Color Pattern Variation Research Articles

Integrative systematics of Cheguevaria Kazantsev, 2007 (Coleoptera, Lampyridae, Cheguevariinae) identifies genetic stability in color-polymorphic individuals and a disjoint relationship with Amydetinae

Abstract The genus Cheguevaria (Kazantsev 2007) is currently comprised of three species which are endemic to the Greater Antilles: Cheguevaria angusta Kazantsev, 2007 (Hispaniola), Cheguevaria taino Kazantsev, 2007 and Cheguevaria montana Kazantsev, 2008 (both from Puerto Rico). Cheguevaria is the sole member of Cheguevariinae, and a previous morphology-based phylogenetic hypotheses suggested a close relationship with the subfamily Amydetinae. Recent collecting trips to Puerto Rico and the survey of other scientific collections allowed us to identify and locate more specimens of the genus, including several color morphs of specimens tentatively identified as C. montana. We used this material to revise the genus, and to describe Cheguevaria cubensisnew species, the first record of the genus from Cuba. We also produced DNA barcodes based on partial cytochrome c oxidase subunit 1 (COI), which were used to investigate the species limits within color morph species of C. montana. Our results showed a high level of genetic stability in those color morphs, indicating that all specimens belong to the same species. We present a discussion to explain possible scenarios leading to such variation in color patterns. We also generated amplicons for two specimens of Amydetinae, represented by two Amydetes Illiger, 1807 species, and we tested their relationship with Cheguevaria in a phylogenetic context based on a four gene dataset. Our analyzes indicated a disjoint relationship between Cheguevaria and Amydetes, corroborating previously published morphology-based phylogenies, and we maintained the status of Cheguevaria as an independent lineage within Lampyridae.

Diversity and distribution of reed frogs (Hyperolius spp.) on Bioko Island, Equatorial Guinea

Reed frogs (Hyperolius spp.) are the most species-rich genus in the family Hyperoliidae, a group of frogs endemic to sub-Saharan Africa and the Gulf of Guinea islands. Three species are endemic to oceanic islands in the archipelago, but the diversity and distribution of reed frogs on Bioko – a land-bridge island in the archipelago – remains unclear. Two species, H. tuberculatus and H. ocellatus ocellatus, were formally documented on Bioko in the colonial era, however, two additional reed frog species have been detected in recent surveys. In addition, observations of heterospecific amplexus suggest one of the unidentified species and H. o. ocellatus may be hybridising on the island. In this study, we examined mitochondrial DNA (mtDNA), nuclear DNA (nuDNA), morphological and acoustic data to confirm the tentative identifications of the reed frogs and determine if there is any evidence of hybridisation. Our data confirm that the two unidentified species are H. endjami and H. fusciventris, and that Bioko H. endjami have unique genetic diversity not shared with the mainland population. Additionally, our mitochondrial (16S) and nuclear DNA (CMYC) haplotype networks suggest that H. o. ocellatus and H. endjami are not hybridising on Bioko. Finally, we report colour pattern variation in H. endjami and H. o. ocellatus that is unique from populations on the continent, and describe male advertisement calls of Bioko H. endjami and H. o. ocellatus. Our study provides a first look at the diversity and distribution of reed frogs on Bioko Island and sets the stage for future studies of the ecology, evolution, and conservation of this island community. KEYWORDS: hybridisation, polymorphism, mitochondrial and nuclear DNA, morphology, land-bridge island

Staging of post-settlement growth in the nudibranch Hypselodoris festiva

Sea slugs of the family Chromodorididae (Nudibranchia, Gastropoda, Mollusca) have garnered attention by researchers and hobbyists alike for their bright and variable color patterns. However, the chromodorid life cycle has yet to be fully elucidated as there exist no reports of their rearing in the laboratory. Here, we report the rearing of Hypselodoris festiva from eggs to adults, where we categorized their post-settlement growth patterns from juvenile to adult stages. Body coloration appeared around 36 days, and organogenesis of vital adult organs began within 42 days after hatching. The anus of H. festiva was observed to change from a ventral to dorsal position during juvenile growth. Individuals reached sexual maturity after six months post-hatching, with successful mating and spawning observed ex situ. This study outlines comprehensive rearing methods and life cycle staging that could be applied to other chromodorid species. We propose H. festiva as a model organism for chromodorid research, with this research contributing to the progress of developmental and evolutionary research on sea slugs.

