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The nauplius eye complex in ‘conchostracans’(Crustacea, Branchiopoda: Laevicaudata, Spinicaudata, Cyclestherida) and its phylogenetic implications
Arthropod Structure & Development 36 (2007) 408e419 www.elsevier.com/locate/asd
The nauplius eye complex in ‘conchostracans’ (Crustacea, Branchiopoda: Laevicaudata, Spinicaudata, Cyclestherida) and its phylogenetic implications Andre´ Reimann a,b, Stefan Richter a,c,* a Humboldt Universita¨t zu Berlin, Vergleichende Zoologie, Institut fu¨r Biologie, Philippstr. 13, 10115 Berlin, Germany Staatliche Naturhistorische Sammlungen Dresden, Museum fu¨r Tierkunde, Ko¨nigsbru¨cker Landstr. 159, 01109 Dresden, Germany c Allgemeine & Spezielle Zoologie, Institut fu¨r Biowissenschaften, Universita¨t Rostock, Universita¨tsplatz 2, 18055 Rostock, Germany b
Received 15 April 2007; accepted 18 August 2007
Abstract The nauplius eye in Cyclestherida, Laevicaudata and Spinicaudata (previously collectively termed Conchostraca) consists of four cups of inverse sensory cells separated by a pigment layer and a tapetum layer. There are two lateral and two medial cups, a ventral medial cup and a posterior medial cup. The pigment and tapetum layers contain two different kinds of pigment granules, the inner pigment layer relatively large, dark (and electron dense) granules, and the outer tapetum layer light, reflective pigment granules. The presence of four cups and two different kinds of pigment granules are interpreted as autapomorphies of Phyllopoda. The position and shape of the nauplius eye in Spinicaudata is very distinct and herein interpreted as an autapomorphy of this taxon. Additional frontal eyes might be present dorsally or ventrally in varying proximity to the nauplius eye, but they have separate nerves from their sensory cells to the nauplius eye centre in the protocerebrum. Rhabdomeric structures are present in all these frontal eyes, evidencing their light sensitivity. In Lynceus biformis and L. tatei (Laevicaudata), two pairs of frontal eyes were found. In Cyclestheria hislopi (Cyclestherida), an unpaired ventral frontal eye is present. We did not find additional frontal eyes in Limnadopsis parvispinus and Caenestheriella sp. (Spinicaudata). Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Nauplius eye; Frontal organ; Frontal eye; Tapetum; Pigment cells; Phyllopoda
1. Introduction In addition to compound eyes, many crustaceans possess other light sensitive organs, which together have been summarized as ‘‘frontal eyes’’ (Elofsson, 2006). Some of these frontal eyes form the nauplius eye, in which three or four eye cups are separated by pigment cells. During ontogeny, the nauplius eye develops before the compound eyes appear. It is the only light sensitive organ in the nauplius larvae (Claus, 1891). In many crustaceans it persists in the adult animals, and in copepods * Corresponding author. Allgemeine & Spezielle Zoologie, Institut fu¨r Biowissenschaften, Universita¨t Rostock, Universita¨tsplatz 2, 18055 Rostock, Germany. Tel.: þ49 381 498 6260; fax:þ49 381 498 6262. E-mail address: [email protected] (S. Richter). 1467-8039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2007.08.005
for example it is the only eye (Elofsson, 1966). The nauplius eye is completely absent in some major crustacean taxa such as Mystacocarida (Elofsson and Hessler, 2005), Cephalocarida (Elofsson and Hessler, 1990) and Remipedia (Gruner, 1993). The nauplius eye as a uniform structure has been interpreted as a product of the fusion of median eyes also present in other (eu)arthropods (Hanstro¨m, 1926; Paulus, 1972, 1979). It has been suggested to be one of the few potential autapomorphies of the Crustacea (Lauterbach, 1983), but differences between various nauplius eye types have been stressed and the independent evolution of nauplius eyes has also been suggested (Elofsson, 1965, 1966, 2006). One such significant difference is the orientation of the sensory cells, which are directed towards the light in Malacostraca (everse eyes) but towards the pigment layer (inverse eyes) in other Crustacea (Elofsson, 2006).
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Apart from the nauplius eye, there are additional structures with light receptive characteristics that are innervated by the nauplius eye centre, and these are called frontal organs or frontal eyes (Elofsson, 1966, 2006). In Crustacea, frontal eyes are distinguished by their position relative to the nauplius eye and termed either ventral, dorsal, or posterior medial frontal eyes. They can be paired or unpaired. In the previous nomenclature some of these organs were not recognized as eyes, a failing which has caused some confusion. Parts of the cups of the nauplius eyes sometimes appear to be detached from the major part of their respective cups, but they possess inverse sensory cells and their axons still fuse with the nerve of the major cup that they belong to. Structures that lack pigment and tapetum cells and with rhabdomeric elements directed towards the cuticle (‘everse’ sensory cells) are recognized as distinct frontal eyes. They have a separate nerve connection to the nauplius eye centre, i.e. nerves that are not part of those of any of the cups of the nauplius eye. Crustacean frontal eyes have been largely ignored for the past 40 years. Most of our knowledge regarding crustacean frontal eyes, and this is particularly true for branchiopods, dates back to the comprehensive studies by Elofsson (1963, 1965, 1966), who also recently provided a detailed review (Elofsson, 2006; see also Martin, 1992). In the present study, we describe the nauplius eye and additional frontal eyes of several representatives of the Phyllopoda that were previously combined as ‘‘Conchostraca’’. For the first time, we provide a detailed description of the frontal eyes in Cyclestheria hislopi (a member of the monotypic Cyclestherida), and additional details on representatives of the Laevicaudata and Spinicaudata. We show that there are significant differences between the three taxa with regard to nauplius and additional frontal eyes. Finally, we discuss our findings in the light of branchiopod evolution, where different phylogenetic analyses are now approaching a consensus (summarized in Richter et al., 2007). 2. Materials and methods The following species were examined: Laevicaudata e Lynceus tatei (Brady, 1886) (Bloodwood Station, Paroo area, NSW, Australia), Lynceus biformis (Ishikawa, 1895) (Lake Biwa Region, Japan); Spinicaudata e Caenestheriella sp. (Bloodwood Station, Paroo area, NSW, Australia), Limnadopsis parvispinus (Henry, 1924) (Bloodwood Station, Paroo area, NSW, Australia); Cyclestherida e Cyclestheria hislopi (Baird, 1859) (Jabiru, Northern Territory, Australia). The specimens were preserved in Bouin’s solution for embedding in Technovit medium (Kulzer) or in Karnovsky’s solution (Karnovsky, 1965) for embedding in araldite. Sections were made with a Zeiss microtome HM 35 (Technovit embedded specimens; 1e5 mm sections) or with a Leica Ultracut (araldite embedded specimens; 0.5e1 mm sections). The Technovit embedded tissues were stained after sectioning with a mixture of Methylene Blue and Azur II (1:1, 1% in aqua dest.) and counterstained with Fuchsin Red (1% in 1% Borax solution, alkaline) and finally differentiated in alcohol
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(>60%). The araldite objects were stained only with Methylene Blue/Azur II (1:1, 1% in aqua dest.) at 80 C. After staining, the objects were studied with a Zeiss Axiophot 1 microscope and photographed with a Nikon D1 digital camera. For TEM, heads of Cyclestheria hislopi were fixed in a glutaraldehyde/paraformaldehyde mixture according to Karnovsky (1965). After several washes in So¨rensen phosphate buffer, the heads were postfixed for 2 h in 2% OsO4 solution at 4 C. The heads were embedded in araldite. Gold or silver sections cut on a Leica Ultracut were stained with uranyl acetate (1 h) and lead citrate (7 min). The sections were studied using a Zeiss EM 10.
3. Results 3.1. Laevicaudata e Lynceus tatei, L. biformis The nauplius eye of L. tatei is situated ventrally to the compound eyes (Fig. 2A), between the dorsal edges of the ‘frontal sensory fields’ (sensu Cash-Clark and Martin, 1994). The nauplius eye consists of four cups, which are clearly separated by pigment and tapetum layers (Fig. 1A, B). The inner pigment layer contains dark pigment granules, whereas the outer tapetum layer houses light pigment granules (Fig. 2C). The ventral medial cup comprises two rounded sensory cells; the posterior medial cup contains eight to nine cells (Fig. 2C). The lateral cups are divided into a ventral portion of five larger and a dorsal portion of approximately 15 smaller sensory cells. Both cell groups have a separate connection to the nauplius eye centre of the protocerebrum (Fig. 2H). All sensory cells of the four cups are inverse (Fig. 2F, G). All cups are fixed at the integument by strands of connective tissue. The lateral cups have two such ligaments extending towards the neuropile masses of the compound eyes, but these are very short because of the short distance between the structures. There is another paired ligament between the lateral cups and the anterior integument. The posterior (or dorsal) medial cup is also connected with the epidermis via a connective tissue strand that ends directly in front of the canal leading to the ‘compound eye chamber’ (which is the result of the internalization of the compound eye). In addition to the nauplius eye, two paired frontal eyes are present in the head of L. tatei (Fig. 1A, B). The ventral frontal eyes are built up of four to five large sensory cells each and are situated anteriorly and ventrally to the nauplius eye (Fig. 2A, D). The rhabdomeres lie in the most apical part of the cells directly beneath the epidermis (Fig. 2E). The nerves of the ventral frontal eyes pass the two sensory cells of the ventral medial cup ventrally (Fig. 2C) and enter the nauplius eye centre more posteriorly. The dorsal frontal eyes, each consisting of about 30 cells, lie more dorsally on the level of the lateral cups of the nauplius eye (Fig. 2B). Each dorsal frontal eye has a separate connection to the nauplius eye centre. They touch the dorsal edge of the ‘setose fields’ on each side but are not associated with this structure. The cells in the dorsal frontal eyes possess rhabdomeres similar to those of the ventral frontal eyes (Fig. 2F). The cells connected to the setae in the
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Fig. 1. Lynceus tatei (Laevicaudata, Lynceidae) e schematic diagram of the nauplius eye and frontal organs. (A) Lateral view (anterior is left). (B) Frontal view (dorsal is up). Abbreviations: dfo e dorsal frontal organ (dorsal frontal eye), dplc e dorsal portion of the lateral cup, lc e lateral cup, ndfo e nerve connection between nauplius eye centre and dorsal frontal organ (dorsal frontal eye), ndplc e nerve connection between nauplius eye centre and dorsal portion of the lateral cup, npmc e nerve connection between nauplius eye centre and posterior medial cup, nvfo e nerve connection between nauplius eye centre and ventral frontal organ (ventral frontal eye), nvmc e nerve connection between nauplius eye centre and ventral medial cup, nvplc e nerve connection between nauplius eye centre and ventral portion of the lateral cup, pl e pigment layer, pmc e posterior medial cup, tap e tapetum layer, vfo e ventral frontal organ (ventral frontal eye), vmc e ventral medial cup, vplc e ventral portion of the lateral cup.
