Background How does intraspecific variation relate to macroevolutionary change in morphology? This question can be addressed in species in which derived characters are present but not fixed. In rhabditid nematodes, the arrangement of the nine bilateral pairs of peripheral sense organs (rays) in tails of males is often the most highly divergent character between species. The development of ray pattern involves inputs from hometic gene expression patterns, TGFβ signalling, Wnt signalling, and other genetic pathways. In  Caenorhabditis briggsae , strain-specific variation in ray pattern has provided an entrée into the evolution of ray pattern. Some strains were fixed for a derived pattern. Other strains were more plastic and exhibited derived and ancestral patterns at equal frequencies.
Background
How does intraspecific variation relate to macroevolutionary change in morphology? This question can be addressed in species in which derived characters are present but not fixed. In rhabditid nematodes, the arrangement of the nine bilateral pairs of peripheral sense organs (rays) in tails of males is often the most highly divergent character between species. The development of ray pattern involves inputs from hometic gene expression patterns, TGFβ signalling, Wnt signalling, and other genetic pathways. In  Caenorhabditis briggsae , strain-specific variation in ray pattern has provided an entrée into the evolution of ray pattern. Some strains were fixed for a derived pattern. Other strains were more plastic and exhibited derived and ancestral patterns at equal frequencies.Caenorhabditis briggsaeResults Recombinant inbred lines (RILs) constructed from crosses between the variant  C. briggsae  AF16 and HK104 strains exhibited a wide range of phenotypes including some that were more extreme than either parental strain. Transgressive segregation was significantly associated with allelic variation in the  C. briggsae  homolog of  abdominal B ,  Cb-egl-5 . At least two genes that affected different elements of ray pattern, ray position and ray fusion, were linked to a second gene,  mip-1 . Consistent with this, the segregation of ray position and ray fusion phenotypes were only partially correlated in the RILs.
Results
Recombinant inbred lines (RILs) constructed from crosses between the variant  C. briggsae  AF16 and HK104 strains exhibited a wide range of phenotypes including some that were more extreme than either parental strain. Transgressive segregation was significantly associated with allelic variation in the  C. briggsae  homolog of  abdominal B ,  Cb-egl-5 . At least two genes that affected different elements of ray pattern, ray position and ray fusion, were linked to a second gene,  mip-1 . Consistent with this, the segregation of ray position and ray fusion phenotypes were only partially correlated in the RILs.C. briggsaeC. briggsaeabdominal BCb-egl-5mip-1Conclusions The evolution of ray pattern has involved allelic variation at multiple loci. Some of these loci impact the specification of ray identities and simultaneously affect multiple ray pattern elements. Others impact individual characters and are not constrained by covariance with other ray pattern elements. Among the genetic pathways that may be involved in ray pattern evolution is specification of anteroposterior positional information by homeotic genes.
Conclusions
The evolution of ray pattern has involved allelic variation at multiple loci. Some of these loci impact the specification of ray identities and simultaneously affect multiple ray pattern elements. Others impact individual characters and are not constrained by covariance with other ray pattern elements. Among the genetic pathways that may be involved in ray pattern evolution is specification of anteroposterior positional information by homeotic genes.

Background A fundamental problem of morphological evolution is how does microevolution relate to macroevolution [ 1 ]. Are variation and selection within species sufficient to account for genetic divergence between species? One approach to this problem has been comparative developmental genetic studies in closely related species. This approach is complicated by developmental redundancy and cryptic variation [ 2 - 4 ]. Developmental redundancy occurs in species pairs in which similar adult morphologies are attained through distinct developmental processes. For example, in the nematodes  Caenorhabditis elegans  and  Oscheius tipulae , cells in the vulval equivalence group that do not participate in vulval development are eliminated through fusion with the multinucleated syncytial epidermis [ 5 - 8 ]. In  Pristionchus pacificus , that same outcome is achieved through apoptosis [ 9 ]. Cryptic variation occurs in species pairs in which the same developmental process is regulated through divergent genetic mechanisms. For example,  Drosophila melanogaster  and  D. simulans  have similar numbers and patterns of bristles. Hybrids have fewer bristles than either parental species [ 10 , 11 ]. Thus, the conserved bristle phenotypes of  D. melanogaster  and  D. simulans  mask cryptic variation in the genetic regulation of bristle pattern. Another approach has been the study of morphological variation within species. In the butterfly  Bicyclus anynana , expression patterns of  distalless  ( dll ),  engrailed  ( en ), and  spalt  ( sal ) coincide with eyespot coloration patterns [ 12 , 13 ]. Moreover, allelic variation in  distalless  has been associated with size variation in eyespots [ 12 ]. Thus, evolution of eyespot patterns may have resulted from selection for variant alleles of  dll ,  en , and  sal . Consistent with this model, conservation of the expression patterns of these genes in developing eyespots has been demonstrated in multiple butterfly species [ 13 ]. Developmental variation also has been characterized in several nematode species. For example, variant patterns of cell division in the ventral epidermis have been described in  C. elegans ,  C. briggsae , and  O. tipulae  [ 14 ]. Another promising model for microevolutionary studies of morphological evolution is ray pattern variation in rhabditid nematodes. The rays are male-specific peripheral sense organs that mediate mating behavior [ 15 - 17 ]. In most rhabditid species there are nine bilateral pairs of rays embedded within the copulatory bursa of the tail [ 17 ]. The pattern of rays; their anteroposterior placement and the dorsoventral position of their sensory endings, generally are constant within species but often are the most divergent character between species [ 18 ]. Ray pattern in adult males is determined by cell contacts that form between ray structural cells and the surrounding cells of the lateral epidermis in L4 larvae [ 19 , 20 ]. These contacts in turn are determined by the specification of 'ray identities' [ 21 ]. In  Caenorhabditis elegans , ray identities are specified through the integration of several regulatory inputs including: positional information from homeotic genes [ 22 - 26 ]; TGF-beta-like signalling [ 27 - 29 ]; Wnt signalling [ 25 ]; and ephrin/semaphorin signalling [ 30 ]. The ray pattern of  Caenorhabditis briggsae  differs from that of  C. elegans  in the placement of ray 3 [ 31 , 32 ]. In  C. elegans  males, ray 3 is located at the cloaca and is separate from from all other rays. In the canonical  C. briggsae  ray pattern, ray 3 is located in a posterior cluster of rays 3–6 and frequently is fused with ray 4. The  C. elegans  ray pattern is shared with several other  Caenorhabditis  species and is ancestral to the Elegans-group [ 33 ], a monophyletic clade that includes  C. briggsae  [ 34 - 36 ]. The  C. briggsae  ray pattern is mimicked by several  C. elegans  mutations including mutations in homeotic genes or in genes that regulate homeotic gene expression patterns [ 21 , 22 , 26 , 37 , 38 ]. Thus, the derived  C. briggsae  ray pattern may have arisen through changes in the specification of anteroposterior positional identities. It now is possible to address this hypothesis as variant  C. briggsae  strains that express an ancestral ray pattern at high frequencies have been identified [ 39 ]. In this paper, the segregation of ray pattern phenotypes in crosses between  C. briggsae  strains is described.
Background
A fundamental problem of morphological evolution is how does microevolution relate to macroevolution [ 1 ]. Are variation and selection within species sufficient to account for genetic divergence between species? One approach to this problem has been comparative developmental genetic studies in closely related species. This approach is complicated by developmental redundancy and cryptic variation [ 2 - 4 ]. Developmental redundancy occurs in species pairs in which similar adult morphologies are attained through distinct developmental processes. For example, in the nematodes  Caenorhabditis elegans  and  Oscheius tipulae , cells in the vulval equivalence group that do not participate in vulval development are eliminated through fusion with the multinucleated syncytial epidermis [ 5 - 8 ]. In  Pristionchus pacificus , that same outcome is achieved through apoptosis [ 9 ]. Cryptic variation occurs in species pairs in which the same developmental process is regulated through divergent genetic mechanisms. For example,  Drosophila melanogaster  and  D. simulans  have similar numbers and patterns of bristles. Hybrids have fewer bristles than either parental species [ 10 , 11 ]. Thus, the conserved bristle phenotypes of  D. melanogaster  and  D. simulans  mask cryptic variation in the genetic regulation of bristle pattern.Caenorhabditis elegansOscheius tipulaePristionchus pacificusDrosophila melanogasterD. simulansD. melanogasterD. simulansAnother approach has been the study of morphological variation within species. In the butterfly  Bicyclus anynana , expression patterns of  distalless  ( dll ),  engrailed  ( en ), and  spalt  ( sal ) coincide with eyespot coloration patterns [ 12 , 13 ]. Moreover, allelic variation in  distalless  has been associated with size variation in eyespots [ 12 ]. Thus, evolution of eyespot patterns may have resulted from selection for variant alleles of  dll ,  en , and  sal . Consistent with this model, conservation of the expression patterns of these genes in developing eyespots has been demonstrated in multiple butterfly species [ 13 ]. Developmental variation also has been characterized in several nematode species. For example, variant patterns of cell division in the ventral epidermis have been described in  C. elegans ,  C. briggsae , and  O. tipulae  [ 14 ].Bicyclus anynanadistallessdllengrailedenspaltsaldistallessdllensalC. elegansC. briggsaeO. tipulaeAnother promising model for microevolutionary studies of morphological evolution is ray pattern variation in rhabditid nematodes. The rays are male-specific peripheral sense organs that mediate mating behavior [ 15 - 17 ]. In most rhabditid species there are nine bilateral pairs of rays embedded within the copulatory bursa of the tail [ 17 ]. The pattern of rays; their anteroposterior placement and the dorsoventral position of their sensory endings, generally are constant within species but often are the most divergent character between species [ 18 ]. Ray pattern in adult males is determined by cell contacts that form between ray structural cells and the surrounding cells of the lateral epidermis in L4 larvae [ 19 , 20 ]. These contacts in turn are determined by the specification of 'ray identities' [ 21 ]. In  Caenorhabditis elegans , ray identities are specified through the integration of several regulatory inputs including: positional information from homeotic genes [ 22 - 26 ]; TGF-beta-like signalling [ 27 - 29 ]; Wnt signalling [ 25 ]; and ephrin/semaphorin signalling [ 30 ].Caenorhabditis elegansThe ray pattern of  Caenorhabditis briggsae  differs from that of  C. elegans  in the placement of ray 3 [ 31 , 32 ]. In  C. elegans  males, ray 3 is located at the cloaca and is separate from from all other rays. In the canonical  C. briggsae  ray pattern, ray 3 is located in a posterior cluster of rays 3–6 and frequently is fused with ray 4. The  C. elegans  ray pattern is shared with several other  Caenorhabditis  species and is ancestral to the Elegans-group [ 33 ], a monophyletic clade that includes  C. briggsae  [ 34 - 36 ]. The  C. briggsae  ray pattern is mimicked by several  C. elegans  mutations including mutations in homeotic genes or in genes that regulate homeotic gene expression patterns [ 21 , 22 , 26 , 37 , 38 ]. Thus, the derived  C. briggsae  ray pattern may have arisen through changes in the specification of anteroposterior positional identities. It now is possible to address this hypothesis as variant  C. briggsae  strains that express an ancestral ray pattern at high frequencies have been identified [ 39 ]. In