Spatial and temporal patterns of phlebotomine sand flies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus in northern Argentina.

Phlebotomine sand flies (Diptera: Psychodidae) were captured in an area of Argentina endemic for American cutaneous leishmaniasis (ACL). A total of 44,944 flies were collected during a 130-wk interepidemic period from 1990 through 1993. These sand flies included Lutzomyia neivai (Pinto) (97.8%), Lutzomyia migonei (Franca) (1.2%), Lutzomyia cortelezzii (Brèthes) (0.8%), Lutzomyia shannoni (Dyar) (0.1%), and Lutzomyia punctigeniculata (Floch and Abonnenc) (0.1%). Lutzomyia neivai was more abundant in secondary forests and peridomestic environments associated with human cases than in primary forest or xeric thorn scrub areas. Time series analyses of species densities suggested a bimodal or trimodal annual pattern related to rainfall peaks, a 5-wk reproductive cycle, and peridomestic local populations that were located adjacent to secondary forests. In general, sand fly abundance was correlated with the rainfall of the previous year. Lutzomyia neivai spatial distributions were consistent with ACL incidence patterns during the study and in the recent outbreaks in Argentina. However, Lu. migonei also may be involved in peridomestic transmission. Our results suggest that there is a need for improved, long-term surveillance of sand flies and ACL cases, as well as development of effective intervention strategies.

Temporal and geographic genetic variation in Culex pipiens quinquefasciatus (Diptera: Culicidae) from Florida.

Culex (Culex) pipiens quinquefasciatus Say field population from Vero Beach, FL, sampled monthly over a period of 8 mo, a colony sample, and six geographic samples were analyzed for genetic variation at 12 enzymes (10 "neutral" gene enzymes with 11 putative loci and two "complex" gene enzymes) by using polyacrylamide gel electrophoresis. The analysis of the 11 putative loci in both temporal and geographic samples showed that the four loci (Gpi, Hk, Mdhp-2, and Pgm) diagnostic of Cx. p. quinquefasciatus in the southern United States are present in similar frequencies in Florida samples. The Cx. p. quinquefasciatus colony sample showed significantly lower genetic variation than the temporal field samples, measured by mean number of alleles per locus (colony 1.2 +/- 0.1 versus field 1.44 +/- 0.03), percentage of polymorphic loci (colony 18.2% versus field 28.4%), mean observed heterozygosity (H(o) = colony 0.027 +/- 0.02 versus field 0.09 +/- 0.01), and mean Hardy-Weinberg expected heterozygosity (H(e) = colony 0.025 +/- 0.02 versus field 0.085 +/- 0.01). Three of the 11 loci (Acoh, Pgd, and Pgm) from the Vero Beach field samples showed bimodal patterns in their frequencies of the most common allele during peak density of the population. The low value of F(st) of 0.058 indicated minimum population substructuring among the temporal samples. Genetic variability values between geographic samples from the Florida panhandle and south Florida were not significant. Gene flow estimates based on F(ST), = 0.05, indicating low levels of gene flow among the geographic samples of Cx. p. quinquefasciatus. The average Nei's and modified Rogers' genetic distances among the six populations were 0.005 +/- 0.001 and 0.077 +/- 0.007, respectively. The cluster analysis did not suggest geographic clustering. The analysis of the "complex" gene enzymes in both temporal and geographic samples of Cx. p. quinquefasciatus from Florida showed the presence of two highly amplified esterases (Estbeta1 and Estalpha2\Estbeta2), indicating resistance to organophosphate insecticides and highly amplified Aldox enzyme (an enzyme that indicates resistance to at least one insecticide and a herbicide). Comparison of our results with previous studies on Cx. p. quinquefasciatus populations in the United States indicates that the genetic characteristics of the Florida populations of Cx. p. quinquefasciatus are very similar to populations from areas where ecological conditions are very different.

Temporal and geographic genetic variation in Culex nigripalpus theobald (Culicidae: Diptera), a vector of St. Louis encephalitis virus, from Florida.

A field population of Culex (Culex) nigripalpus Theobald from Vero Beach, FL sampled monthly over a period of 24 mo, a colony sample and 10 geographic samples were analyzed for genetic variation at 14 enzyme loci using polyacrylamide gel electrophoresis. The Cx. nigripalpus colony sample showed significantly lower genetic variation than the field-collected samples, measured by mean number of alleles per locus (colony 1.4 +/- 0.1 versus field 2.1 +/- 0.22), percentage of polymorphic loci (colony 35.7% versus field 54.8 +/- 7.7%), but mean observed heterozygosity (Ho = colony 0.16 +/- 0.07 versus field 0.17 +/- 0.03) and mean Hardy-Weinberg expected heterozygosity (He = colony 0.14 +/- 0.06 versus field 0.18 +/- 0.02) did not differ significantly. Three of the 14 loci (Aldox, Gpd, and Gpi) from the Vero Beach field samples showed distinct temporal patterns in the frequency of the most common allele. Higher mean observed heterozygosity (Ho) occurred during months following high rainfall in the Vero Beach field samples than during months following low rainfall. The average Nm value of 3.6 indicated high gene flow among the temporally distributed samples of the Vero Beach population. Genetic variability values between geographic samples from Panhandle, FL and south Florida were not significant. Gene flow estimates based on F(ST) = 0.039 provided a Nm of 6.2 indicating high levels of gene flow among the geographic samples of Cx. nigripalpus. The average Nei's and modified Rogers' genetic distances among the 10 populations were 0.009 +/- 0.001 and 0.081 +/- 0.004, respectively. The cluster analysis did not suggest geographic clustering, Because Cx. nigripalpus is the vector of St. Louis encephalitis (SLE) in Florida, temporal and geographic genetic variation in this species is discussed in relation to the seasonal and geographic SLE virus activity in Florida.