sensory fields do not show any similarities to the sensory cells of the nauplius eye or the frontal eyes. The nauplius eye of L. biformis resembles that found in L. tatei, but is located closer to the compound eyes. The ventral medial cup contains two sensory cells, while the dorsal medial cup has seven and both lateral cups approximately 20 sensory cells. The ventral frontal eyes are like those of L. tatei. The dorsal frontal eyes are each composed of only 20 sensory cells, but these are larger than in L. tatei. 3.2. Cyclestherida e Cyclestheria hislopi The nauplius eye of Cyclestheria hislopi is situated ventrally to the fused compound eyes (Fig. 4A, C). Between the two eyes, a canal connects the ‘compound eye chamber’ with the surface of the head via the eye pore (Fig. 4D). The nauplius eye consists of four well-developed eye cups and, in between, a tapetum and a pigment layer (Figs. 3A, B; 4A). The inner pigment layer consists of a few large cells,
which contain dark pigment granules. The tapetum is also cellular and it contains light, reflective pigment granules (Fig. 4E). The nauplius eye is egg-shaped with the broader end to the nauplius eye centre. The shape is the result of the arrangement of the two medial cups that form the posterior part of the eye, each of which contains about 20 cells. These cups are attached at the posterior end and diverge anteriorly, so that they appear to be ‘‘V’’ shaped (Fig. 4C). In between the two branches of the ‘V’, the lateral cups are situated. They are composed of 40 cells each and are triangular in shape. In a frontal and a lateral view, they cover the medial cups completely. All cups have their own nerve connection, but the medial ones fuse just before they enter the nauplius eye centre together. The lateral cups are not divided into two parts as in Lynceus. All the axons of one lateral cup give rise to only one nerve (Fig. 4B). The rhabdomeres of the sensory cells in the lateral cups are fused to a conspicuously large rhabdom (Fig. 4D). Long strands of connective tissue to the epidermis are not present. The nauplius eye takes up almost
Fig. 2. Lynceus tatei (Laevicaudata, Lynceidae) e sections through the head region. (A) Medial section through the head (anterior is left) showing the arrangement of the structures. (B) Horizontal section through the nauplius eye and the dorsal frontal organ, (anterior is at the upper left corner). (C) Frontal section through the nauplius eye and the optical tracts of the compound eyes (dorsal is up). (D) Frontal section through the nauplius eye and the ventral frontal organ (dorsal is up). (E) Frontal section through the ventral frontal organ of the right side showing the orientation of the rhabdomeres towards the cuticle side (‘everse’) of the organ. (F) Frontal section through the nauplius eye and the dorsal frontal organ showing the ‘everse’ orientation of the sensory cells of the dorsal frontal organ and the inverse orientation (towards the tapetum) of the sensory cells of the lateral cup (dorsal is up). (G) Frontal section through the nauplius eye showing a closer view on the sensory cells and the tapetum and pigment layers. (H) Sagittal section through the head region showing the two parts of a lateral cup and the orientation of the nauplius eye in respect to the compound eyes (anterior is left). Scale bars in mm. Abbreviations: bpl e border (cell membranes) between pigment cells, ce e compound eye, cu e cuticle, dfo e dorsal frontal organ (dorsal frontal eye), dplc e dorsal portion of the lateral cup, ech e eye chamber, lc e lateral cup, nc e nerve connections from the two parts of the lateral cup, nce e neuropiles of the compound eyes, ndfo e nerve connection between nauplius eye centre and dorsal frontal organ (dorsal frontal eye), nec e nauplius eye centre, nlc e nerve connection between nauplius eye centre and lateral cup, npc e nucleus of a pigment cell, nsc e nucleus of a sensory cell, ntap e nucleus of a tapetal cell, nvfo e nerve connection between nauplius eye centre and ventral frontal organ (ventral frontal eye), oes e oesophagus, pc e protocerebrum, pl e pigment layer, pmc eposterior medial cup, rh e rhabdomers, sc e sensory cell, sf e setose field, tap e tapetum layer, vfo e ventral frontal organ (ventral frontal eye), vmc e ventral medial cup, vplc e ventral portion of the lateral cup.
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Fig. 3. Cyclestheria hislopi (Cyclestherida) e schematic diagram of the nauplius eye and frontal organs. (A) Lateral view (anterior is left). (B) Frontal view (dorsal is up). Abbreviations: lc e lateral cup, nlc e nerve connection between nauplius eye centre and lateral cup, npmc e nerve connection between nauplius eye centre and posterior medial cup, nvfo e nerve connection between nauplius eye centre and ventral frontal organ (ventral frontal eye), nvmc e nerve connection between nauplius eye centre and ventral medial cup, pl e pigment layer, pmc e posterior medial cup, tap e tapetum layer, vfo e ventral frontal organ (ventral frontal eye), vmc e ventral medial cup.
the whole space ventrally to the compound eye and touches the integument at several points, where it is fixed. In Cyclestheria hislopi, an unpaired ventral frontal eye is present (Fig. 3A, B). It is situated anteriorly and ventrally to the nauplius eye. It is club-shaped and contains 12 sensory cells, which are attached to the epidermis distally. The rhabdomeres are fused to a rhabdom close to the integument. The axons form a nerve leading to the nauplius eye centre (Fig. 4C).
elongated branched bands (Fig. 6D). The lateral cups cover the medial ones completely and have short, very broad nerve connections to the nauplius eye centre. The four cups are separated by a tapetum layer and a pigment layer (Figs. 5B; 6C). There are ligamentous connections to the epidermis in the whole region of the lateral cups. Two of them originate on the dorsal edges of the lateral cups and run parallel to the nerves of the compound eyes and enter the epidermis ventral to the eye chamber. They are flanked proximally by parts of the tapetum and pigment (Fig. 6B), but there are not any sensory cells. No separate frontal eyes were found.
3.3. Spinicaudata (Cyzicidae) e Caenestheriella sp. The conditions in the head of Caenestheriella sp. differ from those described above. Diverticula of the midgut separate the nauplius and compound eyes (Fig. 6A), so that there is not any contact between them unlike in Lynceus or Cyclestheria. The four-cup nauplius eye (Fig. 5A, B) is attached to the ventral epidermis and is surrounded frontally and laterally by the diverticula of the midgut. The ventral medial cup consists of 14 sensory cells with their axons ending in a short nerve to the nauplius eye centre, which is located directly posterior to the eye. The posterior medial cup sits right on the nauplius eye centre, with no noticeable nerve connecting both structures (Fig. 6B). It consists of about 30 sensory cells. The lateral cups are very large (Figs. 5A; 6C) and are formed by approximately 60 cells. Their rhabdomeres are fused to form
3.4. Spinicaudata (Limnadiidae) e Limnadopsis parvispinus The nauplius eye in Limnadopsis parvispinus is the largest in terms of both size and number of sensory cells in the cups (Fig. 8A, D). The nauplius eye lies in a ventral projection of the rostrum. It is long and cone-shaped with a curved tip, and the thicker end of the cone attaches to the ventral epidermis (Fig. 7A, B). The tip reaches the epidermis close to the compound eyes. Between the two contact points to the epidermis, diverticula of the midgut gland are present, as in the Cyzicidae (Fig. 8D). The four eye cups are separated by the layers of tapetum and pigment (Fig. 7A, B). The tapetum partially extends between the distal parts of the sensory cells so
Fig. 4. Cyclestheria hislopi (Cyclestherida) e sections through the head region. (A) Frontal section through the compound eye and the nauplius eye showing the close vicinity of both structures (dorsal is up). (B) Frontal section through the medial cups of the nauplius eye (dorsal is up). (C) Medial section through the head region (anterior is left, dorsal is up) showing the medial cups of the nauplius eye and the unpaired ventral frontal organ. (D) Sagittal section through the nauplius eye and the ventral frontal organ (anterior is left, dorsal is up) showing the rhabdomeric elements of both structures. (E) TEM picture of the nauplius eye showing the two lateral cups and in between the two different kinds of pigment granules in the tapetum and pigment layer. (F) TEM picture of the distal region of a sensory cell in the ventral frontal organ. Scale bars in mm. Abbreviations: ce e compound eye, cec e canal from the anterior cuticle to the eye chamber of the compound eye, cu e cuticle, lc e lateral cup, nce e neuropiles of the compound eyes, nec e nauplius eye centre, nlc e nerve connection between nauplius eye centre and lateral cup, nvfo e nerve connection between nauplius eye centre and ventral frontal organ (ventral frontal eye), pc e protocerebrum, pl e pigment layer, pmc e posterior medial cup, rh e rhabdomers, tap e tapetum layer, vfo e ventral frontal organ (ventral frontal eye), vmc e ventral medial cup.
