The diversification of Heliconius butterflies: what have we learned in 150 years?

Research into Heliconius butterflies has made a significant contribution to evolutionary biology. Here, we review our understanding of the diversification of these butterflies, covering recent advances and a vast foundation of earlier work. Whereas no single group of organisms can be sufficient for understanding life's diversity, after years of intensive study, research into Heliconius has addressed a wide variety of evolutionary questions. We first discuss evidence for widespread gene flow between Heliconius species and what this reveals about the nature of species. We then address the evolution and diversity of warning patterns, both as the target of selection and with respect to their underlying genetic basis. The identification of major genes involved in mimetic shifts, and homology at these loci between distantly related taxa, has revealed a surprising predictability in the genetic basis of evolution. In the final sections, we consider the evolution of warning patterns, and Heliconius diversity more generally, within a broader context of ecological and sexual selection. We consider how different traits and modes of selection can interact and influence the evolution of reproductive isolation.

Contrasting patterns of genetic diversity at three different genetic markers in a marine mammal metapopulation.

Many studies use genetic markers to explore population structure and variability within species. However, only a minority use more than one type of marker and, despite increasing evidence of a link between heterozygosity and individual fitness, few ask whether diversity correlates with population trajectory. To address these issues, we analysed data from the Steller's sea lion, Eumetiopias jubatus, where three stocks are distributed over a vast geographical range and where both genetic samples and detailed demographic data have been collected from many diverse breeding colonies. To previously published mitochondrial DNA (mtDNA) and microsatellite data sets, we have added new data for amplified fragment length polymorphism (AFLP) markers, comprising 238 loci scored in 285 sea lions sampled from 23 natal rookeries. Genotypic diversity was low relative to most vertebrates, with only 37 loci (15.5%) being polymorphic. Moreover, contrasting geographical patterns of genetic diversity were found at the three markers, with Nei's gene diversity tending to be higher for AFLPs and microsatellites in rookeries of the western and Asian stocks, while the highest mtDNA values were found in the eastern stock. Overall, and despite strongly contrasting demographic histories, after applying phylogenetic correction we found little correlation between genetic diversity and either colony size or demography. In contrast, we were able to show a highly significant positive relationship between AFLP diversity and current population size across a range of pinniped species, even though equivalent analyses did not reveal significant trends for either microsatellites or mtDNA.

Pinniped phylogenetic relationships inferred using AFLP markers.

Amplified fragment length polymorphisms (AFLPs) are widely used for phylogenetic reconstruction in plants but their use in animal taxa has been limited, and in mammals rare. In addition, their use has been largely limited to shallow relationships amongst species or subspecies. Here, we genotype 23 pinniped species for 310 AFLP markers and find a strong phylogenetic signal, with individuals coclustering within species, and overall a good agreement between our phylogeny and those constructed using mitochondrial DNA and nuclear sequences even at nodes approximately 15 million years old. Although supporting the existing ideas about pinniped relationships, our data shed light on relationships within the hitherto relatively unresolved Phocine species group, and provide further supporting evidence for raising two subspecies of Zalophus californianus, Z. c. californianus and Z. c. wollebaeki, to species level. Plotting AFLP divergence time estimates against those based on both mtDNA and nuclear sequences we find strong linear relationships, suggesting that the different markers are evolving in a clocklike fashion. These data further emphasize the utility of AFLP markers as general tools for phylogenetic reconstruction.

Estimating levels of inbreeding using AFLP markers.

In the absence of detailed pedigree records, researchers have attempted to estimate individuals' levels of inbreeding using molecular markers, generally making use of heterozygosity measures based on microsatellite markers. Here we report and validate a method for estimating an individual's inbreeding coefficient, f, using amplified fragment length polymorphism (AFLP) markers. We use simulations to confirm that our measure scales appropriately with f when allele frequencies can be estimated from a subset of outbred individuals. We also present an approach for obtaining satisfactory estimates even in the absence of an independent set of known outbred individuals from which to estimate allele frequencies. We then test our method against empirical data from 179 wild and captive-bred old-field mice, Peromyscus polionotus subgriseus, comprising pedigree-based estimates of f, along with genetic data from 94 AFLP markers and 12 microsatellites. Inbreeding estimates based on both AFLP and microsatellite markers were found to correlate strongly with pedigree-based inbreeding coefficients. Owing to their ease of amplification in any species, AFLP markers may prove to be a valuable new tool for estimating f in natural populations and for examining correlations between heterozygosity and fitness.