this paper, the segregation of ray pattern phenotypes in crosses between  C. briggsae  strains is described.Caenorhabditis briggsaeC. elegansC. elegansC. briggsaeC. elegansCaenorhabditisC. briggsaeC. briggsaeC. elegansC. briggsaeC. briggsaeC. briggsaeResults The elements of ray pattern The male-specific rays of rhabditid nematodes are embedded within a lateral fold of cuticle called the bursa or tail fan [ 19 ] (Figure  1 ). The sensory endings of most rays are attached to and open through the surface of the bursa. Ray pattern refers collectively to the anteroposterior positions of each ray, the surface (dorsal, ventral or medial) through which their sensory endings are exposed, and whether or not individual rays are fused with each other. Rays also differ in regard to neurotransmitter usage [ 5 , 21 , 40 ]. Mutations that affect ray pattern also affect neurotransmitter usage indicating that multiple properties of rays arise through the specification of 'ray identities' [ 21 , 24 ]. Figure 1 Ray patterns of  C. briggsae  and  C. elegans . Ventral views of a)  C. elegans  and b)  C. briggsae  male tails. Anterior to the left. Left side up. Bilateral pairs of rays are numbered from anterior to posterior. a)  C. elegans  pattern in which ray 3 is separate from all other rays. This pattern is referred as a 2(1)3+3 pattern. b)  C. briggsae  pattern in which ray 3 is clustered with rays 4 – 6. This pattern is referred to as a 2/4+3 pattern. In this pattern ray 3 may be either free (right side) or fused with ray 4 (left side). The 2(1)3+3 pattern is ancestral to the Elegans-group, a monophyletic clade that includes  C. elegans  and  C. briggsae  [34]. The derived ray pattern of  C. briggsae  entails the posterior displacement of ray 3 from a position level with the cloaca to the post-cloacal cluster of rays 4 – 6 and the frequent fusion of ray 3 with ray 4 [ 31 , 32 , 34 ]. This ray pattern is exhibited in nearly all males of  C. briggsae  strains AF16 and VT847 [ 39 ]. Ray pattern in  C. briggsae  strains HK104, HK105, and PB800 is more variable. These strains exhibit the derived and ancestral ray patterns at approximately equal frequencies [ 39 ]. Segregation of  C. briggsae  ray pattern phenotypes The genetic basis of ray pattern variation in  C. briggsae  was characterized through the segregation of ray pattern phenotypes in a set of recombinant inbred lines (RILs). These RILs were constructed from a cross between strains AF16 and HK104. Individual RILs were established from F2 progeny of this cross. RILs were inbred, one hermaphrodite per generation, through F11. Based on this degree of inbreeding, 99.9% of loci in each RIL should be homozygous. RILs were scored for two aspects of ray pattern; the position of ray 3 and the fusion of rays 3 and 4 (Figure  2 ). Figure 2 Segregation of ray pattern phenotypes in  C. briggsae  RILs. a) Scatter plot comparing frequencies of derived character states for the position of ray 3 (Y-axis) and fusion of rays 3 and 4 (X-axis) in RILs. Each point represents an individual strain for which a minimum of 100 sides were scored for ray pattern. Parental strains, AF16 and HK104 represented by red square and red diamond, respectively. RILs represented by closed circles. Correlation coefficient, r, for ray position and fusion equaled 0.80. b) Frequency distributions for derived character states of ray 3 position (black bars) and ray 3–4 fusion (red bars) among RILs. Several RILs exhibited ancestral or derived phenotypes at frequencies more extreme than either parental strain (Figure  2a ). This was true for both the position of ray 3 and for fusions of rays 3 and 4. For ray 3 position, one quarter of the RILs exhibited phenotypes more extreme than either parental strain. For ray fusions, half of the RILs were more extreme than the parental strains. Such transgressive segregation is best explained through the presence, in both AF16 and HK104, of alleles at some loci that promoted ancestral character states and alleles at other loci that promoted derived character states. The high frequency of RILs with extreme phenotypes indicated that both parental strains likely were fixed for antagonistic alleles at multiple loci. A relatively strong correlation was observed between variation in the position of ray 3 and its fusion with ray 4 (r = 0.797; Figure  2a ). This correlation was highly significant (p = 2.5 × 10 -23 ). This was expected as rays 3 and 4 could not have fused unless they were adjacent to each other [ 19 ] and because the placement of rays and fusion between rays have been considered to be pleiotropic phenotypes derivative of the specification of ray identities [ 19 , 22 - 25 , 27 - 30 ]. If anything, it was surprising that the correlation was not stronger. Based on the observed correlation coefficient, approximately one third of the variation in ray fusion frequency was independent of variation in the position of ray 3. This was readily apparent when the frequency distributions of these two ray pattern elements were compared (Figure  2b ). The frequency distribution for the placement of ray 3 was highly skewed toward the derived phenotype. The frequency distribution for fusions of rays 3 and 4 was much broader and was only slightly skewed. The differences in the shapes of these distributions were significant (Kolmgorov-Smirnov, D = 0.4396, p < 0.001). Neither the frequency distribution for the placement of ray 3 nor that for the fusion of ray3 to ray 4 were normal (p = 0.00 and p = 0.03, respectively; Figure  2b ). The frequency distribution for the placement of ray 3 was highly skewed toward the derived phenotype. This result was inconsistent with the placement of ray 3 being governed strictly by additive affects. More likely, the placement of ray 3 was regulated by epistasis. Twentynine per cent of RILs exhibited the most extreme phenotype (Figure  2b ). Fixation of derived alleles at as few as two major-effect genes could have been sufficient to ensure a posterior localization of ray 3 at this frequency. The frequency distribution for fusions of rays 3 and 4 was very broad and and only slightly skewed. This was consistent with ray fusion being a complex character with inputs from multiple developmental processes. At least one of these processes was shared with ray positioning; rays 3 and 4 could not have fused if they were not adjacent. Association of ray pattern with allelic variation in homeotic genes The derived  C. briggsae  ray pattern may have evolved through changes in homeotic gene expression patterns [ 33 , 39 ]. The homeotic genes most likely to affect ray pattern are  mab-5  and  egl-5 . These genes are expressed in the posterior body and tail regions of  C. elegans  [ 40 ] and mutations in them can alter ray pattern [ 22 ]. Allelic variants that affect the size of the first intron of  Cb-egl-5  have been identified (Figure  3 ). The AF16::HK104 RILs were genotyped for these variants to test  Cb-egl-5  for association with ray pattern variation [see  Additional File 1 ]. Figure 3 Allelic variation of  Cb-egl-5 .  Cb-egl-5  amplification products of 1) AF16; 2) PB800; 3) HK104; 4–17) selected RILs; 18) an HK104/AF16 heterozygote. The expected amplification product size based on the  C. briggsae  AF16 genome sequence was 1,723 bp. m) Marker DNA, sizes of selected markers as indicated. Significant associations were observed between  Cb-egl-5  allelic variants and both the position of ray 3 and the fusion of ray 3 to ray 4 (Table  1 ). These associations were transgressive; the HK104 allele of  Cb-egl-5  cosegregated with derived phenotypes, the AF16 allele with ancestral phenotypes. Allelic variants in  Cb-mab-5  have not been identified. However, the  Cb-mab-5  (=CBG00029) and  Cb-egl-5  (=CBG00023) are located within 60 kb of each other [ 41 , 42 ]. Because of this close physical linkage, allelic variants in these genes very likely cosegregated. Thus, allelic variation in  Cb-mab-5 ,  Cb-egl-5 , and/or other linked loci may have been responsible for observed transgressive associations. Table 1 Cosegregation of  Cb-egl-5  with ray pattern phenotypes. allele 1 phenotype AF16 HK104 p value ray position 0.81 0.90 6.2 × 10 -4 ray fusion 0.42 0.68 4.2 × 10 -5 1  Mean frequency of derived character states exhibited in RILs homozygous for either parental  Cb-egl-5  allele. mip-1  marker-assisted introgression The segregation of phenotypes in the RILs indicated that multiple major-effect and additive loci were involved in ray pattern variation in  C. briggsae . Because a high density recombination map for  C. briggsae  has not yet been constructed, it is not possible to identify these loci through QTL analyses. However, a limited number of mutations with visible phenotypes have been mapped to  C. briggsae  chromosomes [B. Gupta, pers. comm., [ 43 ]]. These mutations all were generated in an AF16 background. One such mutation defines the gene  mip-1 .  mip-1  very likely is the  C. briggsae  homolog of  C. elegans unc-22  [D. Baillie, pers. comm.]. In  C. elegans ,  unc-22  is located on chromosome IV and is linked to several genes known to regulate homeotic gene expression patterns, e.g.  lin-49  which encodes a bromodomain protein thought to be involved in chromatin remodeling [ 26 ].  mip-1  marker-assisted introgression was used to determine if any genes linked to it were involved in ray pattern variation in  C. briggsae . Four  C. briggsae  strains were constructed in which HK104 DNA, in the region of  mip-1 , has been introgressed into an otherwise AF16 background. This was accomplished through a series of backcrosses that were initiated by mating HK104 males to  mip-1  (AF16) hermaphrodites. For each introgressed strain, F1 through F6 males were crossed to  mip-1  (AF16) hermaphrodites. Wild-type F7 hermaphrodites were selected and propagated by self-fertilization. From each F7 hermaphrodite, multiple wild-type F8 hermaphrodites were picked. Homozygous strains were established from F8 hermaphrodites that segregated only wild-type progeny. From each set of backcrosses, a single introgressed strain was retained. These introgressed strains were scored for the position of ray 3 and for ray 3–4 fusions (Figure  4 ). The ray patterns of all four introgressed strains differed significantly from AF16 both in the placement of ray 3 and in the frequency with which ray 3 fused to ray 4 (Table  2 ). Significant differences also were apparent between some pairs of introgressed strains, most notably between PB1060 and PB1065. PB1060 and PB1065 did not differ in the placement of ray 3 but only in the frequency with which ray 3 fused to ray 4 (Figure  4 ; Table  2 ). Thus, it appears that allelic variation in at least two genes linked to  mip-1  is involved in ray pattern variation. One of these genes affects the position of ray 3 and possibly the frequency of ray fusion. The HK104 allele of this gene was present in all  mip-1  introgressed strains. The other gene affects only the frequency of ray fusion. The HK104 allele of this gene was present in PB1065 but not in PB1060. As more  C. briggsae  mutant strains become available, it should be possible to identify these genes through additional introgression studies coupled with genotypic characterizations of introgressed DNA. Table 2 Comparison of  mip-1  introgressed lines. p values for reciprocal chi-squared tests 1 AF16 PB1060 PB1061 PB1062 PB1065 ray pattern 2 AF16 --- 9.6 × 10 -30 3.3 × 10 -54 6.5 × 10 -32 5.3 × 10 -46 PB1060 7.9 × 10 -4 --- 0.031 0.093 1.2 × 10 -5 PB1061 2.2 × 10 -7 0.026 --- 0.367 0.011 PB1062 2.8 × 10 -6 0.092 0.293 --- 0.091 PB1065 2.3 × 10 -9 1.3 × 10 -4 0.038 0.151 --- ray position 3 AF16 --- 6.9 × 10 -4 1.5 × 10 -5 3.6 × 10 -4 3.2 × 10 -4 PB1060 1.32 × 10 -29 --- 0.068 0.72 0.67 PB1061 6.4 × 10 -53 0.053 --- 0.12 0.14 PB1062 8.3 × 10 -30 0.73 0.16 --- 0.95 PB1065 1.65 × 10 -36 0.66 0.14 0.95 --- ray fusion 4 AF16 --- 0.13 5.3 × 10 -3 3.1 × 10 -3 9.8 × 10 -6 PB1060 0.013 --- 0.061 0.033 3.1 × 10 -5 PB1061 4.74 × 10 -6 0.057 --- 0.80 0.029 PB1062 9.37 × 10 -7 0.030 0.80 --- 0.052 PB1065 1.33 × 10 -16 1.59 × 10 -6 0.013 0.028 --- 1  Probabilities in each column were determined using frequencies of different ray patterns in each strain (column head) as the null hypothesis. 2  p values for the complete ray pattern (3 posterior and fused, 3 posterior not fused, 3 anterior). 3  p values for ray placement (anterior vs posterior). 4  p values for fusion of rays 3 and 4 when ray 3 is in a posterior position. Figure 4 Comparisons of ray pattern phenotypes in  mip-1  introgressed strains. Frequencies of ray pattern phenotypes exhibited in AF16 and four  mip-1  introgressed strains. Black bars represents ray 3 in a posterior position and fused with ray 4. Red bars represents ray 3 in a posterior position but not fused with ray 4. White bars represents ray 3 in an anterior postion.