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Fig. 5. Caenestheriella sp. (Spinicaudata, Cyzicidae) e schematic diagram of the nauplius eye. (A) Lateral view (anterior is left). (B) Frontal view (dorsal is up). Abbreviations: lc e lateral cup, nec e nauplius eye centre, nlc e nerve connection between nauplius eye centre and lateral cup, pl e pigment layer, pmc eposterior medial cup, tap e tapetum layer, vmc e ventral medial cup.
that the ventral medial cup appears to be divided, but this is not the case. The ventral medial cup is very compact and consists of approximately 30 cells. The nerve connection to the nauplius eye centre is very long because of the large distance to the protocerebrum (Fig. 8E). The posterior medial cup is made up of 120 sensory cells arranged in two groups, one above and one below the point at which the cup connects to the nauplius eye centre (Fig. 8D, E). In the lateral regions no division is visible. The lateral cups are very large, extending from the ventral to the anterior contact point with the epidermis, and consist of about 200 sensory cells each, which are connected with the nauplius eye centre via a broad nerve. All the sensory cells are approximately of the same size. The lateral cups form the curved tip of the nauplius eye, and occasionally the tapetum separates groups of sensory cells (Fig. 8B, C). These cells belong to the nauplius eye because their axons are part of the nerve from the lateral cups, and they are clearly inverse with the rhabdomeres directed towards the tapetum (Fig. 8C). Ligamentous connections to the epidermis are found in all regions of the nauplius eye (Fig. 8B, C). Additionally, there are two connections leading to the very elongated nerve strands from the compound eyes to the protocerebrum (Fig. 8A). There are not any additional frontal eyes in the head region of Limnadopsis parvispinus. 4. Discussion 4.1. Comparison with previous findings The nauplius eye in Cyclestheria hislopi was first described by Sars (1887), and our description agrees with his in general aspects such as size, shape and position. Sars (1887) also recognized the ventral frontal eye. The only other laevicaudatan species previously studied is Lynceus brachyurus O.F. Mu¨ller, 1776 (Zograf, 1904; Nowikoff, 1905; Elofsson, 1966). As in L. tatei and L. biformis, the nauplius eye in
L. brachyurus consists of four cups (Nowikoff, 1905; Elofsson, 1966). Some differences exist with regard to the number of cells found in each cup. Elofsson (1966) described only four cells in the posterior median cup in L. brachyurus (8e9 in L. tatei and 7 in L. biformis in the present study) and a few more in the lateral cups (approximately 30 in L. tatei and 20 in L. biformis). The number of cells in the ventral medial cup has consistently been determined as two in all three species. In L. brachyurus, a paired ‘dorsal frontal organ’ (Nowikoff, 1905; ‘distal frontal organ’ in Elofsson, 1966) as well as an unpaired ‘ventral frontal organ’ (Nowikoff, 1905) have been described; the latter, however, was questioned by Elofsson (1966). The presence of the paired ‘dorsal frontal eyes’ and of paired ‘ventral frontal eyes’ is supported, at least for the two species we studied. Elofsson (1966) described the presence of an additional ‘posterior medial frontal organ’ attached to a connective tissue strand from the posterior medial cup to the epidermis. However, we could not find any sensory cells on this strand in L. tatei and L. biformis. A potential source of misinterpretation is the apparent division of the posterior medial cup in the two Lynceus species into a ventral part with four cells and a dorsal part with four to five. However, this ‘division’ only appears in some frontal sections and is caused by a little kink in the cup. In general, our description of Caenestheriella sp. concurs with that of Caenestheria var. salberghi by Elofsson (1966), including the absence of any frontal eyes apart from the nauplius eye. An important difference is the double innervation of the lateral cups and the corresponding division of these cups into two parts described by Elofsson (1966), which we did not find in the Caenestheriella sp. we studied. Here, all the sensory cells in the lateral cups are very similar and there is no division into a ventral part with larger cells and a dorsal part with smaller cells. Several frontal organs have previously been described for the single representative of Limnadiidae studied so far,
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Fig. 6. Caenestheriella sp. (Spinicaudata, Cyzicidae) e sections through the head region. (A) Sagittal section through the whole head region showing the separation of the compound eye and the nauplius eye by the diverticles of the midgut gland (anterior is left). (B) Sagittal section through the nauplius eye and the nauplius eye centre showing the close positions of the posterior medial cup and the nauplius eye centre (anterior is left). (C) Frontal section through the nauplius eye showing the four cups and two of the connective tissue strands that fix the position of the nauplius eye (dorsal is up). (D) Sagittal section through the rhabdomeres of the sensory cells of one lateral cup near to the tapetum layer. Scale bars in mm. Abbreviations: ce e compound eye, cts e connective tissue strand, cu e cuticle, ech e eye chamber, ep e epidermis, lc e lateral cup, mgd e diverticles of the midgut gland, nce e neuropiles of the compound eyes, ne e nauplius eye, nec e nauplius eye centre, pc e protocerebrum, pl e pigment layer, pmc eposterior medial cup, rh e rhabdomers, tap e tapetum layer, vmc e ventral medial cup.
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cell in phyllopod crustaceans. Although the reflective granules certainly function as a tapetum, this kind of tapetum is very different from that found in maxillopodans (Fahrenbach, 1964; Elofsson, 1966; Andersson and Nilsson, 1981). 4.2. Comparison with other Phyllopoda
Fig. 7. Limnadopsis parvispinus (Spinicaudata, Limnadiidae) e schematic diagram of the nauplius eye. (A) Lateral view (anterior is left). (B) Frontal view (dorsal is up). Abbreviations: lc e lateral cup, nec e nauplius eye centre, nlc e nerve connection between nauplius eye centre and lateral cup, npmc e nerve connection between nauplius eye centre and posterior medial cup, nvmc e nerve connection between nauplius eye centre and ventral medial cup, pl e pigment layer, pmc eposterior medial cup, tap e tapetum layer, vmc e ventral medial cup.
Limnadia lenticularis (Linne, 1761). Nowikoff (1905) described a ‘dorsal frontal organ’ in anterior-dorsal elongation of the lateral cups (see also Hanstro¨m, 1934) as well as a ‘ventral frontal organ’ divided into two portions. One portion of the latter is Elofsson’s (1966) ‘distal frontal organ’, the other portion he described as a ‘posterior medial frontal organ’ located at the connective tissue strand from the posterior medial cup to the epidermis. We agree with Elofsson (1966) that the structure interpreted by Nowikoff (1905) as a ‘dorsal frontal organ’ in L. lenticularis consists of connective tissue only. In Limnadopsis, sensory cells are not present and these ligaments serve only to fix the nauplius eye to the epineurium of the neuropile masses of the compound eyes. The ‘distal frontal organ’ described by Elofsson (1966) in L. lenticularis (‘main portion of the ventral frontal organ’, Nowikoff, 1905; see also Hanstro¨m, 1934) is not present in Limnadopsis. Although sensory cells are certainly present in the attenuated rostrum of Limnadia lenticularis (our own observations), these cells might better be considered to be detached parts of the lateral cups of the nauplius eye, an idea which has already been discussed by Elofsson (1966). Furthermore, we were not able to find in Limnadopsis the ‘posterior medial frontal organ’ described by Elofsson (1966). As in Lynceus, the presence or absence of such a ‘posterior medial frontal eye’ needs to be confirmed using TEM. The most remarkable difference between our findings and the most detailed study so far (Elofsson, 1966) is the presence of two different kinds of pigment cells, i.e. tapetum cells with light, reflective pigment granules and pigment cells with dark granules. Elofsson (1966) described just one type of pigment
Recent phylogenetic (mostly molecular) analyses have almost reached a consensus on branchiopod phylogenetic relationships. Anostraca is the sister group to Phyllopoda (e.g., Giribet et al., 2005; Olesen, 2007). Within Phyllopoda, Spinicaudata, Cyclestherida and Cladocera constitute a monophylum (Braband et al., 2002; Richter, 2004; deWaard et al., 2006; Stenderup et al., 2006; Olesen, 2007; Richter et al., 2007), with the two latter as sister groups (e.g., Olesen et al., 1997; Ax, 1999; Taylor et al., 1999; Spears and Abele, 2000; Braband et al., 2002). Morphological analyses tend to result in Notostraca as a sister group to Diplostraca (Olesen, 1998, 2000, 2007; Richter et al., 2007) but recent molecular based analyses suggest Laevicaudata to be the most basal offshoot within Phyllopoda (deWaard et al., 2006; Stenderup et al., 2006; Richter et al., 2007). At any rate, a comparison of the conditions in Laevicaudata and Notostraca seems most likely to give the strongest insights into the ancestral conditions, i.e. the ground pattern of Phyllopoda. Within Notostraca, frontal eyes have been described in Lepidurus apus, L. arcticus and Triops cancriformis (Zograf, 1904; Wenke, 1908; Holmgren, 1916; Elofsson, 1966). Both Notostraca and Laevicaudata possess nauplius eyes made of four distinct cups. Another correspondence concerns the lateral cups that are separated into two portions, each with its own nerve. Elofsson (1966) noticed within Notostraca that the two nerves are sometimes fused. A potential explanation for the partition of the lateral cups is given by Dahl (1959), who has provided the only detailed study so far on the development of the nauplius eyes in phyllopods. According to Dahl (1959), the newly hatched larvae in Triops possess a small nauplius eye consisting of two pigment cells and three cups. These three cups apparently correspond (as in the interpretation by Elofsson, 1966) to the ventral medial cup and the ventral portions of the lateral cups. The dorsal portions of the lateral cups and the posterior medial cup were added later in development by the proliferation of two zones, the ‘‘dorso-lateral lobes of the brain’’ (Dahl, 1959). This means that the two partitions of the lateral cups in adult Notostraca and Laevicaudata are of separate ontogenetic origin, a state which might even be persistent in certain representatives of Spinicaudata (Caenestheria salberghi, Elofsson, 1966) but not in others (present study). The developmental findings are obviously also of importance for a comparison with the nauplius eye in Anostraca (see Elofsson, 1966 for Branchinecta paludosa, Tanymastix stagnalis, Polyartemia forcipata and Chirocephalus sp. and Moroff, 1912; Anado´n and Anado´n, 1980; Criel, 1991, and Martin, 1992 for Artemia salina). The nauplius eye in all studied species consists of two lateral cups and one ventral medial cup, which correspond exactly to the larva in Triops. This might indeed provide an argument that the
Fig. 8. Limnadopsis parvispinus (Spinicaudata, Limnadiidae) e sections through the head region. (A) Frontal section through the nauplius eye (dorsal is up). (B) Sagittal section through the anterior-dorsal part of the nauplius eye (elongations of the lateral cups and the posterior medial cup) showing the continuity of the cups and the associated tapetum layer (anterior is left). (C) Same as B showing the inverse orientation of the sensory cells towards the tapetum in the elongation of cups. (D) Sagittal section through the head region showing the orientation of the nauplius eye and the diverticles of the midgut gland in between the nauplius eye and the compound eye. (E) Sagittal section through the ventral part of the nauplius eye showing the innervations of the medial cups and the close positions of the nauplius eye centre and the dorsal medial cup (anterior is left). (F) Frontal section through the antero-ventral part of the nauplius eye showing the close attachment to the cuticle in the rostral region (dorsal is up). Scale bars in mm. Abbreviations: asc e axons of the sensory cells of the lateral cup, ce e compound eye, cts e connective tissue strand, cu e cuticle, elo e elongations of the lateral cups, lc e lateral cup, mgd e diverticles of the midgut gland, mu e muscle fibers, nce e neuropiles of the compound eyes, nec e nauplius eye centre, npmc e nerve connection between nauplius eye centre and posterior medial cup, nvmc e nerve connection between nauplius eye centre and ventral medial cup, oes e oesophagus, pc e protocerebrum, pl e pigment layer, pmc eposterior medial cup, rh e rhabdomers, tap e tapetum layer, to e tractus opticus, vmc e ventral medial cup.