Genetic variability among populations of Lutzomyia (Psathyromyia) shannoni (Dyar 1929) (Diptera: Psychodidae: Phlebotominae) in Colombia.

Polyacrylamide gel electrophoresis was used to elucidate genetic variation at 13 isozyme loci among forest populations of Lutzomyia shannoni from three widely separated locations in Colombia: Palambí (Nariño Department), Cimitarra (Santander Department) and Chinácota (Norte de Santander Department). These samples were compared with a laboratory colony originating from the Magdalena Valley in Central Colombia. The mean heterozygosity ranged from 16 to 22%, with 2.1 to 2.6 alleles detected per locus. Nei's genetic distances among populations were low, ranging from 0.011 to 0.049. The estimated number of migrants (Nm=3.8) based on Wright's F-Statistic, F ST, indicated low levels of gene flow among Lu. shannoni forest populations. This low level of migration indicates that the spread of stomatitis virus occurs via infected host, not by infected insect. In the colony sample of 79 individuals, the Gpi locus was homozygotic (0.62/0.62) in all females and heterozygotic (0.62/0.72) in all males. Although this phenomenon is probably a consequence of colonization, it indicates that Gpi is linked to a sex determining locus.

Wolbachia infections of phlebotomine sand flies (Diptera: Psychodidae).

Old and New World phlebotomine sand fly species were screened for infection with Wolbachia, intracellular bacterial endosymbionts found in many arthropods and filarial nematodes. Of 53 samples representing 15 species, nine samples offour species were found positive for Wolbachia by polymerase chain reaction amplification using primers for the Wolbachia surface protein (wsp) gene. Five of the wsp gene fragments from four species were cloned, sequenced, and used for phylogenetic analysis. These wsp sequences were placed in three different clades within the arthropod associated Wolbachia (groups A and B), suggesting that Wolbacia has infected sand flies on more than one occasion. Two distantly related sand fly species, Lutzomyia (Psanthyromyia) shannoi (Dyar) and Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho), infected with an identical Wolbachia strain suggest a very recent horizontal transmission.

Distribution of phlebotomine sand fly genotypes (Lutzomyia shannoni, Diptera: Psychodidae) across a highly heterogeneous landscape.

Genetic variability of eight Colombian field populations and two laboratory colonies of a tropical forest sand fly, Lutzomyia shannoni Dyar, was assessed by comparing allozyme frequencies at 20 enzyme loci. Substantial genetic variability was noted in all strains, with mean heterozygosities of 13-21% and alleles per locus of 2.0-2.8. Four loci were monomorphic. Six populations in north and central Colombia showed close genetic similarity (Nei's distances, 0.01-0.09), despite mountainous environment, discontinuous forest habitat, and elevation differences from 125 to 1,220 m. Two samples representing the Orinoco (near Villavicencio) and Amazon (near Leticia) river basins were similar (Nei's distance, 0.08) but diverged substantially from the central six samples (Nei's distances, 0.26-0.40). Although the range of L. shannoni extends from the southeastern United States to northern Argentina, three genetically distinct, geographically discrete, groups were discerned by the current analysis: Orinoco-Amazon river basins, north-central Colombia, and eastern United States.

Discovery, distribution, and abundance of the newly introduced mosquito Ochlerotatus japonicus (Diptera: Culicidae) in Connecticut, USA.

The earliest documented specimen of an exotic east Asian mosquito Ochlerotatus (Finlaya) japonicis japonicus (Theobald) in the Western Hemisphere is reported along with the results of a state wide survey to determine the distribution and abundance of this mosquito in Connecticut. Ochlerotatus japonicus was collected from 87 locations in eight counties. It is established throughout the state and occurs in a variety of natural and artificial container habitats including discarded tire casings, bird baths, wooden barrels, porcelain bath tubs (used for watering animals), plastic milk cartons, toys, vinyl tarpaulins (covering wood piles and swimming pools), exposed rock holes in stream beds, tree holes, subterranean catch basins, surface water rain pools, and spring-fed depressions. Larvae were particularly common in containers with water, decaying leaves, and algae, in shaded and sunlit areas and, in rock-pool habitats along streambeds, in association with Ochlerotatus atropalpus (Coquillett). Adult females were collected in sod grass-infused gravid and CO2- baited light traps, from early June through October, with peak collections in September. Biting females were collected by human bait method augmented with CO2, verifying its capacity to feed on humans. The ovitraps used in this study were not effective for recovering this species. Our results suggest that Oc. japonicus was introduced into Connecticut between 1992 and 1998. Because of the ability of Oc. japonicus to transmit West Nile virus, and because of the recent detection of this virus in field-collected specimens, the introduction of Oc. japonicus is considered a significant public health development.

