Morphological and Body Color Variation in Thai <Emphasis Type="Italic">Macaca fascicularis fascicularis</Emphasis> North and South of the Isthmus of Kra

Long-tailed macaques (Macaca fascicularis fascicularis) are widely distributed in Southeast Asia and are morphologically and genetically (Tosi et al. in International Journal of Primatology 23:161–178, 2002) distinguishable on either side of the Isthmus of Kra (ca. 10.5°N). We compared the somatometry and body color of 15 local populations of long-tailed macaques in Thailand distributed over areas from 6.5°N to 16.3°N and also a Thai rhesus macaque population at 17.2°N. Limb proportions and body color variation follow the geographical trend. However, contrary to a previous report, body size does not decrease with latitude in the northern group and also in the southern (southerly distributed) rhesus macaque. Relative tail length (RTL) and color contrast in yellow between the back and thigh are the sole traits that distinctively separate the 2 groups: the southern group has a long relative tail length (RTL >125%) and small color contrast, whereas the northern group has a short RTL (<120%) and large color contrast. The southern rhesus macaques appear to have somatometric and body color traits that follow the geographical trend in long-tailed macaques, though they maintain their distinctive species-specific traits of shorter RTL (ca. 55%), shorter relative facial length, and a bipartite body color pattern. Researchers assume that the northern group of long-tailed macaques and the southern rhesus macaques had undergone partial introgression with each other. Montane refugia present during the glacial period are localities in which introgression occurred in long-tailed macaques.     

BIOCHEMICAL BASIS OF FLORAL COLOR POLYMORPHISM IN A HETEROCYANIC POPULATION OF TRILLIUM SESSILE (LILIACEAE)

A study of flavonoids occurring within a heterocyanic population of Trillium sessile was made to determine the chemical basis of a common floral color polymorphism in this species. In the study population, three floral color phenotypes (red, pink, yellow) are determined primarily by the presence or absence of anthocyanin compounds in the petal tissue, and secondarily by quantitative differences in the concentration of several flavonol glycosides. Petals of red phenotypes contain both cyanidin 3-arabinoside and 3-diarabinoside, petals of pink phenotypes contain only cyanidin 3-arabinoside, and petals of yellow phenotypes lack cyanidin entirely. Quercetin 3-0-glucoside, quercetin 3-0-arabinoglucoside, quercetin 3–0-arabinogalactoside, and quercetin 3-0-arabinogalactosyl, 7-0-glucoside occur in petals of all three phenotypes but differ in relative amounts. Petals of the red phenotype have mostly 3-0-biosides, but lesser amounts of both quercetin 3-0-glucoside and the 3,7-0-triglycoside. Petals of the pink phenotype contain relatively equal amounts of quercetin mono-, di-, and triglycosides. Petals of the yellow phenotypes contain mostly quercetin 3,7-0-triglycosides, and less mono- and di-glycosides. Small amounts of a quercetin tetraglycoside were detected in petals of both yellow and pink phenotypes, but not in red phenotypes. The enhancement of quercetin polyglycoside biosynthesis in yellow petal phenotypes is attributed to the shunting of dihydroflavonol precursors to synthesis of quercetin compounds when their conversion to anthocyanins is blocked genetically.     

Induced mutation in β-CAROTENE HYDROXYLASE results in accumulation of β-carotene and conversion of red to orange color in pepper fruit

Pepper fruit is typically red, but green, orange and yellow cultivars are gaining consumer acceptance. This color variation is mainly due to variations in carotenoid composition. Orange color in pepper can result from a number of carotenoid profiles, but its genetic basis is only partly known. We identified an EMS-induced orange-fruited mutant using the wild-type blocky red-fruited cultivar ‘Maor’ as progenitor. This mutant accumulates mainly β-carotene in its fruit, instead of the complex pattern of red and yellow carotenoids in ‘Maor’. We identified an A⁷⁰⁹ to G transition in the cDNA of β-CAROTENE HYDROXYLASE2 in the orange pepper and complete co-segregation of this single-nucleotide polymorphism with the mutated phenotype. We therefore hypothesized that β-CAROTENE HYDROXYLASE2 controls the orange mutation in pepper. Interestingly, the expression of β-CAROTENE HYDROXYLASE2 and additional carotenogenesis genes was elevated in the orange fruit compared with the red fruit, indicating possible feedback regulation of genes in the pathway. Because carotenoids serve as precursors for volatile compounds, we compared the volatile profiles of the two parents. The orange pepper contained more volatile compounds than ‘Maor’, with predominant elevation of norisoprenoids derived from β-carotene degradation, while sesquiterpenes predominated in the red fruit. Because of the importance of β-carotene as a provitamin A precursor in the human diet, the orange-fruited mutant might serve as a natural source for pepper fruit biofortification. Moreover, the change in volatile profile may result in a fruit flavor that differs from other pepper cultivars.     

