Convergent Evolution of the Cavefish Astyanax (Characidae, Teleostei)

Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066 The Linnean Society of London, 2003? 2003 80? 545554 Original Article

CONVERGENCE OF ASTYANAX CAVEFISH EVOLUTION Biological Journal of the Linnean Society, 2003, 80, 545–554. With 6 figures H. WILKENS and U. STRECKER

Convergent evolution of the caveﬁsh Astyanax (Characidae, Teleostei): genetic evidence from reduced eye-size and pigmentation

HORST WILKENS* and ULRIKE STRECKER Downloaded from https://academic.oup.com/biolinnean/article/80/4/545/2636155 by guest on 29 September 2021 Zoological Institute and Zoological Museum, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany

Received 7 June 2002; accepted for publication 7 March 2003

More than 20 populations of the cave-dwelling characid Astyanax occur within a restricted karst area in Mexico. The ﬁsh possess reduced eyes without lenses and visual cells. It is still an open question as to whether this condition evolved convergently after multiple entries of the surface ancestor into the different caves or whether a single cave ancestor, already characterized by reduced eyes, spread secondarily within them. In the crosses between speciﬁc populations, specimens appear that deviate considerably from those of the parents. They possess larger and better-developed eyes with histologically intact lenses and visual cells; they thus have the structural potential for vision. This indicates that in different cave populations, different mutations in the eye gene system have occurred. In cases where these non-functional rudimentary genes are recombined in hybrid specimens, gene expression may be restored. This is the result of separate evolution of several Astyanax cave populations. © 2003 The Linnean Society of London, Bio- logical Journal of the Linnean Society, 2003, 80, 545-554.

ADDITIONAL KEYWORDS: Anoptichthys - blindness - crossing experiments - melanophore - mutation pressure - relaxed selection - retina - rudimentation - speciation - troglobite.

INTRODUCTION south. These forms (some of which are described as belonging to a separate genus Anoptichthys) are char- Life in continuous darkness, as exhibited by cave liv- acterized by different degrees of rudimentation of ing animals, has stimulated the thinking of biologists eyes and pigmentation. Three populations show an in many ways (Wilkens, Culver & Humphreys, 2000); intermediate stage of reduction, while more than 20 the reduction of biologically functionless traits such as others exhibit an extreme stage (Mitchell, Russell & eyes and pigmentation has been of particular interest. Elliott, 1977). All are closely related to a fully eyed Analysis of the genetic basis of eye and pigment and pigmented ﬁsh common in Mexican surface regression provides insight into the process of adapta- waters, and are interfertile with each other as well as tion. Information is obtained as to whether reduction with the surface form (Wilkens, 1988; Culver & Wilk- in eye-size (and regressive evolution in general) is a ens, 2000). constructive process driven by selection or whether it Troglobitic faunas may speciate in two ways. First, a relies on the accumulation of random mutations single, widespread ancestral surface species may (Kosswig, 1960; for a review see Culver & Wilkens, simultaneously invade separate karst or cave systems 2000). by multiple entries. The adaptations to darkness in In north-eastern Mexico, a series of cave popu- this case would be by convergence. Second, a species lations of the characid Astyanax fasciatus already adapted to a subterranean environment may (= A. mexicanus) occurs within a geographically small multiply within it (Holsinger, 2000). The question as karst limestone area, extending about 150 km north– to whether there was a single or multiple invasion by the different Astyanax cave populations is still *Corresponding author. E-mail: disputed (Wilkens, 1971; Avise & Selander, 1972; [email protected] Mitchell et al., 1977; Espinasa & Borowsky, 2001;