Chemical defences indicate bold colour patterns with reduced variability in aposematic nudibranchs.

The selective factors that shape phenotypic diversity in prey communities with aposematic animals are diverse and coincide with similar diversity in the strength of underlying secondary defences. However, quantitative assessments of colour pattern variation and the strength of chemical defences in assemblages of aposematic species are lacking. We quantified colour pattern diversity using quantitative colour pattern analysis (QCPA) in 13 dorid nudibranch species (Infraorder: Doridoidei) that varied in the strength of their chemical defences. We accounted for the physiological properties of a potential predator's visual system (a triggerfish, Rhinecanthus aculeatus) and modelled the appearance of nudibranchs from multiple viewing distances (2 and 10 cm). We identified distinct colour pattern properties associated with the presence and strength of chemical defences. Specifically, increases in chemical defences indicated increases in colour pattern boldness (i.e. visual contrast elicited via either or potentially coinciding chromatic, achromatic and/or spatial contrast). Colour patterns were also less variable among species with chemical defences when compared to undefended species. Our results indicate correlations between secondary defences and diverse, bold colouration while showing that chemical defences coincide with decreased colour pattern variability among species. Our study suggests that complex spatiochromatic properties of colour patterns perceived by potential predators can be used to make inferences on the presence and strength of chemical defences.

Patterned, plain, and in-between: An assessment of ecogeographic divergence between colour pattern morphs of the common egg-eater Dasypeltis scabra

ABSTRACT Intraspecific variation in colour patterns may reflect adaptive responses to local environmental regimes that favour selection of different trade-offs between visual communication, thermoregulation, and anti-predatory functions. Understanding the drivers of colour pattern variation within species can therefore provide valuable insights about adaptation. However, the ecogeographic correlates, and thus the ecological drivers, associated with the segregation of different colour morphs for most reptile species, including snakes, are not well known. Here, we examined differences in environmental niches associated with occurrence records of preserved museum specimens and georeferenced photographic images of common egg-eaters (Dasypeltis scabra) to characterise ecogeographic divergence between typically patterned, patternless, and faintly marked individuals. We gathered 1707 records that we assigned as either patterned, plain, or intermediate morphs. Most records were of patterned individuals, which were widely distributed in southern Africa. Climate niches differed between morphs as plain and intermediate snakes were restricted to high-elevation areas in the Highveld grasslands of South Africa, and woodland-dominated areas in the central and eastern parts of both Zambia and Zimbabwe, but there were no areas where only plain or intermediate snakes occurred in isolation from patterned snakes. Environmental niche modelling predicted minimal areas suitable for co-habitation of colour morphs. We speculate that plain or weakly-marked colour patterns likely developed as an adaptation to increase camouflage ability in open areas in grassland and certain woodland habitats.

Environmental colour pattern variation in Mediterranean Podarcis

BackgroundLizards of the genus Podarcis are widespread in the Mediterranean region, including islands and island archipelagos. These small-bodied lizards have a predominantly protective green-brown colouration. However, some populations display unusual patterns, in which the colouration is predominantly blue or uniformly black. This study explores the factors that influence this chromatic variation, whether environmental (climate and island conditions) or evolutionary (phylogenetic trait conservatism). The colouration of 1400 individuals (27 species) was analysed in the CIELAB colour space.ResultsPagel’s λ indicated that colouration is weakly conserved within phylogenetic lineages. Although the island surface plays a key role in the chromatic variability of these lacertids, geographic isolation and climate hold less influence. The colouration of some small island populations tends to be uniform and dark, possibly due to intense intraspecific competition and lower predatory pressure.ConclusionsThis study highlights the importance of island populations in understanding the processes that favour the emergence of extreme phenotypes in small ectothermic vertebrates.

Deep divergences among inconspicuously colored clades of Epipedobates poison frogs

Poison frogs (Dendrobatidae) are famous for their aposematic species, having a combination of diverse color patterns and defensive skin toxins, yet most species in this family are inconspicuously colored and considered non-aposematic. Epipedobates is among the youngest genus-level clades of Dendrobatidae that includes both aposematic and inconspicuous species. Using Sanger-sequenced mitochondrial and nuclear markers, we demonstrate deep genetic divergences among inconspicuous species of Epipedobates but relatively shallow genetic divergences among conspicuous species. Our phylogenetic analysis includes broad geographic sampling of the inconspicuous lineages typically identified as E. boulengeri and E. espinosai, which reveals two putative new species, one in west-central Colombia (E. sp. 1) and the other in north-central Ecuador (E. aff. espinosai). We conclude that E. darwinwallacei is a junior subjective synonym of E. espinosai. We also clarify the geographic distributions of inconspicuous Epipedobates species including the widespread E. boulengeri. We provide a qualitative assessment of the phenotypic diversity in each nominal species, with a focus on the color and pattern of inconspicuous species. We conclude that Epipedobates contains eight known valid species, six of which are inconspicuous. A relaxed molecular clock analysis suggests that the most recent common ancestor of Epipedobates is ∼11.1 million years old, which nearly doubles previous estimates. Last, genetic information points to a center of species diversity in the Chocó at the southwestern border of Colombia with Ecuador. A Spanish translation of this text is available in the supplementary materials.

DNA Barcoding Supports "Color-Pattern''-Based Species of Stictochironomus from China (Diptera: Chironomidae).

The genus Stictochironomus (Diptera: Chironomidae) has an almost worldwide distribution, with more than 30 species. However, species delimitation and identification based on the markings on the wings and legs are controversial and uncertain. In this study, we focused on color patterns to review the adults of the genus from China, and two new species (S. trifuscipes sp. nov. and S. quadrimaculatus sp. nov.) are described and figured. DNA barcodes can accurately separate the two new species with specific color patterns. However, heterospecific individuals form a monophyletic cluster in the phylogeny tree. For example, S. maculipennis (Meigen) and S. pictulus (Meigen), which have a lower interspecific genetic divergence, form a single clade. Sequences with the same species name but with high intraspecific distance form more than one phylogenetic clade, such as S. sticticus (Fabricius) of three clades, S. pictulus of four clades, S. akizukii (Tokunaga) and S. juncaii Qi, Shi, and Wang of two clades, might have potential cryptic species diversity. Species delimitation analysis using ASAP, PTP, and GMYC clearly delineated them as separate species. Consequently, color patterns are a good diagnostic characteristic for species delimitation in Stictochironomus. The distance-based analysis shows that a threshold of 4.5-7.7% is appropriate for species delimitation in Stictochironomus. Additionally, an updated key including color pattern variation for male adults of known Stictochironomus species from China is provided.

Pollen, anther, stamen, and androecium mimicry.

Floral colours represent a highly diverse communication signal mainly involved in flower visitors' attraction and guidance, but also flower discrimination, filtering non-pollinators and discouraging floral antagonists. The divergent visual systems and colour preferences of flower visitors, as well as the necessity of cues for flower detection and discrimination, foster the diversity of floral colours and colour patterns. Despite the bewildering diversity of floral colour patterns, a recurrent component is a yellow UV-absorbing floral centre, and it is still not clear why this pattern is so frequent inangiosperms. The pollen, anther, stamen, and androecium mimicry (PASAM) hypothesis suggests that the system composed of the flowers possessing such yellow UV-absorbing floral reproductive structures, the flowers displaying central yellow UV-absorbing structures as floral guides, and the pollen-collecting, as well as pollen-eating, flower visitors responding to such signals constitute the world's most speciose mimicry system. In this review, we call the attention of researchers to some hypothetical PASAM systems around the globe, presenting some fascinating examples that illustrate their huge diversity. We will also present new and published data on pollen-eating and pollen-collecting pollinators' responses to PASAM structures supporting the PASAM hypothesis and will discuss how widespread these systems are around the globe. Ultimately, our goal is to promote the idea that PASAM is a plausible first approach to understanding floral colour patterns in angiosperms.

Predator selection on phenotypic variability of cryptic and aposematic moths

Natural selection generally favours phenotypic variability in camouflaged organisms, whereas aposematic organisms are expected to evolve a more uniform warning coloration. However, no comprehensive analysis of the phenotypic consequences of predator selection in aposematic and cryptic species exists. Using state-of-the-art image analysis, we examine 2800 wing images of 82 moth species accessed via three online museum databases. We test whether anti-predator strategy (i.e., camouflage or aposematism) explains intraspecific variation in wing colour and pattern across northern hemisphere moths. In addition, we test two mutually non-exclusive, ecological hypotheses to explain variation in colour pattern: diel-activity or dietary-niche. In this work, taking into account phylogenetic relationships, moth phenotypic variability is best explained by anti-predator strategy with camouflaged moths being more variable in wing patterning than aposematic species.

The dwarf neon rainbowfish Melanotaenia praecox, a small spiny-rayed fish with potential as a new Acanthomorpha model fish: I. Fin ray ontogeny and postembryonic staging.

Fish fins with highly variable color patterns and morphologies have many functions. In Actinopterygii, the free parts of fins are supported by "soft rays" and "spiny rays." Spiny rays have various functions and are extremely modified in some species, but they are lacking in popular model fish such as zebrafish and medaka. Additionally, some model fish with spiny rays are difficult to maintain in ordinary laboratory systems. Characteristics of the small, spiny-rayed rainbowfish Melanotaenia praecox render it useful as an experimental model species. Neither fish age nor body size correlate well with fin development during postembryonic development in this species. A four-stage developmental classification is proposed that is based on fin ray development. Melanotaenia praecox is an ideal species to rear in laboratories for developmental studies. Our classification allows for postembryonic staging of this species independent of individual age and body size. Development of each fin ray may be synchronized with dorsal fin development. We discuss the differences in mechanisms regulating soft, spiny, and procurrent ray development.

Revision of the Chironius bicarinatus complex (Serpentes: Colubridae): Redefined species boundaries and description of a new species

Abstract Currently, the proposed diagnoses for the Chironius bicarinatus complex reflect a wide variation in color pattern and pholidosis. Herein, we review the Chironius bicarinatus complex based on morphological and molecular data from a sample of 485 specimens covering the species distribution. Our results corroborate the recognition of C. bicarinatus and C. gouveai, and diagnose a distinct lineage without an available name. Thus, here we describe this new species restricted to the Baturité Massif, a relictual rainforest isolated in the Caatinga xerophytic domain, in the state of Ceará, northeastern Brazil. The new species can be distinguished from its congeners by its unique combination of qualitative and quantitative morphological characters (scale counts, morphometric, color pattern), and is also supported by molecular and ecological evidence. Additionally, we rectify data on the distribution and morphological variability of C. gouveai to accurately infer the boundaries between this taxon and C. bicarinatus, which was not properly addressed. Finally, we discuss our results in the light of previous studies that suggest diversification hypotheses in the Atlantic Forest already detected for other taxa, highlighting the importance of conserving the areas of “Brejos de Altitude”, in northeastern Brazil, and the southern limit of Serra do Mar up to Serra do Tabuleiro, in southern Brazil.

Diving into the diversity of colour patterns in reef fishes.

Colours and associated patterns are probably some of the most obvious phenotypic traits in animals and reef teleost fishes are often cited as a textbook example for illustrating this type of diversity. Even if it is well established that colour patterns play a central role in the ecology and evolution of reef fishes, we still lack the necessary toolkits to fully grasp the mechanisms driving the diversification of this obvious phenotypic trait. On the one hand, genotyping power seems now limitless thanks to current DNA sequencing technologies. Today, entire genomes of fishes can be easily produced for large sets of species. On the other hand, the description of colour patterns and the quantification of their variation across reef fishes might be highly challenging. In a cover manuscript in this issue of Molecular Ecology, Coulmance etal.(2023) introduced an innovative approach for extracting and quantifying the major colour pattern elements present in the hamlets (Hypoplectrus spp., Serranidae), a recent reef fish radiation from the Caribbean. Then, they intelligently used the quantified colour pattern variation as a phenotypic trait for a genome-wide association study (GWAS). Interestingly, using a method that required no a priori knowledge, they were able to recover well-established marks (e.g., vertical bars) and to highlight less expected colour pattern elements (e.g., dark to light gradient on ventral part as well as caudal and anal fins), which show strong association peaks on linkage group (LG) 12 and 04. Beyond the demonstration of the potential of their new quantitative analysis of colour pattern variation in reef fishes combined with GWAS, their findings offer new perspectives on our understanding of the intrinsic and extrinsic factors generating this outstanding diversity of the fish world.

Genetic and behavioural factors affecting interpopulation colour pattern variation in two congeneric chameleon species.