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four-cup nauplius eye is an autapomorphy of Phyllopoda (e.g., Elofsson, 1966; Walossek, 1993) and does not represent the plesiomorphic condition of the crustacean ground pattern (e.g., Paulus, 1972, 1979; Lauterbach, 1983). This is also corroborated by other crustaceans possessing a nauplius eye consisting of only three cups, if a nauplius eye is present at all (Elofsson, 1965, 1966, 1992). Another interesting aspect concerns the presence of two different kinds of pigment granules, forming distinct tapetum and pigment layers. These two types of granules also seem to be present in Triops and Lepidurus (Claus, 1891; Zograf, 1904; our own observations) whereas Artemia (Anostraca) only seems to have one kind of pigment cell (Anado´n and Anado´n, 1980), which implies that the reflective pigment granules in phyllopodan nauplius eyes might represent another autapomorphy of this taxon, particularly considering that a tapetum structured completely differently (the tapetal cells are filled with refractive crystals in the form of platelets, e.g. Fahrenbach, 1964; Andersson and Nilsson, 1981) is present in maxillopodan crustaceans. In addition to the nauplius eye, a paired ventral frontal eye formed by two strands of sensory cells which fuse in front of the nauplius eye is present in Anostraca (e.g., Elofsson, 1966). Based on its position ventrally to the medial cup of the nauplius eye, this paired ventral frontal eye can be considered homologous to that in Laevicaudata (also paired) and Cyclestherida (unpaired) and therefore might belong to the phyllopod ground pattern, which, however, implies that it has been reduced in Notostraca. The paired dorsal frontal eyes seem to be restricted to Laevicaudata. The position of the nauplius eye in Spinicaudata, separated from the compound eyes by the midgut diverticula, is very distinct, as is its triangular shape, and in comparison with Notostraca, Laevicaudata and Cyclestherida, this can be most easily interpreted as an autapomorphy of Spinicaudata (see also Richter et al., 2007). In comparison to the descriptions above, the nauplius eye in Cladocera is much reduced (Claus, 1891; Elofsson, 1966). Four cups are only discernible in some Anomopoda, and even then they consist of very few cells. For an overview we refer to Elofsson (2006). It is worth mentioning here the conditions in Eurycercus lamellatus (O.F. Mu¨ller, 1785), where the nauplius eye is comparably well developed. There are four cells in each of the two medial cups and three cells in each of the lateral cups, with each cup having its own nerve to the nauplius eye centre (Elofsson, 1966 and our own observations). Paired ventral and dorsal frontal eyes are present but their homology with those in other branchiopods is difficult to establish. Acknowledgments We would like to thank Dr. Mark Grygier for supplying us with Lynceus biformis from Japan as part of the project at Lake Biwa Museum ‘Research on Large Branchiopods Inhabiting Rice Paddies’, and Dr. Brian Timms for assisting SR in collecting material from Australia in 1999. The collection in Australia was made possible by a Visiting Fellowship of the
Australia Museum, Sydney to SR. We would like to thank Drs. Christopher Tudge and Gerhard Scholtz for critical reading an earlier version of the manuscript and Lucy Cathrow for improving the English. We are particularly grateful to three anonymous reviewers and Drs. Doekele Stavenga and Steffen Harzsch, guest editors of this issue, for helpful suggestions improving the manuscript. References Anado´n, A., Anado´n, E., 1980. Nauplius eye and adjacent organs of adult Artemia. In: Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E. (Eds.), The Brine Shrimp Artemia. Universa Press, Belgium, pp. 41e60. Andersson, A., Nilsson, D.-E., 1981. Fine structure and optical properties of an ostracode (Crustacea) nauplius eye. Protoplasma 107, 361e374. Ax, P., 1999. Phyllopodomorpha. Das System der Metazoa II. Ein Lehrbuch der phylogenetischen Systematik. G. Fischer, Stuttgart, pp. 156e168. Braband, A., Richter, S., Hiesel, R., Scholtz, G., 2002. Phylogenetic relationships within the Phyllopoda (Crustacea, Branchiopoda) based on mitochondrial and nuclear markers. Molecular Phylogenetics and Evolution 25, 229e244. Cash-Clark, C.E., Martin, J.W., 1994. Ultrastructure of the frontal sensory fields in the Lynceidae (Crustacae, Branchiopoda, Laevicaudata). Journal of Morphology 221, 153e160. Claus, C., 1891. Das Medianauge der Crustaceen. Arbeiten des Zoologischen Instituts der Universita¨t Wien 9, 267e284. Criel, G.R.J., 1991. Morphology of Artemia. In: Browne, R.A., Sorgeloos, P.A., Trotman, C.N.A. (Eds.), Artemia Biology. CRC Press, Boca Raton, FL, pp. 119e153. Dahl, E., 1959. The ontogeny and comparative anatomy of some protocerebral sense organs in notostracan phyllopods. Quarterly Journal of Microscopical Science 100, 445e462. deWaard, J.R., Sacherova, V., Cristescu, M.E.A., Remigio, E.A., Crease, T.J., Hebert, P.D.N., 2006. Probing the relationships of the branchiopod crustaceans. Molecular Phylogenetics and Evolution 39, 491e502. Elofsson, R., 1963. The nauplius eye and frontal organs in Decapoda (Crustacea). Sarsia 12, 1e68. Elofsson, R., 1965. The nauplius eye and frontal organs in Malacostraca (Crustacea). Sarsia 19, 1e54. Elofsson, R., 1966. The nauplius eye and frontal organs of the nonMalacostraca (Crustacea). Sarsia 25, 1e128. Elofsson, R., 1992. To the question of eyes in primitive crustaceans. Acta Zoologica 73, 369e372. Elofsson, R., Hessler, R.R., 1990. Central nervous system of Hutchinsoniella macracantha (Cephalocarida). Journal of Crustacean Biology 10, 423e439. Elofsson, R., Hessler, R.R., 2005. The tegumental sensory organ and nervous system of Derocheilocaris typica (Crustacea: Mystacocarida). Arthropod Structure & Development 34, 139e152. Elofsson, R., 2006. The frontal eyes of crustaceans. Arthropod Structure & Development 35, 275e291. Fahrenbach, W.H., 1964. The fine structure of a nauplius eye. Zeitschrift fu¨r Zellforschung 62, 182e197. Giribet, G., Richter, S., Edgecombe, G.D., Wheeler, W.C., 2005. The position of Crustacea within Arthropoda: evidence from nine molecular loci and morphology. Crustacean Issues 16, 307e352. Gruner, H.-E., 1993. Crustacea. In: Gruner, H.-E. (Ed.), Arthropoda (ohne Insecta). Lehrbuch der Speziellen Zoologie. Gustav Fischer, Jena, pp. 448e 1030. Bd I, 4. Teil. Hanstro¨m, B., 1926. Eine genetische Studie u¨ber die Augen und Sehzentren von Turbellarien, Anneliden und Arthropoden (Trilobiten, Xiphosuren, Eurypteriden, Arachnoiden, Myriapoden, Crustaceen und Insekten). Kungliga Svenska Vetenskapsakademiens Handlingar 4, 1e176. Hanstro¨m, B., 1934. Neue Untersuchungen u¨ber Sinnesorgane und Nervensystem der Crustaceen IV. Arkiv fo¨r Zoologi 26A (24), 1e65. Holmgren, N., 1916. Zur vergleichenden Anatomie des Gehirns von Polychaeten, Onychophoren, Xiphosuren, Arachniden, Crustaceen, Myriapoden und
A. Reimann, S. Richter / Arthropod Structure & Development 36 (2007) 408e419 Insekten. Kungliga Svenska Vetenskapsakademiens Handlingar 56, 1e303. Karnovsky, M.J., 1965. A formaldehydeeglutaraldehyde fixative of high osmolarity for use in electron microscopy. Journal of Cell Biology 27, 137Ae138A. Lauterbach, K.-E., 1983. Zum Problem der Monophylie der Crustacea. Verhandlungen des Naturwissenschaftlichen Vereins in Hamburg (NF) 26, 293e320. Martin, J.W., 1992. Branchiopoda. In: Humes, A.G., Harrison, F.W. (Eds.), Microscopic Anatomy of Invertebrates. Crustacea, vol. 9. Wiley-Liss, New York, pp. 25e224. Moroff, T., 1912. Entwicklung und phylogenetische Bedeutung des Medianauges bei Crustacea (Artemia). Zoologischer Anzeiger 40, 11e25. ¨ ber die Augen und Frontalorgane der Branchiopoden. Nowikoff, M., 1905. U Zeitschrift fu¨r wissenschaftliche Zoologie 89, 432e465. Olesen, J., 1998. A phylogenetic analysis of the Conchostraca and Cladocera (Crustacea, Branchiopoda, Diplostraca). Zoological Journal of the Linnean Society 122, 491e536. Olesen, J., 2000. An updated phylogeny of the Conchostraca-Cladocera clade (Branchiopoda, Diplostraca). Crustaceana 73, 869e886. Olesen, J., Martin, J.W., Roessler, E.W., 1997. Description of external morphology of the male of Cyclestheria hislopi (Baird, 1859) (Crustacea, Branchiopoda, Spinicaudata), with comparison of male claspers among the Conchostraca and Cladocera and its bearing on phylogeny of the ‘bivalved’ Branchiopoda. Zoologica Scripta 25, 291e316. Olesen, J., 2007. Monophyly and phylogeny of Branchiopoda, with focus on morphology and homologies of branchiopod phyllopodous limbs. Journal of Crustacean Biology 27, 165e183. Paulus, H.F., 1972. Die Feinstruktur der Stirnaugen einiger Collembolen (Insecta, Entognatha) und ihre Bedeutung fu¨r die Stammesgeschichte der Insekten. Zeitschrift fu¨r Zoologische Systematik und Evolutionsforschung 10, 81e122.