Dowling, Martasian & Jeffery, 2002; Strecker, Ber- RESULTS natchez & Wilkens, 2003). We investigated these issues by crossing fish from JUVENILE EYEBALL SIZE IN THE CAVE FISH AND THEIR populations with extreme eye and pigment reduction CROSSINGS from all over the distribution area: the northernmost Measurement of eyeball size in juveniles (2.5 cm SL) population from Sótano de el Molino (Sierra de Gua- of the various populations revealed differences. It was temala), a more central population from Cueva de El smallest in Piedras, Yerbaniz and Curva and largest in Pachón and southern populations from Sótano de Yer- Molino (which has a distribution range separate from baniz, Sótano de las Piedras and Cueva de la Curva, all the other forms). Pachón overlapped with Molino all of which occur in the Sierra de El Abra (Fig. 1). The and Curva (Figs 2, 3). genetic basis of reduced eye-size and pigmentation in Size in the F1 and F2 crosses fell into two groups. The first contained the crosses of Molino with Pachón, the different populations was analysed by studying Downloaded from https://academic.oup.com/biolinnean/article/80/4/545/2636155 by guest on 29 September 2021 eye-size, histology and melanophore density in the Piedras, Yerbaniz and Curva, in which size surpassed hybrid offspring. The results will contribute to the dis- that of the parental generation (Fig. 2). In F2 the vari- cussion as to whether, or to what degree, these evolu- ability was also considerably increased, with smaller tionary processes derive from convergence. eyed parental forms and sizes in offspring which surpassed even those of Molino. The percentage of large eyes varied: highest in the crosses with Pachón (27% MATERIAL AND METHODS larger than F1), lowest in those with Piedras (8.7%), Curva (8%) and Yerbaniz (10%). The size range was POPULATIONS STUDIED 0.6–1.3 mm in crosses with Piedras and Yerbaniz and The eyed fish were caught in a surface creek draining 0.5–1.5 mm in crosses with Pachón and Curva. into the Micos Cave (Cueva del Río Subtérraneo). The In the second group (the El Abra group) formed by fish from El Sótano de El Molino (1), La Cueva de El crosses between Pachón, Piedras, Yerbaniz and Curva, Pachón (2), El Sótano de Yerbaniz (3), El Sótano de las the size ranges of both F1 and F2 did not surpass those Piedras (4) and La Cueva de la Curva (5) (nomencla- of the parental forms. The values were intermediate ture according to Mitchell et al., 1977) were offspring where the parental eyes were of different sizes; other- of previously caught specimens (2–4 caught in 1971, 1 wise they were similar or even smaller (Fig. 3). and 5 caught in the 1990s by Borowsky (New York Distributions in F1 and F2 crosses of Molino with University)). All were maintained and bred under day- the eyed surface fish corresponded to earlier findings light conditions at 24∞C. in crosses of cave fish with the epigean form (Wilkens, 1988), with mean eye-sizes nearly intermediate between them. The range of the F2 crosses was larger EYES than that of F1 and lay between the parental forms. The horizontal diameter of the enucleated eyeball was The backcross with Molino ranged between F1 and measured in juvenile fish (preservation in 4% formal- Molino. dehyde, 2.5 cm SL, 1 MU = 0.25 mm) with a dissection microscope. For histological analysis, paraffin embed- ded slides of adult specimens were prepared and EYE HISTOLOGY IN ADULT FISH stained in Pasini dye. Adult eye-size was measured in Cave fish these slides. The rudimentary eyes of adult Curva, Piedras and Yerbaniz fish are histologically comparable to those of Pachón and Sabinos (Wilkens, 1970, 1988) and char- MELANOPHORE PATTERN acterized by a variable degree of differentiation. The The total number of melanophores was counted within best developed contain retinal rudiments consisting of an area of 1.77 mm2 from a region in front of the ganglion cell, inner nuclear and inner plexiform layers dorsal fin using a microscope (magnification 35¥). For as well as vitreous bodies, whereas retinal tissue in this purpose the specimens (2.5 cm SL) were narco- the less well developed is not layered and may be tized and adrenalin (L-Noradrenalin, 1 : 1000 in NaCl almost completely reduced. Rudiments of visual cells solution) was distributed in the area with a thin (outer plexiform and nuclear layers, outer segments) pipette. Adrenalin induces a physiological colour never occur in adult specimens. The anterior eye change and the melanophores contract within a few chamber is filled with a derivative of the ligamentum minutes. The fish were kept and bred over black annulare, the spongiosum; the lens and pupil do not underlay to provide equal environmental conditions exist (Wilkens, 1970, 1988). influencing the number of melanophores (morpholog- The Molino fish have similar histological character- ical colour change). istics of eye reduction (Wilkens, 1988). However,

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Figure 1. Distribution of Astyanax cave forms.