We conducted a study on interpopulation variation of colour patterns in two congeneric chameleon species, which have an analogous life history. Both species are able to rapidly change colour pattern, and their context-dependent colour patterns often vary across a wide geographical range. Specifically, we tested four hypotheses that can explain the observed interpopulation variation of colour patterns by a series of behavioural field trials where the colour patterns of individuals were recorded and later analysed by a deep neural network algorithm. We used redundancy analysis to relate genetic, spectral and behavioural predictors to interpopulation colour pattern distance. Our results showed that both isolation by distance (IBD) and alternative mating tactics were significant predictors for interpopulation colour pattern variation in Chamaeleo chamaeleon males. By contrast, in Chamaeleo dilepis, the interpopulation colour pattern variation was largely explained by IBD, and evidence for alternative mating tactics was absent. In both chameleon species, the environmental colours showed no evidence of influencing chameleon interpopulation colour pattern variation, regardless of sex or behavioural context. This contrasting finding suggests that interpopulation context-dependent colour pattern variations in each species are maintained under a different set of selective pressures or circumstances.

First Morphological Description of <i>Rhacophorus orlovi</i> (Anura: Rhacophoridae) from Yunnan, China

Although Rhacophorus orlovi from China was once sampled in previous phylogenetic studies, up to now it has never been formally included in any list of Chinese amphibians, which could lead to a misunderstanding that R. orlovi has no accurate distribution in China. Here, we describe R. orlovi for the first time from China based on one specimen collected from Yunnan Province and compare it with the holotype of the species. Molecular evidence supports it belonging to R. orlovi and genetic divergence within R. orlovi ranges from 0 to 1.85%. Compared to the holotype, the specimen from Hekou, Yunnan, China shows some variations in both morphometric characters and color pattern.

Evidence for a selective link between cooperation and individual recognition

The ability to recognize others is a frequent assumption of models of the evolution of cooperation. At the same time, cooperative behavior has been proposed as a selective agent favoring the evolution of individual recognition abilities. Although theory predicts that recognition and cooperation may co-evolve, data linking recognition abilities and cooperative behavior with evidence of selection are elusive. Here, we provide evidence of a selective link between individual recognition and cooperation in the paper wasp Polistes fuscatus through a combination of clinal, common garden, and population genomics analyses. We identified latitudinal clines in both rates of cooperative nesting and color pattern diversity, consistent with a selective link between recognition and cooperation. In behavioral experiments, we replicated previous results demonstrating individual recognition in cooperative and phenotypically diverse P.fuscatus from New York. In contrast, wasps from a less cooperative and phenotypically uniform Louisiana population showed no evidence of individual recognition. In a common garden experiment, groups of wasps from northern populations formed more stable and individually biased associations, indicating that recognition facilitates group stability. The strength of recent positive selection on cognition-associated loci likely to mediate individual recognition is substantially greater in northern compared with southern P.fuscatus populations. Collectively, these data suggest that individual recognition and cooperative nesting behavior have co-evolved in P.fuscatus because recognition helps stabilize social groups. This work provides evidence of a specific cognitive phenotype under selection because of social interactions, supporting the idea that social behavior can be a key driver of cognitive evolution.

Genetic modification of a Hox locus drives mimetic color pattern variation in a highly polymorphic bumble bee.

Müllerian mimicry provides natural replicates ideal for exploring mechanisms underlying adaptive phenotypic divergence and convergence, yet the genetic mechanisms underlying mimetic variation remains largely unknown. The current study investigates the genetic basis of mimetic color pattern variation in a highly polymorphic bumble bee, Bombus breviceps (Hymenoptera, Apidae). In South Asia, this species and multiple comimetic species converge onto local Müllerian mimicry patterns by shifting the abdominal setal color from orange to black. Genetic crossing between the orange and black phenotypes suggested the color dimorphism being controlled by a single Mendelian locus, with the orange allele being dominant over black. Genome-wide association suggests that a locus at the intergenic region between two abdominal fate-determining Hox genes, abd-A and Abd-B is associated with the color change. This locus is therefore in the same intergenic region but not the same exact locus as found to drive red-black mid-abdominal variation in a distantly related bumble bee species, B. melanopygus. Gene expression analysis and RNA interferences suggest that differential expression of an intergenic long non-coding RNA between abd-A and Abd-B at the onset setal color differentiation may drive the orange black color variation by causing a homeotic shift late in development. Analysis of this same color locus in comimetic species reveals no sequence association with the same color shift, suggesting that mimetic convergence is achieved through distinct genetic routes. Our study establishes Hox regions as genomic hotspots for color pattern evolution in bumble bees, and demonstrates how pleiotropic developmental loci can drive adaptive radiations in nature.

Development shapes the evolutionary diversification of rodent stripe patterns.

Vertebrate groups have evolved strikingly diverse color patterns. However, it remains unknown to what extent the diversification of such patterns has been shaped by the proximate, developmental mechanisms that regulate their formation. While these developmental mechanisms have long been inaccessible empirically, here we take advantage of recent insights into rodent pattern formation to investigate the role of development in shaping pattern diversification across rodents. Based on a broad survey of museum specimens, we first establish that various rodents have independently evolved diverse patterns consisting of longitudinal stripes, varying across species in number, color, and relative positioning. We then interrogate this diversity using a simple model that incorporates recent molecular and developmental insights into stripe formation in African striped mice. Our results suggest that, on the one hand, development has facilitated pattern diversification: The diversity of patterns seen across species can be generated by a single developmental process, and small changes in this process suffice to recapitulate observed evolutionary changes in pattern organization. On the other hand, development has constrained diversification: Constraints on stripe positioning limit the scope of evolvable patterns, and although pattern organization appears at first glance phylogenetically unconstrained, development turns out to impose a cryptic constraint. Altogether, this work reveals that pattern diversification in rodents can in part be explained by the underlying development and illustrates how pattern formation models can be leveraged to interpret pattern evolution.

Psychedelics sea slugs: Observations on colour ontogeny in two nudibranch species from the genus Nembrotha (Doridina: Polyceridae)

In recent decades, thanks to the use of integrated taxonomy, the traditional recognition of a nudibranch species based on observation and colour pattern variation has become increasingly questioned, mainly due to the presence of cryptic and pseudocryptic species complexes. Individuals with the same colour pattern can be genetically identical, but individuals with different colour patterns may also be genetically identical and this variation may instead represent different life stages. But things can get even more complicated. What happens when the same species changes its colour pattern radically as it ages? Here we present two extraordinary examples in species of the genus Nembrotha based on laboratory observation. Specimens of Nembrotha livingstonei Allan, 1933 and Nembrotha yonowae Goethel and Debelius, 1992 were collected in Mozambique and kept in captivity as long as feeding was possible. The results showed that colour patterns in both species changed over time and that this change was linked to diet. Furthermore, species delimitation analysis and comparison of the uncorrected COI pairwise distances of examined specimens from Mozambique and others downloaded from GenBank confirmed that N. yonowae Goether and Debelius, 1992 is a junior synonym of N. cristata Bergh, 1877. Similar studies with laboratory observations are needed on other species of the genus, as they were described on the basis of different colouration, but integrated taxonomy may show different results.

Taxonomic inflation and a reconsideration of speciation in the Andes: the case of the high-elevation tree frog Dendropsophus molitor (Anura: Hylidae)

Abstract Dendropsophus molitor is a hylid frog endemic to the Eastern Cordillera of the Colombian Andes, where it exhibits extensive geographic variation in size and colour pattern. Previous multivariate analyses of acoustic and genetic data suggested that northern and southern populations of D. molitor were distinct lineages, and consequently, the northern populations were described as Dendropsophus luddeckei. In this study, we conducted morphometric and genetic analyses of populations of D. molitor and D. luddeckei to test the validity of this recent taxonomic split. We sequenced the mitochondrial genes 12S, 16S, and COI, and the nuclear marker POMC, and also tested whether variation in the MC1R gene was associated with colour polymorphism in these frogs. Phylogenetic analyses recovered D. molitor and D. luddeckei as polyphyletic and species delimitation tests failed to recover them as separate lineages. Genetic differentiation between populations was mostly explained by high intra- and interpopulation variation in the absence of a north-south split, and we found no differences in morphometry between northern and southern populations. In addition, the coding region of MC1R is not associated with colour polymorphism. Thus, multiple lines of evidence suggest that D. luddeckei is not a valid species and D. molitor should be considered a single species. Our study highlights the danger of taxonomic inflation in the face of limited geographic sampling and a lack of clear diagnostic characters.