茶树品种(品系)芽叶色泽表型遗传数量分类研究

为探讨茶树春梢芽叶色泽的数量遗传特性,应用色差仪测定68份茶树品种(品系)春梢芽叶的色泽表型值并进行数量分类研究。结果表明,对叶色差异度较高的第1叶位L~*、a~*、b~*、C、H值进行分层聚类分析,可将芽叶色泽划分为紫色系、黄色系和绿色系等7个色系。色系分类与CIELab颜色体系的对应性较强,色系划分符合茶树品种(品系)叶色表型特点。茶树品系的叶色表型较丰富,叶色在L~*、a~*、b~*值三维坐标空间整体呈带域分布,色系由紫色系、紫红色系、紫绿色系向黄色系、黄绿色系、绿色系变化。春梢芽叶色泽表型数据的遗传数量分类与色系判定为茶树特异芽叶色泽育种提供科学依据。     

Intensity of male‐male competition predicts morph diversity in a color polymorphic lizard

Sexual selection is one of the main processes involved in the emergence and maintenance of heritable color polymorphisms in a variety of taxa. Here, we test whether the intensity of sexual selection, estimated from population sex ratio, predicts morph diversity in Podarcis muralis, a color polymorphic lizard with discrete white, yellow, orange, white‐orange, and yellow‐orange male and female phenotypes (i.e., morphs). In a sample of 116 Pyrenean populations and 5421 lizards, sex ratios (m/f) vary from 0.29 to 2.5, with the number of morphs for each sex ranging from 2 to 5. Male‐biased sex ratios are associated with increased morph diversity as measured with Shannon's diversity index. The main factor accounting for this relationship is male morph richness (i.e., the number of morphs). In contrast, female morph diversity is not related to sex ratio. These results suggest a relationship between the intensity of male intrasexual competition and male morph diversity. While other selective forces may interact with sexual selection in maintaining the color polymorphisms in P. muralis, this evidence suggests a complex evolutionary scenario possibly involving frequency‐dependent selection of alternative reproductive tactics and/or complex balancing selection.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

Development of a codominant CAPS marker for allelic selection between canary yellow and red watermelon based on SNP in lycopene β-cyclase (<Emphasis Type="Italic">LCYB</Emphasis>) gene

Flesh color of watermelon is an agronomically important trait that is predominantly determined by a network of the carotenoid biosynthetic pathway, which also contributes to the nutritional value of the fruit through the health-promoting function of carotenoids. We have identified a key gene, lycopene β-cyclase (LCYB) that may determine canary yellow and red flesh color of watermelon and developed a zero-distance molecular marker that identifies a critical single nucleotide polymorphism (SNP) that distinguishes different alleles of the LCYB gene. Analysis of the flesh color inheritance in segregating populations indicated that a single gene determines the color difference between canary yellow and red flesh in watermelon. The sequence comparison of full-length cDNA of LCYB, which was isolated using degenerate PCR and RACE, identified three SNPs in the coding region of LCYB between canary yellow and red. These SNPs showed perfect co-segregation with flesh color phenotypes. One of the SNPs introduces an amino acid replacement of evolutionarily conserved Phe226 to Val, which may impair the catalytic function of LCYB. This SNP was used to develop a cleaved amplified polymorphic sequence (CAPS) marker, which perfectly cosegregated with flesh color phenotype. Our results strongly suggest that LCYB may be the genetic determinant for canary yellow or red flesh color and our CAPS marker will allow breeders to economically distinguish between canary yellow and red watermelon fruit color at the seedling stage.     

