Brief communication: Blue eyes in lemurs and humans: Same phenotype,different genetic mechanism

Almost all mammals have brown or darkly‐pigmented eyes (irises), but among primates, there are some prominent blue‐eyed exceptions. The blue eyes of some humans and lemurs are a striking example of convergent evolution of a rare phenotype on distant branches of the primate tree. Recent work on humans indicates that blue eye color is associated with, and likely caused by, a single nucleotide polymorphism (rs12913832) in an intron of the gene HERC2, which likely regulates expression of the neighboring pigmentation gene OCA2. This raises the immediate question of whether blue eyes in lemurs might have a similar genetic basis. We addressed this by sequencing the homologous genetic region in the blue‐eyed black lemur (Eulemur macaco flavifrons; N = 4) and the closely‐related black lemur (Eulemur macaco macaco; N = 4), which has brown eyes. We then compared a 166‐bp segment corresponding to and flanking the human eye‐color‐associated region in these lemurs, as well as other primates (human, chimpanzee, orangutan, macaque, ring‐tailed lemur, mouse lemur). Aligned sequences indicated that this region is strongly conserved in both Eulemur macaco subspecies as well as the other primates (except blue‐eyed humans). Therefore, it is unlikely that this regulatory segment plays a major role in eye color differences among lemurs as it does in humans. Although convergent phenotypes can sometimes come about via the same or similar genetic changes occurring independently, this does not seem to be the case here, as we have shown that the genetic basis of blue eyes in lemurs differs from that of humans. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

A preliminary population viability analysis of the critically endangered blue‐eyed black lemur (Eulemur flavifrons)

Population viability analysis is an important tool to assess the extinction risk in small populations of highly specialized primates. The blue‐eyed black lemur (Eulemur flavifrons) is critically endangered with a restricted range in the north‐western dry deciduous forest of Madagascar, where habitat fragmentation and loss of forest connectivity threaten its survival. We performed a population viability analysis (PVA) of this lemur in Ankarafa Forest in the Sahamalaza Peninsula National Park, north‐western Madagascar, to determine the demographic parameters most influential for population persistence and to assess extinction probabilities. We conducted PVA analyses using different demographic parameters which characterize the species including reproduction, lifespan and population size using the software VORTEX for six scenarios with 100 iterations and simulated over 100 years. The simulations suggested the first extinction within 13 years when the percentage of habitat destruction increased up to 12%. Severe habitat destruction such as fire and logging was the major cause which led to the risk of population extinction. Conservation strategies, in particular measures to reduce habitat destruction, are proposed to ensure the survival of this critically endangered lemur.     

The convergent evolution of blue iris pigmentation in primates took distinct molecular paths

How many distinct molecular paths lead to the same phenotype? One approach to this question has been to examine the genetic basis of convergent traits, which likely evolved repeatedly under a shared selective pressure. We investigated the convergent phenotype of blue iris pigmentation, which has arisen independently in four primate lineages: humans, blue‐eyed black lemurs, Japanese macaques, and spider monkeys. Characterizing the phenotype across these species, we found that the variation within the blue‐eyed subsets of each species occupies strongly overlapping regions of CIE L*a*b* color space. Yet whereas Japanese macaques and humans display continuous variation, the phenotypes of blue‐eyed black lemurs and their sister species (whose irises are brown) occupy more clustered subspaces. Variation in an enhancer of OCA2 is primarily responsible for the phenotypic difference between humans with blue and brown irises. In the orthologous region, we found no variant that distinguishes the two lemur species or associates with quantitative phenotypic variation in Japanese macaques. Given the high similarity between the blue iris phenotypes in these species and that in humans, this finding implies that evolution has used different molecular paths to reach the same end. Am J Phys Anthropol 151:398–407, 2013.© 2013 Wiley Periodicals, Inc.     

Maintenance of female dominance in blue‐eyed black lemurs (Eulemur macaco flavifrons) and gray bamboo lemurs (Hapalemur griseus griseus) under semi‐free‐ranging and captive conditions

Several species of Malagasy prosimians are characterized by female dominance, an unusual trait among mammals. We compare the extent to which female dominance is displayed and the mechanisms that are used to maintain dominance in the frugivorous blue‐eyed black lemurs (Eulemur macaco flavifrons) and the folivorous gray bamboo lemurs (Hapalemur griseus griseus) housed at the Duke Lemur Center. All dominant–submissive interactions were recorded during 448 hr of focal animal observations. Both species of lemurs exhibited clear patterns of female dominance. However, the two species used aggressive dominance (defined as aggression+submission) and social dominance (defined as submission in the absence of aggression) to different extents in maintaining hierarchies within each group. The adult female blue‐eyed black lemurs used aggressive dominance (e.g., chase, cuff, bite) in more of their dominance interactions (66%) than did the adult female gray bamboo lemurs (40%). In both species, rates of aggressive dominance interactions were higher during feeding versus nonfeeding periods and while in smaller outdoor runs versus larger natural habitat enclosures, but the differences were not significant. Overall blue‐eyed black lemurs exhibit a more aggressive form of female dominance compared to the gray bamboo lemur. Zoo Biol 26:345–361, 2007. © 2007 Wiley‐Liss, Inc.     