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Paulus, H.F., 1979. Eye structure and the monophyly of the Arthropoda. In: Gupta, A.P. (Ed.), Arthropod Phylogeny. VNR van Nostrand Reinhold Company, New York, pp. 299e386. Richter, S., 2004. A comparison of the mandibular gnathal edges in branchiopod crustaceans e implications for the phylogenetic position of the Laevicaudata (Crustacea, Branchiopoda). Zoomorphology 123, 31e44. Richter, S., Olesen, J., Wheeler, W.C., 2007. Branchiopod phylogeny e a combined analysis using morphology and six molecular loci. Cladistics 23, 301e336. Sars, G.O., 1887. On Cyclestheria hislopi (Baird), a new generic type of bivalve Phyllopoda raised from dried Australian mud. Forhandlinger i Videnskabs-Selskabet i Christiania 1, 223e239. Spears, T., Abele, L.G., 2000. Branchiopod monophyly and interordinal phylogeny inferred from 18S ribosomal DNA. Journal of Crustacean Biology 20, 1e24. Stenderup, J.T., Olesen, J., Glenner, H., 2006. Molecular phylogeny of the Branchiopoda (Crustacea) e multiple approaches suggest a ‘diplostracan’ ancestry of the Notostraca. Molecular Phylogenetics and Evolution 41, 182e194. Taylor, D.J., Crease, T.J., Brown, W.M., 1999. Phylogenetic evidence for a single long-lived clade of crustacean cyclic parthenogens and its implications for the evolution of sex. Proceedings of the Royal Society of London, B, Biological Sciences 266, 794e797. Walossek, D., 1993. The Upper Cambrian Rehbachiella and the phylogeny of Branchiopoda and Crustacea. Fossil and Strata 32, 1e202. Wenke, W., 1908. Die Augen von Apus productus. Zeitschrift fu¨r wissenschaftliche Zoologie 91, 236e265. Zograf, N., 1904. Das unpaare Auge, die Frontalorgane und das Nackenorgan einiger Branchiopoden. Friedla¨nder und Sohn, Berlin.
The nauplius eye complex in ‘conchostracans’ (Crustacea, Branchiopoda: Laevicaudata, Spinicaudata, Cyclestherida) and its phylogenetic implications Andre´ Reimann a,b, Stefan Richter a,c,* a Humboldt Universita¨t zu Berlin, Vergleichende Zoologie, Institut fu¨r Biologie, Philippstr. 13, 10115 Berlin, Germany Staatliche Naturhistorische Sammlungen Dresden, Museum fu¨r Tierkunde, Ko¨nigsbru¨cker Landstr. 159, 01109 Dresden, Germany c Allgemeine & Spezielle Zoologie, Institut fu¨r Biowissenschaften, Universita¨t Rostock, Universita¨tsplatz 2, 18055 Rostock, Germany b
Received 15 April 2007; accepted 18 August 2007
Abstract The nauplius eye in Cyclestherida, Laevicaudata and Spinicaudata (previously collectively termed Conchostraca) consists of four cups of inverse sensory cells separated by a pigment layer and a tapetum layer. There are two lateral and two medial cups, a ventral medial cup and a posterior medial cup. The pigment and tapetum layers contain two different kinds of pigment granules, the inner pigment layer relatively large, dark (and electron dense) granules, and the outer tapetum layer light, reflective pigment granules. The presence of four cups and two different kinds of pigment granules are interpreted as autapomorphies of Phyllopoda. The position and shape of the nauplius eye in Spinicaudata is very distinct and herein interpreted as an autapomorphy of this taxon. Additional frontal eyes might be present dorsally or ventrally in varying proximity to the nauplius eye, but they have separate nerves from their sensory cells to the nauplius eye centre in the protocerebrum. Rhabdomeric structures are present in all these frontal eyes, evidencing their light sensitivity. In Lynceus biformis and L. tatei (Laevicaudata), two pairs of frontal eyes were found. In Cyclestheria hislopi (Cyclestherida), an unpaired ventral frontal eye is present. We did not find additional frontal eyes in Limnadopsis parvispinus and Caenestheriella sp. (Spinicaudata). Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Nauplius eye; Frontal organ; Frontal eye; Tapetum; Pigment cells; Phyllopoda
1. Introduction In addition to compound eyes, many crustaceans possess other light sensitive organs, which together have been summarized as ‘‘frontal eyes’’ (Elofsson, 2006). Some of these frontal eyes form the nauplius eye, in which three or four eye cups are separated by pigment cells. During ontogeny, the nauplius eye develops before the compound eyes appear. It is the only light sensitive organ in the nauplius larvae (Claus, 1891). In many crustaceans it persists in the adult animals, and in copepods * Corresponding author. Allgemeine & Spezielle Zoologie, Institut fu¨r Biowissenschaften, Universita¨t Rostock, Universita¨tsplatz 2, 18055 Rostock, Germany. Tel.: þ49 381 498 6260; fax:þ49 381 498 6262. E-mail address: [email protected] (S. Richter). 1467-8039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2007.08.005
for example it is the only eye (Elofsson, 1966). The nauplius eye is completely absent in some major crustacean taxa such as Mystacocarida (Elofsson and Hessler, 2005), Cephalocarida (Elofsson and Hessler, 1990) and Remipedia (Gruner, 1993). The nauplius eye as a uniform structure has been interpreted as a product of the fusion of median eyes also present in other (eu)arthropods (Hanstro¨m, 1926; Paulus, 1972, 1979). It has been suggested to be one of the few potential autapomorphies of the Crustacea (Lauterbach, 1983), but differences between various nauplius eye types have been stressed and the independent evolution of nauplius eyes has also been suggested (Elofsson, 1965, 1966, 2006). One such significant difference is the orientation of the sensory cells, which are directed towards the light in Malacostraca (everse eyes) but towards the pigment layer (inverse eyes) in other Crustacea (Elofsson, 2006).