% 50 40 B to Molino (66) 30 20 10 0

40 B to Pachon´ (48) 30 20 % 10 0 40 30 F2 (51) 20 F2 (52) 20 10 10 Downloaded from https://academic.oup.com/biolinnean/article/80/4/545/2636155 by guest on 29 September 2021 0 0

50 50 F1 (44) F1 (20) 40 40 30 30 20 20 10 10 0 0

90 90 Yerbaniz (18) Molino (13) 80 80 Pachon´ (22) Molino (13) 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 mm mm Eye ball size Eye ball size % % 30 F2 (69) 30 F2 (84) 20 20 10 10 0 0

90 90 80 F1 (20) 80 70 70 F1 (20) 60 60 50 50 40 40 30 30 20 20 10 10 0 0

90 Piedras (13) Molino (13) 90 Curva (28) 80 80 Molino (13) 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4

mm mm Eye ball size Eye ball size

Figure 2. Eye-size of the cave forms from the Sierra de El Abra (Pachón, Yerbaniz, Piedras, Curva) and their crosses with the Molino ﬁsh from the Sierra de Guatemala (B = backcrossing).

% % 60 60

50 50 F1 - Yerbaniz F1 (17) x 40 40 Piedras

30 30

20 20

10 10

0 0

Piedras (15) Pachon´ (22) 70

80 Downloaded from https://academic.oup.com/biolinnean/article/80/4/545/2636155 by guest on 29 September 2021 60 Yerbaniz (18) 50 60 40 40 30 20 20 10 0 0

% %

F2 (58) 40 50 F1 (20) 40 30 30

20 20

10 10 0 0 Curva (23) 80 Piedras (15) 40 F1 (30) 60 30 40 20 20 10 0 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 mm 60 Curva (23) Pachon´ (22) Eye ball size 50

10 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 mm Eye ball size

Figure 3. Eye-size of the cave forms from the Sierra de El Abra and the crosses between them.

© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 545–554 550 H. WILKENS and U. STRECKER there are slight differences. When compared with the MELANOPHORE PIGMENTATION Curva fish, the mean eye rudiment size (horizontal No phenotypic differences are found between the Pie- diameter) is about 40% larger. In addition to a loose dras and Curva fish, the crosses between them and network of tissue derived from part of the chorioid, with the Yerbaniz and the Pachón fish. They are char- the scleral bulbus encloses a much smaller dense acterized by the development of a strongly reduced col- complex of rudimentary chorioid, retina and other oration caused by a recessive allele, the ‘brown gene’ eye structures forming only 40% of its volume. This mutation, which reduces the melanin content of the part is also enlarged in the Molino fish (11% in com- melanophores (Sadoglu & McKee, 1969; Wilkens, parison with the Curva fish). All eye rudiments, even 1988; Culver & Wilkens, 2000). In contrast, all F1 the smallest, contain retinal nervous tissue. The vit- Molino/Curva as well as F1 Molino/Piedras resemble reous body, however, which is still formed in most of the surface fish phenotypically because they are able the eyes of the Curva, Pachón and Sabinos fish, to develop the full melanin content. In the F2 crosses Downloaded from https://academic.oup.com/biolinnean/article/80/4/545/2636155 by guest on 29 September 2021 which are larger, is nearly completely displaced a 3 : 1 ratio of dark to pale specimens is found devel- (Fig. 4). oping independently from the number of melanophores. This is due to the Molino fish possessing the Crosses intact gene for full melanin production; they appear The adult eyes of all F1 crosses between Molino and pale, however, because, like the Pachón and Yerbaniz the El Abra group are larger, but also sunken beneath fish, they are albino. This destructive mutation com- the body surface. The mean horizontal eye diameter is pletely disturbs the production of melanin. about 16% larger than Molino and about 65% larger In the Piedras and Curva populations only 20% of than Curva. The dense complex of rudimentary chori- the number of melanophores of the surface form oid, retina and other eye structures is also larger and develop. Crossings revealed that the same percentage fills about 64% of the scleral lumen. In comparison to is characteristic of the albino Yerbaniz and Pachón Molino and Curva, it has increased by 150% and forms. As has already been shown for eye-size, two 180%, respectively. groupings of melanophore densities can also be found In adult F1 crosses, a clearly layered retinal rudi- in F1 and F2. First, no differences in mean densities ment is developed, consisting of ganglion cell, inner and ranges exist in either set of crosses between nuclear and inner plexiform layers and pigment epi- Pachón, Piedras, Yerbaniz and Curva or in comparison thelium. In some specimens, the outer nuclear layer as to the parental generation. Second, in the Molino/ well as outer plexiform layer may be partially devel- Curva and Molino/Piedras crosses, the mean number oped. However, the outer segments of the visual cells of melanophores is enhanced when compared with the are always reduced. The vitreous body, processus fal- respective pigmented parental form (Fig. 6). ciformis, rudimentary lenses at the capsule stage or In the F1 backcross with Molino, the mean melan- opaque ball-like structures at the secondary fibre ophore density is higher than in the backcross with stage may develop (Fig. 4). Curva. This shows that the genetic system responsible In the F2 Molino/ Pachón crosses, extremely large for the number of melanophores in Molino is more eyes are developed, reaching 56% of the size (Molino powerful than that of the Curva and other cave fish 36%, F1 40%) of those of surface fish (2.5 cm SL). (Fig. 6). They are well differentiated and contain all the struc- The number of melanophores in the Molino/sur- tures typical of the surface form. Even in adults, face fish crosses resembles those found in previous visual cells and large translucent lenses are devel- studies of crossings between surface and either oped (Fig. 4) so that the structural potential for vision Pachón or Sabinos cave fish (Wilkens, 1988). The is restored . means and ranges of F2 lie between those of the sur- In the F1 Molino/El Abra crosses, which all devel- face and cave fish, and are double those of the back- oped larger and better differentiated eyes, mean eye- cross (Fig. 6). size was also found to be enlarged, but to a lesser extent in hybrids from crossings within the El Abra group (Fig. 5). Only a few specimens developed consid- DISCUSSION erable structural improvements such as a rudimentary lens capsule. Already seen in the F1 Pachón/ COMPARISON OF REDUCTION IN EYE-SIZE AND Sabinos crosses (Wilkens, 1971), such a structure was PIGMENTATION IN CAVE FISH also detected among the largest-eyed F1 Pachón/ Among the Astyanax cave forms, the Molino fish are Curva specimens. Eyes which were structurally clearly distinct, showing a slightly lesser degree of slightly improved compared to those of the parental reduction in eye-size and pigmentation. It is the only generation were found in both the F2 Molino/El Abra one of the strongly reduced populations lacking the and within El Abra crosses. ‘brown gene’ mutation.

© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 545–554 CONVERGENCE OF ASTYANAX CAVEFISH EVOLUTION 551 Downloaded from https://academic.oup.com/biolinnean/article/80/4/545/2636155 by guest on 29 September 2021

Figure 4. Histological sections of the eyes of the Molino and Curva ﬁsh, their F1 cross and large-eyed Molino/Pachón F2- hybrid specimen. 1. Cornea, 2. Anterior eye chamber/spongiosum. 3. Lens/lens capsule. 4. Vitreous body. 5. Retina rudiment: (a) inner limiting membrane (b) ganglionic layer (c) inner plexiform (d) inner nuclear (e) outer plexiform (f) outer nuclear layers (g) outer segments (h) pigment epithelium. 6. Pigment epithelium. 7. Tapetum lucidum. 8. Chorioid. 9. Sclera. 10. Optic nerve.