Results
The elements of ray pattern The male-specific rays of rhabditid nematodes are embedded within a lateral fold of cuticle called the bursa or tail fan [ 19 ] (Figure  1 ). The sensory endings of most rays are attached to and open through the surface of the bursa. Ray pattern refers collectively to the anteroposterior positions of each ray, the surface (dorsal, ventral or medial) through which their sensory endings are exposed, and whether or not individual rays are fused with each other. Rays also differ in regard to neurotransmitter usage [ 5 , 21 , 40 ]. Mutations that affect ray pattern also affect neurotransmitter usage indicating that multiple properties of rays arise through the specification of 'ray identities' [ 21 , 24 ]. Figure 1 Ray patterns of  C. briggsae  and  C. elegans . Ventral views of a)  C. elegans  and b)  C. briggsae  male tails. Anterior to the left. Left side up. Bilateral pairs of rays are numbered from anterior to posterior. a)  C. elegans  pattern in which ray 3 is separate from all other rays. This pattern is referred as a 2(1)3+3 pattern. b)  C. briggsae  pattern in which ray 3 is clustered with rays 4 – 6. This pattern is referred to as a 2/4+3 pattern. In this pattern ray 3 may be either free (right side) or fused with ray 4 (left side). The 2(1)3+3 pattern is ancestral to the Elegans-group, a monophyletic clade that includes  C. elegans  and  C. briggsae  [34]. The derived ray pattern of  C. briggsae  entails the posterior displacement of ray 3 from a position level with the cloaca to the post-cloacal cluster of rays 4 – 6 and the frequent fusion of ray 3 with ray 4 [ 31 , 32 , 34 ]. This ray pattern is exhibited in nearly all males of  C. briggsae  strains AF16 and VT847 [ 39 ]. Ray pattern in  C. briggsae  strains HK104, HK105, and PB800 is more variable. These strains exhibit the derived and ancestral ray patterns at approximately equal frequencies [ 39 ].
The elements of ray pattern
The male-specific rays of rhabditid nematodes are embedded within a lateral fold of cuticle called the bursa or tail fan [ 19 ] (Figure  1 ). The sensory endings of most rays are attached to and open through the surface of the bursa. Ray pattern refers collectively to the anteroposterior positions of each ray, the surface (dorsal, ventral or medial) through which their sensory endings are exposed, and whether or not individual rays are fused with each other. Rays also differ in regard to neurotransmitter usage [ 5 , 21 , 40 ]. Mutations that affect ray pattern also affect neurotransmitter usage indicating that multiple properties of rays arise through the specification of 'ray identities' [ 21 , 24 ].The derived ray pattern of  C. briggsae  entails the posterior displacement of ray 3 from a position level with the cloaca to the post-cloacal cluster of rays 4 – 6 and the frequent fusion of ray 3 with ray 4 [ 31 , 32 , 34 ]. This ray pattern is exhibited in nearly all males of  C. briggsae  strains AF16 and VT847 [ 39 ]. Ray pattern in  C. briggsae  strains HK104, HK105, and PB800 is more variable. These strains exhibit the derived and ancestral ray patterns at approximately equal frequencies [ 39 ].C. briggsaeC. briggsaeC. briggsaeSegregation of  C. briggsae  ray pattern phenotypes The genetic basis of ray pattern variation in  C. briggsae  was characterized through the segregation of ray pattern phenotypes in a set of recombinant inbred lines (RILs). These RILs were constructed from a cross between strains AF16 and HK104. Individual RILs were established from F2 progeny of this cross. RILs were inbred, one hermaphrodite per generation, through F11. Based on this degree of inbreeding, 99.9% of loci in each RIL should be homozygous. RILs were scored for two aspects of ray pattern; the position of ray 3 and the fusion of rays 3 and 4 (Figure  2 ). Figure 2 Segregation of ray pattern phenotypes in  C. briggsae  RILs. a) Scatter plot comparing frequencies of derived character states for the position of ray 3 (Y-axis) and fusion of rays 3 and 4 (X-axis) in RILs. Each point represents an individual strain for which a minimum of 100 sides were scored for ray pattern. Parental strains, AF16 and HK104 represented by red square and red diamond, respectively. RILs represented by closed circles. Correlation coefficient, r, for ray position and fusion equaled 0.80. b) Frequency distributions for derived character states of ray 3 position (black bars) and ray 3–4 fusion (red bars) among RILs. Several RILs exhibited ancestral or derived phenotypes at frequencies more extreme than either parental strain (Figure  2a ). This was true for both the position of ray 3 and for fusions of rays 3 and 4. For ray 3 position, one quarter of the RILs exhibited phenotypes more extreme than either parental strain. For ray fusions, half of the RILs were more extreme than the parental strains. Such transgressive segregation is best explained through the presence, in both AF16 and HK104, of alleles at some loci that promoted ancestral character states and alleles at other loci that promoted derived character states. The high frequency of RILs with extreme phenotypes indicated that both parental strains likely were fixed for antagonistic alleles at multiple loci. A relatively strong correlation was observed between variation in the position of ray 3 and its fusion with ray 4 (r = 0.797; Figure  2a ). This correlation was highly significant (p = 2.5 × 10 -23 ). This was expected as rays 3 and 4 could not have fused unless they were adjacent to each other [ 19 ] and because the placement of rays and fusion between rays have been considered to be pleiotropic phenotypes derivative of the specification of ray identities [ 19 , 22 - 25 , 27 - 30 ]. If anything, it was surprising that the correlation was not stronger. Based on the observed correlation coefficient, approximately one third of the variation in ray fusion frequency was independent of variation in the position of ray 3. This was readily apparent when the frequency distributions of these two ray pattern elements were compared (Figure  2b ). The frequency distribution for the placement of ray 3 was highly skewed toward the derived phenotype. The frequency distribution for fusions of rays 3 and 4 was much broader and was only slightly skewed. The differences in the shapes of these distributions were significant (Kolmgorov-Smirnov, D = 0.4396, p < 0.001). Neither the frequency distribution for the placement of ray 3 nor that for the fusion of ray3 to ray 4 were normal (p = 0.00 and p = 0.03, respectively; Figure  2b ). The frequency distribution for the placement of ray 3 was highly skewed toward the derived phenotype. This result was inconsistent with the placement of ray 3 being governed strictly by additive affects. More likely, the placement of ray 3 was regulated by epistasis. Twentynine per cent of RILs exhibited the most extreme phenotype (Figure  2b ). Fixation of derived alleles at as few as two major-effect genes could have been sufficient to ensure a posterior localization of ray 3 at this frequency. The frequency distribution for fusions of rays 3 and 4 was very broad and and only slightly skewed. This was consistent with ray fusion being a complex character with inputs from multiple developmental processes. At least one of these processes was shared with ray positioning; rays 3 and 4 could not have fused if they were not adjacent.