Morphological development of the structures related to annualism in the ovarian follicle of the killifish Millerichthys robustus (Costa,1995) (Teleostei: Cyprinodontiformes)

Ovaries of five females of the annual fish teleost species Millerichthys robustus were processed, and the development of the cortical alveoli, zona pellucida and secondary envelope during oogenesis were described. We also documented the origin of the cortical alveoli in time and space similar to the Balbiani body; the synthesis of three generations of cortical alveoli and an active zona pellucida prior to vitellogenesis, which is implicated in the entry of oils to the interior of the oocyte. We found that in this species, the diameter of the alveoli is greater than in the other teleost fish species reported in the literature, except for Fundulus heteroclitus, in which the diameter is similar. The thickness of the zona pellucida recorded in M. robustus is the greatest reported to date. Likewise, two periods of secondary envelope deposition were documented: filaments during pre‐vitellogenesis and, subsequently, trapeze‐shaped projections during the maturation of the oocytes. We report about development of structures that are considered key for the survival of embryos in annual fish during the long periods of diapause in their extreme habitats. The development of peripheral structures described here probably reflects the changes in the physiology of the oocytes in M. robustus. J. Morphol. 277:1219–1230, 2016. © 2016 Wiley Periodicals, Inc.     

SOCIAL CAUSES OF CORRELATIONAL SELECTION AND THE RESOLUTION OF A HERITABLE THROAT COLOR POLYMORPHISM IN A LIZARD

Abstract When selection acts on social or behavioral traits, the fitness of an individual depends on the phenotypes of its competitors. Here, we describe methods and statistical inference for measuring natural selection in small social groups. We measured selection on throat color alleles that arises from microgeographic variation in allele frequency at natal sites of side‐blotched lizards (Uta stansburiana). Previous game‐theoretic analysis indicates that two color morphs of female side‐blotched lizards are engaged in an offspring quantity‐quality game that promotes a density‐and frequency‐dependent cycle. Orange‐throated females are r‐strategists. They lay large clutches of small progeny, which have poor survival at high density, but good survival at low density. In contrast, yellow‐throated females are K‐strategists. They lay small clutches of large progeny, which have good survival at high density. We tested three predictions of the female game: (1) orange progeny should have a fitness advantage at low density; (2) correlational selection acts to couple color alleles and progeny size; and (3) this correlational selection arises from frequency‐dependent selection in which large hatchling size confers an advantage, but only when yellow alleles are rare. We also confirmed the heritability of color, and therefore its genetic basis, by producing progeny from controlled matings. A parsimonious cause of the high heritability is that three alleles (o, b, y) segregate as one genetic factor. We review the physiology of color formation to explain the possible genetic architecture of the throat color trait. Heritability of color was nearly additive in our breeding study, allowing us to compute a genotypic value for each individual and thus predict the frequency of progeny alleles released on 116 plots. Rather than study the fitness of individual progeny, we studied how the fitness of their color alleles varied with allele frequency on plots. We confirmed prediction 1: When orange alleles are present in female progeny, they have higher fitness at low density when compared to other alleles. Even though the difference in egg size of the female morphs was small (0.02 g), it led to knife‐edged survival effects for their progeny depending on local social context. Selection on hatchling survival was not only dependent on color alleles, but on a fitness interaction between color alleles and hatchling size, which confirmed prediction 2. Sire effects, which are not confounded by maternal phenotype, allowed us to resolve the frequency dependence of correlational selection on egg size and color alleles and thereby confirmed prediction 3. Selection favored large size when yellow sire alleles were rare, but small size when they were common. Correlational selection promotes the formation of a self‐reinforcing genetic correlation between the morphs and life‐history variation, which causes selection in the next density and frequency cycle to be exacerbated. We discuss general conditions for the evolution of self‐reinforcing genetic correlations that arise from social selection associated with frequency‐dependent sexual and natural selection.     

Identity of an endangered grasshopper (Acrididae: Brachaspis): Taxonomy, molecules and conservation

