Mitochondrial phylogeography and molecular evolution of the rhodopsin visual pigment in troglobitic populations of Astyanax mexicanus (De Filippi, 1853).

Cave-adapted animals provide a unique opportunity to study the evolutionary mechanisms underlying phenotypic, metabolic, behavioral, and genetic evolution in response to cave environments. The Mexican tetra ( Astyanax mexicanus) is considered a unique model system as it shows both surface and cave-dwelling morphs. To date, at least 33 different cave populations have been identified, with phylogenetic studies suggesting an origin from at least two independent surface lineages, thereby providing a unique opportunity to study parallel evolution. In the present study, we carried out the most exhaustive phylogeographic study of A. mexicanus to date, including cave and surface localities, using two mitochondrial markers (cytochrome b (cyt b) and cytochrome c oxidase subunit I ( COI)) and nuclear rhodopsin visual pigment ( rho). Additionally, we inferred the molecular evolution of rho within the two contrasting environments (cave and surface) and across three geographic regions (Sierra de El Abra, Sierra de Guatemala, and Micos). In total, 267 individuals were sequenced for the two mitochondrial fragments and 268 individuals were sequenced for the rho visual pigment from 22 cave and 46 surface populations. Phylogeographic results based on the mitochondrial data supported the two-lineage hypothesis, except for the Pachón and Chica caves, whose introgression has been largely documented. The Sierra de El Abra region depicted the largest genetic diversity, followed by the Sierra de Guatemala region. Regarding the phylogeographic patterns of rho, we recovered exclusive haplogroups for the Sierra de El Abra (Haplogroup I) and Sierra de Guatemala regions (Haplogroup IV). Moreover, a 544 bp deletion in the rho gene was observed in the Escondido cave population from Sierra de Guatemala, reducing the protein from seven to three intramembrane domains. This change may produce a loss-of-function (LOF) but requires further investigation. Regarding nonsynonymous ( dN) and synonymous ( dS) substitution rates (omega values ω), our results revealed the prevailing influence of purifying selection upon the rho pigment for both cave and surface populations (ω<1), but relaxation at the El Abra region. Notably, in contrast to the other two regions, we observed an increase in the number of dN mutations for Sierra de El Abra. However, given that a LOF was exclusively identified in the Sierra de Guatemala region, we cannot dismiss the possibility of a pleiotropic effect on the Rho protein.

Host-parasite interactions in perpetual darkness: Macroparasite diversity in the cavefish Astyanax mexicanus.

Astyanax mexicanus has repeatedly colonized cave environments, displaying evolutionary parallelisms in many troglobitic traits. Despite being a model system for the study of adaptation to life in perpetual darkness, the parasites that infect cavefish are practically unknown. In this study, we investigated the macroparasite communities in 18 cavefish populations from independent lineages and compared them with the parasite diversity found in their sister surface fish populations, with the aim of better understanding the role that parasites play in the colonization of new environments. Within the cavefish populations, we identified 13 parasite taxa, including a subset of 10 of the 27 parasite taxa known for the surface populations. Parasites infecting the cavefish belong to five taxonomic groups, including trematodes, monogeneans, nematodes, copepods, and acari. Monogeneans are the most dominant group, found in 14 caves. The macroparasites include species with direct life cycles and trophic transmission, including invasive species. Surprisingly, paired comparisons indicate higher parasite richness in the cavefish than in the surface fish. Spatial variation in parasite composition across the caves suggests historical and geographical contingencies in the host-parasite colonization process and potential evolution of local adaptations. This base-line data on parasite diversity in cavefish populations of A. mexicanus provides a foundation to explore the role of divergent parasite infections under contrasting ecological pressures (cave vs. surface environments) in the evolution of cave adaptive traits.

Ecological factors drive the divergence of morphological, colour and behavioural traits in cactus wrens (Aves, Troglodytidae).

