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| Figure 1. Time-calibrated phylogeny of Percopsiformes including cave and surface species of amblyopsid cavefishes inferred from a multilocus dataset. Taxa in gray are cave lineages. Loss of function (red) and nonsynonymous (white) mutations in the photoreceptor gene rhodopsin are indicated on the branches. Cavefish images courtesy of Dante Fenolio. |
Despite the long recognition of regressive evolution in subterranean organisms, there has been surprisingly little evidence for regressive evolution at the molecular in genes associated with regressive characters (e.g., genes involved in the development and function of the eye). The best examples include studies of eye genes in subterranean diving beetles [1], marsupial moles [2], naked mole rats [3], Astanyax cavefish [4], and cave-roosting bats [5]. Several studies of perhaps the most well-studied eye genes, the retinal photoreceptor gene rhodopsin, in subterranean organisms have yet to uncover compelling evidence for loss of gene functionality at the molecular level. For example, Kim et al. [3] discovered at least 19 genes associated with visual perception that were either lost or showed evidence for loss of function in the naked mole rat but rhodopsin was not one of these loci.
What might account for apparent functionality of rhodopsin in the subterranean organisms studied previously? One hypothesis is weak selection for the retention of light-sensing abilities [3, 6]. Another possible explanation is that rhodopsin has other pleiotropic functions, such as in circadian rhythms [7-9]. Support for this latter hypothesis was demonstrated in a recent study by Shen et al. [10] who discovered that invertebrate rhodopsin was important in initiating thermosensory-signaling cascades in Drosophila fruit flies.
An alternative hypothesis to explain presumed functionality in rhodopsin is that the gene no longer has a function in aphotic environments but insufficient time has passed for the accumulation of loss of function (LOF) mutations to accumulate and render the gene nonfunctional. This hypothesis assumes that selection is not the mechanism behind loss of functionality [1]. If neutral processes are responsible for loss of functionality, then integrity of a gene may persist for a considerable period of time due to chance alone [11]. Consequently, inferred maintenance of a visual gene may be the result of recent subterranean colonization rather than retained or pleiotropic functionality.
My colleagues (Drs. Ben Fitzpatrick, Premal Shah, Lars Schmitz and Tom Near) and I investigated this latter hypothesis by testing for loss of selective constraint in rhodopsin of amblyopsid cavefishes in a recently accepted paper in the journal Evolution [12]. We first generated a fossil-calibrated, multilocus molecular phylogeny based on nine genes to provide a temporal and phylogenetic context for elucidating the evolutionary history and potential loss of rhodopsin functionality. Our sampling included every recognized species of amblyopsid cavefish (family Amblyopsidae) as well as related outgroup taxa within the order Percopsiformes (families Aphredoderidae and Percopsidae). The resulting phylogeny (Figure 1) differed considerably from previously phylogenetic hypotheses [13-15], with the most notable difference being the phylogenetic placement of the surface/facultative cave-dwelling genus Forbesichthys. Forbesichthys was nested within a clade of all obligate cave-dwelling lineages and diverged some 5.7 Mya. The most recent common ancestor (MRCA) of all amblyopsids (cave and surface lineages) dates to 12.2 Mya in the Miocene while the MRCA of all cave lineages dates to 10.3 Mya. However, much of the diversification within genera occurred in the Pleistocene.
The intriguing phylogenetic placement of Forbesicthys suggests that a surface-dwelling life history and eye functionality may have reevolved from a cave-dwelling ancestor with degenerate eyes. In fact, ancestral character reconstructions of eye functionality in amblyopsid cavefishes strongly support reevolution in Forbesichthys from a subterranean ancestor. Cave-dwelling organisms are often viewed as "evolutionary dead-ends" that are incapable of recolonizing or adapting to surface habitats. However, the reevolution of a surface form is not novel, as this hypothesis has been proposed for eyed Gammarus amphipods living in karst windows [16] as well as in cave scorpions [17], cavefishes [18] and cave salamanders [19].
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| Figure 2. Rhodopsin gene tree of percopsiform fishes with nonsynonymous mutations (white square), deletions (red square), insertions (green square), and premature stop codons (red octagon) mapped onto branches. Cave lineages are in gray. The size of indels (in base pairs) is indicated within the red or green square. The inset shows two-dimensional models of rhodopsin with cumulative nonsynonymous mutations, deletions, insertions, and premature stop codons indicated for surface and cave lineages. |
However, patterns of molecular evolution in rhodopsin suggest a different evolutionary history (Figure 2). We found no evidence of loss of selective constraint or functionality in rhodopsin of surface lineages, which was to be expected given that surface-dwelling species actively respond to photic stimuli. However, three cave lineages (Troglichthys rosae, Amblyopsis spelaea and Typhlichthys cf. subterraneus TN) exhibited strong evidence for loss of rhodopsin functionality at the molecular level including ten novel LOF mutations (six deletions, one insertion, and three mutations resulting in premature stop codons). These LOF mutations were highlighted by an 111 amino acid deletion in the federally listed Troglichthys rosae. In addition, rhodopsin in cave lineages (even those without LOF mutations) showed increased rates of nonsynonymous (amino acid changing) mutations compared to surface lineages. These mutations were more likely to have a significant deleterious effect on the structure and subsequently function of rhodopsin in cave lineages, as inferred from several physicochemical protein properties.
As a final test to determine if rhodopsin in cave lineages was evolving at a different evolutionary rate (i.e., under neutrality versus balancing or positive selection) compared to surface lineages, we compared a series of branch-based models of selection using the multilocus phylogeny. Interestingly, the best model corresponded to independent evolution of relaxation of selection and loss of functionality in cave lineages rather than reevolution of rhodopsin functionality in Forbesichthys that was suggested based on ancestral character reconstructions. In fact, not a single nonsynonymous mutation was observed in the 4.6 My interval from the MRCA of all cave amblyopsids and the MRCA of Forbesichthys and Amblyopsis (node b-e in Figure 1), as would be expected if there was a single subterranean colonization event and subsequent reevolution of eyes in Forbesichthys. Other lines of evidence also support independent evolution of loss of eye (and rhodopsin) functionality over a reevolution scenario, including patterns of degeneration of individual eye structures based on histological data, biogeographical and geological evidence, and phylogenetic studies of other cave organisms.
In summary, our study provides compelling evidence for repeated loss of selective constraint in rhodopsin in cave amblyopsids. Although we cannot rule out selection as the primary mechanism behind eye degeneration (i.e., selection might act upstream of rhodopsin or at other important eye developmental loci), our results are consistent with the neutral accumulation of mutations responsible of loss of functionality in rhodopsin. Rather than some unknown pleiotropic function, we hypothesize that presumed functionality of rhodopsin in other cave organisms is due to recent subterranean colonization and the random nature of mutation accumulation.
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