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Apart from the nauplius eye, there are additional structures with light receptive characteristics that are innervated by the nauplius eye centre, and these are called frontal organs or frontal eyes (Elofsson, 1966, 2006). In Crustacea, frontal eyes are distinguished by their position relative to the nauplius eye and termed either ventral, dorsal, or posterior medial frontal eyes. They can be paired or unpaired. In the previous nomenclature some of these organs were not recognized as eyes, a failing which has caused some confusion. Parts of the cups of the nauplius eyes sometimes appear to be detached from the major part of their respective cups, but they possess inverse sensory cells and their axons still fuse with the nerve of the major cup that they belong to. Structures that lack pigment and tapetum cells and with rhabdomeric elements directed towards the cuticle (‘everse’ sensory cells) are recognized as distinct frontal eyes. They have a separate nerve connection to the nauplius eye centre, i.e. nerves that are not part of those of any of the cups of the nauplius eye. Crustacean frontal eyes have been largely ignored for the past 40 years. Most of our knowledge regarding crustacean frontal eyes, and this is particularly true for branchiopods, dates back to the comprehensive studies by Elofsson (1963, 1965, 1966), who also recently provided a detailed review (Elofsson, 2006; see also Martin, 1992). In the present study, we describe the nauplius eye and additional frontal eyes of several representatives of the Phyllopoda that were previously combined as ‘‘Conchostraca’’. For the first time, we provide a detailed description of the frontal eyes in Cyclestheria hislopi (a member of the monotypic Cyclestherida), and additional details on representatives of the Laevicaudata and Spinicaudata. We show that there are significant differences between the three taxa with regard to nauplius and additional frontal eyes. Finally, we discuss our findings in the light of branchiopod evolution, where different phylogenetic analyses are now approaching a consensus (summarized in Richter et al., 2007). 2. Materials and methods The following species were examined: Laevicaudata e Lynceus tatei (Brady, 1886) (Bloodwood Station, Paroo area, NSW, Australia), Lynceus biformis (Ishikawa, 1895) (Lake Biwa Region, Japan); Spinicaudata e Caenestheriella sp. (Bloodwood Station, Paroo area, NSW, Australia), Limnadopsis parvispinus (Henry, 1924) (Bloodwood Station, Paroo area, NSW, Australia); Cyclestherida e Cyclestheria hislopi (Baird, 1859) (Jabiru, Northern Territory, Australia). The specimens were preserved in Bouin’s solution for embedding in Technovit medium (Kulzer) or in Karnovsky’s solution (Karnovsky, 1965) for embedding in araldite. Sections were made with a Zeiss microtome HM 35 (Technovit embedded specimens; 1e5 mm sections) or with a Leica Ultracut (araldite embedded specimens; 0.5e1 mm sections). The Technovit embedded tissues were stained after sectioning with a mixture of Methylene Blue and Azur II (1:1, 1% in aqua dest.) and counterstained with Fuchsin Red (1% in 1% Borax solution, alkaline) and finally differentiated in alcohol
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(>60%). The araldite objects were stained only with Methylene Blue/Azur II (1:1, 1% in aqua dest.) at 80 C. After staining, the objects were studied with a Zeiss Axiophot 1 microscope and photographed with a Nikon D1 digital camera. For TEM, heads of Cyclestheria hislopi were fixed in a glutaraldehyde/paraformaldehyde mixture according to Karnovsky (1965). After several washes in So¨rensen phosphate buffer, the heads were postfixed for 2 h in 2% OsO4 solution at 4 C. The heads were embedded in araldite. Gold or silver sections cut on a Leica Ultracut were stained with uranyl acetate (1 h) and lead citrate (7 min). The sections were studied using a Zeiss EM 10.
3. Results 3.1. Laevicaudata e Lynceus tatei, L. biformis The nauplius eye of L. tatei is situated ventrally to the compound eyes (Fig. 2A), between the dorsal edges of the ‘frontal sensory fields’ (sensu Cash-Clark and Martin, 1994). The nauplius eye consists of four cups, which are clearly separated by pigment and tapetum layers (Fig. 1A, B). The inner pigment layer contains dark pigment granules, whereas the outer tapetum layer houses light pigment granules (Fig. 2C). The ventral medial cup comprises two rounded sensory cells; the posterior medial cup contains eight to nine cells (Fig. 2C). The lateral cups are divided into a ventral portion of five larger and a dorsal portion of approximately 15 smaller sensory cells. Both cell groups have a separate connection to the nauplius eye centre of the protocerebrum (Fig. 2H). All sensory cells of the four cups are inverse (Fig. 2F, G). All cups are fixed at the integument by strands of connective tissue. The lateral cups have two such ligaments extending towards the neuropile masses of the compound eyes, but these are very short because of the short distance between the structures. There is another paired ligament between the lateral cups and the anterior integument. The posterior (or dorsal) medial cup is also connected with the epidermis via a connective tissue strand that ends directly in front of the canal leading to the ‘compound eye chamber’ (which is the result of the internalization of the compound eye). In addition to the nauplius eye, two paired frontal eyes are present in the head of L. tatei (Fig. 1A, B). The ventral frontal eyes are built up of four to five large sensory cells each and are situated anteriorly and ventrally to the nauplius eye (Fig. 2A, D). The rhabdomeres lie in the most apical part of the cells directly beneath the epidermis (Fig. 2E). The nerves of the ventral frontal eyes pass the two sensory cells of the ventral medial cup ventrally (Fig. 2C) and enter the nauplius eye centre more posteriorly. The dorsal frontal eyes, each consisting of about 30 cells, lie more dorsally on the level of the lateral cups of the nauplius eye (Fig. 2B). Each dorsal frontal eye has a separate connection to the nauplius eye centre. They touch the dorsal edge of the ‘setose fields’ on each side but are not associated with this structure. The cells in the dorsal frontal eyes possess rhabdomeres similar to those of the ventral frontal eyes (Fig. 2F). The cells connected to the setae in the
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Fig. 1. Lynceus tatei (Laevicaudata, Lynceidae) e schematic diagram of the nauplius eye and frontal organs. (A) Lateral view (anterior is left). (B) Frontal view (dorsal is up). Abbreviations: dfo e dorsal frontal organ (dorsal frontal eye), dplc e dorsal portion of the lateral cup, lc e lateral cup, ndfo e nerve connection between nauplius eye centre and dorsal frontal organ (dorsal frontal eye), ndplc e nerve connection between nauplius eye centre and dorsal portion of the lateral cup, npmc e nerve connection between nauplius eye centre and posterior medial cup, nvfo e nerve connection between nauplius eye centre and ventral frontal organ (ventral frontal eye), nvmc e nerve connection between nauplius eye centre and ventral medial cup, nvplc e nerve connection between nauplius eye centre and ventral portion of the lateral cup, pl e pigment layer, pmc e posterior medial cup, tap e tapetum layer, vfo e ventral frontal organ (ventral frontal eye), vmc e ventral medial cup, vplc e ventral portion of the lateral cup.
sensory fields do not show any similarities to the sensory cells of the nauplius eye or the frontal eyes. The nauplius eye of L. biformis resembles that found in L. tatei, but is located closer to the compound eyes. The ventral medial cup contains two sensory cells, while the dorsal medial cup has seven and both lateral cups approximately 20 sensory cells. The ventral frontal eyes are like those of L. tatei. The dorsal frontal eyes are each composed of only 20 sensory cells, but these are larger than in L. tatei. 3.2. Cyclestherida e Cyclestheria hislopi The nauplius eye of Cyclestheria hislopi is situated ventrally to the fused compound eyes (Fig. 4A, C). Between the two eyes, a canal connects the ‘compound eye chamber’ with the surface of the head via the eye pore (Fig. 4D). The nauplius eye consists of four well-developed eye cups and, in between, a tapetum and a pigment layer (Figs. 3A, B; 4A). The inner pigment layer consists of a few large cells,
which contain dark pigment granules. The tapetum is also cellular and it contains light, reflective pigment granules (Fig. 4E). The nauplius eye is egg-shaped with the broader end to the nauplius eye centre. The shape is the result of the arrangement of the two medial cups that form the posterior part of the eye, each of which contains about 20 cells. These cups are attached at the posterior end and diverge anteriorly, so that they appear to be ‘‘V’’ shaped (Fig. 4C). In between the two branches of the ‘V’, the lateral cups are situated. They are composed of 40 cells each and are triangular in shape. In a frontal and a lateral view, they cover the medial cups completely. All cups have their own nerve connection, but the medial ones fuse just before they enter the nauplius eye centre together. The lateral cups are not divided into two parts as in Lynceus. All the axons of one lateral cup give rise to only one nerve (Fig. 4B). The rhabdomeres of the sensory cells in the lateral cups are fused to a conspicuously large rhabdom (Fig. 4D). Long strands of connective tissue to the epidermis are not present. The nauplius eye takes up almost
Fig. 2. Lynceus tatei (Laevicaudata, Lynceidae) e sections through the head region. (A) Medial section through the head (anterior is left) showing the arrangement of the structures. (B) Horizontal section through the nauplius eye and the dorsal frontal organ, (anterior is at the upper left corner). (C) Frontal section through the nauplius eye and the optical tracts of the compound eyes (dorsal is up). (D) Frontal section through the nauplius eye and the ventral frontal organ (dorsal is up). (E) Frontal section through the ventral frontal organ of the right side showing the orientation of the rhabdomeres towards the cuticle side (‘everse’) of the organ. (F) Frontal section through the nauplius eye and the dorsal frontal organ showing the ‘everse’ orientation of the sensory cells of the dorsal frontal organ and the inverse orientation (towards the tapetum) of the sensory cells of the lateral cup (dorsal is up). (G) Frontal section through the nauplius eye showing a closer view on the sensory cells and the tapetum and pigment layers. (H) Sagittal section through the head region showing the two parts of a lateral cup and the orientation of the nauplius eye in respect to the compound eyes (anterior is left). Scale bars in mm. Abbreviations: bpl e border (cell membranes) between pigment cells, ce e compound eye, cu e cuticle, dfo e dorsal frontal organ (dorsal frontal eye), dplc e dorsal portion of the lateral cup, ech e eye chamber, lc e lateral cup, nc e nerve connections from the two parts of the lateral cup, nce e neuropiles of the compound eyes, ndfo e nerve connection between nauplius eye centre and dorsal frontal organ (dorsal frontal eye), nec e nauplius eye centre, nlc e nerve connection between nauplius eye centre and lateral cup, npc e nucleus of a pigment cell, nsc e nucleus of a sensory cell, ntap e nucleus of a tapetal cell, nvfo e nerve connection between nauplius eye centre and ventral frontal organ (ventral frontal eye), oes e oesophagus, pc e protocerebrum, pl e pigment layer, pmc eposterior medial cup, rh e rhabdomers, sc e sensory cell, sf e setose field, tap e tapetum layer, vfo e ventral frontal organ (ventral frontal eye), vmc e ventral medial cup, vplc e ventral portion of the lateral cup.