Visual acuity in the cathemeral strepsirrhine Eulemur macaco flavifrons

Studies of visual acuity in primates have shown that diurnal haplorhines have higher acuity (30–75 cycles per degree (c/deg)) than most other mammals. However, relatively little is known about visual acuity in non‐haplorhine primates, and published estimates are only available for four strepsirrhine genera (Microcebus, Otolemur, Galago, and Lemur). We present here the first measurements of visual acuity in a cathemeral strepsirrhine species, the blue‐eyed black lemur (Eulemur macaco flavifrons). Acuity in two subjects, a 3‐year‐old male and a 16‐year‐old female, was assessed behaviorally using a two‐alternative forced choice discrimination task. Visual stimuli consisted of high contrast square wave gratings of seven spatial frequencies. Acuity threshold was determined using a 70% correct response criterion. Results indicate a maximum visual acuity of 5.1 c/deg for the female (1718 trials) and 3.8 c/deg for the male (846 trials). These values for E. macaco are slightly lower than those reported for diurnal Lemur catta, and are generally comparable to those reported for nocturnal Microcebus murinus and Otolemur crassicaudatus. To examine ecological sources of variation in primate visual acuity, we also calculated maximum theoretical acuity for Cheirogaleus medius (2.8 c/deg) and Tarsius syrichta (8.9 c/deg) using published data on retinal ganglion cell density and eye morphology. These data suggest that visual acuity in primates may be influenced by activity pattern, diet, and phylogenetic history. In particular, the relatively high acuity of T. syrichta and Galago senegalensis suggests that visual predation may be an important selective factor favoring high visual acuity in primates. Am. J. Primatol. 71:343–352, 2009. © 2009 Wiley‐Liss, Inc.     

Frugivory and Seed Dispersal by Brown Lemurs in a Malagasy Tropical Dry Forest

In the Ankarafantsika tropical dry forest (northwestern Madagascar), the common brown lemur (Eulemur fulvus fulvus) is the largest frugivore and probably the sole disperser of large‐seeded plants (seed diameter > 10 mm). To investigate seed dispersal by this primate, I recorded the feeding activities of a troop; also conducted fecal analyses, germination trials on defecated seeds, and a vegetation survey over 1 yr (beginning Dec 2006). Brown lemurs mostly consumed fruit (68%). The fruit of Vitex beraviensis was the most exploited resource (21% of total feeding time). Among dung samples, 1126 contained intact seeds of 70 plant species, with a median of six seeds and two species per sample. These data indicate that the brown lemur population dispersed approximately 9854 seeds/km²/d. Although the number of annually defecated seeds was overwhelmingly the largest in Grewia triflora, many of the small seeds were often clumped in dung piles. In contrast, large seeds of V. beraviensis occurred in the largest number of dung samples. The rate and time of seed germination in V. beraviensis were improved by passage through brown lemur guts. Therefore, V. beraviensis may readily establish seedlings in sites of brown lemur fecal deposition. Vitex beraviensis and brown lemurs are probably involved in a strong mutualism. Twenty‐three large‐seeded plants were probably dependent on brown lemurs for seed dispersal and some of these species were common trees in the forest. Maintenance of these key plant–animal interactions will probably contribute to the conservation of species diversity and intact regeneration of the Ankarafantsika forest.     

Genotyping of five single nucleotide polymorphisms in the OCA2 and HERC2 genes associated with blue‐brown eye color in the Japanese population

Human eye color is a polymorphic phenotype influenced by multiple genes. It has recently been reported that three single nucleotide polymorphisms (SNPs) within intron 1 of the OCA2 gene (rs7495174, rs4778241, rs4778138) and two SNPs in intron 86 (rs12913832) and the 3′ UTR region (rs1129038) of the HERC2 gene—located in the upstream of the OCA2 locus —have a high statistical association with human eye color. The present study is the first to examine in detail the genotype and haplotype frequencies for these five SNPs in an Asian (Japanese) population (n = 523) comprising solely brown‐eyed individuals. Comparison of the genotype and haplotype distributions in Japanese with those in African and European subjects revealed significant differences between Japanese and other populations. Analysis of haplotypes consisting of four SNPs at the HERC2‐OCA2 locus (rs12913832/rs7495174/rs4778241/rs4778138) showed that the most frequent haplotype in the Japanese population is A‐GAG (0.568), while the frequency of this haplotype is rather low in the European population, even in the brown‐eyed group (0.167). The haplotype distribution in the Japanese population was significantly different from that in the brown‐eyed European group (F_ST = 0.18915). Copyright © 2009 John Wiley & Sons, Ltd.     