The study of ecological mechanisms influencing organisms' phenotypic variation is a central subject of evolutionary biology. In this study, we characterized morphological, plumage colour and acoustic variation in cactus wrens Campylorhynchus brunneicapillus throughout its distribution. We assessed whether Gloger's, Allen's and Bergmann's ecogeographical rules, and the acoustic adaptation hypothesis relate to geographical trait variation. We analysed specimen coloration in belly and crown plumage, beak shape and structural song characteristics. We tested whether the subspecific classification or the peninsular/mainland groups mirrored the geographical variation in phenotypes and whether ecological factors were associated with patterns of trait variation. Our results suggest that colour, beak shape and acoustic traits varied across the range, in agreement with two lineages described by genetics. The simple versions of Gloger's and Allen's rules are related to variations in colour traits and morphology. Conversely, patterns of phenotypic variation did not support Bergmann's rule. The acoustic adaptation hypothesis supported song divergence for frequency-related traits. Phenotypic variation supports the hypothesis of two taxa: C. affinis in the Baja California peninsula and C. brunneicapillus in the mainland. The ecological factors are associated with phenotypic trait adaptations, suggesting that divergence between lineages could result from ecological divergence.

Bergmann's Rule under Rocks: Testing the Influence of Latitude and Temperature on a Chiton from Mexican Marine Ecoregions.

Bergmann's rule relates the trend of increasing body size with higher latitudes, where colder climates are found. In the Mexican Pacific, three marine ecoregions are distinguishable across a latitudinal gradient. Stenoplax limaciformis is an abundant chiton species that is distributed on rocky shores in these ecoregions. Geometric morphometric analyses were performed to describe the shape and size variation of S. limaciformis between marine ecoregions that vary in sea surface temperature with latitude, thus testing Bergmann's rule. Individuals' body shape ranged from elongated to wide bodies. Although there was variation in chitons' body shape and size, the was no evidence of allometry among localities. The Gulf of California is the northernmost ecoregion evaluated in this work, where larger chitons were observed and lower sea surface temperature values were registered. The results suggest that S. limaciformis follows a trend to Bergmann's rule, such as endotherms. These mollusks do not need heat dissipation, but they do need to retain moisture. In addition, larger chitons were observed in zones with high primary productivity, suggesting that chitons do not delay their maturation due to food shortage.

Selection-driven trait loss in independently evolved cavefish populations.

Laboratory studies have demonstrated that a single phenotype can be produced by many different genotypes; however, in natural systems, it is frequently found that phenotypic convergence is due to parallel genetic changes. This suggests a substantial role for constraint and determinism in evolution and indicates that certain mutations are more likely to contribute to phenotypic evolution. Here we use whole genome resequencing in the Mexican tetra, Astyanax mexicanus, to investigate how selection has shaped the repeated evolution of both trait loss and enhancement across independent cavefish lineages. We show that selection on standing genetic variation and de novo mutations both contribute substantially to repeated adaptation. Our findings provide empirical support for the hypothesis that genes with larger mutational targets are more likely to be the substrate of repeated evolution and indicate that features of the cave environment may impact the rate at which mutations occur.

Hybridization underlies localized trait evolution in cavefish.

Introgressive hybridization may play an integral role in local adaptation and speciation (Taylor and Larson, 2019). In the Mexican tetra Astyanax mexicanus, cave populations have repeatedly evolved traits including eye loss, sleep loss, and albinism. Of the 30 caves inhabited by A. mexicanus, Chica cave is unique because it contains multiple pools inhabited by putative hybrids between surface and cave populations (Mitchell et al., 1977), providing an opportunity to investigate the impact of hybridization on complex trait evolution. We show that hybridization between cave and surface populations may contribute to localized variation in traits associated with cave evolution, including pigmentation, eye development, and sleep. We also uncover an example of convergent evolution in a circadian clock gene in multiple cavefish lineages and burrowing mammals, suggesting a shared genetic mechanism underlying circadian disruption in subterranean vertebrates. Our results provide insight into the role of hybridization in facilitating phenotypic evolution.

Genetic diversity and structure of one of the most endangered freshwater fish species in Mexico: Tlaloc hildebrandi (Miller, 1950) and recognition of its evolutionarily significant units.