The use of a biodegradable mesh to prevent radiation-associated small-bowel injury.

Radiation-associated small bowel injury occurs in up to 50% of patients receiving postoperative radiotherapy following pelvic cancer surgery. We describe our experience using a biodegradable mesh that allows the small bowel to be supported above the pelvic inlet and is totally absorbed following radiation therapy. Between 1985 and 1989, 45 procedures were performed in patients with carcinoma of the rectum (anterior resection in 15 patients, abdominoperineal resection in 23 patients, pelvic exenteration in six patients, and proctocolectomy in one patient). In 30 patients a polyglycolic acid (Dexon) mesh was used, and in 15 patients a polyglactin 910 (Vicryl) mesh was used. Forty-four patients received postoperative radiotherapy. The mean (+/- SEM) dose was 56.8 +/- 18.4 Gy. There were no immediate complications related to the mesh. Follow-up ranged from 12 to 53 months (median follow-up, 34 months). With the exception of two patients who had a polyglactin 910 mesh and who developed bowel obstruction due to adhesions under the anterior abdominal wall, there has been no documented incidence of clinical radiation-associated small bowel injury. The use of the absorbable mesh may permit us to use higher doses of postoperative radiotherapy without the associated hazard of radiation-associated small bowel injury.

alpha-Difluoromethylornithine inhibits liver metastasis produced by intrasplenic injection of human tumor cells into nude mice.

The purpose of these studies was to examine metastatic potentials of a human colon tumor xenograft (T6) and three different human tumor cell lines (LS174T, HT29 and A549) using the intrasplenic-nude mouse model system (ISMS model system). A further objective was to study the activity of alpha-difluoromethyl-ornithine (DFMO) against primary and metastatic growth of the xenograft and the three cell lines. DFMO is an irreversible inhibitor of ornithine decarboxylase, a rate-limiting step in polyamine biosynthesis. Tumor burdens in the liver of nude mice were observed 6 weeks after the intrasplenic injection with LS174T and 12-14 weeks after intrasplenic injections with T6, HT29 and A549. Most of the mice developed primary tumor growth in the spleens. DFMO showed significant activity against liver metastases but had little or no activity against primary tumor growth in the spleens of the ISMS model and against s.c. growth of the xenograft. The studies demonstrated that the ISMS model system is an excellent system for studying metastatic behavior of human tumors and for studying the antimetastatic activity of experimental drugs.

Inflammatory pseudotumor of the liver.

Inflammatory pseudotumors (plasma cell granulomas) are localized lesions consisting of a fibrous stroma, chronic inflammatory infiltrate with a predominance of plasma cells, and an absence of anaplasia, which can mimic malignant disease by their gross appearance. Reported in many sites, they are most frequently described in the lung. Twelve cases involving the liver and biliary tract have previously been reported. Here, a 77-year-old man who presented with fever and malaise was found to have a lesion in the left lobe of the liver, which, on exploration, was found to be an inflammatory pseudotumor. This patient, and each of those previously reported, has recovered uneventfully. Because of the benign course of this lesion, hepatectomy should be considered only in those instances in which the mass effect of the lesion is producing symptoms or in those cases in which a firm histologic diagnosis cannot be made preoperatively or by frozen section intraoperatively.

Rationale for aggressive colonoscopy in patients with colorectal neoplasia.

The role of colonoscopy in patients with colorectal neoplasia is not well established. The results of colonoscopy, from 1982 through 1987, in 42 patients with cancers who underwent preoperative colonoscopy (group 1), 64 patients with benign polyps (group 2), and 51 patients who were examined only postoperatively (group 3) were reviewed. These results indicated that (1) approximately one third of all findings would have been missed if endoscopy had been performed to only 60 cm; (2) there was a high incidence of synchronous lesions (33.3%) in group 1 and 34.4% in group 2); (3) 57% of patients with synchronous cancer and 63.6% of patients with synchronous polyps developed metachronous lesions, compared with 10.7% and 11.9% of patients with a single lesion; (4) there was a higher incidence of metachronous lesions seen in group 3, compared with group 1; and (5) the median interval for noting metachronous lesions in patients who underwent colonoscopy preoperatively was approximately 18 months. These findings endorsed preoperative colonoscopy and aggressive follow-up in patients with colorectal tumors.

