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.     

Parallel morphological evolution and habitat‐dependent sexual dimorphism in cave‐ vs. surface populations of the Asellus aquaticus (Crustacea: Isopoda: Asellidae) species complex

Studying parallel evolution (repeated, independent evolution of similar phenotypes in similar environments) is a powerful tool to understand environment‐dependent selective forces. Surface‐dwelling species that repeatedly and independently colonized caves provide unique models for such studies. The primarily surface‐dwelling Asellus aquaticus species complex is a good candidate to carry out such research, because it colonized several caves in Europe. By comparing 17 functional morphological traits between six cave and nine surface populations of the A. aquaticus species complex, we investigated population divergence in morphology and sexual dimorphism. We found habitat‐dependent population divergence in 10 out of 17 traits, likely reflecting habitat‐driven changes in selection acting on sensory systems, feeding, grooming, and antipredator mechanisms. Sexual dimorphism was present in 15 traits, explained by sexual selection acting on male traits important in male–male agonistic behavior or mate guarding and fecundity selection acting on female traits affecting offspring number and nursing. In eight traits, the degree of sexual dimorphism was habitat dependent. We conclude that cave‐related morphological changes are highly trait‐ and function‐specific and that the strength of sexual/fecundity selection strongly differs between cave and surface habitats. The considerable population variation within habitat type warrants further studies to reveal cave‐specific adaptations besides the parallel patterns.     

Is foraging innovation lost following colonization of a less variable environment? A case study in surface‐ vs. cave‐dwelling Asellus aquaticus

Behavioral innovation is a key process for successful colonization of new habitat types. However, it is costly due to the necessary cognitive and neural demands and typically connected to ecological generalism. Therefore, loss of behavioral innovativeness is predicted following colonization of new, simple, and invariable environments. We tested this prediction by studying foraging innovativeness in the freshwater isopod Asellus aquaticus. We sampled its populations along the route of colonizing a thermokarstic water‐filled cave (simple, stable habitat with only bacterial mats as food) from surface habitats (variable environment, wide variety of food). The studied cave population separated from the surface populations at least 60,000 years ago. Animals were tested both with familiar and novel food types (cave food: bacterial mats; surface food: decaying leaves). Irrespective of food type, cave individuals were more likely to feed than surface individuals. Further, animals from all populations fed longer on leaves than on bacteria, even though leaves were novel for the cave animals. Our results support that cave A. aquaticus did not lose the ability to use the ancestral (surface) food type after adapting to a simple, stable, and highly specialized habitat.     

Latitudinal Variation of Allozyme Frequencies in Populations of Two Freshwater Isopods in Western Europe

Populations of the freshwater isopods, Proasellus meridianus Racovitza and P. coxalis Dollfus were found to be polymorphic at loci coding for the enzymes glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM). PGM allele frequencies in populations of both species were significantly associated with latitude as has been previously reported for the closely related Asellus aquaticus (L.). Co-variation of PGM allele frequencies was also found in sympatric populations of P. meridianus and A. aquaticus. Significant interpopulation variation in GPI allele frequencies occurred in P. coxalis but not P. meridianus, however, this variation was not associated with latitude.     

Silver staining analysis of nucleolar-organizer activity during spermatogenesis of Asellus aquaticus (Crustacea,Isopoda)

The Ag-staining technique was employed to investigate the activity of the nucleolar organizer during spermatogenesis of the isopod crustacean Asellus aquaticus. The most interesting results of this investigation were: (1) The NORs remain continuously Ag-stained for the whole of spermatogenesis until maturation of the sperm, contrary to the situation in the other species so far described; (2) In the gonial mitotic cells the NORs of a single pair, in the meiotic cells of both pairs of chromosomes that have the NORs, are Ag-stained and therefore active; (3) Some of the individuals examined exhibited additional NORs.     

Random Amplified Polymorphic DNA Diversity among Surface and Subterranean Populations of Asellus aquaticus (Crustacea: Isopoda)

The ecological and evolutionary processes leading to isolation and adaptation of cave animals compared to their surface ancestors are not yet unequivocally understood. In this study the genetic relations of four cave and three surface population of the freshwater crustacean Asellus aquaticus in the Karst region of SW Slovenia and NE Italy were assessed using RAPDs as genetic markers. The results suggest that specialized populations from two caves invaded their subterranean habitat independently, and that their morphological similarity is a result of convergent evolution. Another, less specialized cave population seems to originate from a later colonization of a cave system already inhabited by a more specialized population, but the two populations do not interbreed. This series of temporally and spatially independent invasions has generated a diversity hotspot of non-interbreeding populations of a ubiquitous freshwatercrustacean, which is uniform over most of its range. Genetic variability estimated by the percentage of polymorphic RAPD fragments was similar (86–91%) in most cave and surface populations. Substantially lower values (as low as 49%) were found in two cave populations affected by heavy pollution. Two a priori groupings of populations, traditional subspecies and hydrologically connected groups, were rejected as not significant by means of nested analysis of molecular variance (AMOVA). On the other hand, groupings revealed by UPGMA clustering displayed a significant component of among-group variance. An analysis of gene flow between populations using estimated migration rates confirmed these findings.     