The endangered Chiapas killifish Tlaloc hildebrandi is an endemic freshwater species that lives in four subbasins of the Grijalva and Usumacinta basins, and one of the most geographically restricted species of the Produndulidae family. The species was originally described as endemic to springs in the high limestone plateau in San Cristóbal de Las Casas in the Río Amarillo subbasin (upper Grijalva basin). However, it was recently recorded in the Jataté and Tzaconejá subbasins in the upper Usumacinta basin, thereby expanding its known distribution range. The discovery of these populations is relevant not only for the conservation of the species but also for a better understanding of its evolutionary history. Currently, the scarce populations of T. hildebrandi, found in only a few localities in the Grijalva and Usumacinta basins, are fragmented and living under unfavorable conditions. Here, we analyzed three mitochondrial (mt-atp8&6 and mt-nd2) and one nuclear (nuc-s7) marker in order to assess the genetic diversity and population structure of T. hildebrandi. We found that, in comparison with other endangered freshwater fish species from Mexico, T. hildebrandi showed a lower level of genetic diversity (mt-nd2: h = 0.469, π = 0.0009; mt-atp8&6: h = 0.398, π = 0.001; and nuc-S7: h = 0.433, π = 0.001). Moreover, the analyzed populations exhibited a strong genetic structure in accordance with their geographic distribution, and can be placed into three genetic clusters: (1) Amarillo plus Chenhaló in the upper Grijalva basin, (2) Jataté, and (3) Tzaconejá, both in the upper Usumacinta basin. On the basis of our results, we propose the recognition of at least three evolutionarily significant units (ESUs) for the species and the urgent implementation of ex situ and in situ conservation and management efforts that consider the genetic background of the species.

Genetic and ecomorphological divergence between sympatric Astyanax morphs from Central America.

Intraspecific ecological and morphological polymorphism can promote ecological speciation and the build-up of reproductive isolation. Here, we evaluate correlations among morphology, trophic ecology and genetic differentiation between two divergent morphs (elongate and deep-body) of the fish genus Astyanax in the San Juan River basin in Central America, to infer the putative evolutionary mechanism shaping this system. We collected the two morphs from three water bodies and analysed: (1) the correlation between body shape and the shape of the premaxilla, a relevant trophic morphological structure, (2) the trophic level and niche width of each morph, (3) the correspondence between trophic level and body and premaxillary shape, and (4) the genetic differentiation between morphs using mitochondrial and nuclear markers. We found a strong correlation between the body and premaxillary shape of the morphs. The elongate-body morph had a streamlined body, a premaxilla with acuter angles and a narrower ascending process, and a higher trophic level, characteristic of species with predatorial habits. By contrast, the deep-body morph had a higher body depth, a premaxilla with less acute angles and a broader trophic niche, suggesting generalist habits. Despite the strong correlation between morphological and ecological divergence, the morphs showed limited genetic differentiation, supporting the idea that morphs may be undergoing incipient ecological speciation, although alternative scenarios such as stable polymorphism or plasticity should also be considered. This study provides support for the role of ecological factors promoting diversification in both lake and stream-dwelling freshwater fish.

Trophic specialization and morphological divergence between two sympatric species in Lake Catemaco, Mexico.

The association of morphological divergence with ecological segregation among closely related species could be considered as a signal of divergent selection in ecological speciation processes. Environmental signals such as diet can trigger phenotypic evolution, making polymorphic species valuable systems for studying the evolution of trophic-related traits. The main goal of this study was to analyze the association between morphological differences in trophic-related traits and ecological divergence in two sympatric species, Astyanax aeneus and A. caballeroi, inhabiting Lake Catemaco, Mexico. The trophic differences of a total of 70 individuals (35 A. aeneus and 35 A. caballeroi) were examined using stable isotopes and gut content analysis; a subset of the sample was used to characterize six trophic and six ecomorphological variables. In our results, we recovered significant differences between both species in the values of stable isotopes, with higher values of δ15N for A. caballeroi than for A. aeneus. Gut content results were consistent with the stable isotope data, with a higher proportion of invertebrates in A. caballeroi (a consumption of invertebrates ten times higher than that of A. aeneus, which in turn consumed three times more vegetal material than A. caballeroi). Finally, we found significant relationship between ecomorphology and stable isotopes (r = .24, p < .01), hence, head length, preorbital length, eye diameter, and δ15N were all positively correlated; these characteristics correspond to A. caballeroi. While longer gut and gill rakers, deeper bodies, and vegetal material consumption were positively correlated and corresponded to A. aeneus. Our results are consistent with the hypothesis that morphological divergence in trophic-related traits could be associated with niche partitioning, allowing the coexistence of closely related species and reducing interspecific competition.

Functional modularity in lake-dwelling characin fishes of Mexico.

Modular evolution promotes evolutionary change, allowing independent variation across morphological units. Recent studies have shown that under contrasting ecological pressures, patterns of modularity could be related to divergent evolution. The main goal of the present study was to evaluate the presence of modular evolution in two sister lacustrine species, Astyanax aeneus and A. caballeroi, which are differentiated by their trophic habits. Two different datasets were analyzed: (1) skull X-rays from 73 specimens (35 A. aeneus and 38 A. caballeroi) to characterize skull variation patterns, considering both species and sex effects. For this dataset, three different modularity hypotheses were tested, previously supported in other lacustrine divergent species; (2) a complete body shape dataset was also tested for four modularity hypotheses, which included a total of 196 individuals (110 Astyanax aeneus and 86 A. caballeroi). Skull shape showed significant differences among species and sex (P < 0.001), where Astyanax caballeroi species showed an upwardly projected mandible and larger preorbital region. For the skull dataset, the modularity hypothesis ranked first included three partitioning modules. While for the complete body dataset the best ranked hypothesis included two modules (head vs the rest of the body), being significant only for A. caballeroi.

Phylogenetic position of Magnivitellinum Kloss, 1966 and Perezitrema Barus & Moravec, 1967 (Trematoda: Plagiorchioidea: Macroderoididae) inferred from partial 28S rDNA sequences, with the establishment of Alloglossidiidae n. fam.

The systematic position of two genera of Macroderoididae McMullen, 1937, Perezitrema Barus & Moravec, 1967 and Magnivitellinum Kloss, 1966 is reviewed based on a phylogenetic analysis of the interrelationships of 15 species of the family allocated into six genera, along with 44 species of plagiorchioid trematodes, using partial sequences of the 28S rRNA gene. Sequences were analysed through parsimony, maximum likelihood and Bayesian inference. The obtained topologies show Perezitrema as the sister taxon of three species of Macroderoides Pearse, 1924; the latter genus appears to be paraphyletic since another three species are not included in this group. Instead, Magnivitellinum was placed as the sister taxon of Alloglossidium Simer, 1929. These relationships are well supported by high bootstrap and posterior probability values. The resulting trees demonstrate that the family Macroderoididae, as currently conceived in taxonomic treatments, is not monophyletic. Magnivitellinum simplex Kloss, 1966 and Alloglossidium spp. were nested as sister taxa of members of the family Leptophallidae Dayal, 1938, whereas Perezitrema bychowskii Barus & Moravec, 1967 and species of Macroderoides and Paramacroderoides Venard, 1941 were grouped with Auridistomum chelydrae (Stafford, 1900), a monotypic member of Auridistomidae Stunkard, 1924. Based on our results, a new family, Alloglossidiidae n. fam. was established to accommodate the genera Magnivitellinum and Alloglossidium.

Phylogenetic relationships and evolutionary history of the Mesoamerican endemic freshwater fish family Profundulidae (Cyprinodontiformes: Actinopterygii).

Freshwater fishes of Profundulidae, which until now was composed of two subgenera, represent one of the few extant fish families endemic to Mesoamerica. In this study we investigated the phylogenetic relationships and evolutionary history of the eight recognized extant species (from 37 populations) of Profundulidae using three mitochondrial and one nuclear gene markers (∼2.9 Kbp). We applied a Bayesian species delimitation method as a first approach to resolving speciation patterns within Profundulidae considering two different scenarios, eight-species and twelve-species models, obtained in a previous phylogenetic analysis. Based on our results, each of the two subgenera was resolved as monophyletic, with a remarkable molecular divergence of 24.5% for mtDNA and 7.8% for nDNA uncorrected p distances, and thus we propose that they correspond to separate genera. Moreover, we propose a conservative taxonomic hypothesis with five species within Profundulus and three within Tlaloc, although both eight-species and twelve-species models were highly supported by the bayesian species delimitation analysis, providing additional evidence of higher taxonomic diversity than currently recognized in this family. According to our divergence time estimates, the family originated during the Upper Oligocene 26 Mya, and Profundulus and Tlaloc diverged in the Upper Oligocene or Lower Miocene about 20 Mya.