Interposition spiral saphenous vein graft bypass in bilateral simultaneous radical neck dissection.

We advocate the use of the spiral saphenous vein bypass of the IJV when bilateral radical neck dissections are performed with the sacrifice of both IJV. The technique we have described herein is uniformly successful in this setting and associated with only 30 minutes to one hour of increased operative time. This bypass will decrease the postoperative facial edema and associated complications and allow the patient to go on to radiation and chemotherapy in an expeditious and safe manner.

Red blood cell deformability in human and experimental sepsis.

Red blood cell (RBC) "deformability" is necessary for maintenance of normal microcirculation. To determine whether RBC deformability was affected in human or murine sepsis, a deformability index was determined in a human study and a murine model. Deformability was decreased postoperatively in patients with sepsis (0.49 +/- 0.12) compared with patients without sepsis (1.62 +/- 0.13) and normal control volunteers (1.51 +/- 0.17). Deformability was decreased in rats that had undergone cecal ligation and puncture (0.37 +/- 0.06) compared with that of sham-operated rats (0.76 +/- 0.12), as well as in endotoxemic rats (0.38 +/- 0.4) compared with control rats (0.82 +/- 0.11). These data suggest that RBC deformability decreases in both human and murine sepsis. This effect could be an important factor in the disordered oxygen utilization noted in human sepsis, and its correction could lead to better tissue oxygenation and preserved organ function.

Unsuspected perforation in bleeding duodenal ulcers.

A combination of bleeding and perforation rarely occurs simultaneously in peptic ulcer disease. The charts of 127 patients undergoing surgery for either complication were reviewed (bleeding, 91; perforation, 36). Nine of 91 (9.9%) patients in the bleeding group were found at operation to have a unsuspected perforated duodenal ulcer. The operative mortality in the patients with the combined complications (44%, 4/9) was significantly higher than that in patients with bleeding alone (8/82, 9.8, P less than 0.001) or those with perforation alone (4/36, 11.00, P less than 0.025). The mean age of nonsurvivors was significantly higher than that of the survivors (74 +/- 8.01 vs 50.4 +/- 5.65 years, P less than 0.005). The duration of symptoms until operation was longer in patients who died (63 +/- 12.7 hours) than in survivors (40.2 +/- 6.02 hours, P = NS). All patients who died, and three of five survivors, had preoperative fever (greater than 99.0 F), leukocytosis (greater than 12,000/mm3), and persistent tachycardia despite adequate hydration and blood replacement. Perforation in bleeding peptic ulcers is not an uncommon finding, and was present in 9.9 per cent of patients. The presenting symptom of bleeding may obscure signs of perforation, delay surgery, and contribute to the higher mortality rate. The presence of fever, leukocytosis, and tachycardia despite adequate fluid and blood replacement warrants a suspicion of perforation in patients with bleeding peptic ulcer.

Lung cancer in patients with head and neck cancer. Incidence and long-term survival.

Of the 3,907 cases of primary head and neck or lung cancer diagnosed between 1961 and 1984, 94 patients were identified with a history of cancer at both sites. The total incidence of lung cancer in our head and neck cancer patients was 5.4 percent. Of the 94 patients, 73 had both cancers diagnosed at our institution. These 73 patients were further analyzed. Squamous cell carcinoma accounted for 63 percent of the lung cancers. Twenty of the lung cancers were synchronous and 47 were metachronous after head and neck cancer. Of the synchronous lung cancers, 50 percent were postoperative stage I, whereas only 11 percent of the metachronous cancers were postoperative stage I. The lung cancer survival rate was significantly better for the synchronous cancer group at 5 years (34 percent) than for the metachronous cancer group (5 percent). The better survival rate was evidently due to the greater proportion of early-stage lung lesions. The relatively large number of advanced-stage lung lesions in the metachronous cancer group suggests that aggressive screening of head and neck cancer patients for lung cancer may detect more metachronous lung cancers at an earlier stage and thus improve the survival rate of these patients.

An assessment of monocyte procoagulant activity in patients with solid tumors.