Segregation of  C. briggsae  ray pattern phenotypes
C. briggsaeThe genetic basis of ray pattern variation in  C. briggsae  was characterized through the segregation of ray pattern phenotypes in a set of recombinant inbred lines (RILs). These RILs were constructed from a cross between strains AF16 and HK104. Individual RILs were established from F2 progeny of this cross. RILs were inbred, one hermaphrodite per generation, through F11. Based on this degree of inbreeding, 99.9% of loci in each RIL should be homozygous. RILs were scored for two aspects of ray pattern; the position of ray 3 and the fusion of rays 3 and 4 (Figure  2 ).C. briggsaeSeveral RILs exhibited ancestral or derived phenotypes at frequencies more extreme than either parental strain (Figure  2a ). This was true for both the position of ray 3 and for fusions of rays 3 and 4. For ray 3 position, one quarter of the RILs exhibited phenotypes more extreme than either parental strain. For ray fusions, half of the RILs were more extreme than the parental strains. Such transgressive segregation is best explained through the presence, in both AF16 and HK104, of alleles at some loci that promoted ancestral character states and alleles at other loci that promoted derived character states. The high frequency of RILs with extreme phenotypes indicated that both parental strains likely were fixed for antagonistic alleles at multiple loci.A relatively strong correlation was observed between variation in the position of ray 3 and its fusion with ray 4 (r = 0.797; Figure  2a ). This correlation was highly significant (p = 2.5 × 10 -23 ). This was expected as rays 3 and 4 could not have fused unless they were adjacent to each other [ 19 ] and because the placement of rays and fusion between rays have been considered to be pleiotropic phenotypes derivative of the specification of ray identities [ 19 , 22 - 25 , 27 - 30 ]. If anything, it was surprising that the correlation was not stronger. Based on the observed correlation coefficient, approximately one third of the variation in ray fusion frequency was independent of variation in the position of ray 3. This was readily apparent when the frequency distributions of these two ray pattern elements were compared (Figure  2b ). The frequency distribution for the placement of ray 3 was highly skewed toward the derived phenotype. The frequency distribution for fusions of rays 3 and 4 was much broader and was only slightly skewed. The differences in the shapes of these distributions were significant (Kolmgorov-Smirnov, D = 0.4396, p < 0.001).Neither the frequency distribution for the placement of ray 3 nor that for the fusion of ray3 to ray 4 were normal (p = 0.00 and p = 0.03, respectively; Figure  2b ). The frequency distribution for the placement of ray 3 was highly skewed toward the derived phenotype. This result was inconsistent with the placement of ray 3 being governed strictly by additive affects. More likely, the placement of ray 3 was regulated by epistasis. Twentynine per cent of RILs exhibited the most extreme phenotype (Figure  2b ). Fixation of derived alleles at as few as two major-effect genes could have been sufficient to ensure a posterior localization of ray 3 at this frequency. The frequency distribution for fusions of rays 3 and 4 was very broad and and only slightly skewed. This was consistent with ray fusion being a complex character with inputs from multiple developmental processes. At least one of these processes was shared with ray positioning; rays 3 and 4 could not have fused if they were not adjacent.Association of ray pattern with allelic variation in homeotic genes The derived  C. briggsae  ray pattern may have evolved through changes in homeotic gene expression patterns [ 33 , 39 ]. The homeotic genes most likely to affect ray pattern are  mab-5  and  egl-5 . These genes are expressed in the posterior body and tail regions of  C. elegans  [ 40 ] and mutations in them can alter ray pattern [ 22 ]. Allelic variants that affect the size of the first intron of  Cb-egl-5  have been identified (Figure  3 ). The AF16::HK104 RILs were genotyped for these variants to test  Cb-egl-5  for association with ray pattern variation [see  Additional File 1 ]. Figure 3 Allelic variation of  Cb-egl-5 .  Cb-egl-5  amplification products of 1) AF16; 2) PB800; 3) HK104; 4–17) selected RILs; 18) an HK104/AF16 heterozygote. The expected amplification product size based on the  C. briggsae  AF16 genome sequence was 1,723 bp. m) Marker DNA, sizes of selected markers as indicated. Significant associations were observed between  Cb-egl-5  allelic variants and both the position of ray 3 and the fusion of ray 3 to ray 4 (Table  1 ). These associations were transgressive; the HK104 allele of  Cb-egl-5  cosegregated with derived phenotypes, the AF16 allele with ancestral phenotypes. Allelic variants in  Cb-mab-5  have not been identified. However, the  Cb-mab-5  (=CBG00029) and  Cb-egl-5  (=CBG00023) are located within 60 kb of each other [ 41 , 42 ]. Because of this close physical linkage, allelic variants in these genes very likely cosegregated. Thus, allelic variation in  Cb-mab-5 ,  Cb-egl-5 , and/or other linked loci may have been responsible for observed transgressive associations. Table 1 Cosegregation of  Cb-egl-5  with ray pattern phenotypes. allele 1 phenotype AF16 HK104 p value ray position 0.81 0.90 6.2 × 10 -4 ray fusion 0.42 0.68 4.2 × 10 -5 1  Mean frequency of derived character states exhibited in RILs homozygous for either parental  Cb-egl-5  allele.
Association of ray pattern with allelic variation in homeotic genes
The derived  C. briggsae  ray pattern may have evolved through changes in homeotic gene expression patterns [ 33 , 39 ]. The homeotic genes most likely to affect ray pattern are  mab-5  and  egl-5 . These genes are expressed in the posterior body and tail regions of  C. elegans  [ 40 ] and mutations in them can alter ray pattern [ 22 ]. Allelic variants that affect the size of the first intron of  Cb-egl-5  have been identified (Figure  3 ). The AF16::HK104 RILs were genotyped for these variants to test  Cb-egl-5  for association with ray pattern variation [see  Additional File 1 ].C. briggsaemab-5egl-5C. elegansCb-egl-5Cb-egl-5Significant associations were observed between  Cb-egl-5  allelic variants and both the position of ray 3 and the fusion of ray 3 to ray 4 (Table  1 ). These associations were transgressive; the HK104 allele of  Cb-egl-5  cosegregated with derived phenotypes, the AF16 allele with ancestral phenotypes. Allelic variants in  Cb-mab-5  have not been identified. However, the  Cb-mab-5  (=CBG00029) and  Cb-egl-5  (=CBG00023) are located within 60 kb of each other [ 41 , 42 ]. Because of this close physical linkage, allelic variants in these genes very likely cosegregated. Thus, allelic variation in  Cb-mab-5 ,  Cb-egl-5 , and/or other linked loci may have been responsible for observed transgressive associations.Cb-egl-5Cb-egl-5Cb-mab-5Cb-mab-5Cb-egl-5Cb-mab-5Cb-egl-5Cosegregation of  Cb-egl-5  with ray pattern phenotypes.Cb-egl-51  Mean frequency of derived character states exhibited in RILs homozygous for either parental  Cb-egl-5  allele.Cb-egl-5mip-1  marker-assisted introgression The segregation of phenotypes in the RILs indicated that multiple major-effect and additive loci were involved in ray pattern variation in  C. briggsae . Because a high density recombination map for  C. briggsae  has not yet been constructed, it is not possible to identify these loci through QTL analyses. However, a limited number of mutations with visible phenotypes have been mapped to  C. briggsae  chromosomes [B. Gupta, pers. comm., [ 43 ]]. These mutations all were generated in an AF16 background. One such mutation defines the gene  mip-1 .  mip-1  very likely is the  C. briggsae  homolog of  C. elegans unc-22  [D. Baillie, pers. comm.]. In  C. elegans ,  unc-22  is located on chromosome IV and is linked to several genes known to regulate homeotic gene expression patterns, e.g.  lin-49  which encodes a bromodomain protein thought to be involved in chromatin remodeling [ 26 ].  mip-1  marker-assisted introgression was used to determine if any genes linked to it were involved in ray pattern variation in  C. briggsae . Four  C. briggsae  strains were constructed in which HK104 DNA, in the region of  mip-1 , has been introgressed into an otherwise AF16 background. This was accomplished through a series of backcrosses that were initiated by mating HK104 males to  mip-1  (AF16) hermaphrodites. For each introgressed strain, F1 through F6 males were crossed to  mip-1  (AF16) hermaphrodites. Wild-type F7 hermaphrodites were selected and propagated by self-fertilization. From each F7 hermaphrodite, multiple wild-type F8 hermaphrodites were picked. Homozygous strains were established from F8 hermaphrodites that segregated only wild-type progeny. From each set of backcrosses, a single introgressed strain was retained. These introgressed strains were scored for the position of ray 3 and for ray 3–4 fusions (Figure  4 ). The ray patterns of all four introgressed strains differed significantly from AF16 both in the placement of ray 3 and in the frequency with which ray 3 fused to ray 4 (Table  2 ). Significant differences also were apparent between some pairs of introgressed strains, most notably between PB1060 and PB1065. PB1060 and PB1065 did not differ in the placement of ray 3 but only in the frequency with which ray 3 fused to ray 4 (Figure  4 ; Table  2 ). Thus, it appears that allelic variation in at least two genes linked to  mip-1  is involved in ray pattern variation. One of these genes affects the position of ray 3 and possibly the frequency of ray fusion. The HK104 allele of this gene was present in all  mip-1  introgressed strains. The other gene affects only the frequency of ray fusion. The HK104 allele of this gene was present in PB1065 but not in PB1060. As more  C. briggsae  mutant strains become available, it should be possible to identify these genes through additional introgression studies coupled with genotypic characterizations of introgressed DNA. Table 2 Comparison of  mip-1  introgressed lines. p values for reciprocal chi-squared tests 1 AF16 PB1060 PB1061 PB1062 PB1065 ray pattern 2 AF16 --- 9.6 × 10 -30 3.3 × 10 -54 6.5 × 10 -32 5.3 × 10 -46 PB1060 7.9 × 10 -4 --- 0.031 0.093 1.2 × 10 -5 PB1061 2.2 × 10 -7 0.026 --- 0.367 0.011 PB1062 2.8 × 10 -6 0.092 0.293 --- 0.091 PB1065 2.3 × 10 -9 1.3 × 10 -4 0.038 0.151 --- ray position 3 AF16 --- 6.9 × 10 -4 1.5 × 10 -5 3.6 × 10 -4 3.2 × 10 -4 PB1060 1.32 × 10 -29 --- 0.068 0.72 0.67 PB1061 6.4 × 10 -53 0.053 --- 0.12 0.14 PB1062 8.3 × 10 -30 0.73 0.16 --- 0.95 PB1065 1.65 × 10 -36 0.66 0.14 0.95 --- ray fusion 4 AF16 --- 0.13 5.3 × 10 -3 3.1 × 10 -3 9.8 × 10 -6 PB1060 0.013 --- 0.061 0.033 3.1 × 10 -5 PB1061 4.74 × 10 -6 0.057 --- 0.80 0.029 PB1062 9.37 × 10 -7 0.030 0.80 --- 0.052 PB1065 1.33 × 10 -16 1.59 × 10 -6 0.013 0.028 --- 1  Probabilities in each column were determined using frequencies of different ray patterns in each strain (column head) as the null hypothesis. 2  p values for the complete ray pattern (3 posterior and fused, 3 posterior not fused, 3 anterior). 3  p values for ray placement (anterior vs posterior). 4  p values for fusion of rays 3 and 4 when ray 3 is in a posterior position. Figure 4 Comparisons of ray pattern phenotypes in  mip-1  introgressed strains. Frequencies of ray pattern phenotypes exhibited in AF16 and four  mip-1  introgressed strains. Black bars represents ray 3 in a posterior position and fused with ray 4. Red bars represents ray 3 in a posterior position but not fused with ray 4. White bars represents ray 3 in an anterior postion.