Brachaspis robustus is an endangeredgrasshopper endemic to South Island, NewZealand. It is both rare and localised;occupying low altitude floodplain terraces andbraided riverbeds of the Mackenzie Basin. Thisis in stark contrast to the two other speciesin this genus (B. nivalis and B.collinus) which occupy montane habitats.Mitochondrial and nuclear sequence data wereemployed to explore genetic diversity andphylogenetic relationships of populations ofBrachaspis with a view to establishingthe status of B. robustus. Molecularevidence indicates that Brachaspisprobably radiated during the Pliocene and thatdivisions within the genus relate more tospatial distribution developed during thePleistocene than to ecology. The mitochondrial(Cytochrome oxidase I) and nuclear (ITS)sequence data indicate that Brachaspisnivalis is divided into northern and southernpopulations. The northern clade is furthersubdivided geographically. The southern cladecomprises alpine populations of B.nivalis and includes the lowland B.robustus. Additionally, it is observed thatsome morphological features previously thoughtto be specific to B. robustus also occurin members of the southern B. nivalisclade. It is suggested that the taxon B.robustus should include all of the southernBrachaspis populations. But it is arguedthat the absence of genetic evidencedistinguishing the endangered population doesnot preclude it from conservation effort. Acombination of morphological and habitatpeculiarities indicate that the survival ofB. robustus (sensu lato) isimportant to the maintenance of diversity.     

Offshore cephalopods (Mollusca: Cephalopoda) collected off the west coast of Mexico during the TALUD cruises

Deep-sea cephalopods are still poorly studied worldwide. In the case of the Mexican Pacific there is a general lack of basic information concerning their distribution and biology. A series of 132 specimens of pelagic and benthic deep-water cephalopods was obtained during the TALUD project. Samples were mostly obtained with benthic sampling gear that operated as mid-water trawls during the ascent of the nets. Micronekton and Isaacs-Kidd samplers were also occasionally used. The specimens (77 lots in total) were obtained at localities off western Mexico at depths between 122 m and 2200 m and belong to 31 species. Considering material identified to species level only, a total of 13 species were found only at a single station, while five others occurred in 5–6 stations (i.e., Leachia dislocata, Abraliopsis [Pfefferiteuthis] falco, Pterygioteuthis giardi, P. holeyi and Benthoctopus robustus). The most widespread species was Japetella diaphana, collected in 11 stations. Twenty-two of the 31 species are strictly pelagic, eight strictly benthic and one is benthopelagic. Significant new distribution records were obtained for seven species: Doryteuthis opalescens, Gonatus berryi, Todarodes pacificus, Opistoteuthis californiana, Benthoctopus leioderma, B. robustus and Graneledone boreopacifica.     

RIBOSOMAL DNA LENGTH POLYMORPHISMS WITHIN POPULATIONS OF XYLARIA MAGNOLIAE (ASCOMYCOTINA)

Xylaria magnoliae forms abundant conidial and sexual fruiting structures on the fallen strobili of Magnolia spp. throughout the southeastern United States. Populations of X. magnoliae may be identified as isolates originating from a single strobilus, among different strobili from different host trees, and different host tree localities. We examined the stratified structure of X. magnoliae populations by analysis of their ribosomal DNA restriction patterns. A sample of 48 single-conidial isolates was obtained from strobili of both Magnolia grandiflora and M. fraseri in the piedmont and mountains of North Carolina and Georgia. Southern blotting experiments revealed considerable variation in rDNA lengths between and within different isolates. Although a single rDNA phenotype usually dominates within a particular collecting locality, different phenotypes were also observed within a locality and even between isolates obtained from a single strobilus. Some rDNA restriction phenotypes are distributed over localities separated by up to 400 km. These results suggest a fine-grained population structure for X. magnoliae, together with the potential for genetic exchange and dispersal between widely separated localities.     

CHROMOPLAST ULTRASTRUCTURE AS AFFECTED BY GENES CONTROLLING GRANA RETENTION AND CAROTENOIDS IN FRUITS OF CAPSICUM ANNUUM

Plastids in the fruits of isogenic lines of pepper (Capsicum annuum) were examined by electron microscopy with reference to four genotypes determining the carotenoid composition and the colors red, yellow, brown, and green of the ripe fruit. One gene pair (y⁺/y) influences carotenoid content and the other pair (cl⁺/cl) controls the chlorophyll. The retention of the grana and chlorophyll in the ripe fruits of the brown and green phenotypes is correlated with the cl cl genotype. The y⁺ gene increases the total carotenoids and promotes the formation of red pigments. Giant grana were found in the yellow and green phenotypes, but during ripening these disappeared in the yellow. Unusual dichotomous and concentric grana were observed in the green. Globule-associated carotenoids forming fibrillar crystalloids were present in all color types, although to a lesser degree in the yellow fruit. Membrane-associated carotenoids occurred only in the yellow and green phenotypes.     