Response of the waterlouse Asellus aquaticus to multiple stressors: effects of current velocity and mineral substratum

Experiments were performed to study theindividual and combined effects of currentvelocity and substratum composition on thewaterlouse Asellus aquaticus (L.). Bothfactors affected growth, mortality, behavior,and food consumption of A. aquaticus.Short-term effects of increasing currentvelocity depended on the type of substratum. Critical currentvelocity for detachment was almost the same onsand as on a polished surface whereas on gravelA. aquaticus could withstand highercurrent velocities by hiding in interstitialspaces. Long-term experiments with differentcombinations of current velocity and substratumcomposition showed that current velocity had agreater effect than substratum on survival andgrowth. Substratum, however, had a greaterinfluence on the distribution of individuals inthe experimental units. Since growth wasreduced at high current velocity and no changesin levels of food intake were observed it isconcluded that a substantial amount of energyis required for withstanding current at higherflow rates. Furthermore, mortality showed astrong inverse correlation to growth. Theinteraction of effects of natural habitatfactors may be better understood using anexperimental and modeling approach focusing onenergy budgets.     

Incipient sex chromosome differentiation in an isopod crustacean species,Asellus aquaticus

Asellus aquaticus, a sexually dimorphic isopod crustacean, has no morphologically distinguishable sex chromosome pair. In 25% of the males from a natural population the fluorochrome chromomycin A₃ revealed a pair of metacentric chromosomes that was heteromorphic for two heterochromatic areas. One chromosome had a fluorescent band in each of the two arms, whereas the other chromosome lacked both bands. Chromosomal analysis was performed on 50 sibships produced by known parents. The heterochromosome was inherited only by the male offspring of males bearing it. Its distribution led us to postulate the existence of an incipient differentiating sex chromosome pair in the A. aquaticus population.This paper is dedicated to Prof. Guiseppe Montalenti on the occasion of his 80th birthday.     

Shared and unique patterns of embryo development in extremophile poeciliids

Background

Closely related lineages of livebearing fishes have independently adapted to two extreme environmental factors: toxic hydrogen sulphide (H₂S) and perpetual darkness. Previous work has demonstrated in adult specimens that fish from these extreme habitats convergently evolved drastically increased head and offspring size, while cave fish are further characterized by reduced pigmentation and eye size. Here, we traced the development of these (and other) divergent traits in embryos of Poecilia mexicana from benign surface habitats (“surface mollies”) and a sulphidic cave (“cave mollies”), as well as in embryos of the sister taxon, Poecilia sulphuraria from a sulphidic surface spring (“sulphur mollies”). We asked at which points during development changes in the timing of the involved processes (i.e., heterochrony) would be detectible.

Methods and Results

Data were extracted from digital photographs taken of representative embryos for each stage of development and each type of molly. Embryo mass decreased in convergent fashion, but we found patterns of embryonic fat content and ovum/embryo diameter to be divergent among all three types of mollies. The intensity of yellow colouration of the yolk (a proxy for carotenoid content) was significantly lower in cave mollies throughout development. Moreover, while relative head size decreased through development in surface mollies, it increased in both types of extremophile mollies, and eye growth was arrested in mid-stage embryos of cave mollies but not in surface or sulphur mollies.

Conclusion

Our results clearly demonstrate that even among sister taxa convergence in phenotypic traits is not always achieved by the same processes during embryo development. Furthermore, teleost development is crucially dependent on sufficient carotenoid stores in the yolk, and so we discuss how the apparent ability of cave mollies to overcome this carotenoid-dependency may represent another potential mechanism explaining the lack of gene flow between surface and cave mollies.     

Albinism in phylogenetically and geographically distinct populations of Astyanax cavefish arises through the same loss-of-function Oca2 allele

The Mexican tetra, Astyanax mexicanus, comprises 29 populations of cave-adapted fish distributed across a vast karst region in northeastern Mexico. These populations have a complex evolutionary history, having descended from ‘old'' and ‘young'' ancestral surface-dwelling stocks that invaded the region ∼6.7 and ∼2.8 MYa, respectively. This study investigates a set of captive, pigmented Astyanax cavefish collected from the Micos cave locality in 1970, in which albinism appeared over the past two decades. We combined novel coloration analyses, coding sequence comparisons and mRNA expression level studies to investigate the origin of albinism in captive-bred Micos cavefish. We discovered that albino Micos cavefish harbor two copies of a loss-of-function ocular and cutaneous albinism type II (Oca2) allele previously identified in the geographically distant Pachón cave population. This result suggests that phylogenetically young Micos cavefish and phylogenetically old Pachón cave fish inherited this Oca2 allele from the ancestral surface-dwelling taxon. This likely resulted from the presence of the loss-of-function Oca2 haplotype in the ‘young'' ancestral surface-dwelling stock that colonized the Micos cave and also introgressed into the ancient Pachón cave population. The appearance of albinism in captive Micos cavefish, caused by the same loss-of-function allele present in Pachón cavefish, implies that geographically and phylogenetically distinct cave populations can evolve the same troglomorphic phenotype from standing genetic variation present in the ancestral taxon.