mip-1  marker-assisted introgression
mip-1The segregation of phenotypes in the RILs indicated that multiple major-effect and additive loci were involved in ray pattern variation in  C. briggsae . Because a high density recombination map for  C. briggsae  has not yet been constructed, it is not possible to identify these loci through QTL analyses. However, a limited number of mutations with visible phenotypes have been mapped to  C. briggsae  chromosomes [B. Gupta, pers. comm., [ 43 ]]. These mutations all were generated in an AF16 background. One such mutation defines the gene  mip-1 .  mip-1  very likely is the  C. briggsae  homolog of  C. elegans unc-22  [D. Baillie, pers. comm.]. In  C. elegans ,  unc-22  is located on chromosome IV and is linked to several genes known to regulate homeotic gene expression patterns, e.g.  lin-49  which encodes a bromodomain protein thought to be involved in chromatin remodeling [ 26 ].  mip-1  marker-assisted introgression was used to determine if any genes linked to it were involved in ray pattern variation in  C. briggsae .C. briggsaeC. briggsaeC. briggsaemip-1mip-1C. briggsaeC. elegans unc-22C. elegansunc-22lin-49mip-1C. briggsaeFour  C. briggsae  strains were constructed in which HK104 DNA, in the region of  mip-1 , has been introgressed into an otherwise AF16 background. This was accomplished through a series of backcrosses that were initiated by mating HK104 males to  mip-1  (AF16) hermaphrodites. For each introgressed strain, F1 through F6 males were crossed to  mip-1  (AF16) hermaphrodites. Wild-type F7 hermaphrodites were selected and propagated by self-fertilization. From each F7 hermaphrodite, multiple wild-type F8 hermaphrodites were picked. Homozygous strains were established from F8 hermaphrodites that segregated only wild-type progeny. From each set of backcrosses, a single introgressed strain was retained. These introgressed strains were scored for the position of ray 3 and for ray 3–4 fusions (Figure  4 ). The ray patterns of all four introgressed strains differed significantly from AF16 both in the placement of ray 3 and in the frequency with which ray 3 fused to ray 4 (Table  2 ). Significant differences also were apparent between some pairs of introgressed strains, most notably between PB1060 and PB1065. PB1060 and PB1065 did not differ in the placement of ray 3 but only in the frequency with which ray 3 fused to ray 4 (Figure  4 ; Table  2 ). Thus, it appears that allelic variation in at least two genes linked to  mip-1  is involved in ray pattern variation. One of these genes affects the position of ray 3 and possibly the frequency of ray fusion. The HK104 allele of this gene was present in all  mip-1  introgressed strains. The other gene affects only the frequency of ray fusion. The HK104 allele of this gene was present in PB1065 but not in PB1060. As more  C. briggsae  mutant strains become available, it should be possible to identify these genes through additional introgression studies coupled with genotypic characterizations of introgressed DNA.C. briggsaemip-1mip-1mip-1mip-1mip-1C. briggsaeComparison of  mip-1  introgressed lines.mip-11  Probabilities in each column were determined using frequencies of different ray patterns in each strain (column head) as the null hypothesis.2  p values for the complete ray pattern (3 posterior and fused, 3 posterior not fused, 3 anterior).3  p values for ray placement (anterior vs posterior).4  p values for fusion of rays 3 and 4 when ray 3 is in a posterior position.Discussion Ray pattern in  C. briggsae  varies with respect to the placement of ray 3 and fusions of ray 3 to ray 4. Ancestral states for these characters in the Elegans-group of  Caenorhabditis  are an anterior location, level with the cloaca, and the absence of ray fusions [ 34 ]. Derived states are a posterior location, clustered with rays 4 through 6, and fusion of rays 3 and 4. An unexpected result was the incomplete correlation between the position or ray 3 and its fusion with ray 4. In  C. elegans , mutations that alter ray position have been accompanied by ray fusion. For this reason, ray position and ray fusion were thought to be pleiotropic phenotypes that arose from the specification of ray identities [ 19 , 22 - 25 , 30 ]. Because of this, it was expected that ray pattern evolution would involve changes in suites of covariant characters rather than independent modification of individual ray pattern elements. However, only two-thirds of variation in ray fusion resulted from variation in ray position (r 2  = 0.635). Moreover, evidence for a gene affecting only ray fusion was obtained from comparisons of ray patterns exhibited by the  mip-1  introgressed strains. Hence, variation in ray pattern evolution is not wholey constrained by the specification of ray identities and other ray pattern elements, such as neurotransmitter usage, also may vary independently. A similar pattern of partial constraint and flexibility has been observed for variation in eyespots size in the butterfly  Bicyclus anynana  [ 12 ]. Two haplotypes of  C. briggsae  have been described [ 44 ] and variation in ray pattern follows haplotype structure [ 39 ]. Strains in haplotype 1, including AF16, exhibit almost exclusively a derived ray pattern. Strains in haplotype 2, including HK104, exhibit derived and ancestral ray patterns at equal frequencies. One interpretation of these results is that full expression of the derived ray pattern never became fixed within haplotype 2. However, a  C. briggsae -like ray pattern also has been reported for  C. clavopapillata  [ 45 ], another species within the Elegans-group.  C. clavopapillata  has not been observed since its first description and some authors have considered it and  C. briggsae  to be synonymous [ 34 ]. If  C. clavopapillata  is distinct from  C. briggsae , the posterior position of ray 3 and its frequent fusion with ray 4 may be ancestral for these two species. This would make the  C. elegans -like ray pattern in haplotype 2 an atavistic character. It should be possible to discriminate between these two models either through the characterization of additional  C. briggsae  strains or through the redescription and molecular characterization of  C. clavopapillata . Regardless of their evolutionary history, haplotypes 1 and 2 provided an entrée to the genetics of morphological variation of ray pattern in  C. briggsae . The relatively slight difference between the AF16 and HK104 phenotypes hid a wealth of genetic variation. This was evident in the nearly continuous variation exhibited in RILs for both the position of ray 3 and its fusion with ray 4. This variation included many RILs with phenotypes more extreme than either parental strain. Such transgressive segregation is common in both plants and animals and is thought to arise through fluctuating selection and/or through stabilizing selection at minor QTL when directional selection at major QTL overshoots a phenotypic optimum [ 46 ]. The transgressive segregation of ray pattern phenotypes may have resulted from selection on homeotic gene expression patterns. In  C. elegans , ray 3 precursor cells are born at the junction of the  mab-5  and  egl-5  expression domains [ 23 , 47 ]. These homeotic genes are required for the specification of positional identities in the posterior body and tail regions, respectively [ 47 , 48 ] and some mutations in them cause the  C. elegans  ray pattern to phenocopy that of  C. briggsae  [ 22 , 26 ]. We have demonstrated a significant transgressive association between allelic variation at  Cb-egl-5  and variation in ray pattern. As  Cb-egl-5  and  Cb-mab-5  are closely linked, variation in either or both of these genes may be responsible for the observed association. Alternatively, linkage to  Cb-egl-5  may be coincidental, and ray pattern variation in  C. briggsae  may not result from variation in homeotic gene expression patterns. The best test of these alternative models will be the high resolution mapping of the allelic variants responsible for ray pattern variation. Loci not linked to the homeotic gene cluster also must be involved in ray pattern variation. Direct evidence for this was obtained from the  mip-1  marker assisted introgression studies. The  C. elegans  homolog of  mip-1 ,  unc-22 , is not linked to the the homeotic gene cluster and linkage of  mip-1  to  Cb-egl-5  and  Cb-mab-5  is unlikely. There appear to be at least two loci that affect ray pattern linked to  mip-1 . One of these affected both the placement of ray 3 and its fusion with ray 4. The other affected only ray fusion. These associations were not transgressive, i.e. the allelic variants linked to  mip-1  antagonized the allelic variants linked to  Cb-egl-5 . If the transgressive segregation linked to  Cb-egl-5  does result from allelic variation in one or more homeotic genes, then the antagonistic variation linked to  mip-1  may be in genes that regulate homeotic gene expression patterns. A direct test of this hypothesis is possible. Several genes required for proper regulation of homeotic gene expression patterns have been identified in  C. elegans  [ 25 , 26 , 37 , 38 , 49 ].  C. briggsae  homologs of these genes could be tested for association with ray pattern variation in the RILs. Ideally, these tests would be integrated into a genome wide screen for variant loci with effects on ray pattern. This will require the enhancement of genetic resources available for  C. briggsae .
Discussion
Ray pattern in  C. briggsae  varies with respect to the placement of ray 3 and fusions of ray 3 to ray 4. Ancestral states for these characters in the Elegans-group of  Caenorhabditis  are an anterior location, level with the cloaca, and the absence of ray fusions [ 34 ]. Derived states are a posterior location, clustered with rays 4 through 6, and fusion of rays 3 and 4. An unexpected result was the incomplete correlation between the position or ray 3 and its fusion with ray 4. In  C. elegans , mutations that alter ray position have been accompanied by ray fusion. For this reason, ray position and ray fusion were thought to be pleiotropic phenotypes that arose from the specification of ray identities [ 19 , 22 - 25 , 30 ]. Because of this, it was expected that ray pattern evolution would involve changes in suites of covariant characters rather than independent modification of individual ray pattern elements. However, only two-thirds of variation in ray fusion resulted from variation in ray position (r 2  = 0.635). Moreover, evidence for a gene affecting only ray fusion was obtained from comparisons of ray patterns exhibited by the  mip-1  introgressed strains. Hence, variation in ray pattern evolution is not wholey constrained by the specification of ray identities and other ray pattern elements, such as neurotransmitter usage, also may vary independently. A similar pattern of partial constraint and flexibility has been observed for variation in eyespots size in the butterfly  Bicyclus anynana  [ 12 ].C. briggsaeCaenorhabditisC. elegansmip-1Bicyclus anynanaTwo haplotypes of  C. briggsae  have been described [ 44 ] and variation in ray pattern follows haplotype structure [ 39 ]. Strains in haplotype 1, including AF16, exhibit almost exclusively a derived ray pattern. Strains in haplotype 2, including HK104, exhibit derived and ancestral ray patterns at equal frequencies. One interpretation of these results is that full expression of the derived ray pattern never became fixed within haplotype 2. However, a  C. briggsae -like ray pattern also has been reported for  C. clavopapillata  [ 45 ], another species within the Elegans-group.  C. clavopapillata  has not been observed since its first description and some authors have considered it and  C. briggsae  to be synonymous [ 34 ]. If  C. clavopapillata  is distinct from  C. briggsae , the posterior position of ray 3 and its frequent fusion with ray 4 may be ancestral for these two species. This would make the  C. elegans -like ray pattern in haplotype 2 an atavistic character. It should be possible to discriminate between these two models either through the characterization of additional  C. briggsae  strains or through the redescription and molecular characterization of  C. clavopapillata .C. briggsaeC. briggsaeC. clavopapillataC. clavopapillataC. briggsaeC. clavopapillataC. briggsaeC. elegansC. briggsaeC. clavopapillataRegardless of their evolutionary history, haplotypes 1 and 2 provided an entrée to the genetics of morphological variation of ray pattern in  C. briggsae . The relatively slight difference between the AF16 and HK104 phenotypes hid a wealth of genetic variation. This was evident in the nearly continuous variation exhibited in RILs for both the position of ray 3 and its fusion with ray 4. This variation included many RILs with phenotypes more extreme than either parental strain. Such transgressive segregation is common in both plants and animals and is thought to arise through fluctuating selection and/or through stabilizing selection at minor QTL when directional selection at major QTL overshoots a phenotypic optimum [ 46 ]. The transgressive segregation of ray pattern phenotypes may have resulted from selection on homeotic gene expression patterns. In  C. elegans , ray 3 precursor cells are born at the junction of the  mab-5  and  egl-5  expression domains [ 23 , 47 ]. These homeotic genes are required for the specification of positional identities in the posterior body and tail regions, respectively [ 47 , 48 ] and some mutations in them cause the  C. elegans  ray pattern to phenocopy that of  C. briggsae  [ 22 , 26 ]. We have demonstrated a significant transgressive association between allelic variation at  Cb-egl-5  and variation in ray pattern. As  Cb-egl-5  and  Cb-mab-5  are closely linked, variation in either or both of these genes may be responsible for the observed association. Alternatively, linkage to  Cb-egl-5  may be coincidental, and ray pattern variation in  C. briggsae  may not result from variation in homeotic gene expression patterns. The best test of these alternative models will be the high resolution mapping of the allelic variants responsible for ray pattern variation.C. briggsaeC. elegansmab-5egl-5C. elegansC. briggsaeCb-egl-5Cb-egl-5Cb-mab-5Cb-egl-5C. briggsaeLoci not linked to the homeotic gene cluster also must be involved in ray pattern variation. Direct evidence for this was obtained from the  mip-1  marker assisted introgression studies. The  C. elegans  homolog of  mip-1 ,  unc-22 , is not linked to the the homeotic gene cluster and linkage of  mip-1  to  Cb-egl-5  and  Cb-mab-5  is unlikely. There appear to be at least two loci that affect ray pattern linked to  mip-1 . One of these affected both the placement of ray 3 and its fusion with ray 4. The other affected only ray fusion. These associations were not transgressive, i.e. the allelic variants linked to  mip-1  antagonized the allelic variants linked to  Cb-egl-5 . If the transgressive segregation linked to  Cb-egl-5  does result from allelic variation in one or more homeotic genes, then the antagonistic variation linked to  mip-1  may be in genes that regulate homeotic gene expression patterns. A direct test of this hypothesis is possible. Several genes required for proper regulation of homeotic gene expression patterns have been identified in  C. elegans  [ 25 , 26 , 37 , 38 , 49 ].  C. briggsae  homologs of these genes could be tested for association with ray pattern variation in the RILs. Ideally, these tests would be integrated into a genome wide screen for variant loci with effects on ray pattern. This will require the enhancement of genetic resources available for  C. briggsae .mip-1C. elegansmip-1unc-22mip-1Cb-egl-5Cb-mab-5mip-1mip-1Cb-egl-5Cb-egl-5mip-1C. elegansC. briggsaeC. briggsaeConclusions Ray pattern in  Caenorhabditis  provides a powerful model for the study of morphological evolution. Macroevolutionary comparisons between species and microevolutionary analyses of variation within species are possible. Augmenting these approaches is a detailed understanding of the genetic and cellular basis of ray pattern development in  C. elegans . In  C. briggsae , intraspecific variants have been characterized that affect the expression of ancestral and derived ray patterns. These variants have a complex genetic basis involving multiple genes. Some of these genes exhibit transgenic segregation, some affect all of elements of ray pattern, and some that affect only a subset of ray pattern elements. At least one gene that affects ray pattern variation in  C. briggsae  is linked to the homeotic gene cluster. Thus, ray pattern variation may result from altered expression patterns of homeotic genes. Further characterizations of the genetics of ray pattern variation will test this model and will address interactions between different genes that impact ray pattern in  C. briggsae .
