Cancerous tumours contain a rare subset of cells with stem‐like properties that are termed cancer stem cells (CSCs). CSCs are defined by their ability to divide both symmetrically and asymmetrically, to initiate new tumour growth and to tolerate the foreign niches required for metastatic dissemination. Accumulating evidence suggests that tumours arise from cells with stem‐like properties, the generation of CSCs is therefore likely to be an initiatory event in carcinogenesis. Furthermore, CSCs in established tumours exist in a dynamic and plastic state, with nonstem tumour cells thought to be capable of de‐differentiation to CSCs. The regulation of the CSC state both during tumour initiation and within established tumours is a desirable therapeutic target and is mediated by epigenetic factors. In this review, we will explore the epigenetic parallels between induced pluripotency and the generation of CSCs, and discuss how the epigenetic regulation of CSCs opens up novel opportunities for therapeutic intervention. AMLacute myeloid leukaemiaATPadenosine triphosphateBAFBRG1‐ or BRM‐associated factorsBERbase‐excision repairBETbromodomain and extra terminalBRDbromodomainCIMPCpG island methylator phenotypeCSCcancer stem cellDNAdeoxyribose nucleic acidDNMTDNA methyltransferaseEMTepithelial‐mesenchymal transitionESCembryonic stem cellFACsfluorescence‐activated cell sortingGBMglioblastoma multiformeHDAChistone deacetylaseIL6/17interleukin 6/17iPSCinduced pluripotent stem celllncRNAlong noncoding RNALSD1lysine‐specific demethylase 1METmesenchymal‐epithelial transitionmiRNA/miRmicroRNAncRNAnoncoding RNAPDACpancreatic ductal adenocarcinomaPRCpolycomb repressive complexRISCRNA‐induced silencing complexRNAribose nucleic acidSAMS‐adenosyl methionineSWI/SNFSWItch/sucrose nonfermentableTAMtumour‐associated macrophageTETten‐eleven transcription factorTGFtransforming growth factorTMEtumour microenvironmentVEGFvascular endothelial growth factor1As with adult tissues, cancerous tumours also contain a rare subset of cells with stem‐like properties that can function to regenerate the heterogeneous cell populations observed therein. These cancer stem cells (CSCs) are defined by their ability to divide both symmetrically and asymmetrically, to initiate new tumour growth and to tolerate the foreign niches required for metastatic dissemination. As the tumour‐initiating population, CSCs underpin the very nature of malignancy and studying their regulation is essential for understanding tumour formation, metastasis and relapse after therapy.As it is not possible to isolate CSCs based on functional properties, CSC identification can be achieved by FACs sorting based on surrogate cell surface marker profiles and subsequent transplantation into immune‐compromised mice to demonstrate enhanced tumourigenic potential. Using this strategy, CSCs have been identified in most cancers, first in acute myeloid leukaemia (AML) followed by breast cancer and other solid malignancies such as brain, colon and pancreatic cancer, and are purported to account for only a few per cent of the total cell population.Epigenetics refers to a number of mechanisms that control the reversible regulation of gene expression by changing the chromosome without altering the DNA sequence: DNA can be altered epigenetically by methylation and demethylation of CpG nucleotides. Epigenetic changes in the overall structure of chromatin occur through at least three interrelated mechanisms: posttranslational modifications of histones, ATP‐dependent chromatin remodelling and the incorporation (or replacement) of specialised histone variants into chromatin. Finally, noncoding RNA can interact with transcriptional processes to alter gene expression. In addition to 2D processes, epigenetic regulation can also involve higher‐order chromatin organisation including promoter‐enhancer interactions, regulatory DNA loops and 3D chromatin localisation in the cell nucleus Figure .As these epigenetic mechanisms are important mediators of cellular identity, we will explore how the restructuring of such epigenetic barriers reinforces the stem‐like state in both normal cells and cancer, and their relevance to tumour initiation Figure . Further2DNA methylation is a form of gene silencing that occurs mainly to CpG dinucleotides which cluster in CpG islands; areas of high CpG density usually found at promoters. To methylate cytosines, DNA methyltransferases (DNMTs) catalyse the transfer of a methyl group from cofactor S‐adenosylmethionine to the carbon of the cytosine ring to generate 5‐methylcytosine (5mC). This functions to inhibit gene expression either by recruitment of methyl‐CpG‐binding domain proteins which in turn recruit histone‐modifying and chromatin‐remodelling complexes, or by preventing the recruitment of DNA‐binding proteins, that is, transcription factors. To reactivate expression after silencing, 5‐mCs can be oxidised to 5‐hydroxymethylcytosine (5hmC) and back to the unmodified state by TET proteins and base excision repair (BER), to ultimately restore the unmethylated cytosine. TET protein‐mediated demethylation can occur via either passive or active processes. Passive demethylation results from the failure to maintain 5mC marks across cell divisions (5hmC is not a substrate for DNMTs). Active demethylation is enzymatic, whereby TET proteins further oxidise 5hmC marks to 5‐formylcytosine (5fC) and 5‐carboxylcytosine (5caC) then back to the unmodified state. 5fC bases are recognised by thymine‐DNA‐glycosylases (TDG) that excise mismatched pyrimidines that would then be replaced by an unmodified cytosine by the BER pathway. Intermediate marks (5fC or 5caC) are much less abundant than 5hmC but may also have independent functionsMethylation patterns are altered in cancer; localised hypermethylation occurs in CpG islands of the promoters of tumour suppressor genes , silencing their expression.In general, CpG methylation is required for differentiation whereas demethylation is essential for induction of the pluripotent state.Some hypomethylation also continues to be associated with CSCs once the tumour has been established. For example, the CpG region of the CD133 gene promoter, a key gene involved in CSCs, was shown to be hypomethylated in several cancer types including breast, ovarian, colorectal and glioblastoma.However, opposing roles for DMNT1 have been demonstrated as it can both promote or inhibit CSC formation. For example, DNMT1 was required for the initiation of colon cancer.The importance of both hypomethylation and DNMT1 in CSCs suggests that specific localization of 5mC and 5hmC marks is likely to be a more important determinant of cellular identity than the overall global patterning. In particular, hypomethylation at pluripotency loci and hypermethylation at tumour suppressor genes or those involved in differentiation is important in the generation of CSCs.3The amino acid residues located on the N‐ and C‐terminal tails of histones can be modified to influence gene expression, including by acetylation, methylation and ubiquitylation. When occurring on promoters or enhancer regions, these modifications confer chromatin states that affect gene expression by altering the ability of protein complexes to bind. Chromatin formation that permits protein binding and gene expression is known as euchromatin, and repressive is known as heterochromatin. These states are mediated by the Trithorax group (TrxG) proteins and Polycomb group (PcG) proteins, respectively. Modifications are named by the histone type  followed by the amino acid and modification, for example, K4 me3 (trimethylation on lysine 4). The most studied modifications are those that occur to histone 3 found primarily at active enhancers , promoters (H3K4me3) and within the bodies of actively transcribed genes (H3K36me3).Alterations of the histone modification landscape are widely associated with SCs with approximately one‐third of the genome differing in chromatin structure between differentiated cells and ESCs.Although there are many parallels between PSCs and CSCs, histone‐modifying enzymes can have tumour‐specific effects. The subunit of the Polycomb group complex 2 (PRC2) that catalyses H3K27me3 is the histone methyltransferase EZH2 (enhancer of zeste homologue 2). EZH2 inhibition has been shown to be sufficient for tumourigenesis and is associated with the generation of CSCs in some cancers.As well as methylation, other modifications have shown importance in CSCs. The BMI1 subunit of the polycomb repressive complex 1 (PRC1), which monoubiquitinates histone H2A on lysine 119 (H2AK119Ub1) was able to promote CSCs in leukaemia and GBM.Nearly all the genes involved in ESC identity, including KLF4, Sox2, Oct4 and Nanog, are regulated by super‐enhancers; large genomic regions with very high levels of transcription factors that are highly important in controlling pluripotency and differentiation.Chromatin at promoters and enhancers can be classed as active, repressed or poised. Poised regions are bivalent in terms of histone modifications as they contain both H3K27me (repressive) and H3K4me (activating) histone marks. In ESCs, the majority of bivalency occurs in the promoters of transcription factors and half of bivalent domains have binding sites for pluripotency transcription factors.4The basic unit of chromatin is the nucleosome, which is formed of 147 bp of DNA wrapped around eight histones, two each of histones H2A, H2B, H3 and H4. These appear like beads on a string of chromatin. Gene expression is regulated at the level of chromatin structure in an ATP‐dependant process by chromatin modifiers which act to remove or slide assembled nucleosomes along with the DNA, and can also exchange histone H2A‐H2B dimers with dimers of histone variants. The presence of nucleosomes naturally represses gene expression by preventing the access of transcription factors. The absence or loss of nucleosomes at a transcription start site (nucleosome‐free region [NFR]) allows for assembly of the transcription machinery and rapid activation of gene expression.Sox2 and Oct4, and promote differentiation to mesoderm, but not ectoderm.The chromatin architecture of SCs differs greatly from that of differentiated cells. While small‐scale chromatin changes at DNA regulatory elements occur during transcriptional regulation in most cells, extensive remodelling of chromatin structure is required for cellular differentiation during embryonic development.SNF5 have been identified in epithelioid sarcoma and renal medullary carcinoma.As nucleosome repositioning is required for the generation of the pluripotent state it may be important for the formation of CSCs. This may occur via aberrant functions of ATP‐dependent chromatin remodelers, which has also been linked to cancer, especially in terms of the SWI/SNF complex, which is frequently found mutated.ATP‐dependent chromatin remodelling complexes can also regulate transcription via incorporation of histone variants into nucleosomes.High mobility and exchange of histone variants is a key feature of SCs and this dynamism is thought to contribute to SC plasticity.These findings suggest that the overexpression of labile histone variants such H2A.Z (and in some cases H3.3), coupled with the reduction of stable histone variants such as macroH2A could contribute to cancer cell plasticity and the generation of CSCs.4.1Noncoding RNAs (ncRNA) are transcribed from regions of the genome that do not encode for proteins. The resulting RNA transcripts function to regulate the expression of protein‐coding genes and are therefore essential for control of cellular function and identity. ncRNAs can be divided into two major groups based on their size: small ncRNAs being 200 nucleotides or less, and long ncRNAs (lncRNAs) 200 nucleotides or more. Small ncRNAs can be further subcategorized based on length, function and subcellular localization and include microRNAs (miRNAs) and short interfering RNAs (siRNAs) amongst others.4.2LncRNAs can physically associate with DNA or proteins to either promote or repress gene expression. To promote transcription, lncRNAs can function as guides or scaffolds for the assembly of protein complexes at specific loci. To repress transcription lncRNAs can function as decoys, binding and preventing functions of RNA or protein targets4.3MiRNAs function by recognising small sequences (6‐8 nucleotides) at the 3′‐untranslated region of multiple target mRNAs.The importance of miRNAs in embryonic stem cells (ESCs) has been established by deletion of Dicer1 and DGCR8  in mouse models. Dicer1 deletion is embryonic lethal whereas DGCR8‐deficient mice have impaired differentiation due to failure to silence the stem markers, Oct4, Sox2 and Nanog.Let7 is also known as a negative regulator of CSCs especially in breast cancer where, as expected, let‐7 was linked with differentiation. Inhibition of let‐7 promoted CSC properties, mediated by increased RAS and HMGA2 expression.As well as directly affecting the transcription of pluripotency‐associated genes, miRNAs can also affect CSC biology via cross‐talk with other CSC pathways and epigenetic mechanisms. For example, the maturation of miR‐21 via a DROSHA and RNA helicase p68 complex is induced by TGF‐β signalling.5Many studies have associated EMT with the generation of CSCs.CSC generation by EMT may also be influenced by the localised tissue environment via epigenetic modification. Cells present in the tumour CSC niche such as stromal and immune cells produce cytokines and growth factors which promote CSC characteristics. For example, tumour‐associated macrophages (TAMs) secrete VEGF and IL6, and T cells secrete IL17 and TGF‐β, all of which are able to promote CSCs via EMT.6Many epigenetic aberrations identified as being involved in the generation of CSCs are likely early markers of cancer and therefore potentially useful as early detection methods. Early cancer detection could be achieved by analysis of patient blood samples, which is considered a relatively noninvasive procedure compared to a biopsy. Patient blood can contain cancer cells and cell‐free DNA, which could be identified by characteristic genetic and epigenetic signatures as biomarkers of the cancerous state. Should such a process be found to be accurate, it may facilitate the detection of tumour signatures even when the tumour is not large enough to be picked up on a scan. This technique was originally designed for the identification of large genomic alterations, however, advances in the understanding of epigenetic aberrations in cancer have led to the pursuit of an epigenetic signature that may define the cancerous state. Although these may differ between cancers, epigenetic signatures associated with the generation of CSCs are likely candidates across cancer types due to the requirement for CSC generation in tumour initiation Figure .BCAT1, CDO1, TRIM58 and ZNF177, has been shown to predict lung cancer.Most approaches have focussed on the methylation status of particular gene promoters but it is likely that the methylation status of several promoters would be required to serve as an accurate and reliable signature. For example, hypermethylation of the combination of four genes: An increase in histones and their associated covalent modifications have also been identified in the blood of cancer patients.A useful form of epigenetic cancer detection is ncRNA. Noncoding RNAs are readily secreted by the tumour into the microenvironment and subsequently reach the bloodstream and even distant tissue sites. LncRNAs and miRNAs have been detected in blood, urine and saliva, all of which facilitate diagnostic testing and many have been identified as potential biomarkers.Epigenetic targeting is an exciting area of translational cancer research offering many novel opportunities for therapeutic intervention. Many studies have explored the use of existing and novel compounds to target epigenetic marks to impair cancer cell growth and elicit an improved clinical response Figure .HDAC inhibitors are already widely considered to have anticancer effects and some are in clinical use. For example, SAHA (vorinostat) is approved for T‐cell lymphoma and many others are in clinical trials for various malignancies either alone or in combination with other therapeutics.Targeting of histone modifications may also be achieved through LSD1 inhibitors. Lysine‐specific demethylase 1A (LSD1) removes methyl groups from the histone 3 proteins (H3K4me2/1 and H3K9me2/1) resulting in transcriptional repression or activation, respectively.Histone modification could also be targeted by inhibition of PRC components BMI‐1 or EZH2. BMI‐1 is a key regulatory component of (PRC1) and its function is strongly associated with the maintenance of CSCs.Although an increasing number of ncRNAs are known to be involved in CSCs and cancer in general, and are therefore attractive drug targets, it is considered difficult to specifically target aberrant mRNAs or RNA‐protein interactions with small molecules. Nevertheless, due to the high promise of such targets, a number of studies have attempted therapeutic modulation. The miRNA lin28 which is very important in CSC generation has been found to be targeted by the compound “N‐methyl‐N‐[3‐phenyl]acetamide”, which acts by blocking the interaction between lin‐28 and let‐7. This compound impaired Lin‐28, rescued let‐7 function and resulted in differentiation in ESCs, and reduced tumorsphere formation in cancer cell lines. It is likely that the reduction in tumoursphere formation was caused by differentiation of CSCs.Another way of targeting DNA and RNA moieties is by using peptide nucleic acids (PNA). These are synthetic analogues of DNA that are highly stable and have a hybridization affinity toward natural DNA and RNA, and are therefore a potential approach for the modification of gene expression and epigenetic states.Despite promising results, therapeutic targeting of epigenetic marks should proceed with caution. A therapeutic strategy designed to target epigenetic readers and writers is by definition not a specific approach. Such an intervention would elicit gene expression changes on a global scale and is likely to incur off‐target and adverse effects. Therefore much further investigation is required to identify a clinically viable approach. Epigenetic targeting that focuses on a specific gene or pathway changes  may be more easily targeted to CSC inhibition and therefore have much greater potential for efficacy.7To obtain a greater understanding of the epigenetic mechanisms which drive CSC formation from normal cells.To determine whether the mechanisms of CSC generation differ when deriving from normal or cancerous cells and their associated stem and nonstem subpopulations. Epigenetic mechanisms involved in the emergence of CSCs will likely differ depending on from which population they are derived, that is, from a normal cell, normal stem cell, pre‐CSC or bulk tumour cell, as each of these identities has distinct epigenetic profiles.To ascertain the relative contribution of genetic and epigenetic effects to tumour initiation; can epigenetic events alone initiate the transformed state?To determine which are the most important environmental signals involved in CSC generation and how these can be modified or targeted for prevention and treatment.The epigenetic processes involved in CSC generation have substantial implications for tumour initiation, metastasis and relapse. Many studies have focussed on the elucidation of mechanistic links between epigenetic processes and CSCs, and have identified attractive targets for cancer prevention, diagnosis and therapy. However, as always, these studies have highlighted many areas in which further investigation is required. In particular:The authors note no conflict of interest.


The enamel microstructure of fossil and extant Geomyoidea  lower incisors incorporates three‐ or two‐layered schmelzmusters with uniserial, transverse Hunter‐Schreger bands having parallel and perpendicular or exclusively perpendicular oriented interprismatic matrix. Phylogenetically, these schmelzmusters are regarded as moderately (enamel type 2) to highly derived (enamel type 3). Our analysis detected a zone of modified radial enamel close to the enamel–dentine junction. Modified radial enamel shows a strong phylogenetic signal within the clade Geomorpha as it is restricted to fossil and extant Geomyoidea and absent in Heliscomyidae, Florentiamyidae, and Eomyidae. This character dates back to at least the early Oligocene , where it occurs in entoptychine gophers. We contend that this specialized incisor enamel architecture developed as a biomechanical adaptation to regular burrowing activities including chisel‐tooth digging and a fiber‐rich diet and was probably present in the common ancestor of the clade. We regard the occurrence of modified radial enamel in lower incisors of scratch‐digging Geomyidae and Heteromyidae as the retention of a plesiomorphic character that is selectively neutral. The shared occurrence of modified radial enamel is a strong, genetically anchored argument for the close phylogenetic relationship of Geomyidae and Heteromyidae on the dental microstructure level. Incisors of fossil and extant Geomyidae (pocket gophers) and Heteromyidae  show enamel with built‐in failure prevention related to chisel‐tooth digging. This distinctive microstructure character strengthens the joint phylogenetic origin of these North American rodents because it is absent in other (extinct) clades within Geomorpha .  GeomyoiGregorymys veloxikua created tooth‐excavated foraging burrows in southern Mexico  with some taxa possibly semi‐fossorial (not subterranean), others more fossorial or, in contrast, adapted to a terrestrial lifestyle  are small‐ to medium‐bodied rodents with anatomies highly adapted to a subterranean‐burrowing  lifestyle in open habitats. They possess a wedge‐shaped, massive skull with a broad, forward‐sloping occipital surface, heavy muscle attachments, and protruding lower incisors that are used as chisels for tunneling by representatives of Thomomyini  and Eodipodomys  and for inflated auditory bullae in the early Miocene  perognathine Bursagnathus  are small‐bodied rodents  of North America  describe the schmelzmuster and lower incisor morphology in a representative sample of fossil and extant Geomyoidea , (b) document the occurrence of modified radial enamel, (c) discuss its assumed biomechanical and higher level phylogenetic implications, and (d) compare these enamel microstructure results with examples in basal geomorphs (Heliscomyidae).Our motivation for this study is a serendipitous discovery of a conspicuous microstructure  in the lower incisors of 2Enamel microstructure analysis is a powerful tool for answering systematic–phylogenetic questions at the genus and higher taxonomic levels. Enamel formation is controlled by genetic and epigenetic factors; as a consequence, few samples of each taxonomic subgroup are sufficient to characterize its main schmelzmuster features.Here, we describe the schmelzmuster of lower incisors of ten species of Geomyidae, ten species of Heteromyidae, and one or two species of Heliscomyidae. The sample from geomyoids covers taxa from the late Oligocene/Early Miocene (Arikareean) to Recent, the two heliscomyid samples come from the late Eocene (Chadronian) and early Oligocene (Orellan) Appendix .In geomyines, the enamel microstructure is similar in upper and lower incisors. However, as in many rodent clades, upper and lower incisors in Dipodomyinae and Perognathinae have different schmelzmuster with the lower incisor being more apomorphic , USA. ZSHD, Zoologische Sammlung Heidelberg, Germany.Institutional abbreviations. EDJ, enamel–dentine junction; HSB, Hunter‐Schreger band(s); IPI, inner portio interna; IPM, interprismatic matrix; MRE, modified radial enamel; OPI, outer portio interna; OES, outer enamel surface; PE, portio externa; PI, portio interna; PLEX, prismless layer.Anatomical Abbreviations. 3All taxa feature uniserial Hunter‐Schreger bands (HSB). Table 3.13.1.1This taxon has a three‐layered schmelzmuster (schmelzmuster type 2) with continuous transversely oriented HSB showing a steep inclination. In the IPI, HSB decussate at high acute angles and the IPM is perpendicular to the prism long axes. In the thin OPI, the IPM turns into a very low angled to parallel direction in respect to the prisms. At the EDJ is a 2–3 prism thick zone, in which the HSB decussate at a low angle forced by the plate‐like IPM in between them. The PE is made up of radial enamel. The incisor cross section is triangular‐shaped with labially flat enamel.Entoptychus sp. has a three‐layered schmelzmuster with transversely oriented HSB showing moderate to steep inclination; HSB are diagonally oriented at the mesial and lateral ends of the enamel (schmelzmuster type 2a). The enamel thickens somewhat at the bend to mesial and lateral. The PI is made up of a thick IPI, in which HSB decussate at a generally high angle except for a 1–2 prism thin zone near the EDJ where the decussation angle is low because of thick plate‐like IPM. In the equally thin OPI (1–2 prisms thick), IPM runs parallel to the prism long axes. The PE has radial enamel. The incisor cross section is subtriangular in shape with a rounded dentine body and labially flat enamel.Pleurolicus sp. shows a three‐layered schmelzmuster (schmelzmuster type 2) with transversal HSB having a moderate to steep inclination. At the bend to mesial and lateral, the enamel thickens somewhat. In the thick IPI, the IPM is perpendicular to the HSB, the latter decussating at a high angle; the OPI with prism‐parallel IPM is 4 to 5 prisms thick. At the EDJ, a zone measuring about 3 prisms shows thick plate‐like IPM forcing the HSB to decussate at a very low angle. The thin OPI shows prism‐parallel IPM over a thickness of 2 to 3 prisms. The PE has radial enamel. The entire incisor cross section could not be evaluated; the labial part of the enamel is less flat than in Gregorymys and Entoptychus.Geomys sp. shows a three‐layered schmelzmuster (schmelzmuster type 2a) with mostly transversely but toward mesial also slightly diagonally oriented HSB and steep inclination. The decussation angle of the HSB is high to perpendicular with the exception of an about two prims thick zone at the EDJ where they have a very low decussation angle because of thick, plate‐like IPM. The plate‐like character of the IPM gets pronounced at the turn of the enamel toward lateral. The IPM is at right angles in the IPI and prism‐parallel in the thin, 1–2 prism thick OPI. The PE shows radial enamel. The incisor cross section is triangular‐shaped with labially flat enamel.Geomys quinni shows a three‐layered schmelzmuster (schmelzmuster type 2) with transversely oriented HSB throughout the entire enamel; the HSB are steeply inclined. The IPI shows HSB with perpendicular IPM, and the OPI measures only 3–4 prisms and has prism‐parallel IPM. There is a conspicuous, 3–4 prism thick zone at the EDJ consisting of thick, plate‐like IPM allowing HSB to decussate only at a very low angle. The HSB decussation angle gets larger toward the PI/PE junction but seems not to be fully perpendicular. The PE consists of radial enamel. The incisor cross section is triangular‐shaped with labially flat enamel.cf. The schmelzmuster is three‐layered (schmelzmuster type 2a) with transversely oriented HSB, which transition to a slightly diagonal orientation to the lateral and mesial parts of the enamel. In the IPI, the IPM is perpendicular to the prism long axes. This angle becomes low acute to parallel in the 2–4 prism thick OPI, which is best discernable in the longitudinal section. Also, HSB layers decussate at varying angles: angles closer to the EDJ are about 45 degrees and rise closer to 90 degrees, never being fully perpendicular. There is a conspicuous zone at the EDJ, where the IPM is thick and plate‐like forcing the first four HSB layers to decussate at a very low angle. Toward the lateral parts of the enamel, this zone measures even 6 prism layers. The PE is made up of radial enamel. The enamel surface is wrinkled resulting in a wavy appearance in transverse sections. The incisor cross section is triangular‐shaped with labially flat enamel in KOE 2379; incisor cross section is subtriangular in shape with somewhat more rounded enamel in KOE 3275.The schmelzmuster is three‐layered (schmelzmuster type 2), and HSB are oriented transversely throughout the entire enamel; the HSB are steeply inclined. The PI is twofold having an IPI, in which IPM is perpendicular to the prisms and a 1–3 prism thick OPI, in which IPM is at low angle or parallel to the prisms. Right at the EDJ is a four prism thick zone with thick, plate‐like IPM, forcing the decussation angle of the HSB to about zero. Toward the junction PI/PE, the HSB decussation angle rises reaching 90 degrees. The PE consists of radial enamel. The OES is wrinkled causing a wavy OES as seen in transverse sections. The incisor cross section is subtriangular in shape with a rounded dentine body and labially flat enamel.Three‐layered schmelzmuster (schmelzmuster type 2) features steeply inclined, transversely oriented HSB decussating at right angles. The thick IPI shows IPM that is mostly perpendicular to the prisms; at the EDJ, however, is a two prism thick zone with plate‐like IPM inducing a quite reduced angle of HSB decussation. The OPI with prism‐parallel IPM measures only 2 to 3 prisms and is best observable in the longitudinal section. The PE is made up of radial enamel. The incisor cross section is triangular‐shaped with labially flat enamel.Thomomys feature a three‐layered schmelzmuster (schmelzmuster type 2a) with transversely to slightly diagonally oriented HSB with steep inclination. The PI is twofold with a thick IPI with perpendicular IPM and an only 4 prism thick OPI with prism‐parallel IPM. There is a conspicuous zone at the EDJ where the IPM is thickened and plate‐like among the HSB, forcing the latter to decussate at low angle. This zone is especially well expressed in T. bottae where it gets even more obvious toward the more lateral and mesial parts of the enamel band in transverse sections. The PE consists of radial enamel that passes into primitive radial enamel in the central part of the enamel band close to the OES. The incisor cross section is triangular‐shaped with labially flat enamel in both Thomomys species.Both species of the genus Schizodontomys sulcidens shows a three‐layered schmelzmuster (schmelzmuster type 2). The PI has transversely oriented, steeply inclined HSB with IPM at right angle to the long axes of the prisms in the IPI; this angle is low or IPM is parallel over 2 to 3 prism rows in the OPI. The PE is made up of radial enamel. The incisor cross section is oval‐shaped with rounded enamel.Cupidinimus species is three‐layered (schmelzmuster type 2) and has transversely oriented, steeply inclined HSB in the entire enamel. The twofold PI consists of an IPI with perpendicular oriented IPM and a 1–3 prism thick IPI with prism‐parallel IPM. At the EDJ is an about three prism zone with thick, plate‐like IPM, allowing the HSB still to decussate, but only at a low angle. The HSB decussation angle is high in the remaining part of the PI but does not reach 90 degrees. The PE is made up of radial enamel. The incisor cross section is oval‐shaped in both species of Cupidinimus, the middle labial part of the enamel is flattened in KOE 3496; this feature cannot be evaluated in KOE 3259.The schmelzmuster in both D. ordii, Oregon) or are parallel . The PE consists of radial enamel that in its outermost portion merges into primitive radial enamel. The incisor cross section is oval‐shaped and the middle labial part of the enamel is flattened.The schmelzmuster in both specimens is two‐layered (schmelzmuster type 3) and shows transversely oriented, moderately to steeply inclined HSB throughout the entire enamel. The HSB decussation angle in the PI is high. The IPM is perpendicular to the prism long axes in most of the PI but angle decreases markedly in a 2 to 3 prism thick zone before the junction PI/PE. At the EDJ, the IPM is thickened and plate‐like in a zone of 2 to 3 prism thickness; the HSB still decussate but at low angle . The thick PI shows IPM at right angles but the angle is markedly reduced over 2 to 3 prisms at the junction PI/PE. At the EDJ, the IPM is thickened and plate‐like in a zone measuring 3 to 4 prisms. The PE is made up of radial enamel. The incisor cross section is pear‐shaped with rounded enamel.Perognathus. The HSB are transversely oriented in the entire enamel; the inclination is steep in the Early Pliocene taxa and moderate in the Pleistocene/Holocene taxa. The IPM is perpendicular to the prism long axes in the PI, yet the angle decreases somewhat near the junction PI/PE. In a zone at the EDJ, the IPM is thickened and plate‐like and does not anastomose between the subparallel HSB; this zone is best expressed at the bending of the enamel laterally (P. rexroadensis) or medially , respectively. The PE consists of radial enamel. The incisor cross section is oval‐shaped in all four species of Perognathus; the middle labial part of the enamel is somewhat flattened in P. bibalii and P. merriami.The schmelzmuster is two‐layered (schmelzmuster type 3) in all four species of The schmelzmuster is two‐layered (schmelzmuster type 3). The HSB are moderately inclined and transversely oriented in the enamel. The PI is thick and the IPM is perpendicular to the prism long axes; this angle decreases close to the junction PI/PE. At the EDJ is a conspicuous, 2–3 prism thick zone with thick, plate‐like IPM, in which HSB decussate at low angle. The PE is made up of radial enamel and a thin PLEX at the OES. The incisor cross section is oval‐shaped and the middle labial part of the enamel is flattened.3.1.2Heliscomys sp. and Heliscomys vetus have a three‐layered schmelzmuster (schmelzmuster type 2a), in which HSB are oriented transversely to diagonally with a high inclination. The IPM in the IPI is perpendicular to the prisms, this angle decreases markedly in the thin OPI, which measures only 1–2 prisms. The PE consists of radial enamel. The incisor cross section is slender and oval‐shaped and the middle labial part of the enamel is flattened.4Pleurolicus, Entoptychus, Gregorymys) and Geomyinae  have a three‐layered schmelzmuster with transversely and, in part, diagonally oriented HSB. The PI is twofold with IPM at different, mostly high angles to the prism long axes in a thick IPI and prism‐parallel IPM in a thin OPI; the PE consists of radial enamel. Enamel is moderate to greatly thick in both Entoptychinae and Geomyinae. From an evolutionary perspective, this schmelzmuster is moderately derived and represents types 2 and 2a of Kalthoff , and the extant Dipodomyinae (Dipodomys) and Heteromyinae (Heteromys) have schmelzmuster type 3 with transversely oriented HSB and angled IPM in an only onefold PI and radial enamel in the PE. The Miocene (Barstovian) dipodomyine Cupidinimus and the Miocene (Arikareean) mioheteromyine Schizodontomys feature a more plesiomorphic, three‐layered schmelzmuster of type 2, similar to that described for geomyids. The thickness of the enamel varies somewhat from very thick in Schizodontomys and Heteromys to moderate/thick in perognathines, and from moderate/thick in Miocene dipodomyines to thick in extant dipodomyines. The incisor cross section is oval with a flattened middle labial part of the enamel with the exception of Schizodontomys, Heteromys, Perognathusrexroadensis, and P. mclaughlini, which all have rounded enamel  define the modified radial enamel of Pfretzschner , 1994. IWe detected modified radial enamel close to the EDJ exclusively within Geomyoidea but not in Geomorpha outgroups, such as Heliscomyidae; nor was modified radial enamel reported to occur in the lower incisors of Eomyidae  or demonstrate ricochetal/saltatorial locomotion . All species excavate burrows, which can vary in architecture from simple tubes (Microdipodops) to extended systems (Heteromys)  or even arboreal (Proheteromys latidens) lifestyle , and the arvicoline Ellobius murinus (Northern Mole Vole) , a terrestrial species with no burrowing activities , which is a scansorial species dependent on forest environments , which has terrestrial adaptations but uses self‐constructed burrows , showing terrestrial and scansorial adaptations but also short legs and strong toes, well‐adapted to digging , Rhizomyinae , Spalacinae (blind mole‐rats), the murine Goodman, ; (c) Bas Perrin, . At presIn the light of the above reported evidence of modified radial enamel in some taxa unrelated to Geomyoidea and among each other, a reviewer suggested that this enamel trait might as well be homoplastic in Geomyidae and Heteromyidae. However, we disagree because modified radial enamel is a character consistently present in both families. This is a clear difference to the above examples, where this character occurs isolated.Octodon “bridgesi” and in Dactylomys boliviensis but we could not evaluate whether the IPM is thick and plate‐like  have transverse to slightly diagonal HSB with a twofold PI with perpendicular IPM in the IPI and prism‐parallel IPM in the OPI; the PE consists of radial enamel (schmelzmuster type 2). The incisors are slender and cross sections are oval‐shaped with a rather flat middle labial part of the enamel  is characterized by the moderately derived schmelzmuster type 2  and the more derived schmelzmuster type 3 .The lower incisor microstructure of Heliscomyidae is characterized by the moderately derived schmelzmuster type 2.In all fossil and extant Geomyoidea, we detected a zone of modified radial enamel close to the enamel–dentine junction (EDJ).Gregorymys,earliest Arikareean, Ar1).The earliest fossil record of this character dates back to at least the early Oligocene , underground feeding, and feeding on abrasive, fiber‐rich plants and plant parts.Within Geomorpha, modified radial enamel is restricted phylogenetically to Geomyoidea, for which it is interpreted as ancestral character.Modified radial enamel does not occur in Heliscomyidae, nor was it reported from Florentiamyidae Wahlert,  or EomyiThe shared occurrence of modified radial enamel is yet another argument for the close phylogenetic relationship of Geomyidae and Heteromyidae, here on the dental microstructure level.We described the schmelzmuster and lower incisor morphology in a representative sample of fossil and extant Geomyoidea  and compared the results with those in basal geomorphs (Heliscomyidae) and with those in other, nonrelated rodents. We came to the following conclusions:The authors have no conflict of interest to declare.Daniela C. Kalthoff: Conceptualization ; Data curation (lead); Formal analysis ; Funding acquisition (lead); Investigation ; Methodology ; Project administration ; Validation ; Visualization (lead); Writing‐original draft ; Writing‐review & editing . Thomas Mörs: Conceptualization ; Data curation (supporting); Formal analysis ; Funding acquisition (supporting); Investigation ; Methodology ; Project administration ; Validation ; Visualization (supporting); Writing‐original draft ; Writing‐review & editing .This article does not contain any studies with human participants or animals performed by any of the authors.We declare that some minor adjustments to brightness or contrast have been made to the scanning electron micrographs. Adjustments apply equally across the entire images.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


Patients with multimorbidity often experience treatment burden as a result of fragmented, specialist‐driven healthcare. The ‘family doctor team' is an emerging service model in China to address the increasing need for high‐quality routine primary care.This study aimed to explore the extent to which treatment burden was associated with healthcare needs and patients' experiences.N = 2160) who had ≥2 clinically diagnosed long‐term conditions (multimorbidity) and had ≥1 clinical encounter in the past 12 months since enrolment registration with the family doctor team. Patients' experiences and treatment burden were measured using a previously validated Chinese version of the Primary Care Assessment Tool (PCAT) and the Treatment Burden Questionnaire, respectively.Multisite surveys were conducted in primary care facilities in Guangdong province, southern China. Interviewer‐administered questionnaires were used to collect data from patients (p < .001) and lower treatment burden scores  when compared to those who often bypassed primary care. Greater healthcare needs were significantly correlated with increased treatment burden , whilst better patients' experiences were associated with lower treatment burden  after adjusting for confounders.The mean age of the patients was 61.4 years, and slightly over half were females. Patients who had a family doctor team as the primary source of care reported significantly higher PCAT scores (mean difference 7.2 points, The inverse association between patients' experiences and treatment burden supports the importance of primary care in managing patients with multimorbidity.Primary care service users were involved in the instrument development and data collection. In a fragmented healthcare system, it is less likely that multiple, episodic healthcare providers will take into account the entirety of a patient's healthcare conundrum including inappropriate polypharmacy, demanding self‐management regimens and competing priorities and more vulnerability to safety issues due to multimorbidity.This study aimed to provide an insight into healthcare needs, patients' experiences and treatment burden from the perspective of process of care. Our key research question is whether there is a significant association between primary care experiences and treatment burden in the context of patients' increasing healthcare needs due to multimorbidity. In the absence of a secondary healthcare gatekeeping function in primary care, we hypothesize that patients who do not consider the family doctor team as their preferred usual source of care will have poorer primary care experiences and greater treatment burden.22.1Multisite cross‐sectional survey data were collected from primary care service users, with a diversity of geographic locations, in 9 out of a total of 21 cities in Guangdong province, southern China. In the first stage, three cities were selected in each of the western, central and eastern areas of Guangdong, respectively. In the second stage, two sites per city were randomly selected from primary care facilities that were organizational members of the Guangdong Primary Healthcare Association to facilitate the fieldwork coordination.2.2The study was conducted on‐site at 18 primary care facilities where free‐of‐charge, annual check‐up, as part of the national BPH service package, was offered to people aged ≥35 years who had hypertension or diabetes.2.3The inclusion criteria of target participants were as follows: (1) patients who had ≥2 clinically diagnosed long‐term conditions including hypertension or type 2 diabetes and (2) had at least one clinical encounter in the past 12 months since enrolment registration with the family doctor team. We excluded those who were passers‐by  to ensure that all study participants had valid exposure to the primary care provider before study participation, and could hence minimize the likelihood of capturing ‘hearsay' information that was not actually experienced by the patients. Patients who were unable to communicate or who were not on regular medications were excluded. Eligible participants were referred to trained interviewers by healthcare staff, following a modified systematic random sampling that was previously used.2.4Patients' experiences were captured by a previously validated, culturally adapted, Mandarin Chinese version of the Primary Care Assessment Tool (PCAT)‐Adult Edition used in our previous research.2.5α was .884, suggesting satisfactory internal consistency reliability. The test–retest intraclass correlation coefficients of individual item scores ranged from .725 to .846, suggesting that the results of assessment were stable through repetition.Treatment burden was defined as the challenges that patients face in coping with everything they have to do to take care of their health, and its impact on functioning and well‐being.2.6The content validity of the entire questionnaire, including both PCAT and TBQ instrument items, was assessed by a panel consisting of two GP professionals (J.‐J. W. and X.‐Y. W.) and two public health specialists (H. H. X. W. and Y.‐T. L.). Each item was rated with regard to the relevancy and clarity by a content validity index using a 4‐point Likert‐type scale. All items were rated as quite (3‐point) or highly (4‐point) relevant and clear by all panel experts. Survey interviewers included on‐site healthcare staff and medical university students. Training sessions were held by the two lead investigators. The survey was pilot‐tested by paired interviewers among 20 primary care service users to improve the interrater reliability.2.7χ2 tests or Student's t tests, where appropriate, were used to compare the differences with regard to categorical and continuous variables between groups. A general linear model analysis was conducted to examine patient‐level factors associated with treatment burden after controlling for confounders. The absence of multicollinearity and plausible interactions among variables were tested to ensure the robustness of the linear regression model. We also performed a series of sensitivity analyses to further explore the relationship between primary care experiences and each treatment burden measure, while controlling for other confounding factors in the multiple linear regression analysis. A p‐value <.05 was considered statistically significant. All statistical analyses were performed in IBM SPSS Statistics 25, and the Complex Samples module was used to account for the multistage sample design.Data entry was independently performed by two trained medical students using EpiData 3.1 with double verification. Sample mean with standard error (SE) or 95% confidence interval (CI), where appropriate, was applied in descriptive analysis. 2.8All study participants provided written consent. Data anonymization was performed by removing all patient identifiers from the data set before data analysis. Ethics approval was granted from the School of Public Health Biomedical Research Ethics Review Committee at Sun Yat‐Sen University (SYSU‐SPH2016027) in accordance with the Declaration of Helsinki 2013.33.1A total of 2160 out of 2471 eligible primary care patients with multimorbidity were included . The mean age of the participants was 61.4 years (95% CI: 60.7–62.2 years), and slightly over half were females. Less than half had completed secondary school education or above. Nearly one in five patients had multimorbidity for over 10 years. Approximately 40% of people had a monthly household income per capita below ¥2000. When compared to China's median disposable personal income (¥2028 per month) in 2018,3.2p = .001) when compared to their counterparts  of the participants considered the CHC family doctor team as their usual source of primary care. The traditional face‐to‐face visit was more common than distance communications for consultations. The family doctor team played a moderate positive role in service delivery perceived by patients, ranging from 53.7% to 68.1%. On average, the duration of CHC enrolment registration was 11.8 months, and each patient received 3.6 follow‐up appointments annually Table . Partici% of the 3.3p < .001) and lower treatment burden  when compared to their counterparts who were in favour of using specialist care over primary care , which was slightly higher than the first quantile of the score range (0–150), whilst a total primary care assessment score of 100.7 (SE: 0.9) was reported on average, falling within the third quantile of the score range 0–132). This implied moderate‐to‐light treatment burden and medium‐to‐optimal primary care experiences overall  and increased counts of healthcare needs  were positively associated with greater treatment burden. Factors negatively associated with treatment burden included frequently delivered follow‐up (p = .001), regular use of primary care (p = .007) and higher primary care assessment scores (p < .001), indicating that better patients' experiences were associated with lower treatment burden overall  remained negative  regular healthcare reminding patients of their health problems; (b) the financial burden of treatment; (c) the burden of arranging and adapting to medical appointments; and (d) difficulties in relationships with healthcare providers.Patients in our study reported higher primary care assessment scores and lower treatment burden scores when directly compared to previous studies,Compared to the United Kingdom and other international countries where a primary care gatekeeping function is in place, the primary care facilities in China are still far from acting as a first‐contact point and regular source of care, despite ongoing development of the ‘family doctor team'. Concerns over the absence of healthcare gatekeeping and a rapid growth of hospital outpatients visits in China have been raised in the past decade.4.4Primary care is expected to lie at the heart of care delivery in response to healthcare needs from patients with multimorbidity. The family doctor team initiative shares similarities to the GP service delivery in the United Kingdom with regard to strengthened relationships between the enrolled individual patients and the primary care providers in a continuous and collaborative manner. This, of course, requires proper knowledge, adequate skills, right professional values and positive attitudes that embrace core attributes of primary care. Our study adds to the evidence favouring the benefits of primary care multidisciplinary team‐based service delivery in coping with multimorbidity. This carries international implications for other countries where primary care transformation is in progress as a vital step towards achieving an improved team‐based approach to population health and multimorbidity management. Further longitudinal investigations are warranted to examine the effects of the family doctor team service delivery on the care for patients with multimorbidity in the long term. Efforts to explore an optimal panel size of registration per primary care team and the ratio of GP clinicians versus allied healthcare professionals linked with health outcomes may also help to inform strategies for multimorbidity care over time in areas of different socioeconomic strata.It has been widely recognized that treatment burden prevents optimal adherence to the provision and management of care for people with long‐term conditions,5Our study suggested that higher healthcare needs were significantly associated with increased treatment burden, whilst better patients' experiences were associated with lower treatment burden in the context of the family doctor team service delivery. This implies the necessity of optimizing the key attributes of primary care in person‐centred service delivery and quality improvement, and is therefore of major relevance to healthcare strategies aiming to deliver less burdensome care for people with multimorbidity.The authors declare that there are no conflict of interests.Harry H. X. Wang and Xiu‐Jing Hu conceived the idea of the study. Harry H. X. Wang, Yu‐Ting Li and Jia‐Ji Wang participated in the fieldwork coordination. Data analysis was conducted by Xiu‐Jing Hu and Yu‐Ting Li. All authors contributed to the literature search and interpretation of the data. Harry H. X. Wang and Xiu‐Jing Hu wrote the first draft. All authors have read, contributed to and approved the final version of the manuscript.


Species‐level environmental niche modeling has been crucial in efforts to understand how species respond to climate variation and change. However, species often exhibit local adaptation and intraspecific niche differences that may be important to consider in predicting responses to climate. Here, we explore whether phylogeographic lineages of the bank vole originating from different glacial refugia  show niche differentiation, which would suggest a role for local adaptation in biogeography of this widespread Eurasian small mammal. We first model the environmental requirements for the bank vole using species‐wide occurrences (210 filtered records) and then model each lineage separately to examine niche overlap and test for niche differentiation in geographic and environmental space. We then use the models to estimate past [Last Glacial Maximum (LGM) and mid‐Holocene] habitat suitability to compare with previously hypothesized glacial refugia for this species. Environmental niches are statistically significantly different from each other for all pairs of lineages in geographic and environmental space, and these differences cannot be explained by habitat availability within their respective ranges. Together with the inability of most of the lineages to correctly predict the distributions of other lineages, these results support intraspecific ecological differentiation in the bank vole. Model projections of habitat suitability during the LGM support glacial survival of the bank vole in the Mediterranean region and in central and western Europe. Niche differences between lineages and the resulting spatial segregation of habitat suitability suggest ecological differentiation has played a role in determining the present phylogeographic patterns in the bank vole. Our study illustrates that models pooling lineages within a species may obscure the potential for different responses to climate change among populations. We explore whether phylogeographic lineages of the bank vole originating from different glacial refugia show environmental niche differentiation. We found that niches were significantly different between all pairs of lineages in geographic and environmental space and these differences cannot be explained by habitat availability within their respective ranges, suggesting that ecological differentiation has played a role in determining the present phylogeographic patterns in the bank vole. Western Siberia is considered as an area with the current climatic conditions resembling those in Europe during the LGM, and its use as the test area helped ensure the transferability of the models , indicating that the present phylogeographic patterns are, to large extent, the product of species’ adaptive niches and habitat availability  curve for the test data  on a relative scale of 0 to 1, with higher values indicating broader niches , which is represented by all the combinations of values of bioclimatic variables observed across the study area  generated by Latin hypercube sampling with a chunk size of 100,000 points and tolerance of 0.00001  lower than expected from the null distribution  on the set of climate rasters used to build the models, separately for Set 1 and Set 2, and plotted the occurrence data together with the background points and a 100,000‐point sample of the multidimensional E‐space , the geographic distribution of habitat suitability for the bank vole under the current climate closely approximates the present distribution range of the species Figure .2. ApplyAlthough the composite prediction from the models constructed for the individual lineages Figure .3–S2.4 cIn general, the habitat suitability for Carpathian, Southern, and Western lineages responded positively to high precipitation levels. In contrast, for Eastern lineage, high habitat suitability was predicted mostly in drier climates, with a strong negative influence of precipitation of the wettest (BIO 16) and driest (BIO 17) quarters Figure .1.The models for the individual lineages showed varying abilities to predict the occurrences of the other lineages Table .1. For e3.3In the mid‐Holocene, the geographic distribution of each lineage's habitat suitability was similar to its present distribution Figure .5. HowevModels for the individual lineages produced considerably overlapping predictions of habitat suitability during the LGM Figure , with Se3.4G‐space and E‐space  and E‐space (D of 0.25–0.55), with the Eastern lineage showing substantially higher overlap than the other lineages in G‐space, but the lowest in E‐space , the background similarity test indicated that niches of the lineages are more similar than expected based on the available environmental conditions , which replaces the bank vole throughout the northern taiga and forest‐tundra zones of Eurasia  ; Investigation (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (supporting). Michaela Horníková: Investigation (supporting); Writing‐review & editing (supporting). Silvia Marková: Investigation (supporting); Writing‐review & editing (supporting). Petr Kotlik: Conceptualization (lead); Investigation (supporting); Supervision (lead); Writing‐original draft (supporting); Writing‐review & editing (lead).Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


Color variation is one of the most obvious examples of variation in nature, but biologically meaningful quantification and interpretation of variation in color and complex patterns are challenging. Many current methods for assessing variation in color patterns classify color patterns using categorical measures and provide aggregate measures that ignore spatial pattern, or both, losing potentially important aspects of color pattern.Colormesh, a novel method for analyzing complex color patterns that offers unique capabilities. Our approach is based on unsupervised color quantification combined with geometric morphometrics to identify regions of putative spatial homology across samples, from histology sections to whole organisms. Colormesh quantifies color at individual sampling points across the whole sample.Here, we present Colormesh using digital images of Trinidadian guppies (Poecilia reticulata), for which the evolution of color has been frequently studied. Guppies have repeatedly evolved in response to ecological differences between up‐ and downstream locations in Trinidadian rivers, resulting in extensive parallel evolution of many phenotypes. Previous studies have, for example, compared the area and quantity of discrete color  between these up‐ and downstream locations neglecting spatial placement of these areas. Using the Colormesh pipeline, we show that patterns of whole‐animal color variation do not match expectations suggested by previous work.We demonstrate the utility of Colormesh can be deployed to address a much wider range of questions about color pattern variation than previous approaches. Colormesh is thus especially suited for analyses that seek to identify the biologically important aspects of color pattern when there are multiple competing hypotheses or even no a priori hypotheses at all. There is considerable interest in quantifying color patterns in order to investigate the role that color plays in ecological and evolutionary processes, including competition, mate choice, kin selection, predation, and speciation. The Colormesh approach is a novel, unsupervised method for sampling color from digital images, enabling highly multivariate quantification of color patterns. We demonstrate the utility of the Colormesh approach by evaluating patterns of color variation in the Trinidadian guppy, a model system for studies of the role of color in local adaptation and in the maintenance of genetic diversity. Many different approaches that often rely on human perception of color have been used to quantify color patterns and how they vary among organisms, populations, and species. Early methods of assessing color in biology relied on categorical schemes, such as assigning patterns to discrete morphs , and spectrophotometry can capture the entire reflectance spectra of specific locations on an organism. In digital image analysis, every pixel can be described by a quantitative value for each color channel. Pixels can then be grouped into discrete color categories using clustering or thresholding to perform image segmentation. Applications such as ImageJ has been used for counting pixels within a specified color range to identify diseased tissue . While colordistance accounts for how much of each color is present in an image and how close those colors are in color space, the location of these colors within the pattern is lost. In order to evaluate whole color pattern variation, measurement of spatially explicit variation in color is needed, and tools to evaluate color patterns in this manner have been lacking.Methods other than segmentation of digital images are necessary if information on whole color pattern variation  is required. The pavo2, provides a framework for processing and combing spectral and spatial data with visual models  enable analysis of color patterns that are highly variable, spatially complex, and/or lack well‐organized color pattern elements , (b) capture the continuous, high‐dimensional nature of color variation, (c) use an unsupervised method to determine points on standard digital images from which to sample color values, and (d) allow for flexibility in color sampling density and the size of the area sampled.Here, we present a new approach for sampling color patterns from digital images using Colormesh using digital images of Trinidadian guppies (Poecilia reticulata), in which the evolution of color is a topic of active study. We photographed male guppies from eight natural and three experimental populations (described below). Complex color patterns in this species are male limited, highly heritable, and highly variable both within and between populations , that preys on juveniles and small adult guppies  Endler, . In natuColormesh to address three questions about color variation and evolution in Trinidadian guppies. We first asked if our method could successfully classify individual fish by their population of origin. We then asked if the direction of color evolution between up‐ and downstream populations was consistent across river drainages. Finally, we asked if the within‐population variance in color differed consistently between up‐ and downstream populations and evaluated the contribution of color at different locations on the fish to within‐population variation in color.Here we use 22.1Colormesh package. To sample color from homologous locations among images, subjects must first be processed to a standardized size and shape. Image processing (landmark placement and image transformation to a consensus shape) within the Colormesh package is described in brief below. Alternatively, image processing may be completed externally using other geometric morphometric software  and theColormesh can be downloaded for free at https://github.com/J0vid/Colormesh. The GitHub site provides instructions for download and a forum for posting questions or issues. Explanatory details and example code for using Colormesh to process images  and color sampling are provided on the Readme page of the GitHub site, as well as in a tutorial provided in the Appendix 2.2Colormesh requires two CSV files to provide information during processing: one having the unique specimen image names and associated identification information and the other to provide the known RGB values of the colors on the standard for the calibration process. To sample RGB color values from pixels, Colormesh requires two sets of images as input: one set of images that have been processed using landmark‐based geometric morphometric methods  for color sampling and the original set of images containing a color standard for the image‐specific color calibration. Lastly, two arrays containing landmark coordinate data are required: one of the consensus shape and another having the locations to sample the color standard. Landmark placement and generation of consensus shape images within Colormesh (described below) will produce the required coordinate data arrays; externally generated landmark coordinate data are easily imported , as well as the raw image formats unique to Canon, Nikon, and Olympus brand cameras  are compatible with ge Ooms,  is used landmark.images function within Colormesh. This presents each image to the user and utilizes the user‐friendly digitization capabilities provided by the R package geomorph ; for this array, a scale is not defined since color calibration does not use shape analysis.For the color calibration process, the tps.unwarp function which employs the utilities of two R packages: geomorph  with a reduced number of points that function as a concise representation of the shape  best differentiates among specimens in the Multivariate classification and differentiation among populations section below.The 2.2.3Colormesh to correct the sampled color values. Calibration is performed by the rgb.calibrate function which calculates an image‐specific color channel correction vector. First, Colormesh reads in the array of coordinate data from landmarks placed on the color standard in the images. The RGB values are then sampled from the pixels contained within a sampling circle centered on the coordinates provided by the array. The mean deviation from the known color channel values of the standard included in each image is calculated and the channel‐specific correction is applied to the measured values for that image.Providing the known RGB values of the color standard enables 2.32.3.1To compare color patterns in a diverse set of populations, we photographed wild male guppies captured during the dry season in May of 2016 and 2017. These fish were collected from six rivers belonging to three major drainage systems in the Northern Range in Trinidad  and converted to the .TIF format with an output color space of sRGB in Photoshop CC 2018. Each digital photograph of a male guppy included a size and color standard. After setting the image scale (pixels/cm), a total of 62 landmarks were placed around the perimeter of each fish. Seven traditional landmarks were first placed at the following locations: the tip of the snout, anterior and posterior connection points of the dorsal fin to the body, dorsal and ventral connection points of the caudal fin to the caudal peduncle, posterior and anterior connection points of the gonopodium to the body . Color data were extracted from guppy images using the auxiliary package  1 Table .2.3.3adegenet  = 100. We compared the lowest RMSE and proportion of successful placements for each of the 12 sampling schemes to determine which sampling scheme would be used for the remaining analyses.We used Discriminant Analysis of Principal Components (DAPC) in the R package, Jombart,  to deterJombart,  to asses2.3.4Colormesh data to compare the direction of evolutionary divergence between populations with known ancestor‐descendant relationships. This analysis tests the hypothesis that color pattern evolution has evolved convergently each time high‐predation guppies have moved upstream to invade low‐predation habitats. For four of the six rivers that we sampled , the paired high‐ and low‐predation populations were sampled in the same year. This meant the images had the same pixel resolution prior to the image processing described above, so the high‐ and low‐predation populations within these four rivers could be compared directly. For this analysis, each fish had R, G, and B values measured at 2,462 positions for a color vector of length = 7,386 variables.We used the guppy r), which is mathematically equivalent to Pearson's correlation coefficient.To produce an average color pattern for a given population, we calculated the mean value of each color channel at each sampling point. Thus, each population had a mean color vector consisting of 2,462 values for each color channel. Because low‐predation populations are known to be descendants of high‐predation populations within each river, the difference between the population mean color vectors measures the direction of color evolution within a river. If color pattern evolution was similar among rivers, these difference vectors should be more similar than that expected by chance. We measured the similarity of these vectors by calculating the angle between the vectors of each pair of rivers. If colors evolved similarly in two river drainages, the vectors indicating direction of evolution will have a smaller angle between them than random vectors. The angle between the vectors can also be expressed as a vector correlation . We calculated the trace of the variance‐covariance matrix to determine the phenotypic variance in color for each real population. Our test statistic was the trace of the high‐predation population minus the trace of the low‐predation population.sample function in base R (V3.5.3), created 1,000 permutation samples, and calculated the difference between traces. The distribution of these 1,000 measures of difference in variance was used to represent the null distribution.We used permutation to test if the phenotypic variation differed between high‐ and low‐predation populations within a river. We permuted the label of high predation or low predation for each whole‐fish color pattern within a single river using the x, y coordinate in the data set. We estimated variance components with restricted maximum‐likelihood using Proc Varcomp of SAS version 9.4 , with the color value  at a given x, y position as the dependent variable, year as a fixed effect, and population ID as a random effect.We were also interested in characterizing the whole‐specimen spatial pattern of within‐population variance in color. To do so, we compared the within‐ and between‐population components of variance for each color channel at every 33.1We first sought to determine a combination of sampling density and sampling circle size that would enable us to classify the 11 guppy populations with the highest precision. As the number of rounds of Delaunay triangulations (DT) increased, the root mean squared error (RMSE), averaged over all sampling circle sizes, consistently decreased  and one to the Oropuche (Turure) drainage. This allowed us to ask whether correlations in direction of color evolution between rivers belonging to different drainages were weaker than correlations within drainage. The cross‐drainage correlations were somewhat more variable than the within‐drainage correlations; interestingly, the cross‐drainage Aripo‐Turure correlation is just as strong as the strongest within‐drainage correlation Figure .3.3p‐value <.001 and p = .009, respectively). In the other two rivers (El Cedro and Guanapo), the high‐predation population had nominally higher total phenotypic variance, but these differences were not significant  allows the analysis of variation in chromatically and spatially complex patterns, (b) avoids loss of information by discretizing color or subjective identification of color elements, (c) allows high‐throughput analysis by using digital images of whole color patterns, and (d) provides flexibility with respect to sampling density and area.We used this novel method to determine if it could accurately assign fish from 11 populations to the correct population and to evaluate whether the direction of color pattern evolution was consistent in four different rivers. We also asked if total phenotypic variation differed consistently between up‐ and downstream populations across drainages. Below, we discuss the performance of our approach on these tasks and compare our conclusions to those arrived at using different methods.4.1Colormesh. Regardless of the type of specimen, our approach enables the use of color data to examine the granularity of whole‐pattern sampling, as well as identify regions within the pattern that contribute most to among‐population differentiation and within‐population variation. Colormesh allows the color of an individual to be characterized at different scales by using different numbers of points on the body and averaging over different numbers of pixels around each point. Variation in the number of semilandmarks would also influence the density of sampling points since these landmarks are used in the initial round of Delaunay triangulation. Therefore, an important precursor to analysis is to investigate how varying these parameters  affects the conclusions of any analysis. For our guppy samples, increasing sampling density increased our ability to differentiate populations, and varying the number of pixels averaged around each point made little difference. This is likely because male guppy color pattern is complex, with color changing fairly dramatically at small spatial scales. Organisms with less fine‐grained patterns might be sufficiently sampled at coarser scales.The guppy example highlights the usefulness of spatially explicit sampling of whole color patterns performed by The discriminant analysis of principal components (DAPC) using our selected sampling scheme  separated populations largely by sampling year along the first discriminant function axis. Camera settings differed between years which likely accounts for the variation explained by this axis; however, since different populations were sampled in different years, this axis probably also captured some population differences in color. Although this year effect limited which downstream analyses we could perform, it also indicates that this method is sensitive to unsuspected sources of variation, which suggests that consistency in the image collection process is important.DAPC also allowed us to identify axes of variation that consistently separated high‐ and low‐predation populations. Differences between predation regimes have been reported in previous studies assessing color patterns; however, the nature of these reported differences has varied. For example, in a study of 112 sites sampled from 53 streams located in Trinidad and Venezuela, Endler  reported4.2Colormesh compliments these studies by assessing total phenotypic variation across the entire fish while avoiding the potential constraints imposed by the use of human‐determined color categories. Three of the four rivers  in our comparisons belong to the same drainage system (Caroni), with the El Cedro being a tributary of the Guanapo. Comparisons between pairs of rivers within the Caroni drainage system showed that the El Cedro and Guanapo rivers were more similar in the trajectory of evolution than the other pairs of rivers, but the degree of similarity was small (vector correlation <0.3).With our guppy example, we sought to test whether the repeated transition from high‐ to low‐predation pressure among rivers resulted in parallel directions of color pattern evolution among several rivers. Using the known ancestor‐descendant relationship between high‐ and low‐predation populations, we calculated correlations in direction of multivariate color evolution between rivers. Our results were similar to those reported in two previous studies in that we found inconsistencies in the direction of color evolution among the rivers. In both previous studies, an increase or decrease in quantity and size of individual color pattern elements were compared to determine similarity in evolutionary trajectories  with each of the three rivers belonging to the Caroni drainage. These three correlation measures were highly variable. We expected the Turure‐Guanapo and Turure‐El Cedro comparisons to show greater correlation in direction of evolution than the Turure‐Aprio comparison because the low‐predation Turure population was originally a guppy‐free location where guppies from a high‐predation population in the Arima river  were introduced by Haskins in 1957  to provide more insight into the determinants of variation in this ecologically important trait.Using the guppy system example, we also evaluated differences in total phenotypic variation within and among populations. The processes that promote variation in male guppy color patterns have been investigated extensively ; formal analysis ; funding acquisition (supporting); methodology ; software ; writing‐original draft (lead). Jose David Aponte: Conceptualization ; software . Mitch J. Daniel: Formal analysis (supporting); writing‐review & editing (supporting). Kenna Dwinell: Data curation (supporting). Helen Rodd: Data curation (supporting); funding acquisition ; methodology (supporting); writing‐review & editing (supporting). David Houle: Formal analysis (supporting); methodology (supporting); writing‐review & editing . Kimberly A. Hughes: Conceptualization ; formal analysis ; funding acquisition ; methodology ; writing‐review & editing .https://doi.org/10.1101/2020.07.17.205369).The research presented was described in Animal Research Protocol No. 1442 approved on 29 October 2014 and Protocol No. 1740 approved on 16 October 2017, by the Animal Care and Use Committee of Florida State University. A preprint version of this manuscript was made available through BiorXiv (Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.Appendix S1Click here for additional data file.


Pisum sativum (Pea) plants. This undergraduate research experience, piloted in the fall semester of 2020 in a class with 90 students, was created to help participants learn and practice scientific research skills during the COVID‐19 pandemic. Specifically, the learning outcomes associated with this at‐home research experience were: (1) generate a testable hypothesis, (2) design an experiment to test the hypothesis, (3) explain the importance of biological replication, (4) perform meaningful statistical analyses using R, and (5) compose a research paper to effectively communicate findings to a general biology audience. Students were provided with an at‐home laboratory kit containing the required materials and reagents, which were chosen to be accessible and affordable in case students were unable to access our laboratory kit. Students were guided through all aspects of research, including hypothesis generation, data collection, and data analysis, with video tutorials and live virtual sessions. This at‐home laboratory provided students an opportunity to practice hands‐on research with the flexibility to collect and analyze their own data in a remote setting during the COVID‐19 pandemic. This, or similar laboratories, could also be used as part of distance learning biology courses.The COVID‐19 pandemic prompted a transition to remote delivery of courses that lack immersive hands‐on research experiences for undergraduate science students, resulting in a scientific research skills gap. In this report, we present an option for an inclusive and authentic, hands‐on research experience that all students can perform off‐campus. Biology students in a semester‐long (13 weeks) sophomore plant physiology course participated in an at‐home laboratory designed to study the impacts of nitrogen addition on growth rates and root nodulation by wild nitrogen‐fixing Rhizobia in  Laboratory activities are used as a pedagogical tool for practicing real‐world problem‐solving and critical thinking and often result in greater intellectual curiosity and appreciation for the natural world  and Interactive Laboratory Microbiology (www.interactivelabmicro.com); however, these can be cost‐prohibitive in the long term due to subscription prices and restricted budgets in higher education. Other educators designed data‐focused laboratories, where instructors or teaching assistants collected the relevant data for students to perform guided data analysis  Plant Physiology laboratory course that is designed to be conducted at home over the course of a semester. This authentic, at‐home undergraduate research experience (URE) was designed with the goal of giving students an opportunity to apply scientific processes they will use throughout their undergraduate, graduate, and work careers  and to guide students through quantitative reasoning, as recommended in Vision and Change in Undergraduate Biology Education: A Call to Action (American Association for the Advancement of Science (AAAS), Pisum sativum (Pea) growth rate and root nodulation by wild nitrogen‐fixing Rhizobia using local soils independently sourced by students, and (2) the collected data are pooled and shared among the students so that they can perform guided data analysis using the open‐source statistical software R  students study the effect of nitrogen fertilizer on 1.11.1.1® All‐Purpose Water‐Soluble Plant Food  was provided for two treatment levels (1X and 3X) with preparation instructions detailing how to bring the treatment solutions to the final concentrations using tap water. Students were instructed to prepare a “control” of 0X Miracle Gro® containing only tap water. The kit also contained fluorescent colored Safety Data Sheet sticker labels that the students were instructed to apply to solution storage containers, and instructions for safely storing containers out of reach of children and pets. Additional materials that must be provided by the student include the following: three sealable treatment solution containers, plant pots, and local soil. The kit was mailed out to students who were unable to pick up the kit (Canada only). As previously stated, materials in the at‐home kit were chosen to be affordable and easily sourced if students were unable to access our kit. International students were provided with the list of materials to be sourced. All students were provided with the option to perform an alternative, virtual only experiment if they were unable to perform the experiment for any reason.The at‐home laboratory kit Figure  containe1.1.2P. sativum L.) roots. Pea plants are members the Fabaceae/Leguminosae (the legume family); there are approximately 19,500 legume species, making it the third largest plant family , a form of nitrogen that can be assimilated into organic molecules by plants since plants and other eukaryotes are not able to directly access atmospheric N2  required for plant growth, and agriculture relies on sustained nitrogen provision to maintain continued productivity. There are many legume species that are used in agriculture, and symbiotic BNF by legumes plays a major role in provision of nitrogen in global agriculture, amounting to 33–46 Tg nitrogen per year for the combination of crop and pasture/fodder legumes  to the bacteria , ammonium (NH4+), and amino acids , a 1X treatment, and a 3X treatment . Early in the semester, after the students began the at‐home experiment, they were guided through data analysis in R using a practice data set (data collected in a previous year). Following this learning exercise, students submitted a Results section  and were allowed to resubmit this assessment following a round of feedback. To aid students constructing a Methods section, we used an assessment where students were provided with a poorly written Methods section and asked to critique it. Students submitted their Introduction section for peer review and then re‐submitted the updated Introduction (following peer‐review) for instructor feedback. Grading rubrics for all assessments were provided to the class at the start of the semester. Distribution of scores achieved in the assessments described above is shown in Figure 1.1.6To ensure equitable opportunities for achieving learning outcomes, students that were unable to complete the at‐home laboratory were provided with an overview (background information) of an alternative experiment studying the effect of plant hormone gibberellic acid on the development pattern of wild‐type and dwarf pea cultivars in the accompanying laboratory manual , also called gibberellins, are a well‐characterized class of plant hormones  and data including plant height, number of internodes and fresh weight at end of experiment. Students were also guided through the analysis of this data in rstudio.cloud through video screencasts and a companion HTML document built using RMarkdown  process of science and (2) quantitative reasoning . This requires biologists to be able to appropriately format data, perform data quality control checks, identify data input errors, explore data to identify appropriate statistical tests, and produce graphical representations of experimental results  experiments  suitability for a nonlaboratory setting  and (2) allowing all students the opportunity to achieve associated learning goals and outcomes. In addition, equity should be considered not only in terms of resource accessibility and socioeconomic inequalities, but also in terms of student diversity in learning experiences and educational background. These activities must therefore be designed to reasonably accommodate financial, time, and space limitations for students.To keep student costs low in our at‐home laboratory design, most of the required supplies are provided to students. Students who chose to perform the at‐home experiment need to source soil for the experiment themselves; this can present a challenge for students if they live in an apartment or do not have access to a yard. At‐home laboratories have the potential to require more time than students would otherwise spend performing a tightly scheduled laboratory with instructor setup and maintained experiment, since at‐home UREs require students to set up and monitor the experimental system for the duration of the experiment. Additionally, limited instructor oversight of the daily experimental process, especially with respect to housekeeping items such as seed germination, watering, and ensuring general health of the experimental plants, may lead to unforeseen issues such as overwatering or mold, ultimately leading to loss of experimental plants. In terms of monitoring experimental plants, students are provided with a guide for selecting soils, a recommended watering schedule, and tips monitoring plant health to ensure plants remain healthy over the duration of the experiment. However, these limitations must be taken into consideration when designing assessments and other laboratory‐associated activities.An at‐home laboratory provides flexibility in terms of monitoring and data collection and can better accommodate student work schedules and family obligations. Students can perform the data collection at a time that works for them and without the added costs of travel to an on‐campus laboratory, which itself may require data collection outside of scheduled laboratory time. Space constraint is another limitation that can deter students from having a successful at‐home laboratory experience. In our experiment, for example, students need space in front of a light source (window or grow lights if available) to effectively grow the experimental plants. To maintain equity in cases where laboratory design or requirements prohibit students from performing the laboratory, an alternative experiment and assessment was provided. Alternative assessments help maintain flexibility and accessibility to the learning outcomes of this at‐home laboratory, especially in cases where students may lose experimental plants early in the experimental timeline. In 2020, 4% of the students opted to perform the alternative experiment as they were unable to perform the at‐home laboratory, while 12% opted to use data from the alternative experiment in their final report due to death of plants during the at‐home experiment.3The pivot to online teaching necessitated by the COVID‐19 pandemic has required instructors implement new strategies to help students achieve learning outcomes, especially those associated with scientific research. Though at‐home laboratories come with unique challenges, such as cost (if kits are not provided to students), time, and space to perform the activities, those designed with these challenges in mind can provide students with authentic hands‐on research experiences. In many cases, at‐home laboratories are an opportunity for increased learning equity through flexibility of time, experiment pace, and place as well as increased control and opportunity for more independent data analysis. In this at‐home laboratory, we focused on developing two of the core competencies recommended in Vision and Change in Undergraduate Biology Education: A Call to Action: (1) the ability to apply the process of science, and (2) the ability to use quantitative reasoning AAAS, . With reNone.Laura J. Schnell: Methodology ; Resources (supporting); Writing – original draft (supporting); Writing – review & editing . Gavin L. Simpson: Conceptualization (supporting); Data curation (lead); Methodology (supporting); Resources ; Software (lead); Writing – review & editing . Danae M. Suchan: Conceptualization (supporting); Methodology (supporting); Resources (supporting); Writing – review & editing . William Quere: Methodology (supporting); Resources (supporting); Writing – review & editing (supporting). Harold G. Weger: Resources (supporting); Writing – review & editing . Maria C. Davis: Conceptualization (lead); Methodology ; Resources (lead); Writing – original draft (lead); Writing – review & editing .