Species‐specific coordinated gene expression and trans‐regulation of larval color pattern in three swallowtail butterflies

SUMMARY The diversity of butterfly larval color pattern has been attracted to people since Darwin's time; however, its molecular mechanisms still remain largely unknown. Larval body markings often differ completely between closely related species under natural selection. The final instar larvae of the swallowtail butterflies Papilio xuthus and Papilio polytes show a green camouflage pattern, whereas those of Papilio machaon show a warning color pattern, although P. xuthus and P. machaon are closely related species. To identify the genes that contribute to species divergence, we compared the expression pattern of eight pigment‐associated genes between three Papilio species. The spatial expression pattern of melanin‐related genes coincided with the species‐specific cuticular markings. We newly found that the combination of bilin‐binding protein and yellow‐related gene (YRG) correlated perfectly with larval blue, yellow, and green coloration. To distinguish whether the interspecific differences in pigment‐associated genes are caused by cis‐regulatory changes or distribution differences in trans‐regulatory proteins, we compared species‐specific mRNA expression in an F1 hybrid specimen. Px‐YRG and Pp‐YRG showed a similar expression pattern, suggesting that the change in expression of YRG is caused mainly by changes in the distribution of trans‐regulatory proteins. Our findings shed light on the gene regulatory networks for butterfly larval color pattern.     

Morphological Clines and Weak Drift along an Urbanization Gradient in the Butterfly,Pieris rapae

Urban areas are increasing globally, providing opportunities for biodiversity researchers to study the process in which species become established in novel, highly disturbed habitats. This ecological process can be understood through analyses of morphological and genetic variation, which can shed light on patterns of neutral and adaptive evolution. Previous studies have shown that urban populations often diverge genetically from non-urban source populations. This could occur due to neutral genetic drift, but an alternative is that selection could lead to allele frequency changes in urban populations. The development of genome scan methods provides an opportunity to investigate these outcomes from samples of genetic variation taken along an urbanization gradient. Here we examine morphological variation in wing size and diversity at neutral amplified fragment length polymorphisms in the butterfly Pieris rapae L. (Lepidoptera, Pieridae) sampled from the center to the periphery of Marseille. We utilize established and novel environmental correlation approaches to scan genetic variation for evidence of selection. We find significant morphological differences in urban populations, as well as weak genetic structure and decreased genetic diversity in urban versus non-urban sites. However, environmental correlation tests provide little support for selection in our dataset. Our comparison of different methods and allele frequency clines suggests that loci identified as significant are false positives. Although there is some indication that selection may be acting on wing size in urban butterflies, genetic analyses suggest P. rapae are undergoing neutral drift.     

Purification and Characterization of Biliverdin-binding Protein from Larval Hemolymph of the Swallowtail Butterfly,Papilio xuthus L.

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The N-amino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.     

Butterfly community ecology: the influences of habitat type,weather patterns,and dominant species in a temperate ecosystem

We compared variation in butterfly communities across 3 years at six different habitats in a temperate ecosystem near Boulder, Colorado, USA. These habitats were classified by the local Open Space consortium as Grasslands, Tallgrass, Foothills Grasslands, Foothills Riparian, Plains Riparian, and Montane Woodland. Rainfall and temperature varied considerably during these years. We surveyed butterflies using the Pollard‐Yates method of invertebrate sampling and compared abundance, species richness, and diversity across habitats and years. Communities were most influenced by habitat, with all three quantitative measures varying significantly across habitats but only two measures showing variation across years. Among habitats, butterfly abundance was higher in Plains Riparian sites than in Montane Woodland or Grassland sites, though diversity was lowest in Plains Riparian areas. Butterfly species richness was higher in Foothills Riparian sites than it was in all but one other habitat (Tallgrass). Among years, butterfly abundance and species richness were lower during the year of least rainfall and highest temperatures, suggesting a substantial impact of the hot, dry conditions. Across habitats and years, butterfly abundance was consistently high at Plains Riparian and Foothills Riparian sites, and richness and diversity were consistently high in Foothills Riparian areas. These two habitats may be highly suitable for butterflies in this ecosystem, regardless of weather conditions. Generally low abundance and species richness in Montane Woodlands sites, particularly in 2002, suggested low suitability of the habitat to butterflies in this ecosystem, and this may be especially important during drought‐like conditions. Finally, to examine the effect that the presence of the very abundant non‐native species Pieris rapae L. (Lepidoptera: Pieridae) has on these communities, we re‐analyzed the data in the absence of this species. Excluding P. rapae dramatically reduced variation of both butterfly abundance and diversity across habitats, highlighting the importance of considering community membership in analyses like ours.     

The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses

The nearly complete mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) was sequenced for its nucleotide sequence of 13,964 bp. The genome has a typical gene order identical to other lepidopteran species. All tRNAs showed same stable canonical clover-leaf structure as those of other insects, except for tRNA^Ser (AGN), in which the dihydrouracil arm (DHU arm) could not form stable stem–loop structure. Anomalous initiation codons have been observed for the cox1 gene, where the ATTACG hexa-nucleotide was believed to be involved in the initiation signaling. Twelve mitochondrial protein-coding gene sequence data were used to infer the phylogenetic relationships among the insect orders. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the Homometabola was not supported in this paper. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, Papilionoidea and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea, Pyraloidea and Papilionoidea are monophyletic (Obtectomera). Bombycoidea (Bombyx mandarina and Antheraea pernyi) and Papilionoidea (P. xuthus and Coreana raphaelis) formed a sister group.     

Caterpillar color patterns are determined by a two‐phase melanin gene prepatterning process: new evidence from tan and laccase2

SUMMARY The larval color patterns in Lepidoptera exhibit splendid diversity, and identifying the genes responsible for pigment distribution is essential to understanding color‐pattern evolution. The swallowtail butterfly, Papilio xuthus, is a good candidate for analyzing marking‐associated genes because its body markings change dramatically at the final molt. Moreover, the silkworm Bombyx mori is most suitable for identification of lab‐generated color mutants because genome information and many color mutants are available. Here, we analyzed the expression pattern of 10 melanin‐related genes in P. xuthus, and analyzed whether these genes were responsible for Bombyx larval color mutants. We found that seven genes correlated strongly with the stage‐specific larval cuticular markings of P. xuthus, suggesting that, compared with Drosophila, more genes showed marking specificity in lepidopteran larvae. We newly found that the expression of both tan and laccase2 is strongly correlated with the larval black markings in both P. xuthus and B. mori. The results of F2 linkage analysis and mutant analysis strongly suggest that tan is the responsible gene for Bombyx larval color mutant rouge, and that tan is important in emphasizing black markings of lepidopteran larvae. Detailed comparison of temporal and spatial expression patterns showed that larval cuticular markings were regulated at two different phases. Marking‐specific expression of oxidizing enzymes preceded the marking‐specific expression of melanin synthesis enzymes at mRNA level, which is the reverse of the melanin synthesis step.     

Revealing the non-overlapping characteristics between original centers and genetic diversity of Purpureocillium lilacinum

Purpureocillium lilacinum is a free-living, multitrophic, cosmopolitan fungus. This study estimated the genetic diversity of P. lilacinum and its geographic distribution pattern based on a global ITS dataset. At the intercontinental levels, the highest genetic diversity was in Asia. Divergence time estimation indicated that Hap 5, Hap 9, Hap 31, and Hap 32 from the Hainan, Guangdong, Jiangxi, and Fujian regions of China in Asia were the earliest divergence haplotypes. The neutrality test indicated that P. lilacinum is undergoing population expansion. These results support that the southeastern coastal region of China is the original center of P. lilacinum, while the East Asian region adjacent to this origin is the center of the genetic diversity of P. lilacinum. The geographical distribution pattern of P. lilacinum showed that only one haplotype (Hap 1) was globally distributed and that most haplotypes were distributed in Asia, while the few remaining haplotypes were scattered on other continents. The results provide valuable information to elucidate the origin, genetic diversity, and evolution of fungi.     

Historical biogeography of two alpine butterflies in the Rocky Mountains: broad-scale concordance and local-scale discordance

Aim We inferred the phylogeography of the alpine butterfly Colias meadii Edwards (Pieridae) and compared its genetic structure with that of another high elevation, co‐distributed butterfly, Parnassius smintheus Doubleday (Papilionidae), to test if the two Rocky Mountain butterflies responded similarly to the palaeoclimatic cycles of the Quaternary. Location Specimens were collected from 18 alpine sites in the Rocky Mountains of North America, from southern Colorado to northern Montana. Methods We sequenced 867 and 789 nucleotides of cytochrome oxidase I from an average of 19 and 20 individuals for C. meadii and P. smintheus, respectively, from each of the same 18 localities. From the sequence data, we calculated measures of genetic diversity within each population (H, θ), genetic divergence among populations (F_ST), and tested for geographic structure through an analysis of molecular variance (amova ). Population estimates were compared against latitude and between species using a variety of statistical tests. Furthermore, nested clade analysis was implemented to infer historic events underlying the geographic distribution of genetic variation in each species. Then, we compared the number of inferred population events between species using a nonparametric Spearman's rank correlation test. Finally, we ran coalescent simulations on each species’ genealogy to test whether the two species of Lepidoptera fit the same model of population divergence. Results Our analyses revealed that: (1) measures of within‐population diversity were not correlated with latitude for either species, (2) within‐site diversity was not correlated between species, (3) within a species, nearly all populations were genetically isolated, (4) both species exhibited significant and nearly identical partitioning of genetic variation at all hierarchical levels of the amova , including a strong break between populations across the Wyoming Basin, (5) both species experienced similar cycles of expansion and contraction, although fewer were inferred for C. meadii, and (6) data from both species fit a model of three refugia diverging during the Pleistocene. Main conclusions While our findings supported a shared response of the two butterfly species to historic climate change across coarse spatial scales, a common pattern was not evident at finer spatial and temporal scales. The shared demographic history of the two species is consistent with an expanding–contracting archipelago model, suggesting that populations persisted across the geographic range throughout the climate cycles, experiencing isolation on ‘sky islands’ during interglacial periods and becoming connected as they migrated down‐slope during cool, wet climates.     

Phylogeography and population expansion of Western Himalayan highly-erratically migratory painted lady butterfly (Lepidoptera: Nymphalidae: Vanessa cardui)

Understanding population genetics, genetic diversity and the migration pattern of ecologically important species is crucial for effective conservation plans. We studied mitochondrial cytochrome c oxidase subunit-I (COI) gene variation in painted lady butterfly (Vanessa cardui) from different altitudinal gradients of Uttarakhand (Western Himalayas), India. We sequenced COI) gene of 91 samples and 22 COI sequences retrieved from GenBank as representatives of different localities for comparative analysis. We observed a total of 16 haplotypes, of which seven haplotypes were observed in Uttarakhand with intra-species sequence divergence ranges from 0.001 to 0.009. Haplotype (Hd) and nucleotide diversity (π) were comparatively low and averaging 0.635 and 0.002, respectively. The neutrality test, mismatch distribution graph and observed pattern of diversity indices (Hd and π) revealed population expansion. It was further confirmed by the Median Joining Network, which exhibited a star-like topology from the core haplotype, while the Bayesian phylogenetic tree displayed a mixing of haplotypes from different localities to an identical clade. Haplotype organization in the Bayesian tree and Median Joining Network was not clustered as per their topographical affinity, indicating incomplete lineage sorting and late species population development or colonization in the novel area.     

Phylogeography suggest the Yili Valley being the glacial refuge of the genus Ixiolirion (Amaryllidaceae) in China

The Pleistocene climatic oscillations had profound effects on the demographic history and genetic diversification of plants in arid north-west China where some glacial refugia have been recognized. The genus Ixiolirion comprises three species, of which two, I. tataricum and I. songaricum (endemic), occur in China. In some locations they are sympatric. We investigated their population structure and population history in response to past climatic change using a sample of 619 individuals in 34 populations with nITS and ptDNA sequences. A significant genetic divergence between the two species was supported by a high level of pairwise genetic differentiation, very low gene flow, and phylogenetic analysis showing that I. songaricum haplotypes were monophyletic, whereas those of I. tataricum were polyphyletic. We found significant differentiation and phylogeographic structure in both species. The split of the two species was dated to the late Miocene (～7?Ma), but deep divergence occurred in the mid-late Quaternary. A similar haplotype distribution pattern was found in both species: one to two dominant haplotypes across most populations, with unique haplotypes in a few populations or a geographic group. The genetic diversity, haplotype number, and haplotype diversity decreased from the Yili Valley to the central Tianshan and Barluk Mountains. Additionally, ptDNA analysis showed that I. tataricum diversified in the eastern Tianshan and Barluk Mountains, which might be due to physical barriers to long distance seed dispersal such as desert. In conclusion, our results indicated that the Yili Valley was likely a glacial refuge for Ixiolirion in China, with postglacial dispersal from the Yili Valley eastward to the eastern Tianshan Mountains, and northward to the Barluk Mountains. The climatic changes in the Miocene and Pleistocene and geographic barriers are important factors driving species divergence and differentiation of Ixiolirion and other taxa.     

地理因素对箭竹复合体遗传多样性与遗传分化的影响

地理因素对植物天然居群的物种分布和种内分化具有重要影响。该研究通过对箭竹复合体内39个居群的14对SSR数据进行深入分析,旨在揭示重要地理因素(如海拔、纬度、地理距离)对该复合体内遗传多样性和遗传分化式样的影响。结果表明:(1)糙花箭竹亚支系遗传多样性最高(H_e=0.50),而团竹亚支系的遗传多样性最低(H_e=0.33)。(2)遗传多样性与纬度、海拔在A、B两个支系水平呈显著正相关关系,但在亚支系水平,遗传多样性的变化趋势呈现出更为复杂的局面,部分支系表现为负相关关系,推测纬度和海拔对箭竹复合体内遗传多样性水平具有一定影响,但也需重视其他进化因素的作用。(3)Mantel检验显示,仅在团竹亚支系中检测出较弱的正相关关系,表明地理距离不是影响箭竹复合体内遗传分化的主导因素,后续需结合基因流检测推断杂交事件对其遗传分化的影响。     

Butterfly wing patterns

This paper integrates genetical studies of variation in the wing patterns of Lepidoptera with experimental investigations of developmental mechanisms. Research on the tropical butterfly,Bicyclus anynana, is described. This work includes artificial selection of lines with different patterns of wing eyespots followed by grafting experiments on the lines to examine the phenotypic and genetic differences in terms of developmental mechanisms. The results are used to show how constraints on the evolution of this wing pattern may be related to the developmental organisation. The eyespot pattrn can be envisaged as a set of developmental homologues; a common developmental mechanism is associated with a quantitative genetic system involving high genetic correlations. However, individual genes which influence only subsets of the eyespots, thus uncoupling the interdependence of the eyespots, may be important in evolutionary change. The postulated evolutionary constraints are illustrated with respect to differences in wing pattern found among other species ofBicyclus.     

Horizontal Gene Transfer and Gene Duplication of β-Fructofuranosidase Confer Lepidopteran Insects Metabolic Benefits

Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect β-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of β-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of β-fructofuranosidase were found in Coleoptera and Hymenoptera. β-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran β-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of β-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of β-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.     

Low Genetic Diversity and Strong Geographical Structure of the Critically Endangered White-Headed Langur (Trachypithecus leucocephalus) Inferred from Mitochondrial DNA Control Region Sequences

Many Asian colobine monkey species are suffering from habitat destruction and population size decline. There is a great need to understand their genetic diversity, population structure and demographic history for effective species conservation. The white-headed langur (Trachypithecus leucocephalus) is a Critically Endangered colobine species endemic to the limestone karst forests in southwestern China. We analyzed the mitochondrial DNA (mtDNA) control region sequences of 390 fecal samples from 40 social groups across the main distribution areas, which represented one-third of the total extant population. Only nine haplotypes and 10 polymorphic sites were identified, indicating remarkably low genetic diversity in the species. Using a subset of 77 samples from different individuals, we evaluated genetic variation, population structure, and population demographic history. We found very low values of haplotype diversity (h = 0.570 ± 0.056) and nucleotide diversity (π = 0.00323 ± 0.00044) in the hypervariable region I (HVRI) of the mtDNA control region. Distribution of haplotypes displayed marked geographical pattern, with one population (Chongzuo, CZ) showing a complete lack of genetic diversity (having only one haplotype), whereas the other population (Fusui, FS) having all nine haplotypes. We detected strong population genetic structure among habit patches (Φ _ST = 0.375, P < 0.001). In addition, the Mantel test showed a significant correlation between the pairwise genetic distances and geographical distances among social groups in FS (correlation coefficient = 0.267, P = 0.003), indicting isolation-by-distance pattern of genetic divergence in the mtDNA sequences. Analyses of demographic history suggested an overall stable historical population size and modest population expansion in the last 2,000 years. Our results indicate different genetic diversity and possibly distinct population history for different local populations, and suggest that CZ and FS should be considered as one evolutionarily significant unit (ESU) and two management units (MUs) pending further investigation using nuclear markers.     