Conclusions
Ray pattern in  Caenorhabditis  provides a powerful model for the study of morphological evolution. Macroevolutionary comparisons between species and microevolutionary analyses of variation within species are possible. Augmenting these approaches is a detailed understanding of the genetic and cellular basis of ray pattern development in  C. elegans . In  C. briggsae , intraspecific variants have been characterized that affect the expression of ancestral and derived ray patterns. These variants have a complex genetic basis involving multiple genes. Some of these genes exhibit transgenic segregation, some affect all of elements of ray pattern, and some that affect only a subset of ray pattern elements. At least one gene that affects ray pattern variation in  C. briggsae  is linked to the homeotic gene cluster. Thus, ray pattern variation may result from altered expression patterns of homeotic genes. Further characterizations of the genetics of ray pattern variation will test this model and will address interactions between different genes that impact ray pattern in  C. briggsae .CaenorhabditisC. elegansC. briggsaeC. briggsaeC. briggsaeMethods Strains and strain maintenance C. briggsae  strains AF16, and HK104 are available from the  Caenorhabditis  Genetics Center [ 50 ]. These strains were maintained on agar plates seeded with  E. coli  strain OP50. Recombinant inbred lines (RIL) were constructed starting with HK104 males mated to sperm-depleted AF16 hermaphrodites [ 15 ]. Each RIL was initiated with a single F2 hermaphrodite and propagated through F11, one hermaphrodite per generation. Microscopy Ray pattern phenotypes were scored using differential interference contrast optics at a magnification of 400× [ 5 ]. Right and left sides were scored independently [ 39 ]. Microscopic images of worms anethesized in 0.2% sodium azide were digitally captured using a Spot Camera and Software (Diagnostic Instruments, Inc., Sterling Heights MI). PCR amplification Forward and reverse primers for PCR amplifications  Cb-egl-5 , CAGGGAGCGGACAACTTCAAAGG and GGACACAGCCCAGGATTAGCGAC, respectively, were designed based on genome sequence data from  C. briggsae  strain AF16 [ 41 ]. Amplification products were size fractionated by electrophoresis through 1% agarose. Statistical analyses Pearson's product moment correlation cofficients between ray pattern elements in RILs and Wilcoxon nonparametric tests of the association between  Cb-egl-5  allelic variation with ray pattern variation were determined online at [ 51 ]. Frequency distributions of the segregation of ray 3 positional and fusion phenotypes were compared using the Kolmogorov-Smirnov tests as implemented at [ 52 ]. Ray pattern phenotypes of  mip-1  introgressed strains were compared using reciprocal chi-squared tests using Excel v10.1.0 (Microsoft, Inc., Redman WA).
Methods
Strains and strain maintenance C. briggsae  strains AF16, and HK104 are available from the  Caenorhabditis  Genetics Center [ 50 ]. These strains were maintained on agar plates seeded with  E. coli  strain OP50. Recombinant inbred lines (RIL) were constructed starting with HK104 males mated to sperm-depleted AF16 hermaphrodites [ 15 ]. Each RIL was initiated with a single F2 hermaphrodite and propagated through F11, one hermaphrodite per generation.
Strains and strain maintenance
C. briggsae  strains AF16, and HK104 are available from the  Caenorhabditis  Genetics Center [ 50 ]. These strains were maintained on agar plates seeded with  E. coli  strain OP50. Recombinant inbred lines (RIL) were constructed starting with HK104 males mated to sperm-depleted AF16 hermaphrodites [ 15 ]. Each RIL was initiated with a single F2 hermaphrodite and propagated through F11, one hermaphrodite per generation.C. briggsaeCaenorhabditisE. coliMicroscopy Ray pattern phenotypes were scored using differential interference contrast optics at a magnification of 400× [ 5 ]. Right and left sides were scored independently [ 39 ]. Microscopic images of worms anethesized in 0.2% sodium azide were digitally captured using a Spot Camera and Software (Diagnostic Instruments, Inc., Sterling Heights MI).
Microscopy
Ray pattern phenotypes were scored using differential interference contrast optics at a magnification of 400× [ 5 ]. Right and left sides were scored independently [ 39 ]. Microscopic images of worms anethesized in 0.2% sodium azide were digitally captured using a Spot Camera and Software (Diagnostic Instruments, Inc., Sterling Heights MI).PCR amplification Forward and reverse primers for PCR amplifications  Cb-egl-5 , CAGGGAGCGGACAACTTCAAAGG and GGACACAGCCCAGGATTAGCGAC, respectively, were designed based on genome sequence data from  C. briggsae  strain AF16 [ 41 ]. Amplification products were size fractionated by electrophoresis through 1% agarose.
PCR amplification
Forward and reverse primers for PCR amplifications  Cb-egl-5 , CAGGGAGCGGACAACTTCAAAGG and GGACACAGCCCAGGATTAGCGAC, respectively, were designed based on genome sequence data from  C. briggsae  strain AF16 [ 41 ]. Amplification products were size fractionated by electrophoresis through 1% agarose.Cb-egl-5C. briggsaeStatistical analyses Pearson's product moment correlation cofficients between ray pattern elements in RILs and Wilcoxon nonparametric tests of the association between  Cb-egl-5  allelic variation with ray pattern variation were determined online at [ 51 ]. Frequency distributions of the segregation of ray 3 positional and fusion phenotypes were compared using the Kolmogorov-Smirnov tests as implemented at [ 52 ]. Ray pattern phenotypes of  mip-1  introgressed strains were compared using reciprocal chi-squared tests using Excel v10.1.0 (Microsoft, Inc., Redman WA).
Statistical analyses
Pearson's product moment correlation cofficients between ray pattern elements in RILs and Wilcoxon nonparametric tests of the association between  Cb-egl-5  allelic variation with ray pattern variation were determined online at [ 51 ]. Frequency distributions of the segregation of ray 3 positional and fusion phenotypes were compared using the Kolmogorov-Smirnov tests as implemented at [ 52 ]. Ray pattern phenotypes of  mip-1  introgressed strains were compared using reciprocal chi-squared tests using Excel v10.1.0 (Microsoft, Inc., Redman WA).Cb-egl-5mip-1Abbreviations RIL = recombinant inbred line; QTL = quantitative trait loci;  dll  =  distalless ;  en  =  engrailed ;  sal  =  spalt
Abbreviations
RIL = recombinant inbred line; QTL = quantitative trait loci;  dll  =  distalless ;  en  =  engrailed ;  sal  =  spaltdlldistallessenengrailedsalspaltAuthor's contributions SEB : Planned and supervised all research activities. Participated in construction and genotyping of RILs. Reviewed all statistical analyses. Wrote manuscript. CRD : Constructed, genotyped, and characterized ray pattern phenotypes of RILs. Statistical analyses of the segregation of ray pattern phenotypes and association of  Cb-egl-5  variation with ray pattern variation. Reviewed manuscript. JCB : Constructed and characterized phenotypes of  mip-1 -assisted introgressed lines. Statistical analyses of ray pattern phenotypes of  mip-1  introgressed lines. Reviewed manuscript.
Author's contributions
SEB : Planned and supervised all research activities. Participated in construction and genotyping of RILs. Reviewed all statistical analyses. Wrote manuscript.SEBCRD : Constructed, genotyped, and characterized ray pattern phenotypes of RILs. Statistical analyses of the segregation of ray pattern phenotypes and association of  Cb-egl-5  variation with ray pattern variation. Reviewed manuscript.CRDCb-egl-5JCB : Constructed and characterized phenotypes of  mip-1 -assisted introgressed lines. Statistical analyses of ray pattern phenotypes of  mip-1  introgressed lines. Reviewed manuscript.JCBmip-1mip-1Supplementary Material Additional File 1 AF16 × HK104 RIL ray pattern data.  Cb-egl-5  genotypes and ray pattern phenotypes of RIL derived from AF16 × HK104. Click here for file
Supplementary Material
Additional File 1
AF16 × HK104 RIL ray pattern data.  Cb-egl-5  genotypes and ray pattern phenotypes of RIL derived from AF16 × HK104.Cb-egl-5Click here for file


Background Recent magnetic resonance spectroscopy (MRS) studies report that glutamine is altered in the brains of schizophrenic patients. There were also conflicting findings on glutamate in cerebrospinal fluid (CSF) of schizophrenic patients, and absent for glutamine. This study aims to clarify the question of glutamine and glutamate in CSF of first episode and drug naive schizophrenic patients.