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Fig. 3. Cyclestheria hislopi (Cyclestherida) e schematic diagram of the nauplius eye and frontal organs. (A) Lateral view (anterior is left). (B) Frontal view (dorsal is up). Abbreviations: lc e lateral cup, nlc e nerve connection between nauplius eye centre and lateral cup, npmc e nerve connection between nauplius eye centre and posterior medial cup, nvfo e nerve connection between nauplius eye centre and ventral frontal organ (ventral frontal eye), nvmc e nerve connection between nauplius eye centre and ventral medial cup, pl e pigment layer, pmc e posterior medial cup, tap e tapetum layer, vfo e ventral frontal organ (ventral frontal eye), vmc e ventral medial cup.
the whole space ventrally to the compound eye and touches the integument at several points, where it is fixed. In Cyclestheria hislopi, an unpaired ventral frontal eye is present (Fig. 3A, B). It is situated anteriorly and ventrally to the nauplius eye. It is club-shaped and contains 12 sensory cells, which are attached to the epidermis distally. The rhabdomeres are fused to a rhabdom close to the integument. The axons form a nerve leading to the nauplius eye centre (Fig. 4C).
elongated branched bands (Fig. 6D). The lateral cups cover the medial ones completely and have short, very broad nerve connections to the nauplius eye centre. The four cups are separated by a tapetum layer and a pigment layer (Figs. 5B; 6C). There are ligamentous connections to the epidermis in the whole region of the lateral cups. Two of them originate on the dorsal edges of the lateral cups and run parallel to the nerves of the compound eyes and enter the epidermis ventral to the eye chamber. They are flanked proximally by parts of the tapetum and pigment (Fig. 6B), but there are not any sensory cells. No separate frontal eyes were found.
3.3. Spinicaudata (Cyzicidae) e Caenestheriella sp. The conditions in the head of Caenestheriella sp. differ from those described above. Diverticula of the midgut separate the nauplius and compound eyes (Fig. 6A), so that there is not any contact between them unlike in Lynceus or Cyclestheria. The four-cup nauplius eye (Fig. 5A, B) is attached to the ventral epidermis and is surrounded frontally and laterally by the diverticula of the midgut. The ventral medial cup consists of 14 sensory cells with their axons ending in a short nerve to the nauplius eye centre, which is located directly posterior to the eye. The posterior medial cup sits right on the nauplius eye centre, with no noticeable nerve connecting both structures (Fig. 6B). It consists of about 30 sensory cells. The lateral cups are very large (Figs. 5A; 6C) and are formed by approximately 60 cells. Their rhabdomeres are fused to form
3.4. Spinicaudata (Limnadiidae) e Limnadopsis parvispinus The nauplius eye in Limnadopsis parvispinus is the largest in terms of both size and number of sensory cells in the cups (Fig. 8A, D). The nauplius eye lies in a ventral projection of the rostrum. It is long and cone-shaped with a curved tip, and the thicker end of the cone attaches to the ventral epidermis (Fig. 7A, B). The tip reaches the epidermis close to the compound eyes. Between the two contact points to the epidermis, diverticula of the midgut gland are present, as in the Cyzicidae (Fig. 8D). The four eye cups are separated by the layers of tapetum and pigment (Fig. 7A, B). The tapetum partially extends between the distal parts of the sensory cells so
Fig. 4. Cyclestheria hislopi (Cyclestherida) e sections through the head region. (A) Frontal section through the compound eye and the nauplius eye showing the close vicinity of both structures (dorsal is up). (B) Frontal section through the medial cups of the nauplius eye (dorsal is up). (C) Medial section through the head region (anterior is left, dorsal is up) showing the medial cups of the nauplius eye and the unpaired ventral frontal organ. (D) Sagittal section through the nauplius eye and the ventral frontal organ (anterior is left, dorsal is up) showing the rhabdomeric elements of both structures. (E) TEM picture of the nauplius eye showing the two lateral cups and in between the two different kinds of pigment granules in the tapetum and pigment layer. (F) TEM picture of the distal region of a sensory cell in the ventral frontal organ. Scale bars in mm. Abbreviations: ce e compound eye, cec e canal from the anterior cuticle to the eye chamber of the compound eye, cu e cuticle, lc e lateral cup, nce e neuropiles of the compound eyes, nec e nauplius eye centre, nlc e nerve connection between nauplius eye centre and lateral cup, nvfo e nerve connection between nauplius eye centre and ventral frontal organ (ventral frontal eye), pc e protocerebrum, pl e pigment layer, pmc e posterior medial cup, rh e rhabdomers, tap e tapetum layer, vfo e ventral frontal organ (ventral frontal eye), vmc e ventral medial cup.
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Fig. 5. Caenestheriella sp. (Spinicaudata, Cyzicidae) e schematic diagram of the nauplius eye. (A) Lateral view (anterior is left). (B) Frontal view (dorsal is up). Abbreviations: lc e lateral cup, nec e nauplius eye centre, nlc e nerve connection between nauplius eye centre and lateral cup, pl e pigment layer, pmc eposterior medial cup, tap e tapetum layer, vmc e ventral medial cup.
that the ventral medial cup appears to be divided, but this is not the case. The ventral medial cup is very compact and consists of approximately 30 cells. The nerve connection to the nauplius eye centre is very long because of the large distance to the protocerebrum (Fig. 8E). The posterior medial cup is made up of 120 sensory cells arranged in two groups, one above and one below the point at which the cup connects to the nauplius eye centre (Fig. 8D, E). In the lateral regions no division is visible. The lateral cups are very large, extending from the ventral to the anterior contact point with the epidermis, and consist of about 200 sensory cells each, which are connected with the nauplius eye centre via a broad nerve. All the sensory cells are approximately of the same size. The lateral cups form the curved tip of the nauplius eye, and occasionally the tapetum separates groups of sensory cells (Fig. 8B, C). These cells belong to the nauplius eye because their axons are part of the nerve from the lateral cups, and they are clearly inverse with the rhabdomeres directed towards the tapetum (Fig. 8C). Ligamentous connections to the epidermis are found in all regions of the nauplius eye (Fig. 8B, C). Additionally, there are two connections leading to the very elongated nerve strands from the compound eyes to the protocerebrum (Fig. 8A). There are not any additional frontal eyes in the head region of Limnadopsis parvispinus. 4. Discussion 4.1. Comparison with previous findings The nauplius eye in Cyclestheria hislopi was first described by Sars (1887), and our description agrees with his in general aspects such as size, shape and position. Sars (1887) also recognized the ventral frontal eye. The only other laevicaudatan species previously studied is Lynceus brachyurus O.F. Mu¨ller, 1776 (Zograf, 1904; Nowikoff, 1905; Elofsson, 1966). As in L. tatei and L. biformis, the nauplius eye in
L. brachyurus consists of four cups (Nowikoff, 1905; Elofsson, 1966). Some differences exist with regard to the number of cells found in each cup. Elofsson (1966) described only four cells in the posterior median cup in L. brachyurus (8e9 in L. tatei and 7 in L. biformis in the present study) and a few more in the lateral cups (approximately 30 in L. tatei and 20 in L. biformis). The number of cells in the ventral medial cup has consistently been determined as two in all three species. In L. brachyurus, a paired ‘dorsal frontal organ’ (Nowikoff, 1905; ‘distal frontal organ’ in Elofsson, 1966) as well as an unpaired ‘ventral frontal organ’ (Nowikoff, 1905) have been described; the latter, however, was questioned by Elofsson (1966). The presence of the paired ‘dorsal frontal eyes’ and of paired ‘ventral frontal eyes’ is supported, at least for the two species we studied. Elofsson (1966) described the presence of an additional ‘posterior medial frontal organ’ attached to a connective tissue strand from the posterior medial cup to the epidermis. However, we could not find any sensory cells on this strand in L. tatei and L. biformis. A potential source of misinterpretation is the apparent division of the posterior medial cup in the two Lynceus species into a ventral part with four cells and a dorsal part with four to five. However, this ‘division’ only appears in some frontal sections and is caused by a little kink in the cup. In general, our description of Caenestheriella sp. concurs with that of Caenestheria var. salberghi by Elofsson (1966), including the absence of any frontal eyes apart from the nauplius eye. An important difference is the double innervation of the lateral cups and the corresponding division of these cups into two parts described by Elofsson (1966), which we did not find in the Caenestheriella sp. we studied. Here, all the sensory cells in the lateral cups are very similar and there is no division into a ventral part with larger cells and a dorsal part with smaller cells. Several frontal organs have previously been described for the single representative of Limnadiidae studied so far,
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Fig. 6. Caenestheriella sp. (Spinicaudata, Cyzicidae) e sections through the head region. (A) Sagittal section through the whole head region showing the separation of the compound eye and the nauplius eye by the diverticles of the midgut gland (anterior is left). (B) Sagittal section through the nauplius eye and the nauplius eye centre showing the close positions of the posterior medial cup and the nauplius eye centre (anterior is left). (C) Frontal section through the nauplius eye showing the four cups and two of the connective tissue strands that fix the position of the nauplius eye (dorsal is up). (D) Sagittal section through the rhabdomeres of the sensory cells of one lateral cup near to the tapetum layer. Scale bars in mm. Abbreviations: ce e compound eye, cts e connective tissue strand, cu e cuticle, ech e eye chamber, ep e epidermis, lc e lateral cup, mgd e diverticles of the midgut gland, nce e neuropiles of the compound eyes, ne e nauplius eye, nec e nauplius eye centre, pc e protocerebrum, pl e pigment layer, pmc eposterior medial cup, rh e rhabdomers, tap e tapetum layer, vmc e ventral medial cup.