Generation of thromboplastin by monocytes has been shown to play a vital role in hypercoagulable states seen in malignancy. The purpose of this study was to compare the procoagulant activity in cancer patients and controls. Recalcification times (RT) of whole blood from 19 normal volunteers, 8 patients with benign polyps, 12 patients previously treated by surgery for head and neck (H&N) or colon cancer, and 13 untreated patients with various stages of H&N or colon cancer were determined. Tests were performed with and without stimulation with Escherichia coli endotoxin. The mean RT in saline (RTS) of untreated patients with early cancer (4.58 +/- 0.83 min) and that of patients with advanced cancer (5.23 +/- 1.16 min) were lower than that of controls (6.55 +/- 0.82 min), P less than 0.01 and P less than 0.05, respectively. The RTS of patients previously treated and of those with benign polyps were no different from those of controls. Activation with endotoxin significantly lowered the recalcification times (RTE) in the early (3.90 +/- 0.58 min) and advanced cancer patients (4.23 +/- 0.66 min) compared to the RTE of controls (5.69 +/- 0.75 min, P less than 0.01 for both groups) as well as compared to those with benign tumors, P less than 0.05. The mean RTE of previously treated patients (4.72 +/- 0.58 min) was also lower than that of controls, P less than 0.05. Our results suggest that RT is significantly reduced in cancer patients compared to that of controls. Furthermore, monocyte activation with endotoxin may enable us to distinguish cancer patients from controls as well as from those with benign tumors.

Surgical endoscopy training in a university program.

Recent directives from the American Board of Surgery requires that applicants become familiar with a variety of endoscopic procedures. The purpose of this report is to review the VA Medical Center's two and a half year experience following the establishment of a surgical endoscopy unit. During the first year, July 1982-June 1983, 212 procedures were performed. These included 64 Colonoscopies (C), 98 Gastroscopies (G), 42 Flexible Sigmoidoscopies (S) and 8 Choledochoscopies (CD). The following year 564 procedures were performed (C-201, G-269, S-86, CD-8). Over the last 6 months 210 procedures were performed (C-98, G-86, S-22, CD-4). Senior surgical residents in their fourth or fifth year of training spent 4 months in the endoscopy service as part of their surgical oncology rotation. Technical and diagnostic competency was achieved in 2 to 4 months. This review indicates that the volume of cases, currently averaging 45 per month, is adequate to train surgical residents and support an independent endoscopy service. The establishment of the unit has also been associated with the development of screening programs for colon and esophageal cancers as well as broader applications of some of the newer indications of fiberoptic endoscopy. Future plans include introduction of variceal sclerotherapy and ERCP.

Evaluation of adenosine deaminase activity in patients with head and neck cancer.

ADA is a key enzyme in the mammalian purine salvage pathway. The lack of ADA activity has been linked to a lack of cellular immunity in various immune and myeloproliferative disorders. Data on its role in patients with solid tumors are scant and inconclusive. In this report, we have evaluated the activity of this enzyme in the peripheral lymphocytes of patients with head and neck squamous cell cancer (HNC). The mean ADA activity in Stage IV patients (0.57 +/- 0.08 SEM, n = 12) was significantly lower than that of controls (1.55 +/- 0.25 SEM, P less than 0.05, n = 14) and also significantly lower that the mean ADA activity in patients with Stages I, II, and III (1.14 +/- 0.10 SEM, P less than 0.05, n = 17). Fourteen out of 19 controls, Stage I, II, and III patients had positive skin tests compared to 7 out of 10 Stage IV patients. These differences were not statistically significant. There was also no correlation between ADA activity and the absolute lymphocyte counts. Our results indicate that ADA activity in lymphocytes of patients with advanced HNC is lower than that of controls or patients with earlier stages of the disease. ADA may be a more sensitive indicator of suppressed cellular immunity than delayed cutaneous hypersensitivity reactions, or monitoring absolute lymphocyte counts.

Management of squamous cell carcinoma of unknown origin in cervical lymph nodes.

Forty-three patients with the diagnosis of metastatic squamous cell carcinoma from unknown primary to the cervical lymph nodes were treated in EOVAMC between 1962-1982. Six of these patients were N1, 19 N2, and 18 N3. Five-year survival was 41 per cent for N1, 15 per cent for N2, and 6 per cent for N3 nodes. There was no significant difference in the treatment failure of those patients treated with unimodality versus multimodality. A subsequent primary was detected in the head and neck in 23 per cent of these patients (10/43), and eight of these patients had received previous radiotherapy. The most common site for these subsequent primaries was hypopharynx. Nine of the 43 patients developed distant metastasis with no correlation to stage of disease or treatment modality. These data show that the survival of these patients correlates with the stage of nodal disease, and that radiotherapy did not decrease the subsequent appearance of the primary lesions.