Background
Recent magnetic resonance spectroscopy (MRS) studies report that glutamine is altered in the brains of schizophrenic patients. There were also conflicting findings on glutamate in cerebrospinal fluid (CSF) of schizophrenic patients, and absent for glutamine. This study aims to clarify the question of glutamine and glutamate in CSF of first episode and drug naive schizophrenic patients.Method Levels of glutamine and glutamate in CSF of 25 first episode and drug-naive male schizophrenic patients and 17 age-matched male healthy controls were measured by a high performance liquid chromatography.
Method
Levels of glutamine and glutamate in CSF of 25 first episode and drug-naive male schizophrenic patients and 17 age-matched male healthy controls were measured by a high performance liquid chromatography.Results The ratio (126.1 (median), 117.7 ± 27.4 (mean ± S.D.)) of glutamine to glutamate in the CSF of patients was significantly (z = -3.29, p = 0.001) higher than that (81.01 (median), 89.1 ± 22.5 (mean ± S.D.)) of normal controls although each level of glutamine and glutamate in patients was not different from that of normal controls.
Results
The ratio (126.1 (median), 117.7 ± 27.4 (mean ± S.D.)) of glutamine to glutamate in the CSF of patients was significantly (z = -3.29, p = 0.001) higher than that (81.01 (median), 89.1 ± 22.5 (mean ± S.D.)) of normal controls although each level of glutamine and glutamate in patients was not different from that of normal controls.Conclusion Our data suggests that a disfunction in glutamate-glutamine cycle in the brain may play a role in the pathophysiology of schizophrenia.
Conclusion
Our data suggests that a disfunction in glutamate-glutamine cycle in the brain may play a role in the pathophysiology of schizophrenia.

Background Multiple lines of evidence suggest that a dysfunction in glutamatergic neurotransmission might be involved in the pathophysiology of schizophrenia [ 1 - 6 ]. The amino acid glutamate plays a central role in nitrogen metabolism and participates in multiple biochemical pathways. Released glutamate is taken up by glia, where it is converted to glutamine, transported back to the presynaptic neuron, and reconverted to glutamate [ 6 , 7 ]. Thus, it seems that glutamate-glutamine cycle plays a role in the neuron-glia communication in the synapse, and that impairment of glutamate-glutamine cycle may be implicated in the pathophysiology of schizophrenia [ 1 - 6 ]. By means of  in vivo  proton magnetic resonance spectroscopy (MRS), a significant increase in glutamine level was detected in the medial prefrontal cortex of never-treated schizophrenic patients compared with controls [ 8 ]. In addition, a recent 4.0 T MRS study demonstrated that levels of glutamine in the left anterior cingulate cortex and thalamus of the never-treated patients with schizophrenia were significantly higher than those of healthy controls [ 9 ]. In contrast, significant lower levels of glutamine were found in the left anterior cingulate cortex of medicated patients with chronic schizophrenia than in the healthy controls, suggesting the role of chronic medication [ 10 ]. Thus, it is possible that the glutamate-glutamine cycle between neuron and glia may play a role in the glutamate hypothesis of schizophrenia. Although Kim et al. [ 11 ] first reported reduction of cerebrospinal fluid (CSF) levels of glutamate in patients with schizophrenia, the findings of subsequent studies are inconsistent, with several report of no alteration in CSF levels of glutamate [ 12 - 14 ]. Furthermore, it was reported that a gradient for glutamate and glutamine in CSF was lack, and that there were significant correlations between the CSF and serum levels of glutamate (r = 0.67, p < 0.05) and glutamine (r = 0.84, p < 0.01)[ 15 ]. Moreover, sodium-dependent neutral amino acids transporters, located in the abluminal membranes of the blood brain barrier, are capable of actively removing neutral amino acids from the brain [ 16 ]. These findings suggest that concentration of neutral amino acids in the extracellular fluid of brain are maintained at ~10% of those of the blood [ 15 , 16 ]. In this study, we investigated whether levels of glutamate and glutamine or ratio of glutamine to glutamate in CSF of first episode and drug naive schizophrenic patients are different from those of age-matched healthy normal controls.
Background
Multiple lines of evidence suggest that a dysfunction in glutamatergic neurotransmission might be involved in the pathophysiology of schizophrenia [ 1 - 6 ]. The amino acid glutamate plays a central role in nitrogen metabolism and participates in multiple biochemical pathways. Released glutamate is taken up by glia, where it is converted to glutamine, transported back to the presynaptic neuron, and reconverted to glutamate [ 6 , 7 ]. Thus, it seems that glutamate-glutamine cycle plays a role in the neuron-glia communication in the synapse, and that impairment of glutamate-glutamine cycle may be implicated in the pathophysiology of schizophrenia [ 1 - 6 ].By means of  in vivo  proton magnetic resonance spectroscopy (MRS), a significant increase in glutamine level was detected in the medial prefrontal cortex of never-treated schizophrenic patients compared with controls [ 8 ]. In addition, a recent 4.0 T MRS study demonstrated that levels of glutamine in the left anterior cingulate cortex and thalamus of the never-treated patients with schizophrenia were significantly higher than those of healthy controls [ 9 ]. In contrast, significant lower levels of glutamine were found in the left anterior cingulate cortex of medicated patients with chronic schizophrenia than in the healthy controls, suggesting the role of chronic medication [ 10 ]. Thus, it is possible that the glutamate-glutamine cycle between neuron and glia may play a role in the glutamate hypothesis of schizophrenia.in vivoAlthough Kim et al. [ 11 ] first reported reduction of cerebrospinal fluid (CSF) levels of glutamate in patients with schizophrenia, the findings of subsequent studies are inconsistent, with several report of no alteration in CSF levels of glutamate [ 12 - 14 ]. Furthermore, it was reported that a gradient for glutamate and glutamine in CSF was lack, and that there were significant correlations between the CSF and serum levels of glutamate (r = 0.67, p < 0.05) and glutamine (r = 0.84, p < 0.01)[ 15 ]. Moreover, sodium-dependent neutral amino acids transporters, located in the abluminal membranes of the blood brain barrier, are capable of actively removing neutral amino acids from the brain [ 16 ]. These findings suggest that concentration of neutral amino acids in the extracellular fluid of brain are maintained at ~10% of those of the blood [ 15 , 16 ].In this study, we investigated whether levels of glutamate and glutamine or ratio of glutamine to glutamate in CSF of first episode and drug naive schizophrenic patients are different from those of age-matched healthy normal controls.Methods Twenty-five male patients with schizophrenia (mean age 26.1 years, range 18–39) and 17 age-matched male healthy subjects (mean age 27.3 years, range 22–44) with no psychiatric disease were enrolled in Uppsala University and Linkoping University Hospital, Sweden. All patients diagnosed according to the DSM-III-R were first episode and drug naive, i.e. they had never been treated with antipsychotic drugs. In the morning (8:00–9:00) from May 1997 to January 1998, CSF of subjects was obtained by lumbar puncture (L4-L5), and twelve to eighteen μL of CSF was collected with a 0.9 mm needle and the samples were immediately frozen at -80°C, as reported previously [ 17 ]. The ethics committee of each institute approved the present study, and we received the informed consent from the participants of the study. Measurement of glutamate and glutamine levels were carried out according to established methods [ 18 ] with a slight modification using a high performance liquid chromatography (HPLC) system with fluorescence detection (Shimadzu Corporation, Kyoto, Japan). Ten μL of the human CSF was added with 10 μL of 0.1 M borate buffer (pH 8.0) and 30 μL of 50 mM 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F; Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) in CH 3 CN. The reaction mixture was then heated at 60°C for 2 min, and immediately supplemented with 100 μL of H 2 O/CH 3 CN (90/10) containing 0.1 % trifluoroacetic acid (TFA) to stop the reaction. Ten μL of the resultant solution was injected into the HPLC system. A reversed-phase ODS column (TSKgel ODS-80Ts, Tosoh Corporation, Tokyo, Japan) was used for the separation and quantification of glutamate and glutamine, and the gradient elution of the mobile phase was kept at a constant flow rate of 0.8 mL/min. Mobile phase 1a consisted of H 2 O/CH 3 CN (90/10) containing 0.1 % TFA, and phases 1b and 1c, of H 2 O/CH 3 CN (10/90) containing 0.1 % TFA and CH 3 CN, respectively. The time program for gradient elution was programmed as follows: 0–50.5 min 1a: 1b : 1c = 95 : 5 : 0, 50.5–55.5 min 1a : 1b : 1c = 0 : 100 : 0, and 55.5–57 min, 1a : 1b : 1c = 0 : 0 : 100. The column temperature of all columns was maintained at 35°C. Fluorescence detection was made at 530 nm with an excitation wavelength at 470 nm. Differences between two groups were analyzed using the Mann-Whitney U-test. The relationship between two variables was examined using Spearman correlation coefficients. A p < 0.05 was considered significant.
