioxus studies, it would be helpful to include a short paragraph discussing the introduction of molecular biology (mainly in situ hybridisation) to amphioxus research, citing some representative seminal papers such as Holland PW et al. (Development 116:653-61), which started the amphioxus EvoDevo field.

7) In addition, for transgenic amphioxus lines (line 315), it is necessary to include the Tol2 assay (TALEN and CRISPR are for knock out). More detailed information on TALEN and Tol2 techniques can be found in Chapter1 "Laboratory Culture and Mutagenesis of Amphioxus" by Linda Holland and Guang Li in Developmental Biology of the Sea Urchin and Other Marine Invertebrates (pp1-9, Humana Press), so citing this reference may be useful. The same applies to Su L et al. Genes 2020, 11, 1311; doi:10.3390/genes11111311 for the CRISPR technique.

8) Lines 337-369 ("Amphioxus as a model to understand vertebrate evolution")

This section is shorter than expected and would benefit from a more in-depth description of recent results and their impact on our understanding of vertebrate evolution (including, possibly, one or two extra figures).

9) Please consider adding a figure which shows the global distribution of the species discussed in the article.

10) The reviewers also felt that the title needed attention and the editor will discuss a new title with you if the revised article is accepted for publication.

Essential revisions:

1) Line 83-84; a recent paper (Huang et al. 2023 PNAS 120 e2201504120) has shown that the amphioxus Hox cluster is also divided into two TAD domains from gastrula stage, similar to that in vertebrates. Thus, the 3-D chromatin architecture of amphioxus genome may not be much simpler than that of vertebrates.

We appreciate this comment from the reviewers, however, the results of Huang et al., although interesting, as they open a discussion on the evolution of genomic structure in chordates, present some questionable points concerning the presence of two TADs in the Hox locus in amphioxus. It is one thing to say that there is a TAD boundary in the middle of the amphioxus Hox locus (very debatable on the other hand by looking at the HiC results of Huang et al.) and another very different is to say that the general 3D structure is conserved with vertebrates. The anterior region is syntenically equivalent to vertebrates (as we published in Acemel et al. NatGen). However, the "posterior" TAD of amphioxus (assuming that the TAD from Hox6-7 onwards is a boundary TAD) only includes up to Hox15 and EvxA-B (at most), which in vertebrates is attached to the cluster. The entire syntenic region to the vertebrate posterior TAD is outside in the amphioxus described TAD (i.e. particularly the introns of Jazf where the Hox enhancers are described in vertebrates). Lnp is also out and 80% of the region orthologous to the vertebrate TAD is on another chromosome in amphioxus. Thus, the results, that under our interpretation are weak, concerning the TAD boundary together with the lack of conservation of the composition of the posterior TAD (including the Hox regulatory regions) lead us to disagree with the results presented by Huang and colleagues.

However, since this article is not intended to discuss this point, we have changed the text to include the possible debate between the two studies, so that the reader can access both interpretations.

2) Line 98-100. Here the authors only cite Delsuc et al. 2006 Nature paper in this sentence; however, in this paper the phylogenetic analysis did not recover the monophyletic chordate clade and grouped cephalochordates with echinoderms. A later paper by Bourlat et al., (Nature 444:85-88, 2006) corrected this problem by including more data from invertebrate deuterostome animals for phylogenetic analyses, and further support the Olfactores (tunicates + vertebrates) hypothesis. Subsequently Dulsuc et al. also published another paper (genesis, 46:592-604, 2008) with broader sampling to correct their previous analysis, and they also recovered the monophyletic chordate group and further support cephalochordates as the most basal lineage while tunicates and vertebrates are sister groups. I would suggest the authors to also cite these later publications for completeness of information.

That is correct, thanks, we added the indicated references.

3) Line 102-104; I would suggest the authors to include some appropriate references here about those traits that are shared between tunicates and vertebrates but absent in cephalochordates. About MHB evolution, I think it is still a contentious issue. There are expression data suggesting a MHB-like region exists in amphioxus CNS (Castro 2006 Dev Biol 295:40-51; Albuixech-Crespo 2017 PLoS Biol 15: e2001573), but whether it has secondary organizer function remains unknown. In addition, a MHB-like gene regulatory program appears to be present in hemichordate S. kowalevskii (Pani 2012 Nature 483:289-2924), suggesting a conserved gene module for AP patterning of the embryonic ectoderm was already evolved in the deuterostome common ancestor.