Extensive variability in the gut microbiome of a highly‐specialized and critically endangered lemur species across sites

Deforestation continues to jeopardize Malagasy primates as viable habitats become smaller, more fragmented, and more disturbed. This deforestation can lead to changes in diet, microhabitat, and gene flow between populations of endangered species, and it remains unclear how these changes may affect gut microbiome (GM) characteristics. The black‐and‐white ruffed lemur (Varecia variegata), which is among Madagascar's most threatened lemur species, provides a critical model for understanding the relationships between historical and on‐going deforestation (habitat disturbance), feeding ecology, and GM composition and diversity. We studied four populations inhabiting two rainforests (relatively pristine vs. highly disturbed) in southeastern Madagascar. We conducted full‐day focal animal behavioral follows and collected fecal samples opportunistically across a three‐month period. Our results indicate that lemurs inhabiting sites characterized by habitat disturbance and low dietary diversity exhibited reduced gut microbial alpha diversity. We also show that these same factors were associated with high community dissimilarity using weighted and unweighted UniFrac metrics. Finally, an indicator species analysis showed that the most pristine site was characterized by an abundance of methanogenic archaea. While it is impossible to disentangle the relative contributions of each confounding variable presented by our sampling design, these results provide crucial information about GM variability, thereby underscoring the importance of monitoring endangered species at the population‐level.     

Genetic depletion at adaptive but not neutral loci in an endangered bird species

Many endangered species suffer from the loss of genetic diversity, but some populations may be able to thrive even if genetically depleted. To investigate the underlying genetic processes of population bottlenecks, we apply an innovative approach for assessing genetic diversity in the last known population of the endangered Pale‐headed Brushfinch (Atlapetes pallidiceps) in Ecuador. First, we measure genetic diversity at eleven neutral microsatellite loci and adaptive SNP variation in five Toll‐like receptor (TLR) immune system genes. Bottleneck tests confirm genetic drift as the main force shaping genetic diversity in this species and indicate a 99 % reduction in population size dating back several hundred years. Second, we compare contemporary microsatellite diversity with historic museum samples of A. pallidiceps, finding no change in genetic diversity. Third, we compare genetic diversity in the Pale‐headed Brushfinch with two co‐occurring‐related brushfinch species (Atlapetes latinuchus, Buarremon torquatus), finding a reduction of up to 91% diversity in the immune system genes but not in microsatellites. High TLR diversity is linked to decreased survival probabilities in A. pallidiceps. Low TLR diversity is thus probably an adaptation to the specific selection regime within its currently very restricted distribution (approximately 200 ha), but could severely restrict the adaptive potential of the species in the long run. Our study illustrates the importance of investigating both neutral and adaptive markers to assess the effect of population bottlenecks and for recommending specific management plans in endangered species.     

Change in sexual signalling traits outruns morphological divergence across an ecological gradient in the post‐glacial radiation of the songbird genus Junco

The relative roles of natural and sexual selection in promoting evolutionary lineage divergence remains controversial and difficult to assess in natural systems. Local adaptation through natural selection is known to play a central role in promoting evolutionary divergence, yet secondary sexual traits can vary widely among species in recent radiations, suggesting that sexual selection may also be important in the early stages of speciation. Here, we compare rates of divergence in ecologically relevant traits (morphology) and sexually selected signalling traits (coloration) relative to neutral structure in genome‐wide molecular markers and examine patterns of variation in sexual dichromatism to explore the roles of natural and sexual selection in the diversification of the songbird genus Junco (Aves: Passerellidae). Juncos include divergent lineages in Central America and several dark‐eyed junco (J. hyemalis) lineages that diversified recently as the group recolonized North America following the last glacial maximum (ca. 18,000 years ago). We found an accelerated rate of divergence in sexually selected characters relative to ecologically relevant traits. Moreover, sexual dichromatism measurements suggested a positive relationship between the degree of colour divergence and the strength of sexual selection when controlling for neutral genetic distance. We also found a positive correlation between dichromatism and latitude, which coincides with the geographic axis of decreasing lineage age in juncos but also with a steep ecological gradient. Finally, we found significant associations between genome‐wide variants linked to functional genes and proxies of both sexual and natural selection. These results suggest that the joint effects of sexual and ecological selection have played a prominent role in the junco radiation.     