Methods
Twenty-five male patients with schizophrenia (mean age 26.1 years, range 18–39) and 17 age-matched male healthy subjects (mean age 27.3 years, range 22–44) with no psychiatric disease were enrolled in Uppsala University and Linkoping University Hospital, Sweden. All patients diagnosed according to the DSM-III-R were first episode and drug naive, i.e. they had never been treated with antipsychotic drugs. In the morning (8:00–9:00) from May 1997 to January 1998, CSF of subjects was obtained by lumbar puncture (L4-L5), and twelve to eighteen μL of CSF was collected with a 0.9 mm needle and the samples were immediately frozen at -80°C, as reported previously [ 17 ]. The ethics committee of each institute approved the present study, and we received the informed consent from the participants of the study.Measurement of glutamate and glutamine levels were carried out according to established methods [ 18 ] with a slight modification using a high performance liquid chromatography (HPLC) system with fluorescence detection (Shimadzu Corporation, Kyoto, Japan). Ten μL of the human CSF was added with 10 μL of 0.1 M borate buffer (pH 8.0) and 30 μL of 50 mM 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F; Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) in CH 3 CN. The reaction mixture was then heated at 60°C for 2 min, and immediately supplemented with 100 μL of H 2 O/CH 3 CN (90/10) containing 0.1 % trifluoroacetic acid (TFA) to stop the reaction. Ten μL of the resultant solution was injected into the HPLC system. A reversed-phase ODS column (TSKgel ODS-80Ts, Tosoh Corporation, Tokyo, Japan) was used for the separation and quantification of glutamate and glutamine, and the gradient elution of the mobile phase was kept at a constant flow rate of 0.8 mL/min. Mobile phase 1a consisted of H 2 O/CH 3 CN (90/10) containing 0.1 % TFA, and phases 1b and 1c, of H 2 O/CH 3 CN (10/90) containing 0.1 % TFA and CH 3 CN, respectively. The time program for gradient elution was programmed as follows: 0–50.5 min 1a: 1b : 1c = 95 : 5 : 0, 50.5–55.5 min 1a : 1b : 1c = 0 : 100 : 0, and 55.5–57 min, 1a : 1b : 1c = 0 : 0 : 100. The column temperature of all columns was maintained at 35°C. Fluorescence detection was made at 530 nm with an excitation wavelength at 470 nm.Differences between two groups were analyzed using the Mann-Whitney U-test. The relationship between two variables was examined using Spearman correlation coefficients. A p < 0.05 was considered significant.Results The CSF levels (421.7 μM (median), 468.1 ± 146.1 μM (mean ± S.D.), 254.0–775.1 (range)) of glutamine in the patients were not different (z = -1.038, p = 0.299) from those (410.5 μM (median), 405.6 ± 108.6 μM (mean ± S.D.), 219.8–689.0 (range)) of normal controls. The CSF levels (4.17 μM (median), 4.25 ± 1.77 μM (mean ± S.D.), 2.22–8.88 (range)) of glutamate in the patients were not different (z = -1.307, p = 0.191) from those (5.26 μM (median), 4.73 ± 1.29 μM (mean ± S.D.), 2.54–6.51 (range)) of normal controls. However, the ratio (126.1 (median), 117.7 ± 27.4 (mean ± S.D.), 42.0–161.6 (range)) of glutamine to glutamate in the CSF of patients was significantly (z = -3.29, p = 0.001) higher than that (81.01 (median), 89.1 ± 22.5 (mean ± S.D.), 59.7–134.0 (range)) of controls (Table 1). Furthermore, we found significant correlations between glutamate and glutamine in normal controls (r = 0.549, p = 0.022) or patients (r = 0.780, p < 0.001).
Results
The CSF levels (421.7 μM (median), 468.1 ± 146.1 μM (mean ± S.D.), 254.0–775.1 (range)) of glutamine in the patients were not different (z = -1.038, p = 0.299) from those (410.5 μM (median), 405.6 ± 108.6 μM (mean ± S.D.), 219.8–689.0 (range)) of normal controls. The CSF levels (4.17 μM (median), 4.25 ± 1.77 μM (mean ± S.D.), 2.22–8.88 (range)) of glutamate in the patients were not different (z = -1.307, p = 0.191) from those (5.26 μM (median), 4.73 ± 1.29 μM (mean ± S.D.), 2.54–6.51 (range)) of normal controls. However, the ratio (126.1 (median), 117.7 ± 27.4 (mean ± S.D.), 42.0–161.6 (range)) of glutamine to glutamate in the CSF of patients was significantly (z = -3.29, p = 0.001) higher than that (81.01 (median), 89.1 ± 22.5 (mean ± S.D.), 59.7–134.0 (range)) of controls (Table 1). Furthermore, we found significant correlations between glutamate and glutamine in normal controls (r = 0.549, p = 0.022) or patients (r = 0.780, p < 0.001).Discussion In this study, we found that the ratio of glutamine to glutamate in the CSF of first episode and drug naive schizophrenic patients was significantly higher than that of normal controls although each level of glutamine and glutamate in the CSF of patients was not significantly different from that of normal controls. To our knowledge, this is a first report demonstrating that the ratios of glutamine to glutamate in the first episode and drug naive patients are significantly higher than those of normal controls. In contrast, it was supposed earlier that alterations in CSF levels of glutamate are not so prominent compared with those in the brain [ 14 ]. Therefore, it is likely that a difference in glutamate (or glutamine) levels between our CSF study and MRS studies may be due to the difference between CSF samples and specific corticolimbic regions. However, it should be noted that alterations in the ratio of glutamine to glutamate are detected in the CSF samples of first episode and drug naive schizophrenic patients, suggesting an abnormality of the glia-neuronal glutamate-glutamine cycle in the brain of patients with schizophrenia. In general, glutamine is synthesized in astrocytes from glutamate by the enzyme glutamine synthetase, found exclusively in brain glia cells. Glutamine then crosses the astrocytes to be transported into nerve cell terminals, where it is converted again into the neurotransmitter glutamate by glutaminase. It is reported that activities of glutaminase and glutamic acid decarboxylase (GAD; the rate-limiting enzyme in the synthesis of GABA by decarboxylation of glutamate) are significantly greater in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia than in the control group, whereas activities of glutamate dehydrogenase, glutamine synthetase, and GABA transaminase in the DLPFC of schizophrenia are not different from the control group [ 19 ]. These findings suggest that metabolism of glutamate and GABA might be altered in the DLPFC of schizophrenic patients. Furthermore, it has been reported that activity of glutamine synthetase and glutamate dehydrogenase, the key enzymes involved in glutamate-glutamine cycle between neuron and glia, were markedly altered in the prefrontal cortex of schizophrenic patients, suggesting abnormalities in the function of glutamate-glutamine cycle in schizophrenic brain [ 20 ]. It is also well known that the glutamate-glutamine cycle between neuron and glia is tightly related to glutamate neurotransmission, glutamatergic function, and their regulation in human brain [ 7 ]. Taken together, it is likely that a dysfunction in glutamate-glutamine cycle in the brain may play a role in the pathophysiology of schizophrenia, supporting the glutamate hypothesis of schizophrenia. As described in introduction, sodium-dependent amino acids transporters, located in the abluminal membranes of the blood brain barrier, are capable of actively removing amino acids from the brain [ 16 , 20 , 21 ]. Sodium-dependent amino acids transporter are capable of pumping both glutamine (system N) and glutamate (glutamate transporters EAAT-1, 2, and -3) from the extracellular fluid into endothelial cells [ 20 , 21 ]. The luminal facilitative carriers for both glutamate and glutamine can then transport them to the blood [ 16 , 20 , 21 ]. Therefore, the concentrations of naturally occurring amino acids in the CSF [presumably similar to the extracellular fluid of brain] are ~10% of those of the blood [ 15 , 16 ]. Taken together, it seems that alteration in the transport mechanisms regulating levels of glutamate and glutamine in CSF may be implicated in elevated glutamine/glutamate ratio in CSF of schizophrenic patients although further study is necessary.
Discussion
In this study, we found that the ratio of glutamine to glutamate in the CSF of first episode and drug naive schizophrenic patients was significantly higher than that of normal controls although each level of glutamine and glutamate in the CSF of patients was not significantly different from that of normal controls. To our knowledge, this is a first report demonstrating that the ratios of glutamine to glutamate in the first episode and drug naive patients are significantly higher than those of normal controls. In contrast, it was supposed earlier that alterations in CSF levels of glutamate are not so prominent compared with those in the brain [ 14 ]. Therefore, it is likely that a difference in glutamate (or glutamine) levels between our CSF study and MRS studies may be due to the difference between CSF samples and specific corticolimbic regions. However, it should be noted that alterations in the ratio of glutamine to glutamate are detected in the CSF samples of first episode and drug naive schizophrenic patients, suggesting an abnormality of the glia-neuronal glutamate-glutamine cycle in the brain of patients with schizophrenia.In general, glutamine is synthesized in astrocytes from glutamate by the enzyme glutamine synthetase, found exclusively in brain glia cells. Glutamine then crosses the astrocytes to be transported into nerve cell terminals, where it is converted again into the neurotransmitter glutamate by glutaminase. It is reported that activities of glutaminase and glutamic acid decarboxylase (GAD; the rate-limiting enzyme in the synthesis of GABA by decarboxylation of glutamate) are significantly greater in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia than in the control group, whereas activities of glutamate dehydrogenase, glutamine synthetase, and GABA transaminase in the DLPFC of schizophrenia are not different from the control group [ 19 ]. These findings suggest that metabolism of glutamate and GABA might be altered in the DLPFC of schizophrenic patients. Furthermore, it has been reported that activity of glutamine synthetase and glutamate dehydrogenase, the key enzymes involved in glutamate-glutamine cycle between neuron and glia, were markedly altered in the prefrontal cortex of schizophrenic patients, suggesting abnormalities in the function of glutamate-glutamine cycle in schizophrenic brain [ 20 ]. It is also well known that the glutamate-glutamine cycle between neuron and glia is tightly related to glutamate neurotransmission, glutamatergic function, and their regulation in human brain [ 7 ]. Taken together, it is likely that a dysfunction in glutamate-glutamine cycle in the brain may play a role in the pathophysiology of schizophrenia, supporting the glutamate hypothesis of schizophrenia.As described in introduction, sodium-dependent amino acids transporters, located in the abluminal membranes of the blood brain barrier, are capable of actively removing amino acids from the brain [ 16 , 20 , 21 ]. Sodium-dependent amino acids transporter are capable of pumping both glutamine (system N) and glutamate (glutamate transporters EAAT-1, 2, and -3) from the extracellular fluid into endothelial cells [ 20 , 21 ]. The luminal facilitative carriers for both glutamate and glutamine can then transport them to the blood [ 16 , 20 , 21 ]. Therefore, the concentrations of naturally occurring amino acids in the CSF [presumably similar to the extracellular fluid of brain] are ~10% of those of the blood [ 15 , 16 ]. Taken together, it seems that alteration in the transport mechanisms regulating levels of glutamate and glutamine in CSF may be implicated in elevated glutamine/glutamate ratio in CSF of schizophrenic patients although further study is necessary.Conclusion Our findings suggest that a dysfunction in glutamate-glutamine cycle between neuron and glia may play a role in the pathophysiology of schizophrenia, supporting the glutamate hypothesis of schizophrenia.
Conclusion
Our findings suggest that a dysfunction in glutamate-glutamine cycle between neuron and glia may play a role in the pathophysiology of schizophrenia, supporting the glutamate hypothesis of schizophrenia.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contribution KH conceived of the study, its design and coordination, and edited the manuscript. GE participated in the design of the study. CN and LHL recruited subjects and collected CSF samples. ES and MI assisted HPLC analysis and data analyses. All authors read and approved the final manuscript. Figure 1 Levels of glutamine and glutamate, and ratio of glutamine to glutamate in CSF of normal controls, and first episode and drug naive schizophrenic patients. (A) CSF levels of glutamine in patients were not different from those of normal controls. (B) CSF levels of glutamate in patients were not different from those of normal controls. (C) Ratios of glutamine to glutamate in patients were significantly higher than those of normal controls.
Authors' contribution
KH conceived of the study, its design and coordination, and edited the manuscript. GE participated in the design of the study. CN and LHL recruited subjects and collected CSF samples. ES and MI assisted HPLC analysis and data analyses. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: