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Molecular Phylogenetic Analysis of the Main Lineages of Nymphalinae (Nymphalidae: Lepidoptera) Based on the Partial Mitochondrial COI Gene
Abstract
The phylogenetic relationships of the subfamily Nymphalinae (sensu Chou 1994) were analyzed based on 1 488 bp of mtDNA cytochrome oxidase subunit I (COI) gene sequence data obtained from 24 individuals, along with those of eight species obtained from GenBank. The base compositions of this COI fragment varied among the individuals as follows: T 39.9%, C 14.6%, A 32.2%, and G 13.4%, with a strong AT bias (72.1%), as usually found in insect mitochondrial genomes. The A + T contents of the third, second, and first codon positions of the COI fragments in this study was 92.4, 62.2, and 61.4%, respectively. The phylogenetic trees were reconstructed by neighbor-joining (NJ), maximum likelihood (ML), and Bayesian methods by using Byblia anvatara as outgroup. Phylogenetic analyses based on the COI gene sequence data created very similar topologies, which were producing trees with two main clades A and B, and five subclades. The data indicated that the tribes Nymphalini and Hypolimni (sensu Chou 1994) are not monophyletic groups, and the genus Junonia should be removed from Nymphalini to Hypolimni (= Junoniini). On the basis of the data, the Symbrenthia and Araschnia had a relative distant relationship with the rest of Nymphalini. The relationships of species in the Nymphalini were confirmed via the NJ, ML, and Bayesian methods, namely ((((Nymphalis + Kaniska) + Polygonia) + Aglais) + Vanessa) + (Symbrenthia + Araschnia). This investigation provides a little novel information for Chinese researches of butterflies.
... For the sake of brevity, we will only present the maximum likelihood analysis (HKY model, 10 replicate heuristic searches with random number seeds, tree bisection, and reconnection branch swapping algorithm) [69]. Other previously published Junonia COI sequences were included in this phylogenetic analysis [23,24,51,57,[70][71][72][73][74]. We also conducted a maximum likelihood bootstrap analysis of this dataset (500 fast addition replicates, collapsing all notes with frequency less than 50%). ...
Up to 9 described species of Junonia butterflies occur in the Americas, but authorities disagree due to species similarities, geographical and seasonal variability, and possible hybridization. In dispute is whether Caribbean Junonia are conspecific with South American species. Cytochrome oxidase I (COI) barcodes, wingless (wg) sequences, and Randomly Amplified Fingerprints (RAF) were studied to reveal Junonia population structure in French Guiana, Guadeloupe, Martinique, and Argentina. Phylogenetic analysis of COI recovered 2 haplotype groups, but most Junonia species can have either haplotype, so COI barcodes are ambiguous. Analysis of nuclear wingless alleles revealed geographic patterns, but did not identify Junonia species. Nuclear RAF genotyping distinguished 11 populations of Junonia arranged into 3 clusters. Gene flow occurs within clusters, but is limited between clusters. One cluster included all Argentinian samples. Two clusters included samples from French Guiana, Martinique, and Guadeloupe and appear to be divided by larval host plant use (Lamiales versus Scrophulariales). Many Junonia taxa were distributed across populations, possibly reflecting patterns of genetic exchange. We had difficulty distinguishing between Caribbean forms J. zonalis and J. neildi, but demonstrate that Caribbean Junonia are genetically distinct from South American J. evarete and J. genoveva, supporting the taxonomic hypothesis that they are heterospecific.
... Several different tree reconstruction algorithms were used: Neighbor-Joining (using uncorrected 'p' distances with systematic tie-breaking by taxon order), Parsimony (ten replicate heuristic searches with random number seeds, maximum 1000 trees retained at each replicate, tree bisection and reconnection branch swapping algorithm), Parsimony Bootstrap (1 000 000 fast stepwise addition heuristic search replicates with steepest descent, retaining compatible groupings < 50%), and Maximum Likelihood (HKY model, ten replicate heuristic searches with random number seeds, tree bisection and reconnection branch swapping algorithm). Other previously published Junonia COI sequences were also included in these phylogenetic analyses (Kodandaramaiah & Wahlberg, 2007;Uma Shaanker et al., 2008;Zhang et al., 2008;Janzen & Hajibabaei, 2009;Marcus et al., 2009;Hebert et al., 2010;Qin et al., 2010;Brévignon & Brévignon, 2012;Pfeiler et al., 2012a). A minimum spanning network for COI haplotypes was created by an amova analysis in Arlequin3.5 (Schneider et al., 2000) with the following settings; 1000 permutations, determining the minimum spanning network among haplotypes, computing distance matrix and pairwise difference was selected with a gamma a value equal to 0. The Minimum Spanning tree portion of the generated output from this was used to produce a graphical representation of the network in HapStar-0.7 (Teacher & Griffiths, 2011). ...
There are nine named species of buckeye butterflies (genus Junonia Hübner) in the Western Hemisphere. There is considerable geographic variation within Junonia species, and possible ongoing hybridization between species, suggesting that Junonia may be a ring species, but also making this a very difficult group to define taxonomically. We tried to determine whether two forms of Junonia from Argentina – conventionally referred to as Junonia genoveva hilaris C. & R. Felder, the light buckeye butterfly, and Junonia evarete flirtea (Fabricius), the dark buckeye butterfly – were genetically distinct species or simply colour forms of a single species using morphological characters, mitochondrial cytochrome oxidase I (COI) DNA barcodes, nuclear wingless (wg) locus DNA sequences, and anonymous nuclear Randomly Amplified Fingerprints (RAF). Phylogenetic analysis of COI identified two distinct mitochondrial haplotypes that differ by about 4% sequence divergence; one confined to light-coloured Junonia specimens and one shared between some light-coloured Junonia and all of dark-coloured Junonia specimens. Analysis of nuclear wingless sequences revealed 32 alleles among 22 Junonia specimens and showed significant genetic differentiation between light-coloured and dark-coloured Junonia. Analysis of RAF genotypes suggests that there are actually three genetically distinct Junonia populations in Argentina: two with light wing coloration, and one with dark wing coloration. Genetic evidence of recent hybridization among these populations was also observed, consistent with the ring species hypothesis. Careful comparisons of morphological characters between Argentinian Junonia and Junonia species from elsewhere in South America suggests that the two light-coloured populations correspond to J. genoveva and either a genetically disparate population of the same species or an undescribed cryptic Junonia species, The dark-coloured population may correspond to J. wahlbergi Brévignon. Our data suggest that COI DNA barcodes by themselves are not very useful for studying Junonia taxonomy, population structure or evolution.
... Methodological advances are required to effectively analyze such large amounts of data. Most recently published phylogenetic hypotheses are reconstructed from datamatrices containing few genes, and sometimes only one [41]. A single short gene fragment may well be sufficient depending on the phylogenetic question under investigation. ...
Despite apparently abundant amounts of observable variation and species diversity, the order Lepidoptera exhibits a morphological homogeneity that has provided only a limited number of taxonomic characters and led to widespread use of nucleotides for inferring relationships. This study aims to characterize and develop methods to quantify the value of priority gene regions designated for Lepidoptera molecular systematics. In particular, I assess how the DNA barcode segment of the mitochondrial COI gene performs across a broad temporal range given its number one position of priority, most sequenced status, and the conflicting opinions on its phylogenetic performance.
Gene regions commonly sequenced for lepidoptera phylogenetics were scored using multiple measures across three categories: practicality, which includes universality of primers and sequence quality; phylogenetic utility; and phylogenetic signal. I found that alternative measures within a category often appeared correlated, but high scores in one category did not necessarily translate into high scores in another. The DNA barcode was easier to sequence than other genes, and had high scores for utility but low signal above the genus level.
Given limited financial resources and time constraints, careful selection of gene regions for molecular phylogenetics is crucial to avoid wasted effort producing partially informative data. This study introduces an approach to assessing the value of gene regions prior to the initiation of new studies and presents empirical results to help guide future selections.
We describe and compare the exochorion of six species of Hypanartia (H. paullus, H. bella, H. lethe, H. godmanii, H. dione disjuncta, and H. trimaculata autumna) using specimens collected in the field and deposited in entomological collections. We used a standard staining technique and scanning electron microscopy to visualize and describe the main exochorionic characters, especially the ridges and the differentiation in the micropylar, perimicropylar, and transition zones in the apical region. We included plates with photographs, drawings, and schemes for clearer visualization of these structures, which are tabulated for comparison. For our characterization, we considered the ridges in colonnade as the main exochorionic feature in the Nymphalini. Thus, we made brief comparisons of these features among several species of the tribe, particularly with the genus Antanartia. The exochorionic characters agree with the separation of groups (paullus group and dione group).
Molecular comparison of species belonging to the genus Junonia collected from Myanmar was completed using mtDNA sequence data from 605-bp cytochrome oxidase subunit I (COI). Six species were sequenced, aligned, and used to construct phylogenetic trees. The base composition of the COI sequences was 37.8% T, 15.4% C, 31.4% A, and 15.4% G, revealing strong AT bias (69.2%). The sequence distance of Junonia ranged from 1.5% to 9.0%. Nucleotide substitution primarily occurred through transition rather than transversion.. Phylogenetic trees were constructed by the neighbor-joining (NJ) and maximum likelihood (ML) methods, using Hypolimas misippus as out group. Both trees had almost identical topologies. All COI sequences of each species fell in the same cluster as those of the same species obtained from the NCBI. Species in Junonia exhibited the following relationships: (((J. orithya + J. hierta) + J. lemonias lemonias) + J. almana almana) + (J. atlites + J. iphita). The clustering results were almost identical to current morphological classification.
Fragments of the mitochondrial 16S rRNA gene were amplified and sequenced for 27 species from 7 subfamilies of the Nymphalidae and 2 species of Danaidae. Combining these with the homologous sequences of 6 species of Nymphalidae obtained from the GenBank, their sequence variations were analyzed and their phylogenetic trees were reconstructed, in an attempt to clarify their phylogenetic relationships. The results of sequence variations showed that the length of the aligned fragments was 494 bp, including 206 variable and 145 parsimonious informative sites. The percentages of A + T and C+ G were 781 4% and 2116% , respectively. Using Parantica aglea and Euploea core as outgroups, the phylogenetic trees were reconstructed with NJ and Bayesian methods. Results of phylogenetic analyses showed that the Nymphalinae is not monophyletic, and that Junonia should be classified into Hypolimni. The Apaturinae, Byblinae and Nymphalinae are closely related to each other, and the Argynninae and Heliconniinae should be categorized into one subfamily. The Pseudergolinae and Byblinae should be separated from the Limenitinae [ Acta Zoologica Sinica 53 (1) : 106-115, 2007] .
We present a molecular phylogeny of butterflies belonging to the genus Euphydryas s.l., based on partial sequences of 3 mtDNA genes (COI, ND1, and 16S). Euphydryas s.l. has been divided into 4 genera in an earlier revision. The current results show 3 well-supported groups within the genus, corresponding to 3 of the 4 proposed genera. The pair-wise divergences of 1 of the sequences (COI) are on the order of 3% within the 3 groups and 7% between the groups. This level of variation leads us to suggest that the entire group should be considered to be in the genus Euphydryas with 3 subgenera: Euphydryas, Hypodryas, and Eurodryas.
The Ca2+-dependent cell-cell adhesion molecules, termed cadherins, are subdivided into several subclasses. E (epithelial)-and P (placental)- cadherins are involved in the selective adhesion of epidermal cells. E- cadherin is expressed on the cell surfaces of all epidermal layers and P- cadherin is expressed only on the surfaces of basal cells. Ultrastructural studies have shown that E-cadherin is distributed on the plasma membranes of keratinocytes with a condensation in the intercellular space of the desmosomes. During human skin development P-cadherin expression is spatiotemporally controlled and closely related to the segregation of basal layers as well as to the arrangement of epidermal cells into eccrine ducts. In human skin diseases E-cadherin expression is markedly reduced on the acantholytic cells of tissues in pemphigus and Darier's disease. Cell adhesion molecules are now considered to play a significant role in the cellular connections of cancer and metastatic cells. Reduced expression of E- cadherin on invasive neoplastic cells has been demonstrated for cancers of the stomach, liver, breast, and several other organs. This reduced or unstable expression of E- and P-cadherin is observed in squamous cell carcinoma, malignant melanoma, and Paget's disease, but cadherin expression is conserved in basal cell carcinoma. Keratinocytes cultured in high calcium produce much more intense immunofluorescence of intercellular E- and P- cadherin than those cells grown in low calcium. E-cadherins on the plasma membrane of the keratinocytes are shifted to desmosomes under physiological conditions, and therein may express an adhesion function in association with other desmosomal cadherins. Soluble E-cadherins in sera are elevated in various skin diseases including bullous pemphigoid, pemphigus vulgaris, and psoriasis, but not in patients with burns. Markedly high levels in soluble E- cadherin are demonstrated in patients with metastatic cancers.
Unlabelled:
We have developed a new software package, Molecular Evolutionary Genetics Analysis version 2 (MEGA2), for exploring and analyzing aligned DNA or protein sequences from an evolutionary perspective. MEGA2 vastly extends the capabilities of MEGA version 1 by: (1) facilitating analyses of large datasets; (2) enabling creation and analyses of groups of sequences; (3) enabling specification of domains and genes; (4) expanding the repertoire of statistical methods for molecular evolutionary studies; and (5) adding new modules for visual representation of input data and output results on the Microsoft Windows platform.
Availability:
http://www.megasoftware.net.
Contact:
s.kumar@asu.edu
Tissue concentrations of ATP, glucose, and lactate in cervical cancer biopsies that were taken before a conventional radiation treatment were imaged quantitatively with a bioluminescence technique. Concomitantly, a number of clinically relevant data, such as local tumor control, patient survival, metastatic spread, etc., were documented. There was no correlation between staging or grading and any of the metabolic parameters measured. Local correlations between ATP, glucose, and lactate on a pixel-to-pixel basis were generally positive, with respective Spearman's correlation coefficients being lower in patients without clinically documented metastasis compared to those with metastatic spread. Lactate concentrations were significantly higher and scattered over a wider range in tumors with metastatic spread in comparison to malignancies in patients without metastasis. Thus, high local lactate levels of > or = 20 mumole/g appear to be associated with a high risk of metastasis, at least in the ten human cervical tumors investigated to date.
We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
To study the phylogenetic relationships among Anseriformes, sequences for the complete mitochondrial control region (CR) were determined from 45 waterfowl representing 24 genera, i.e., half of the existing genera. To confirm the results based on CR analysis we also analyzed representative species based on two mitochondrial protein-coding genes, cytochrome b (cytb) and NADH dehydrogenase subunit 2 (ND2). These data allowed us to construct a robust phylogeny of the Anseriformes and to compare it with existing phylogenies based on morphological or molecular data. Chauna and Dendrocygna were identified as early offshoots of the Anseriformes. All the remaining taxa fell into two clades that correspond to the two subfamilies Anatinae and Anserinae. Within Anserinae Branta and Anser cluster together, whereas Coscoroba, Cygnus, and Cereopsis form a relatively weak clade with Cygnus diverging first. Five clades are clearly recognizable among Anatinae: (i) the Anatini with Anas and Lophonetta; (ii) the Aythyini with Aythya and Netta; (iii) the Cairinini with Cairina and Aix; (iv) the Mergini with Mergus, Bucephala, Melanitta, Callonetta, Somateria, and Clangula, and (v) the Tadornini with Tadorna, Chloephaga, and Alopochen. The Tadornini diverged early on from the Anatinae; then the Mergini and a large group that comprises the Anatini, Aythyini, Cairinini, and two isolated genera, Chenonetta and Marmaronetta, diverged. The phylogeny obtained with the control region appears more robust than the one obtained with mitochondrial protein-coding genes such as ND2 and cytb. This suggests that the CR is a powerful tool for bird phylogeny, not only at a small scale (i.e., relationships between species) but also at the family level. Whereas morphological analysis effectively resolved the split between Anatinae and Anserinae and the existence of some of the clades, the precise composition of the clades are different when morphological and molecular data are compared.
MrBayes 3 performs Bayesian phylogenetic analysis combining information from different data partitions or subsets evolving under different stochastic evolutionary models. This allows the user to analyze heterogeneous data sets consisting of different data types—e.g. morphological, nucleotide, and protein—and to explore a wide variety of structured models mixing partition-unique and shared parameters. The program employs MPI to parallelize Metropolis coupling on Macintosh or UNIX clusters.
Availability: http://morphbank.ebc.uu.se/mrbayes
Contact: fredrik.ronquist@ebc.uu.se
*
To whom correspondence should be addressed.
Roles of cell adhesion molecules mediating the interaction of cancer and endothelial cells in tumor angiogenesis were investigated using new in vitro and in vivo model systems with a cultured murine endothelial cell line (F-2) and human cultured epidermoid cancer cells (A431). The A431 cells exhibited typical in vitro cell adhesion to the endothelial F-2 cells. The initial step of adhesion was mediated by sialyl Lewis(x) (Le(x)) and sialyl Le(a), the carbohydrate determinants expressed on the cancer cells, and E-selectin expressed constitutively on F-2 cells. Prolonged culture led to the implantation of cancer cells into the monolayer of the F-2 cells, which was mediated mainly by alpha(3)beta(1)-integrin. F-2 cells cultured on Matrigel showed evident tube formation, and coculture of F-2 cells with A431 cells led to the formation of A431 cell nests constantly surrounded by tube-like networks consisting of F-2 cells. This in vitro morphogenesis was inhibited by the addition of anti-sialyl Le(x)/Le(a) or anti-beta(1)-integrin antibodies, which led to the formation of cancer cell aggregates that were independent from the F-2 cell networks. This in vitro morphological appearance was exactly reproduced in the in vivo tumors, which were formed when the mixture of A431 and F-2 cells at the ratio of 10:1 were cotransplanted s.c. into the back of nude rats. The tumors of A431 supplemented with F-2 cells were profoundly vascularized throughout by the tubular structures formed by F-2 cells, the lumen of which contained the host rat blood cells. The tumor mass thus formed was an average 5.8-fold as large as control A431 tumors that were grown without F-2 cells. The co-injection of anti-Le(x)/Le(a) or anti-beta(1)-integrin antibodies produced a marked reduction in the size of A431 tumors, which were not vascularized and accompanied an independent tiny remnant clump of F-2 cells. The size of these A431 tumors did not differ significantly from those of control A431 tumors raised without F-2 cells. These results indicate that the interaction of tumor cells and endothelial cells in orderly tumor angiomorphogenesis is highly dependent on the action of cell adhesion molecules mediating the adhesion of cancer cells to endothelial cells, inhibition of which remarkably retards tumor growth and angiogenesis.
The HyPhypackage is designed to provide a flexible and unified platform for carrying out likelihood-based analyses on multiple alignments
of molecular sequence data, with the emphasis on studies of rates and patterns of sequence evolution.
Availability: http://www.hyphy.org
Contact: muse{at}stat.ncsu.edu
Supplementary information: HyPhydocumentation and tutorials are available at http://www.hyphy.org
The study of the historical biogeography of butterflies has been hampered by a lack of well-resolved phylogenies and a good estimate of the temporal span over which butterflies have evolved. Recently there has been surge of phylogenetic hypotheses for various butterfly groups, but estimating ages of divergence is still in its infancy for this group of insects. The main problem has been the sparse fossil record for butterflies. In this study I have used a surprisingly good fossil record for the subfamily Nymphalinae (Lepidoptera: Nymphalidae) to estimate the ages of diversification of major lineages using Bayesian relaxed clock methods. I have investigated the effects of varying priors on posterior estimates in the analyses. For this data set, it is clear that the prior of the rate of molecular evolution at the ingroup node had the largest effect on the results. Taking this into account, I have been able to arrive at a plausible history of lineage splits, which appears to be correlated with known paleogeological events. The subfamily appears to have diversified soon after the K/T event about 65 million years ago. Several splits are coincident with major paleogeological events, such as the connection of the African and Asian continents about 21 million years ago and the presence of a peninsula of land connecting the current Greater Antilles to the South American continent 35 to 33 million years ago. My results suggest that the age of Nymphalidae is older than the 70 million years speculated to be the age of butterflies as a whole.
A new species of the genus Ogovea is described and illustrated, Ogovea cameroonensis n. sp. from Yaoundé. This species considerably enlarges the distribution range of the family Ogoveidae towards the Northeast. The new species, only known from its type locality in Cameroon, is the first described cyphophthalmid for that country. A new diagnosis for the family Ogoveidae is provided and a putative relationship of Ogovea with the South American genus Huitaca is rejected.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
The biology of Heliconius butterflies has provided a rich source of data to test theories of ecological genetics, coevolution and community ecology. Many putatively adaptive characters have been discussed with reference to a phylogenetic hypothesis based on a variety of morphological and life-history traits interpreted from an evolutionary taxonomic perspective. Here, alternate interpretations of characters on the traditional tree and a more recent mitochondrial DNA cladogram with a substantially different topology are compared and contrasted. It is shown that many characters ostensibly providing support for the traditional phylogenetic hypothesis are almost equally parsimoniously distributed and in some cases more parsimoniously distributed on the mtDNA tree than on the tree inferred from those characters. Discussion of alternate evolutionary scenarios based on the mDNA-based topology is presented for pupal mating, pollen feeding, foodplant coevolution, and other ecologically significant features.
Cancer is a multistep process and occurs as a result of the loss of control of cell division, leading to the initial tumor formation, which is followed by metastatic spread. Recent years have witnessed a vast improvement in the understanding of the molecular mechanisms regulating cell division and their links to tumorigenesis. The process of metastasis involves an intricate interplay between cell adhesion, proteolysis, migration, and angiogenesis. However, there is little knowledge of how these events are coordinately regulated in the tumor cell. Given that the uncontrolled spread of the tumor to distant organs is usually lethal, a study of the molecular mechanisms regulating metastasis assumes great significance. Recently, several technologies have been developed for analyzing differential gene expression. The current review discusses the importance of these technologies in the molecular analyses of metastasis. Cancer 2002;94:1821–9. © 2002 American Cancer Society.
DOI 10.1002/cncr.10362
We report a cladistic analysis of 77 butterfly species of the tribe Melitaeini (Lepidoptera: Nymphalidae) based on mitochondrial DNA gene sequences. We sequenced ca. 536 bp from the 16S ribosomal DNA (rDNA) and a 1422-bp sequence from the cytochrome oxidase I gene. Alignments are critical to statements of homology, especially when aligning rDNA sequences. We aligned the 16S sequences using conventional methods and direct optimization. We found that direct optimization of the sequences produced the best alignments and our preferred phylogenetic hypothesis. Our results suggest that many of the previously proposed genera are paraphyletic and we conclude that there are four monophyletic groups of species in our cladogram: the Euphydryas group, the Phyciodes group, the Chlosyne group, and the Melitaea group. The following genera are found to be paraphyletic: Castilia, Chlosyne, Didymaeformia, Eresia, Melitaea, and Thessalia. In addition, recognition of the monophyletic genera Cinclidia, Mellicta, and Telenassa would render other genera paraphyletic. Our phylogenetic hypothesis indicates that the melitaeines originated in the Nearctic and have colonized the Neotropics three times and the Palaearctic twice.
DURING the past sixteen years more than three thousand cancers have been removed by means of the microscopically controlled excisions which characterize the method which we have termed "chemosurgery."1 A striking observation repeatedly made during the course of these excisions was that many external cancers exhibited irregular outgrowths which had not been detected by clinical visualization and palpation.
In some cases these "silent" extensions were due to a peculiar affinity of the cancer for some specific tissue structure which it would follow selectively for unexpectedly great distances. Some structures for which certain cancers showed particular affinity were the following: (1) dermis, (2) fascial planes, (3) periosteum, (4) perichondrium, (5) embryologic fusion planes, (6) nerve sheaths, (7) lymphatic vessels, and (8) blood vessels. In other cases no specific affinities were observed, but nevertheless irregular outgrowths were present, apparently as a result of the countless local variations in
Squamous cell skin cancer, though common, remains largely unreported and unstudied, with little known about its incidence and time trends. We have used a unique resource—a continuous population-based registry of cases of squamous cell skin cancer within a single prepaid health plan—to describe basic epidemiologic features of this malignancy and compare it with the more widely studied melanoma. Both malignancies are considerably more common in this population than we expected based on previous reports from the general population. From the 1960s to the 1980s, the incidence of squamous cell skin cancer increased 2.6 times in men and 3.1 times in women, while incidence of melanoma rose 3.5-fold and 4.6-fold in men and women, respectively. Skin cancers of both types involving the head and neck or the extremities increased essentially in parallel over these 27 years. Melanomas of the trunk, however, appeared to increase at a faster rate in both sexes. These observations are consistent with the impression that the rising incidence of both malignancies may be attributable to increased voluntary exposure to the sun over an extended period.
(JAMA. 1989;262:2097-2100)
Part of the mitochondrial Cytochrome Oxidase I gene was sequenced for seven species ofGonepteryx(Pieridae) butterflies. Four of the species are island endemics inhabiting the Canary Island archipelago and Madeira. The remaining three are European and African conspecifics. Sequence data were analysed phylogenetically by maximum parsimony and maximum likelihood methods. The resulting trees were used to deduce Canarian species» ancestry, sequential inter-island colonization and systematics. They suggest African ancestry for the Canary Island taxa and a colonization pattern, within the archipelago, compatible with the geological ages of the islands and other Canarian fauna: a colonization sequence from Africa to Tenerife and Gomera, followed by Tenerife to La Palma. The molecular phylogeny indicated that there are three Canarian endemics,G. cleobule,G. palmaeandG. eversifrom Tenerife, La Palma and Gomera, respectively.
Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. C SIMON, F FRATI, A BECKENBACH, B CRESPI, H LIU, P
The biology of Heliconius butterflies has provided a rich source of data to test theories of ecological genetics, coevolution and community ecology. Many putatively adaptive characters have been discussed with reference to a phylogenetic hypothesis based on a variety of morphological and life-history traits interpreted from an evolutionary taxonomic perspective. Here, alternate interpretations of characters on the traditional tree and a more recent mitochondrial DNA cladogram with a substantially different topology are compared and contrasted. It is shown that many characters ostensibly providing support for the traditional phylogenetic hypothesis are almost equally parsimoniously distributed and in some cases more parsimoniously distributed on the mtDNA tree than on the tree inferred from those characters. Discussion of alternate evolutionary scenarios based on the mDNA-based topology is presented for pupal mating, pollen feeding, foodplant coevolution, and other ecologically significant features.
In reviewing the hostplant associations of nymphalid butterflies, particular emphasis is placed on the intractable problem of nymphalid classification. Although offering few certain conclusions, if used in conjunction with more formal morphological characters, the data presented should contribute toward a resolution of the inter-relationships of the many widely recognized groupings within the Nymphalidae, several of which seem to be broadly characterized by typical host families. As a direct result of this analysis, the presumed association between larval hostplants and unpalatability is re-appraised.
Seasonal polyphenism is studied in a community of five African butterflies of the genus Bicyclus at the transition between a wet and a dry season from May to July.
Butterflies characterized by large eyespots and, especially in B.sufitza (Hewitson), a pale band (the wet season form) are replaced over this period by butterflies lacking conspicuous wing markings (the dry season form, dsf). The latter butterflies also tend to be larger, but more variable in size. Butterflies of an intermediate phenotype are recruited over a comparatively short interim period.
This turnover coincides with a period of declining temperature and drying of the habitat, including the grasses on which larvae feed. Butterflies are progressively more likely to rest on brown leaf litter rather than on green herbage.
A relationship with temperature is supported by laboratory experiments with B.saJitza and B.anynana (Butler) showing that increasingly extreme dsf butterflies develop with decreasing rearing temperature in the final larval instar.
Some differences in behaviour and activity were observed between the seasonal forms. Butterflies of the dsf develop ovarian dormancy and fat bodies. They can survive to reproduce at the beginning of the rains in November.
Capture‐recapture experiments showed that the adult butterflies have a comparatively long life expectancy and are quite sedentary.
The results are discussed in relation to a hypothesis linking the polyphenism to seasonal changes in resting background and selection for crypsis.
Part of the mitochondrial Cytochrome Oxidase I gene was sequenced for seven species of Gonepteryx (Pieridac) butterflies. Four of the species are island endemics inhabiting the Canary Island archipelago and Madeira. The remaining three are European and African conspecifics. Sequence data were analysed phylogenetically by maximum parsimony and maximum likelihood methods. The resulting trees were used to deduce Canarian species' ancestry, sequential inter-island colonization and systematics. They suggest African ancestry for the Canary Island taxa and a colonization pattern, within the archipelago, compatible with the geological ages of the islands and other Canarian fauna: a colonization sequence from Africa to Tenerife and Gomera, followed by Tenerife to La Palma. The molecular phylogeny indicated that there are three Canarian endemics, G. cleobule, G. palmae and G. eversi from Tenerife, La Palma and Gomera, respectively.
We report a cladistic analysis of 77 butterfly species of the tribe Melitaeini (Lepidoptera: Nymphalidae) based on mitochondrial DNA gene sequences. We sequenced ca. 536 bp from the 16S ribosomal DNA (rDNA) and a 1422-bp sequence from the cytochrome oxidase I gene. Alignments are critical to statements of homology, especially when aligning rDNA sequences. We aligned the 16S sequences using conventional methods and direct optimization. We found that direct optimization of the sequences produced the best alignments and our preferred phylogenetic hypothesis. Our results suggest that many of the previously proposed genera are paraphyletic and we conclude that there are four monophyletic groups of species in our cladogram: the Euphydryas group, the Phyciodes group, the Chlosyne group, and the Melitaea group. The following genera are found to be paraphyletic: Castilia, Chlosyne, Didymaeformia, Eresia, Melitaea, and Thessalia. In addition, recognition of the monophyletic genera Cinclidia, Mellicta, and Telenassa would render other genera paraphyletic. Our phylogenetic hypothesis indicates that the melitaeines originated in the Nearctic and have colonized the Neotropics three times and the Palaearctic twice.
We infer for the first time the phylogenetic relationships of genera and tribes in the ecologically and evolutionarily well-studied subfamily Nymphalinae using DNA sequence data from three genes: 1450 bp of cytochrome oxidase subunit I (COI) (in the mitochondrial genome), 1077 bp of elongation factor 1-alpha (EF1-) and 400–403 bp of wingless (both in the nuclear genome). We explore the influence of each gene region on the support given to each node of the most parsimonious tree derived from a combined analysis of all three genes using Partitioned Bremer Support. We also explore the influence of assuming equal weights for all characters in the combined analysis by investigating the stability of clades to different transition/transversion weighting schemes. We find many strongly supported and stable clades in the Nymphalinae. We are also able to identify ‘rogue’ taxa whose positions are weakly supported (the different gene regions are in conflict with each other) and unstable. Our main conclusions are: (1) the tribe Coeini as currently constituted is untenable, and Smyrna, Colobura and Tigridia are part of Nymphalini; (2) ‘Kallimini’ is paraphyletic with regard to Melitaeini and should be split into three tribes: Kallimini s.s., Junoniini and Victorinini; (3) Junoniini, Victorinini, Melitaeini and the newly circumscribed Nymphalini are strongly supported monophyletic groups, and (4) Precis and Junonia are not synonymous or even sister groups. The species Junonia coenia, a model system in developmental biology, clearly belongs in the genus Junonia. A dispersal-vicariance analysis suggests that dispersal has had a major effect on the distributions of extant species, and three biotic regions are identified as being centres of diversification of three major clades: the Palaearctic for the Nymphalis-group, the Afrotropics for Junoniini and the Nearctic for Melitaeini. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 227–251.
Despite the fact that Bicyclus anynana has become an important model species for wing-pattern developmental biology and studies of phenotypic plasticity, little is known of the evolutionary history of the genus Bicyclus and the position of B. anynana. Understanding the evolution of development as well as the evolution of plasticity can be attempted in this species-rich genus that displays a large range of wing patterns with variable degrees of phenotypic responses to the environment. A context to guide extrapolations from population genetic studies within B. anynana to those between closely related species has been long overdue. A phylogeny of 54 of the 80 known Bicyclus species is presented based on the combined 3000-bp sequences of two mitochondrial genes, cytochrome oxidase I and II, and the nuclear gene, elongation factor 1α. A series of tree topologies, constructed either from the individual genes or from the combined data, using heuristic searches under a variety of weighting schemes were compared under the best maximum-likelihood models fitted for each gene separately. The most likely tree topology to have generated the three data sets was found to be a tree resulting from a combined MP analysis with equal weights. Most phylogenetic signal for the analysis comes from silent substitutions at the third position, and despite the faster rate of evolution and higher levels of homoplasy of the mitochondrial genes relative to the nuclear gene, the latter does not show substantially stronger support for basal clades. Finally, moving branches from the chosen tree topology to other positions on the tree so as to comply better with a previous morphological study did not significantly affect tree length.
The striking impression obtained from reviewing the cancer literature is how difficult it is to analyze the data for answers to many important biologic, behavioral, prognostic, and therapeutic questions about squamous cell carcinoma of the skin. This article addresses current concepts, controversies, and management of cutaneous squamous cell carcinoma (excluding the lip and oral mucosa).
We reviewed all studies since 1940 on the prognosis of squamous cell carcinoma (SCC) of the skin and lip. The following variables are correlated with local recurrence and metastatic rates: (1) treatment modality, (2) prior treatment, (3) location, (4) size, (5) depth, (6) histologic differentiation, (7) histologic evidence of perineural involvement, (8) precipitating factors other than ultraviolet light, and (9) host immunosuppression. Local recurrences occur less frequently when SCC is treated by Mohs micrographic surgery. This local recurrence rate differential in favor of Mohs micrographic surgery holds true for primary SCC of the skin and lip (3.1% vs 10.9%), for ear SCC (5.3% vs 18.7%), for locally recurrent (previously treated) SCC (10% vs 23.3%), for SCC with perineural involvement (0% vs 47%), for SCC of size greater than 2 cm (25.2% vs 41.7%), and for SCC that is poorly differentiated (32.6% vs 53.6%).
A metachronous skeletal muscle secondary from a primary squamous cell carcinoma on the back of the hand manifested as an area of cellulitis of the forearm. Such clinical behaviour does not appear to have been previously described.
Cutaneous squamous cell carcinomas (SCC) arising in actinically damaged skin, unassociated with chronic inflammation or injury, are generally regarded as nonaggressive lesions. These tumors occasionally recur or metastasize, however, as do de novo SCC. The authors reviewed 63 patients with cutaneous SCC of the trunk or extremities, excluding lesions that developed in known high risk settings, in order to explore the potential of histologic microstaging as a prognostic indicator. Fifty-four patients (86%) were free of recurrence following primary surgical therapy. Nine patients (14%) had either local recurrence or metastases; five of these (8% of the entire series) died of their tumors. Tumor behavior correlated best with the level of dermal invasion and the vertical tumor thickness. All tumors that recurred were 4 mm or more thick and involved the deep half of the dermis or deeper structures. All tumors that proved fatal were at least 10 mm in maximum thickness, and the four lethal lesions that could be evaluated for level of invasion extended into subcutaneous tissue or deeper structures. The thickness and level of invasion of cutaneous SCC appear to represent important prognostic factors and may be relevant indicators for wide field resection and/or elective lymph node dissection.
In summary, MR imaging is the preferred modality for the evaluation of a soft tissue mass after plain films have been taken. The radiologic appearance of certain soft tissue tumors or tumorlike processes such as myositis ossificans, benign fatty tumors, intramuscular hemangiomas, pigmented villonodular synovitis, and certain hematomas may be sufficiently unique to allow a strong presumptive radiologic diagnosis. It must be emphasized that MR cannot reliably distinguish between benign and malignant lesions, and when radiologic evaluation is nonspecific, one is ill advised to suggest a lesion is benign or malignant solely on the basis of its MR appearance. CT may be useful in specific instances for the identification of subtle soft tissue mineralization in those patients in whom lesions are not adequately evaluated by radiographs. Ultrasonography may be useful in the assessing of recurrent disease as well as in establishing tumor vascularity.
The acquisition of an invasive or metastatic phenotype in malignant neoplasms is often correlated with reduced cellular adhesiveness. We investigated the expression of the adhesion-associated cytoplasmic protein, vinculin, in normal and neoplastic human squamous epithelia, as well as in metastases of squamous cell carcinomas, and correlated the results with invasiveness and metastatic potential. Tissue samples from various tumors were examined, including basal cell carcinomas (BCC), keratoacanthomas, and squamous cell carcinomas (SCC). In addition, lymph node metastases from nine of the SCC were tested in this study. Our results indicate that most BCC, keratoacanthomas, and in situ SCC display strong positive staining for vinculin. The level of immunolabeling for vinculin and its pattern of distribution in the low malignant, nonmetastasizing lesions was similar to those observed in normal squamous epithelia. In contrast, in SCC, which are invasive and possess metastatic potential, as well as in their metastases, vinculin labeling was negative or poor, irrespective of their degree of differentiation. In conclusion, poor vinculin labeling in tumors of squamous epithelial origin examined here appears to be related to the metastatic potential of the tumor. Vinculin immunostaining of primary tumors originating in stratified squamous epithelia may thus be of value in helping to determine the metastatic potential of these neoplasms.