Orange, yellow and white-cream: inheritance of carotenoid-based colour in sunflower pollen

Inheritance of pollen colour was studied in sunflower (Helianthus annuus L.) using three distinct pollen colour morphs: orange, yellow and white‐cream. Orange is the most common colour of sunflower pollen, while the yellow morph is less frequent. These two types were observed in the inbred lines F11 and EF2L, respectively. White‐cream pollen is a rare phenotype in nature, and was identified in a mutant, named white‐cream pollen, recovered in the R₂ generation of an in vitro regenerated plant. The F11 inbred line was used as starting material for in vitro regeneration. The carotenoid content of these three pollen morphs differed, and was extremely reduced in white‐cream pollen. The phenotype of F₁ populations obtained by reciprocal crosses revealed that the orange trait was dominant over both white‐cream and yellow. Segregation of F₂ populations of both crosses, orange × yellow and orange × white‐cream, approached a 3:1 ratio, indicating the possibility of simple genetic control. By contrast, a complementation cross between the two lines with white‐cream and yellow pollen produced F₁ plants with orange pollen. The F₂ populations of this cross‐segregated as nine orange: four white‐cream: four yellow. A model conforming to the involvement of two unlinked genes, here designated Y and O, can explain these results. Accessions with yellow pollen would have the genotype YYoo, the white‐cream pollen mutant would have yyOO and the accession with orange pollen would have YYOO. Within F₂ populations of the cross white‐cream × yellow a new genotype, yyoo, with white‐cream pollen was scored. The results of the cross yyoo × YYoo produced only F₁ plants with yellow pollen, supporting a recessive epistatic model of inheritance between two loci. In this model, yy is epistatic on O and o. In F₂ populations, the distributions of phenotypic classes suggested that the genetic control of carotenoid content is governed by major genes, with large effects segregating in a background of polygenic variation. These three pollen morphs can provide insight into the sequence in which genes act, as well into the biochemical pathway controlling carotenoid biosynthesis in anthers and the transfer of these different pigments into pollenkitt.     

Aggressive Behavior and Courtship Rejection in Brightly and Plainly Colored Female Keeled Earless Lizards (Holbrookia propinqua)

Relationships between bright secondary sexual coloration and behavior were studied in female Holbrookia propinqua, which develop striking orange and yellow colors during the breeding season. In tethered introduction studies, brightly colored females performed aggressive courtship rejection behaviors toward conspecific males; plainly colored females were not aggressive toward males, but attempted to avoid them. Responses of females of the two color patterns toward conspecific females of both color phases were not detectably different. Experimental introductions of lizards with coloration modified by paint showed that females of both color patterns recognize any other lizard bearing the bright female colors as female, regardless of actual sex. Both the orange and yellow components were shown to contribute to sex recognition. The yellow component alone allowed accurate sex identification, but only half the females responded to males painted with only the orange female component as if they were females. Because females did not behave differently toward other females on the basis of coloration, the hypothesis that bright coloration evolved as an adaptive signal between females is rejected. The dark ventrolateral stripes of male and plainly colored females did not appear to affect intraspecific social responses by females.     

Coadapted changes in energy metabolites and body color phenotypes for resistance to starvation and desiccation in latitudinal populations of D. melanogaster

In D. melanogaster, resistance to starvation and desiccation vary in opposite directions across a geographical gradient in India but there is lack of such clinal variation on other continents. However, it is not clear whether these resistance traits or other correlated traits are the target of natural selection. For resistance to starvation or desiccation in D. melanogaster, we tested the hypothesis whether body color phenotypes and energy metabolites show correlated selection response. Our results are interesting in several respects. First, based on within population analysis, assorted darker and lighter flies from a given population showed that darker flies store higher amount of trehalose and confer greater desiccation resistance as compared with lighter flies. By contrast, lighter flies store higher lipids content and confer increased starvation tolerance. Thus, there is a trade-off for energy metabolites as well as body color phenotypes for starvation and desiccation stress. Further, trait associations within populations reflect similar patterns in geographical populations. Second, we found opposite clines for trehalose and body lipids. Third, coadapated phenotypes have evolved under contrasting climatic conditions i.e. drier and colder northern localities select darker flies with higher trehalose as well as desiccation resistance while hot and humid localities favor lighter flies with higher lipids level and greater starvation tolerance. Thus, the evolution of coadapated phenotypes associated with starvation and desiccation resistance might have resulted due to specific ecological conditions i.e. humidity changes on the Indian subcontinent.     