Genetic divergence and evidence for sympatric host-races in the highly polyphagous brown tail moth,Euproctis chrysorrhoea (Lepidoptera: Erebidae)

The brown tail moth (BTM) Euproctis chrysorrhoea (Linnaeus 1758) (Lepidoptera: Erebidae) is a forest and ornamental pest in Europe and the United States. Its extreme polyphagy, and documented phenological shift associated with host use suggest the presence of distinct host-races. To test this hypothesis, we sampled BTM infesting different host species in several locations along its distribution, and used DNA sequence data (a total of 1,672 bp from cytochrome c oxidase subunit I, elongation factor 1-alpha, and wingless) to produce haplotype networks and reconstruct the phylogenetic relationships between individuals. Population genetic diversity indices pointed out a higher genetic diversity in Europe, particularly in the samples from southern Spain and southern England. Lower F _ST values were found between geographically closer populations when compared to more distant ones, but analyses of molecular variance and Mantel tests failed to reveal geographically associated genetic differentiation. However, haplotype networks and phylogenetic reconstructions revealed a previously unknown genetic differentiation within the BTM, with one lineage circumscribed to southern Europe. Although BTM haplotypes did not cluster according to their host plant, host-associated haplotypes were observed within certain geographic regions. Hence, our data support the existence of host-races of BTM within southern Spain and southern England, where populations from different hosts occur in sympatry.     

Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, <Emphasis Type="Italic">Papilio xuthus</Emphasis>

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.     

Genetic diversity and population connectivity of the Asian green mussel Perna viridis in South China Sea,inferred from mitochondria DNA markers

The Asian green mussel Perna viridis is ecologically and economically important in the coastal regions of China. In order to characterize the genetic diversity and population connectivity of P. viridis in South China Sea, a 664 bp region of mitochondrial COI gene and a 293 bp region of 16S rRNA gene were sequenced and analyzed for 78 and 92 individuals from four populations in South China Sea, respectively. A total of 15 haplotypes were defined by 14 variable nucleotide sites in COI gene, and 7 haplotypes by 6 variable nucleotide sites in 16S rRNA gene. High haplotype diversity and low nucleotide diversity were observed in COI gene, while moderate haplotype diversity and low nucleotide diversity were observed in 16S rRNA gene. Pairwise F_ST values of COI gene were all negative and those of 16S rRNA gene ranged from −0.01409 to 0.10289. The results showed that no significant genetic divergence (or shallow genetic structure) and high levels of population connectivity among the four populations of P. viridis in South China Sea.     

Phylogeography and genetic structure of disjunct Salix arbutifolia populations in Japan

Disjunct geographic distributions of boreal plant species at the southern edges of their ranges are expected to result in low genetic diversity and high genetic differentiation in the disjunct populations. This prediction was tested in a riparian willow, Salix arbutifolia, distributed in the northeastern Eurasian continent and the Sakhalin, Hokkaido, and Honshu Islands, using chloroplast DNA haplotypes and nuclear microsatellite genotypes. Hokkaido and Honshu populations shared a chloroplast haplotype identical to a closely related species, S. cardiophylla. This haplotype was divergent from haplotypes in the Eurasian continent (Primorsky) and the Sakhalin Island. In the nuclear microsatellites, most Hokkaido populations were genetically closer to Primorsky populations than to Sakhalin populations in spite of the geographical vicinity between Sakhalin and Hokkaido. The unexpected genetic divergence between Sakhalin and Hokkaido implies a complicated history of migration and colonization. The most peripheral populations in Honshu had the lowest genetic diversity and were most differentiated from the others. Thus, low genetic diversity and high genetic differentiation at the range periphery suggest substantial effects of genetic drift on genetic structure in the disjunct populations of Salix arbutifolia at the southern edge of its range.     

The population biology and ecology of the Homerus swallowtail, <Emphasis Type="Italic">Papilio (Pterourus) homerus</Emphasis>, in the Cockpit Country,Jamaica

The Homerus swallowtail, Papilio (Pterourus) homerus (Lepidoptera: Papilionidae), is an endangered species of butterfly endemic to Jamaica. As the largest species of the genus Papilio in the world and the largest butterfly in the Western Hemisphere, this rare butterfly once inhabited most of Jamaica but has now dwindled into two tiny populations: an eastern population, found where the Blue Mountains and John Crow Mountains merge, and a western population in the Cockpit Country. The present research focused on the previously unstudied Cockpit Country population of P. homerus; most previous information about this species is derived from studies of the eastern population. The purpose was to estimate the size of the remaining population in the Cockpit Country using MRR protocols, while making observations to better understand its ecology. Sampling consisted of carefully netting the butterfly, marking a permanent ink number on the wing (metallic Sharpie® marker), and recording winglength, wing condition, time, and sex. The population was found to be very small, estimated at fewer than 50 flying individuals. Many observations were made about the ecology of the species. These new data suggest a conservation plan is strongly needed, coupled with a breeding program to increase numbers of this extraordinary butterfly.