Untangling the evolutionary history of a highly polymorphic species: introgressive hybridization and high genetic structure in the desert cichlid fish Herichtys minckleyi.

Understanding the origin of biodiversity requires knowledge on the evolutionary processes that drive divergence and speciation, as well as on the processes constraining it. Intraspecific polymorphisms can provide insight into the mechanisms that generate and maintain phenotypic, behavioural and life history diversification, and can help us understand not only the processes that lead to speciation but also the processes that prevent local fixation of morphs. The 'desert cichlid' Herichtys minckleyi is a highly polymorphic species endemic to a biodiversity hotspot in northern Mexico, the Cuatro Ciénegas valley. This species is polymorphic in body shape and trophic apparatus, and eco-morphotypes coexist in small spring-fed lagoons across the valley. We investigated the genetic structure of these polymorphisms and their phylogeographic history by analysing the entire control region of the mitochondrial DNA and 10 nuclear microsatellite markers in several populations from different sites and morphs. We found two very divergent mitochondrial lineages that most likely predate the closing of the valley and are not associated with morphotypes or sites. One of these lineages is also found in the sister species Herichthys cyanoguttatus. Data from neutral microsatellite markers suggest that most lagoons or drainages constitute their own genetic cluster with sympatric eco-morphotypes forming panmictic populations. Alternative mechanisms such as phenotypic plasticity and a few loci controlled traits provide possible explanations for the sympatric coexistence of discrete nonoverlapping eco-morphotypes with apparent lack of barriers to gene flow within multiple lagoons and drainages.

Evolutionary history of the fish genus Astyanax Baird & Girard (1854) (Actinopterygii, Characidae) in Mesoamerica reveals multiple morphological homoplasies.

BACKGROUND: Mesoamerica is one of the world's most complex biogeographical regions, mostly due to its complex geological history. This complexity has led to interesting biogeographical processes that have resulted in the current diversity and distribution of fauna in the region. The fish genus Astyanax represents a useful model to assess biogeographical hypotheses due to it being one of the most diverse and widely distributed freshwater fish species in the New World. We used mitochondrial and nuclear DNA to evaluate phylogenetic relationships within the genus in Mesoamerica, and to develop historical biogeographical hypotheses to explain its current distribution. RESULTS: Analysis of the entire mitochondrial cytochrome b (Cytb) gene in 208 individuals from 147 localities and of a subset of individuals for three mitochondrial genes (Cytb, 16 S, and COI) and a single nuclear gene (RAG1) yielded similar topologies, recovering six major groups with significant phylogeographic structure. Populations from North America and Upper Central America formed a monophyletic group, while Middle Central America showed evidence of rapid radiation with incompletely resolved relationships. Lower Central America lineages showed a fragmented structure, with geographically restricted taxa showing high levels of molecular divergence. All Bramocharax samples grouped with their sympatric Astyanax lineages (in some cases even with allopatric Astyanax populations), with less than 1% divergence between them. These results suggest a homoplasic nature to the trophic specializations associated with Bramocharax ecomorphs, which seem to have arisen independently in different Astyanax lineages. We observed higher taxonomic diversity compared to previous phylogenetic studies of the Astyanax genus. Colonization of Mesoamerica by Astyanax before the final closure of the Isthmus of Panama (3.3 Mya) explains the deep level of divergence detected in Lower Central America. The colonization of Upper Mesoamerica apparently occurred by two independent routes, with lineage turnover over a large part of the region. CONCLUSION: Our results support multiple, independent origins of morphological traits in Astyanax, whereby the morphotype associated with Bramocharax represents a recurrent trophic adaptation. Molecular clock estimates indicate that Astyanax was present in Mesoamerica during the Miocene (approximately 8 Mya), which implies the existence of an incipient land-bridge connecting South America and Central America before the final closure of the Isthmus of Panama (approximately 3.3 Mya).