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cell in phyllopod crustaceans. Although the reflective granules certainly function as a tapetum, this kind of tapetum is very different from that found in maxillopodans (Fahrenbach, 1964; Elofsson, 1966; Andersson and Nilsson, 1981). 4.2. Comparison with other Phyllopoda
Fig. 7. Limnadopsis parvispinus (Spinicaudata, Limnadiidae) e schematic diagram of the nauplius eye. (A) Lateral view (anterior is left). (B) Frontal view (dorsal is up). Abbreviations: lc e lateral cup, nec e nauplius eye centre, nlc e nerve connection between nauplius eye centre and lateral cup, npmc e nerve connection between nauplius eye centre and posterior medial cup, nvmc e nerve connection between nauplius eye centre and ventral medial cup, pl e pigment layer, pmc eposterior medial cup, tap e tapetum layer, vmc e ventral medial cup.
Limnadia lenticularis (Linne, 1761). Nowikoff (1905) described a ‘dorsal frontal organ’ in anterior-dorsal elongation of the lateral cups (see also Hanstro¨m, 1934) as well as a ‘ventral frontal organ’ divided into two portions. One portion of the latter is Elofsson’s (1966) ‘distal frontal organ’, the other portion he described as a ‘posterior medial frontal organ’ located at the connective tissue strand from the posterior medial cup to the epidermis. We agree with Elofsson (1966) that the structure interpreted by Nowikoff (1905) as a ‘dorsal frontal organ’ in L. lenticularis consists of connective tissue only. In Limnadopsis, sensory cells are not present and these ligaments serve only to fix the nauplius eye to the epineurium of the neuropile masses of the compound eyes. The ‘distal frontal organ’ described by Elofsson (1966) in L. lenticularis (‘main portion of the ventral frontal organ’, Nowikoff, 1905; see also Hanstro¨m, 1934) is not present in Limnadopsis. Although sensory cells are certainly present in the attenuated rostrum of Limnadia lenticularis (our own observations), these cells might better be considered to be detached parts of the lateral cups of the nauplius eye, an idea which has already been discussed by Elofsson (1966). Furthermore, we were not able to find in Limnadopsis the ‘posterior medial frontal organ’ described by Elofsson (1966). As in Lynceus, the presence or absence of such a ‘posterior medial frontal eye’ needs to be confirmed using TEM. The most remarkable difference between our findings and the most detailed study so far (Elofsson, 1966) is the presence of two different kinds of pigment cells, i.e. tapetum cells with light, reflective pigment granules and pigment cells with dark granules. Elofsson (1966) described just one type of pigment
Recent phylogenetic (mostly molecular) analyses have almost reached a consensus on branchiopod phylogenetic relationships. Anostraca is the sister group to Phyllopoda (e.g., Giribet et al., 2005; Olesen, 2007). Within Phyllopoda, Spinicaudata, Cyclestherida and Cladocera constitute a monophylum (Braband et al., 2002; Richter, 2004; deWaard et al., 2006; Stenderup et al., 2006; Olesen, 2007; Richter et al., 2007), with the two latter as sister groups (e.g., Olesen et al., 1997; Ax, 1999; Taylor et al., 1999; Spears and Abele, 2000; Braband et al., 2002). Morphological analyses tend to result in Notostraca as a sister group to Diplostraca (Olesen, 1998, 2000, 2007; Richter et al., 2007) but recent molecular based analyses suggest Laevicaudata to be the most basal offshoot within Phyllopoda (deWaard et al., 2006; Stenderup et al., 2006; Richter et al., 2007). At any rate, a comparison of the conditions in Laevicaudata and Notostraca seems most likely to give the strongest insights into the ancestral conditions, i.e. the ground pattern of Phyllopoda. Within Notostraca, frontal eyes have been described in Lepidurus apus, L. arcticus and Triops cancriformis (Zograf, 1904; Wenke, 1908; Holmgren, 1916; Elofsson, 1966). Both Notostraca and Laevicaudata possess nauplius eyes made of four distinct cups. Another correspondence concerns the lateral cups that are separated into two portions, each with its own nerve. Elofsson (1966) noticed within Notostraca that the two nerves are sometimes fused. A potential explanation for the partition of the lateral cups is given by Dahl (1959), who has provided the only detailed study so far on the development of the nauplius eyes in phyllopods. According to Dahl (1959), the newly hatched larvae in Triops possess a small nauplius eye consisting of two pigment cells and three cups. These three cups apparently correspond (as in the interpretation by Elofsson, 1966) to the ventral medial cup and the ventral portions of the lateral cups. The dorsal portions of the lateral cups and the posterior medial cup were added later in development by the proliferation of two zones, the ‘‘dorso-lateral lobes of the brain’’ (Dahl, 1959). This means that the two partitions of the lateral cups in adult Notostraca and Laevicaudata are of separate ontogenetic origin, a state which might even be persistent in certain representatives of Spinicaudata (Caenestheria salberghi, Elofsson, 1966) but not in others (present study). The developmental findings are obviously also of importance for a comparison with the nauplius eye in Anostraca (see Elofsson, 1966 for Branchinecta paludosa, Tanymastix stagnalis, Polyartemia forcipata and Chirocephalus sp. and Moroff, 1912; Anado´n and Anado´n, 1980; Criel, 1991, and Martin, 1992 for Artemia salina). The nauplius eye in all studied species consists of two lateral cups and one ventral medial cup, which correspond exactly to the larva in Triops. This might indeed provide an argument that the
Fig. 8. Limnadopsis parvispinus (Spinicaudata, Limnadiidae) e sections through the head region. (A) Frontal section through the nauplius eye (dorsal is up). (B) Sagittal section through the anterior-dorsal part of the nauplius eye (elongations of the lateral cups and the posterior medial cup) showing the continuity of the cups and the associated tapetum layer (anterior is left). (C) Same as B showing the inverse orientation of the sensory cells towards the tapetum in the elongation of cups. (D) Sagittal section through the head region showing the orientation of the nauplius eye and the diverticles of the midgut gland in between the nauplius eye and the compound eye. (E) Sagittal section through the ventral part of the nauplius eye showing the innervations of the medial cups and the close positions of the nauplius eye centre and the dorsal medial cup (anterior is left). (F) Frontal section through the antero-ventral part of the nauplius eye showing the close attachment to the cuticle in the rostral region (dorsal is up). Scale bars in mm. Abbreviations: asc e axons of the sensory cells of the lateral cup, ce e compound eye, cts e connective tissue strand, cu e cuticle, elo e elongations of the lateral cups, lc e lateral cup, mgd e diverticles of the midgut gland, mu e muscle fibers, nce e neuropiles of the compound eyes, nec e nauplius eye centre, npmc e nerve connection between nauplius eye centre and posterior medial cup, nvmc e nerve connection between nauplius eye centre and ventral medial cup, oes e oesophagus, pc e protocerebrum, pl e pigment layer, pmc eposterior medial cup, rh e rhabdomers, tap e tapetum layer, to e tractus opticus, vmc e ventral medial cup.
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four-cup nauplius eye is an autapomorphy of Phyllopoda (e.g., Elofsson, 1966; Walossek, 1993) and does not represent the plesiomorphic condition of the crustacean ground pattern (e.g., Paulus, 1972, 1979; Lauterbach, 1983). This is also corroborated by other crustaceans possessing a nauplius eye consisting of only three cups, if a nauplius eye is present at all (Elofsson, 1965, 1966, 1992). Another interesting aspect concerns the presence of two different kinds of pigment granules, forming distinct tapetum and pigment layers. These two types of granules also seem to be present in Triops and Lepidurus (Claus, 1891; Zograf, 1904; our own observations) whereas Artemia (Anostraca) only seems to have one kind of pigment cell (Anado´n and Anado´n, 1980), which implies that the reflective pigment granules in phyllopodan nauplius eyes might represent another autapomorphy of this taxon, particularly considering that a tapetum structured completely differently (the tapetal cells are filled with refractive crystals in the form of platelets, e.g. Fahrenbach, 1964; Andersson and Nilsson, 1981) is present in maxillopodan crustaceans. In addition to the nauplius eye, a paired ventral frontal eye formed by two strands of sensory cells which fuse in front of the nauplius eye is present in Anostraca (e.g., Elofsson, 1966). Based on its position ventrally to the medial cup of the nauplius eye, this paired ventral frontal eye can be considered homologous to that in Laevicaudata (also paired) and Cyclestherida (unpaired) and therefore might belong to the phyllopod ground pattern, which, however, implies that it has been reduced in Notostraca. The paired dorsal frontal eyes seem to be restricted to Laevicaudata. The position of the nauplius eye in Spinicaudata, separated from the compound eyes by the midgut diverticula, is very distinct, as is its triangular shape, and in comparison with Notostraca, Laevicaudata and Cyclestherida, this can be most easily interpreted as an autapomorphy of Spinicaudata (see also Richter et al., 2007). In comparison to the descriptions above, the nauplius eye in Cladocera is much reduced (Claus, 1891; Elofsson, 1966). Four cups are only discernible in some Anomopoda, and even then they consist of very few cells. For an overview we refer to Elofsson (2006). It is worth mentioning here the conditions in Eurycercus lamellatus (O.F. Mu¨ller, 1785), where the nauplius eye is comparably well developed. There are four cells in each of the two medial cups and three cells in each of the lateral cups, with each cup having its own nerve to the nauplius eye centre (Elofsson, 1966 and our own observations). Paired ventral and dorsal frontal eyes are present but their homology with those in other branchiopods is difficult to establish. Acknowledgments We would like to thank Dr. Mark Grygier for supplying us with Lynceus biformis from Japan as part of the project at Lake Biwa Museum ‘Research on Large Branchiopods Inhabiting Rice Paddies’, and Dr. Brian Timms for assisting SR in collecting material from Australia in 1999. The collection in Australia was made possible by a Visiting Fellowship of the
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