1.4 Piedras x Molino mm % 30 20 F2 (52) 10 1.2 0

60 50 F1 (20) 1.0 * 40 30 20 10 0.8 0 Downloaded from https://academic.oup.com/biolinnean/article/80/4/545/2636155 by guest on 29 September 2021 60

0.6 40 Piedras (20)

Curva 20 F1 Pachon´ x Curva 0.4 Pachon´ 0 mm 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 mel/unit 0.40.60.81.01.21.4 Curva x Molino % Figure 5. Comparison of eye-size (abscissa = horizontal 30 B to Molino (43) diameter, ordinate = vertical diameter) in adult Pachón and 20 10 Curva cavefish and the F1 cross between them (*occur- 0 rence of lens capsule rudiment). 20 B to Curva (28) 10 0 Eye genes 30 Regression in Astyanax cave fish was calculated to be 20 F2 (147) dependent on at least six genetic factors (‘eye genes’; 10 Wilkens, 1988) which appear to be developmental con- 0 trol genes; only a few of them are structural 40 30 F1 (20) (Yokoyama et al., 1995; Culver & Wilkens, 2000; Parry 20 et al., 2003). In the hybrid offspring of the crosses 10 between the various populations, eyes are found that 0 are larger than, and develop structures never found 50 in, those of the adult parental generation. This phe- 40 nomenon can be explained by the fact that, as a con- 30 20 Curva (20) sequence of the recombination of rudimentary genes 10 deriving from different cave populations that have 0 evolved separately, ‘eye gene’ expression is restored 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 again and eye rudiments may develop to a greater mel/unit Surface x Molino extent than in either parental population. % It has been shown that larger eyes (though only to a 30 B to Molino (60) very slight extent) are developed in only a few hybrid 20 specimens of the F1 crosses within the El Abra group. 10 0 In contrast, in the F1 El Abra/Molino crosses, all the 20 F2 (43) eyes are better differentiated, while in the F2 crosses 10 some specimens even develop eyes with the potential 0 for vision. This can be explained by the fact that the 20 Surface(19) ‘eye genes’ in the Molino fish have been subjected to 10 0 fewer mutations than those of the other blind cave 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 populations, which is also substantiated by the mel/unit slightly lesser degree of histological reduction. Fur- thermore, in contrast to the F2 El Abra within-group Figure 6. Distribution of melanophore densities in and El Abra/Molino crosses, the range of eye-size is Molino/Piedras, Molino/Curva and Molino/surface fish larger than that of the F1 and parental generations. crosses (B = backcross, mel = melanophores).

This could be explained by the fact that the rudimen- (Shh) gene suppresses the Pax6 control gene and tary eye genes in Molino are additionally character- thereby influences the extent of eye development. ized by a high degree of heterozygosity. However, the From this he concludes that constructive (enhanced possibility cannot be excluded that the nature of the midline signalling) rather than regressive mecha- very different mutations in the two parental popula- nisms ultimately explain cave fish eye reduction. This tions additionally or alternatively combine to give a assumption is in concordance with the neodarwinian much greater range of possible phenotypes than is the paradigm of selection, but conflicts with the findings case between more similar populations. presented in this study. We have shown that destruc- It could also be concluded that the rudimentary eye tive mutations in eye and in melanophore genes are genes in the El Abra populations are homozygous to a obviously responsible for rudimentation, while colour higher degree than those of Molino. A higher degree of genes responsible for the formation of melanin, gua-