Chromosomal‐level genomes of three rice planthoppers provide new insights into sex chromosome evolution

The brown planthopper Nilaparvata lugens, white‐backed planthopper Sogatella furcifera, and small brown planthopper Laodelphax striatellus are three major insect pests of rice. They are genetically close; however, they differ in several ecological traits such as host range, migration capacity, and in their sex chromosomes. Though the draft genome of these three planthoppers have been previously released, the quality of genome assemblies need to be improved. The absence of chromosome‐level genome resources has hindered in‐depth research of these three species. Here, we performed a de novo genome assembly for N. lugens to increase its genome assembly quality with PacBio and Illumina platforms, increasing the contig N50 to 589.46 Kb. Then, with the new N. lugens genome and previously reported S. furcifera and L. striatellus genome assemblies, we generated chromosome‐level scaffold assemblies of these three planthopper species using HiC scaffolding technique. The scaffold N50s significantly increased to 77.63 Mb, 43.36 Mb and 29.24 Mb for N. lugens, S. furcifera and L. striatellus, respectively. To identify sex chromosomes of these three planthopper species, we carried out genome re‐sequencing of males and females and successfully determined the X and Y chromosomes for N. lugens, and X chromosome for S. furcifera and L. striatellus. The gene content of the sex chromosomes showed high diversity among these three planthoppers suggesting the rapid evolution of sex‐linked genes, and all chromosomes showed high synteny. The chromosome‐level genome assemblies of three planthoppers would provide a valuable resource for a broad range of future research in molecular ecology, and subsequently benefits development of modern pest control strategies.     

Species‐level view of population structure and gene flow for a critically endangered primate (Varecia variegata)

Lemurs are among the world's most threatened mammals. The critically endangered black‐and‐white ruffed lemur (Varecia variegata), in particular, has recently experienced rapid population declines due to habitat loss, ecological sensitivities to habitat degradation, and extensive human hunting pressure. Despite this, a recent study indicates that ruffed lemurs retain among the highest levels of genetic diversity for primates. Identifying how this diversity is apportioned and whether gene flow is maintained among remnant populations will help to diagnose and target conservation priorities. We sampled 209 individuals from 19 sites throughout the remaining V. variegata range. We used 10 polymorphic microsatellite loci and ~550 bp of mtDNA sequence data to evaluate genetic structure and population dynamics, including dispersal patterns and recent population declines. Bayesian cluster analyses identified two distinct genetic clusters, which optimally partitioned data into populations occurring on either side of the Mangoro River. Localities north of the Mangoro were characterized by greater genetic diversity, greater gene flow (lower genetic differentiation) and higher mtDNA haplotype and nucleotide diversity than those in the south. Despite this, genetic differentiation across all sites was high, as indicated by high average F_ST (0.247) and Φ_ST (0.544), and followed a pattern of isolation‐by‐distance. We use these results to suggest future conservation strategies that include an effort to maintain genetic diversity in the north and restore connectivity in the south. We also note the discordance between patterns of genetic differentiation and current subspecies taxonomy, and encourage a re‐evaluation of conservation management units moving forward.     

Ecological and genetic factors influencing the transition between host‐use strategies in sympatric Heliconius butterflies

Shifts in host‐plant use by phytophagous insects have played a central role in their diversification. Evolving host‐use strategies will reflect a trade‐off between selection pressures. The ecological niche of herbivorous insects is partitioned along several dimensions, and if populations remain in contact, recombination will break down associations between relevant loci. As such, genetic architecture can profoundly affect the coordinated divergence of traits and subsequently the ability to exploit novel habitats. The closely related species Heliconius cydno and H. melpomene differ in mimetic colour pattern, habitat and host‐plant use. We investigate the selection pressures and genetic basis underlying host‐use differences in these two species. Host‐plant surveys reveal that H. melpomene specializes on a single species of Passiflora. This is also true for the majority of other Heliconius species in secondary growth forest at our study site, as expected under a model of interspecific competition. In contrast, H. cydno, which uses closed‐forest habitats where both Heliconius and Passiflora are less common, appears not to be restricted by competition and uses a broad selection of the available Passiflora. However, other selection pressures are likely involved, and field experiments reveal that early larval survival of both butterfly species is highest on Passiflora menispermifolia, but most markedly so for H. melpomene, the specialist on that host. Finally, we demonstrate an association between host‐plant acceptance and colour pattern amongst interspecific hybrids, suggesting that major loci underlying these important ecological traits are physically linked in the genome. Together, our results reveal ecological and genetic associations between shifts in habitat, host use and mimetic colour pattern that have likely facilitated both speciation and coexistence.     