We studied second primary cancer among 25,947 patients diagnosed with squamous cell carcinoma of the skin (SCC) in Sweden between 1958 and 1992. In total, 5,706 patients developed a second primary cancer at any site, compared with an expected number of 2,651 [standardized incidence ratio (SIR) = 2.15; 95% confidence interval (CI) = 2.10-2.21]. Men below 60 years of age at diagnosis of SCC had higher SIR (2.5; CI = 2.2-2.8) with the highest risk during the first year of follow-up (SIR = 9.2; CI = 6.9-12.2). If second primary SCC was excluded, the SIR was reduced to 1.30 (CI = 1.25-1.34); the relationships by sex, age and time since diagnosis remained similar. For skin cancer, the SIR for second SCC was markedly elevated (SIR = 15.6) and the risk of malignant melanoma was elevated 3-fold. Significantly increased risks were found for most second cancers in squamous cell epithelium: lip (SIR = 5.2), respiratory organs (SIR = 1.7), esophagus (SIR = 1.5), cervix uteri (SIR = 2.2), and vulva including vagina (SIR = 2.3). There was a generally increased risk of almost 2-fold for second cancer in hematopoietic or lymphoproliferative tissues. Slightly increased rates (SIR = 1.0-1.5) were seen for second tumors in digestive tissues. Finally, a high SIR (SIR = 5.5) was observed for second primary cancer in salivary glands. In conclusion, patients with SCC are at increased risk to develop new primary cancer, especially in skin, squamous cell epithelial and tobacco-related tissues. Common risk factors among the tumor types might explain our findings, however, an intrinsic susceptibility among SCC patients to develop cancer is also possible.
Metastatic squamous cell carcinoma of the soft tissue is extremely rare. We report two patients with soft tissue metastasis from squamous cell carcinoma of the cervix in one and scalp in another. Case 1: A 63-year-old black woman with a history of cervical cancer presented with a painful mass over the right scapula. An incisional biopsy revealed squamous cell carcinoma. She underwent radiation treatment followed by wide local excision with en bloc resection and a myocutaneous flap closure. Case 2: A 46-year-old white man with a history of squamous cell carcinoma of the scalp and two kidney transplantations requiring long-term immunosuppression presented with a 2-month history of a left proximal arm mass. Magnetic resonance imaging revealed that the mass was within the triceps muscle and fixed to the humerus. Biopsy revealed squamous cell carcinoma. He underwent a shoulder disarticulation for tumor invasion into the adjacent humerus and neurovascular bundles. The patients remain disease-free at 12 and 8 months, respectively. To our knowledge there are no reports of soft tissue squamous cell carcinoma metastatic from the cervix and only rare cases from the lung, head, and neck. The optimal mode of treatment and prognosis is undefined in these patients because of its rare incidence. Surgery and radiation with curative intent were used.
Phylogenetic relationships among Cotesia Cameron (Braconidae) species parasitising Melitaeini butterflies were examined using DNA sequence data (mitochondrial cytochrome oxidase subunit I and NADH1 dehydrogenase genes, nuclear ribosomal DNA internal transcribed spacer region) as well as 12 microsatellite loci. Molecular data were available from ostensibly six species of Cotesia from 16 host butterfly species in Europe, Asia, and North America. Analysis of the combined sequence data using both maximum parsimony and maximum likelihood revealed two distinct Cotesia clades. In one clade (C. acuminata (Reinhard); C. bignellii (Marshall)) host ranges are apparently narrow and, although Euphydryas (s. lato) is well-utilised, permeation of Melitaea (s. lato) has been slight. In the other clade (C. melitaearum (Wilkinson); C. lycophron (Nixon); C. cynthiae (Nixon)) host utilization across the Melitaeini as a whole is more extensive and the data are consistent with more recent, or active, speciation processes. Neighbour-joining trees calculated separately for the two main clades based on chord distance (DCE) of microsatellite allele frequencies were consistent with phylogenetic trees obtained from the sequence data. Our analysis strongly suggests the presence of several additional, previously unrecognised, Cotesia species parasitising this group of butterflies.
The Euptychiina is one of the more diverse lineages of satyrine butterflies, represented by over 300 species. The first phylogenetic analyses of the subtribe is presented based on 2506 aligned nucleotide sequences obtained from 69 individuals spanning 28 ingroup genera and nine outgroup genera. Two genes were used, the mitochondrial gene cytochrome oxidase 1 (1268 bp) and the nuclear gene elongation factor-1alpha (1238 bp). The subtribe is never recovered as monophyletic in analyses using parsimony, maximum likelihood, or Bayesian inference. Several euptychiine genera are placed basal to the ingroup, but support is found only for Euptychia and Oressinoma. Three main lineages within the ingroup were clearly defined and many taxonomic groupings within the clades strongly supported. The majority of genera tested were paraphyletic or polyphyletic. Based on results presented here and novel host use, a close relationship of Euptychia to the Indo-Australian tribe Ragadiini is hypothesized. Origins of the group remain unclear, but the basal position of most of the Nearctic genera is discussed.
The history of the development of statistical hypothesis testing in time series analysis is reviewed briefly and it is pointed out that the hypothesis testing procedure is not adequately defined as the procedure for statistical model identification. The classical maximum likelihood estimation procedure is reviewed and a new estimate minimum information theoretical criterion (AIC) estimate (MAICE) which is designed for the purpose of statistical identification is introduced. When there are several competing models the MAICE is defined by the model and the maximum likelihood estimates of the parameters which give the minimum of AIC defined by AIC = (-2)log-(maximum likelihood) + 2(number of independently adjusted parameters within the model). MAICE provides a versatile procedure for statistical model identification which is free from the ambiguities inherent in the application of conventional hypothesis testing procedure. The practical utility of MAICE in time series analysis is demonstrated with some numerical examples.