Cancer of the tonsil. Improved survival with combination therapy.

One hundred seventy-four cases of squamous cell cancer of the tonsil (SCCT) were reviewed. Radiation therapy (RT) alone was used in 81 patients, surgery alone (S) in 19 patients, preoperative RT + S in 49 patients, and chemotherapy [( C] methotrexate plus bleomycin plus cisplatin) in 25 patients. The 5-year survival was 83% in Stage I (n = 21), 72% in Stage II (n = 19), 23% in Stage III (n = 34), and 15% in Stage IV (n = 100). RT and S were equally effective in Stages I and II. In Stage III, the 5-year survival for RT + S was 31% versus 11% for RT alone; and in Stage IV, the respective 3- and 5-year survivals for RT + S were 24% and 15% versus 6% and 0%, respectively, for RT alone. There was an 84% response rate to C, and the patients who completed C + RT + S had 3- and 5-year survival rates of 41.7% and 32%, respectively. Our results indicate that RT + S appears to offer better survival in Stage III and IV SCCT. The high response rate in early survival data seen with C + RT + S suggests a promising role for this approach.

A prospective evaluation of 5-fluorouracil plus cisplatin in advanced squamous-cell cancer of the head and neck.

Recent studies have shown improved efficacy of chemotherapy in patients with advanced squamous-cell cancer of the head and neck. Our purpose was to evaluate prospectively the activity of cisplatin plus 5-fluorouracil (5FU) in 37 patients with advanced stage IV squamous-cell cancer of the head and neck. There were two groups. Group 1 consisted of 19 previously untreated patients with either T4 or N3 disease. They received 100 mg/m2 cisplatin (days 1 and 28) and 120-hour infusion of 1,000 mg/m2/24 hours 5FU (days 1 to 5 and 28 to 32). They subsequently were offered preoperative radiotherapy (RT) and surgery. Group 2 consisted of 18 previously treated patients. They received 5FU and cisplatin in the same dosage every 28 days for either recurrent or metastatic disease. It was found that in group 1 there was an 84% response rate (five complete responses (CR) and 11 partial responses (PR) ). Three of those with PR achieved a CR after RT. Seven patients have had RT plus surgery and are disease free at 8 to 27 month follow-up. Six patients (one CR, five PR) refused surgery and progressed within 4 months. In group 2 there was an 11% response rate after two cycles (two PR), three patients had a minimal response (MR, less than 50% response) and received a mean of four cycles of treatment. Three patients with stable disease received a mean of four cycles of chemotherapy until progression. Two of 11 patients who had received previous chemotherapy plus RT showed an MR; nine of these patients had shown a response to their previous chemotherapy. Only one of 14 patients who had RT plus chemotherapy had a PR, and three had MR. Of five patients who had previous surgery, only one had a PR. All five had received chemotherapy as well. It was concluded that 5FU plus cisplatin is an effective combination in previously untreated patients. In previously treated patients with recurrent disease, there is a substantially lower response rate.

Preoperative intra-arterial infusion chemotherapy for advanced squamous cell carcinoma of the mouth and oropharynx.

Twelve patients with advanced locoregional (Stage III and IV) squamous cell carcinoma of the oral cavity and oropharynx underwent treatment with cisplatin, vinblastine, bleomycin, and 5-fluorouracil, given by intra-arterial infusion, as primary adjuvant therapy, in preparation for radiation therapy and surgery. Responses were observed during or immediately after infusion therapy in 8 of 12 (67%) of patients (1 complete response, 7 partial responses). Infusion chemotherapy was followed by radiation therapy alone in five patients and by radiation and surgery in six patients. The protocol was initiated in August 1981, and six patients are now free of their primary cancer, at 21 to 36 months, whereas six have died with disease. Arterial infusion of a combination of effective antineoplastic agents is a promising method for the preparation of selected patients for radiation therapy and surgery, as it is less likely to produce serious systemic toxicity and it requires a shorter period than systemic neoadjuvant chemotherapy.