De novo assembly of a chromosome‐level reference genome of red‐spotted grouper (Epinephelus akaara) using nanopore sequencing and Hi‐C

The red‐spotted grouper Epinephelus akaara (E. akaara) is one of the most economically important marine fish in China, Japan and South‐East Asia and is a threatened species. The species is also considered a good model for studies of sex inversion, development, genetic diversity and immunity. Despite its importance, molecular resources for E. akaara remain limited and no reference genome has been published to date. In this study, we constructed a chromosome‐level reference genome of E. akaara by taking advantage of long‐read single‐molecule sequencing and de novo assembly by Oxford Nanopore Technology (ONT) and Hi‐C. A red‐spotted grouper genome of 1.135 Gb was assembled from a total of 106.29 Gb polished Nanopore sequence (GridION, ONT), equivalent to 96‐fold genome coverage. The assembled genome represents 96.8% completeness (BUSCO) with a contig N50 length of 5.25 Mb and a longest contig of 25.75 Mb. The contigs were clustered and ordered onto 24 pseudochromosomes covering approximately 95.55% of the genome assembly with Hi‐C data, with a scaffold N50 length of 46.03 Mb. The genome contained 43.02% repeat sequences and 5,480 noncoding RNAs. Furthermore, combined with several RNA‐seq data sets, 23,808 (99.5%) genes were functionally annotated from a total of 23,923 predicted protein‐coding sequences. The high‐quality chromosome‐level reference genome of E. akaara was assembled for the first time and will be a valuable resource for molecular breeding and functional genomics studies of red‐spotted grouper in the future.     

The brown coat colour of Coppernecked goats is associated with a non‐synonymous variant at the TYRP1 locus on chromosome 8

The recent development of a goat SNP genotyping microarray enables genome‐wide association studies in this important livestock species. We investigated the genetic basis of the black and brown coat colour in Valais Blacknecked and Coppernecked goats. A genome‐wide association analysis using goat SNP50 BeadChip genotypes of 22 cases and 23 controls allowed us to map the locus for the brown coat colour to goat chromosome 8. The TYRP1 gene is located within the associated chromosomal region, and TYRP1 variants cause similar coat colour phenotypes in different species. We thus considered TYRP1 as a strong positional and functional candidate. We resequenced the caprine TYRP1 gene by Sanger and Illumina sequencing and identified two non‐synonymous variants, p.Ile478Thr and p.Gly496Asp, that might have a functional impact on the TYRP1 protein. However, based on the obtained pedigree and genotype data, the brown coat colour in these goats is not due to a single recessive loss‐of‐function allele. Surprisingly, the genotype distribution and the pedigree data suggest that the ⁴⁹⁶Asp allele might possibly act in a dominant manner. The ⁴⁹⁶Asp allele was present in 77 of 81 investigated Coppernecked goats and did not occur in black goats. This strongly suggests heterogeneity underlying the brown coat colour in Coppernecked goats. Functional experiments or targeted matings will be required to verify the unexpected preliminary findings.     

Unexpected species diversity of Malagasy primates (<Emphasis Type="Italic">Lepilemur</Emphasis> spp.) in the same biogeographical zone: a morphological and molecular approach with the description of two new species

Background  

The lemurs of Madagascar provide an excellent mammalian radiation to explore mechanisms and processes favouring species diversity and evolution. Species diversity, in particular of nocturnal species, increased considerably during the last decade. However, the factors contributing to this high diversity are not well understood. We tested predictions derived from two existing biogeographic models by exploring the genetic and morphological divergence among populations of a widely distributed lemur genus, the sportive lemur (Lepilemur ssp.) along a 560 km long transect from western to northern Madagascar.     

Species delimitation in lemurs: multiple genetic loci reveal low levels of species diversity in the genus Cheirogaleus

Background  

Species are viewed as the fundamental unit in most subdisciplines of biology. To conservationists this unit represents the currency for global biodiversity assessments. Even though Madagascar belongs to one of the top eight biodiversity hotspots of the world, the taxonomy of its charismatic lemuriform primates is not stable. Within the last 25 years, the number of described lemur species has more than doubled, with many newly described species identified among the nocturnal and small-bodied cheirogaleids. Here, we characterize the diversity of the dwarf lemurs (genus Cheirogaleus) and assess the status of the seven described species, based on phylogenetic and population genetic analysis of mtDNA (cytb + cox2) and three nuclear markers (adora3, fiba and vWF).