We thank the reviewer for highlighting this point. However, as the idea of this article is not to discuss possible controversial points, but rather, particularly in this paragraph, to indicate that certain morphological characteristics are shared by tunicates and vertebrates but absent in amphioxus, we have preferred to substitute the existence, or not, of a MHB, by the occurrence of placodes, structures that clearly exist in tunicates but not in amphioxus.

4) Line 163-164; the last part of this sentence is incorrect. Both Asymmetron and Epigonichthys have gonads only on the right side of the animals, whereas Branchiostoma has one row of gonads on each side of the body (see references such as: Igawa 2017 Sci Rep 7: 1157; Holland and Holland 2022 Curr Topics Dev Bio 147:563-594).

We agree and modified the text, thanks.

5) Line 318-320; The paper in PNAS by Huang et al. mentioned above has further increased the number of chromosome-level genome assemblies to four amphioxus species, as well as Hi-C data from B. floridae, B. japonicum, and B. belcheri. The authors should include these new results into this review.

In addition, there are quite a few valuable bulk RNA-seq transcriptome datasets from several Branchiostoma and Asymmetron species, which have contributed extensively to many comparative analyses and phylogenomics studies. Again, the authors should consider mention this work.

Right, we now included the chromosome-level genomes available for other species.

6) Lines 286-323 ("Technical advances in amphioxus research")

In this section, the authors first present a chronological overview of amphioxus husbandry and spawning induction techniques. They then summarise the technical advances in functional experiments, most of which have been made in recent years. However, they overlook the descriptive studies, which, although they may appear old-fashioned, have undoubtedly contributed to and still play an important role in amphioxus research. In order to give the readers a context for understanding the whole picture of modern amphioxus studies, it would be helpful to include a short paragraph discussing the introduction of molecular biology (mainly in situ hybridisation) to amphioxus research, citing some representative seminal papers such as Holland PW et al. (Development 116:653-61), which started the amphioxus EvoDevo field.

Thanks. We now added a sentence introducing the importance of molecular biology (mainly in situ hybridization) for the amphioxus research in the 90s.

7) In addition, for transgenic amphioxus lines (line 315), it is necessary to include the Tol2 assay (TALEN and CRISPR are for knock out). More detailed information on TALEN and Tol2 techniques can be found in Chapter1 "Laboratory Culture and Mutagenesis of Amphioxus" by Linda Holland and Guang Li in Developmental Biology of the Sea Urchin and Other Marine Invertebrates (pp1-9, Humana Press), so citing this reference may be useful. The same applies to Su L et al. Genes 2020, 11, 1311; doi:10.3390/genes11111311 for the CRISPR technique.

We thank the reviewer and added the two suggested references.

8) Lines 337-369 ("Amphioxus as a model to understand vertebrate evolution")

This section is shorter than expected and would benefit from a more in-depth description of recent results and their impact on our understanding of vertebrate evolution (including, possibly, one or two extra figures).

We believe that the purpose of the eLife section “Natural History of Model Organism” is mainly to describe the general aspects of the biology of a given animal model. So in agreement with the eLife Editors when they accepted our review proposal, we decided that the important points of the article would focus on the known data on the ecology, life form, distribution and number of known species of amphioxus. The last point, about the usefulness of amphioxus as a model of study in EvoDevo, is simply to give an idea to the reader who does not know this animal of why it is important for the research without going into details, which, moreover, have been discussed in other reviews.

9) Please consider adding a figure which shows the global distribution of the species discussed in the article.

We thank the reviewer, nevertheless we prefer not to include this suggestion because such a figure is already published in Poss and Boschung (1996), that we have referenced in our work.

10) The reviewers also felt that the title needed attention and the editor will discuss a new title with you if the revised article is accepted for publication.

We agree and propose the following new title:

The Natural History of Model Organisms: The amphioxus as a model to study evolution of development in chordates

Author details

1. Salvatore D'Aniello

   Salvatore D'Aniello is in Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Napoli, Italy

   Contribution

   Conceptualization, Data curation, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   salvatore.daniello@szn.it

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7294-1465

2. Stephanie Bertrand

   Stephanie Bertrand is at Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, France

   Contribution

   Conceptualization, Data curation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0689-0126

3. Hector Escriva

   Hector Escriva is at Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, France

   Contribution

   Conceptualization, Data curation, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   hector.escriva@obs-banyuls.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7577-5028

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