Resistance of Common Bean (Phaseolus vulgaris) to Bacterial Wilt Caused by Curtobacterium flaccumfaciens pv. flaccumfaciens

Bacterial wilt caused by Curtobacterium flaccumfaciens pv. flaccumfaciens is an important new disease of common bean (Phaseolus vulgaris) in western Canada. Both yellow and orange variants of the pathogen were found in the region. A controlled environment study was conducted to assess 124 common bean cultivars and lines from eight market classes for resistance to the yellow and orange variants of the pathogen, using the hilum injury/seed inoculation method. Results of the screening tests showed significant (P < 0.05) differences in resistance to bacterial wilt among the cultivars or lines. The great northern line L02E317, the great northern cultivar Resolute and pinto lines L02B662 and 999S‐2A, were highly resistant to both variants of the pathogen, with disease severity indices of 0 on a rating scale of 0 (no wilt symptoms) to 5 (dead seedling). Resistant cultivars or lines were found among black, great northern, pink, pinto, small red and Flor de Mayo bean market classes. The study concludes that new bacterial wilt‐resistant germplasm exists among Canadian bean cultivars and lines, and constitutes a valuable resource for breeding common beans for resistance to both yellow and orange variants of C. flaccumfaciens pv. flaccumfaciens.     

Phenotypic Diversity among Alleles at the PER-1 Locus of NEUROSPORA CRASSA

Comparison of 11 perithecial color mutants suggested that all were alleles at the per-1 locus but nonetheless separable into two groups because of phenotypic differences. Three of the mutant strains produced orange perithecia and black ascospores, and eight produced paler, yellow perithecia and white ascospores. Perithecial phenotype was dependent upon the genotype of the protoperithecial parent; ascospore phenotype, upon the genotype of the individual ascospore. No evidence was found that the white ascospores were due to chromosomal rearrangements. No separation of the perithecial and ascospore phenotypes by recombination was observed in a cross between one of the mutants and a per-1+ strain. However, apparent low levels of recombination in crosses between some of the mutants indicated possible genetic complexity at the per-1 locus. The phase specificity of the per-1 mutations and the possible nature and mode of expression of the orange and yellow perithecial pigments are discussed.     

Standing genetic variation as a potential mechanism of novel cave phenotype evolution in the freshwater isopod,Asellus aquaticus

Novel phenotypes can come about through a variety of mechanisms including standing genetic variation from a founding population. Cave animals are an excellent system in which to study the evolution of novel phenotypes such as loss of pigmentation and eyes. Asellus aquaticus is a freshwater isopod crustacean found in Europe and has both a surface and a cave ecomorph which vary in multiple phenotypic traits. An orange eye phenotype was previously revealed by F₂ crosses and backcrosses to the cave parent within two examined Slovenian cave populations. Complete loss of pigmentation, both in eye and body, is epistatic to the orange eye phenotype and therefore the orange eye phenotype is hidden within the cave populations. Our goal was to investigate the origin of the orange eye alleles within the Slovenian cave populations by examining A. aquaticus individuals from Slovenian and Romanian surface populations and Asellus aquaticus infernus individuals from a Romanian cave population. We found orange eye individuals present in lab raised surface populations of A. aquaticus from both Slovenia and Romania. Using a mapping approach with crosses between individuals of two surface populations, we found that the region known to be responsible for the orange eye phenotype within the two previously examined Slovenian cave populations was also responsible within both the Slovenian and the Romanian surface populations. Complementation crosses between orange eye Slovenian and orange eye Romanian surface individuals suggest that the same gene is responsible for the orange eye phenotype in both surface populations. Additionally, we observed a low frequency phenotype of eye loss in crosses generated between the two surface populations and also in the Romanian surface population. Finally, in a cave population from Romania, A. aquaticus infernus, we found that the same region is also responsible for the orange eye phenotype as the Slovenian cave populations and the Slovenian and Romanian surface populations. Therefore, we present evidence that variation present in the cave populations could originate from standing variation present in the surface populations and/or transgressive hybridization of different surface phylogenetic lineages rather than de novo mutations.