heterozygosity in the latter could be derived from its nine and carotenoids are also functionless because of Downloaded from https://academic.oup.com/biolinnean/article/80/4/545/2636155 by guest on 29 September 2021 possibly younger phylogenetic age. destructive mutations (Culver & Wilkens, 2000). These genes are, at least in part, randomly distributed Melanophore system between the different cave populations. Both facts As with the ‘eye genes’, the formation and number of support the view that the reduction in size of eye and melanophores is also dependent on an additive poly- pigment are not being driven by selective forces, but genic system. Both systems show an almost identical by the random accumulation of neutral mutations manner of phenotypic manifestation (Wilkens, 1988; (Wilkens, 1988). Culver & Wilkens, 2000). In Molino the genetic system responsible for melanophores produces less reduction Multiple origin and convergent evolution than that of the other cave populations. The higher The question as to whether Astyanax cave populations numbers in F2 than F1 could be explained in the same have convergently adapted to the dark environment or way. if they are the result of secondary dispersal of an already cave-adapted ancestor is still being studied Character of eye and melanophore genes (Espinasa & Borowsky, 2001; Dowling et al., 2002; The character of these is still being disputed. The fact Strecker et al., 2003). Based on allozyme studies, that the rudimentation process of the eye is mainly Avise & Selander (1972) concluded that “the eyeless characterized by a diminution in size caused by muta- and unpigmented condition is believed to have evolved tions of developmental control, is a consequence of the in whole or part prior to the present-day subdivision of inductive integration of the eyeball during head for- the populations”. In contrast, Mitchell et al. (1977) mation. Complete eye loss due to structural mutations suggested, on the basis of hydrological data from the with a totally detrimental effect on the eyeball is not Sierra de El Abra, that a multiple colonization hypoth- possible, because the head skeleton would fail to esis is more plausible. Confirmation of convergent evo- develop in an adequate manner (Mathers & Jamrich, lution is also implied by classical genetic studies. They 2000). A similar phenomenon is supposed to take place show that several monogenic colour mutants are not when the melanophore system is reduced, because distributed over all populations, but are limited to spe- melanophore development is integrated with that of cific ones (Culver & Wilkens, 2000). Furthermore, the neural crest. Therefore a rudimentary pattern of although histology has confirmed the degree of eye melanophores is still developed in cave fish. reduction in the cave populations from the Sierra de The characteristic variability in size and degree of El Abra, the genetic basis for that reduction differs differentiation develops during ontogenetic growth between them. This conclusion was reached because (Wilkens, 1988). This may indicate that cave fish eye some rudimentary structures in the F1 and F2 crosses development is not only disturbed at the top of the dif- are better developed than those of the parental gener- ferentiation cascade, as postulated by Jeffery (2001), ation. Furthermore, differences in the respective size but that the genetic basis responsible during later ranges of the F2 crosses of Pachón, Yerbaniz, Piedras ontogeny is in disorder due to mutations in subordi- and Curva with Molino demonstrate that even nate genes as well. This view is substantiated by the between the four El Abra populations (found in rela- allometric growth relationship, which is not linear but tively close proximity), eye genetics diverge to a cer- shows variation of the allometric coefficient (Wilkens, tain extent. 1988). Mutative structural genes, such as opsin genes For the Molino fish, an even greater divergence with in cave fish, which cause the expression of multiple respect to regressive eye and melanophore genes is photopigments in specimens produced by hybridiza- found. Thus multiple origins can be deduced for some tion surface fish, might be one of the reasons for this of the cave populations in the Sierras de Guatemala (Yokoyama et al., 1995; Parry et al., 2003). and de El Abra with strongly reduced eye-size and pig- Jeffery (2001) has shown that the sonic hedgehog mentation. Recent microsatellite analysis of the

Pachón, Sabinos and Tinaja cave populations revealed Kosswig C. 1960. Darwin und die degenerative Evolution. no recent gene flow between Pachón and the two other Abhandlungen und Verhandlungen des Naturwissenschaftli- cave populations studied and only potential gene flow chen Vereins Hamburg N.F. 4: 21–42. between Tinaja and Sabinos (Strecker et al., 2003). Mathers PH, Jamrich M. 2000. Regulation of eye formation by the Rx and pax6 homeobox genes. Cellular and Molecular Life Sciences 57: 186–194. ACKNOWLEDGEMENTS Mitchell RW, Russell WH, Elliott WR. 1977. Mexican eyeless characin fishes, genus Astyanax: Environment, distribu- We thank R. Borowsky for providing the fish from the tion and evolution. Special Publication of the Museum of Molino population and M. Hänel for the drawings. J. Texas Technical University 12: 1–89. K. Bowmaker and two anonymous reviewers are much Parry JL, Peirson SN, Wilkens H, Bowmaker JK. 2003. appreciated for constructive comments. Multiple photopigments from the Mexican blind cavefish,

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