Interspecific hybridization and genetic variability of Phlebotomus sandflies.

The first successful hybridization is reported between Phlebotomus papatasi and P. duboscqi, two important Old World sandfly vectors of leishmaniasis and other diseases. Laboratory strains of P. papatasi and P. duboscqi were separable by six diagnostic enzyme loci: Est-3, Idh-1, Mdh-2, Mpi, Tre-1 and Tre-3. Hybrids between the two species were verified by the recovery of heterozygous isozyme patterns for the diagnostic loci. No F2 or backcross progeny were obtained. P. papatasi was separated from P. bergeroti by three diagnostic enzyme loci: Est-3, Mpi and Pgd. The isozyme patterns of P. bergeroti contain elements of both P. duboscqi and P. papatasi, although seven diagnostic loci (Est-3, Idh-1, Me, Mpi, Pgd, Tre-1 and Tre-3) separated P. bergeroti from P. duboscqi. Genetic variability profiles of the three species were established for 20 enzyme loci. Three geographically distant strains of P. papatasi from Calcutta, Maharashtra and Israel had isozyme genetic distances of < 0.05. The recently established Calcutta strain showed an unexpectedly low genetic variability with only one (Idh-2) of 20 loci being polymorphic (average heterozygosity of 1.9%) in contrast to 5-8 polymorphic loci (10-12% heterozygosity) in the Maharashtra and Israel strains. Mass and single pair crosses between the three P. papatasi strains were fertile with normal progeny numbers. Thus we found no signs of speciation in P. papatasi.

Reproductive biology of Lutzomyia shannoni (Dyar) (Diptera: Psychodidae) under experimental conditions.

Baseline biological growth data of Lutzomyia shannoni (Dyar) were compared under two experimental conditions within insulated styrofoam chests and in standard laboratory incubators. The developmental time from egg to adult was 67 and 52 days, respectively. Based on cohorts of 100 females in each experiment, horizontal life tables were constructed. The following predictive parameters were obtained under each of the two conditions: net rate of reproduction (23.5 and 18.0 females per cohort female), generation time (11.4 and 9.4 weeks), intrinsic rate of population increase (0.27 and 0.30), and finite rate of population increment (1.31 and 1.36). The reproductive value for each class age of the cohort females was calculated. The observed parameters were obtained under each experimental condition: net rate of reproduction (1.9 and 2.5 females per cohort female), generation time (11.7 and 9.6 weeks), intrinsic rate of population increase (0.05 and 0.09), and finite rate of population increment (1.06 and 1.10). Vertical life tables were elaborated and mortality was described for every generation in each cohort. In addition, for two successive generations, additive variance and heritability for fecundity were estimated.

Epidemiology of endemic Oropouche virus transmission in upper Amazonian Peru.

A cross-sectional serosurvey of a rural community near Iquitos, Peru was conducted to determine Oropouche (ORO) virus antibody prevalence and risk factors for human infection. Venous blood samples, and demographic, social, and risk factor data were obtained from people age five years of age and older who lived in the village of Santa Clara on the Nanay River, a tributary of the Amazon River. Sera were tested for ORO viral antibody by an ELISA. The specificity of viral antibody reactivity was determined by a standard plaque-reduction neutralization test. Interview data were analyzed by univariate and multiple logistic regression to determine which variables were statistically associated with previous ORO viral infection, as indicated by the presence of IgG antibody. Final models were evaluated based on log-likelihood and Wald chi-square. Clustering of seropositive residents within houses was analyzed by the method of Walter. Among 1,227 persons sampled, 33.7% (n=414) were positive for ORO viral IgG antibody. Overall, antibody prevalence was similar for males (33.9%) and females (33.6%), and increased significantly with age for both sexes to include more than half of persons more than 25 years of age. The length of residence in the village was positively associated with serologic status; persons who had moved to the village within the past 15 years were less likely to be seropositive than life-long residents of the same age. Antibody prevalence among immigrants who had lived in Santa Clara more than 15 years was similar to that in life-long residents. The activity most predictive of previous ORO viral infection was travel to forest communities and travel to Iquitos. No evidence of spatial heterogeneity in ORO virus antibody distribution was observed. Results suggested that endemic transmission of ORO virus in this region has been ongoing during many decades, and that people are at considerable risk of infection.

Genetic polymorphism of morphological and biochemical characters in a Natal, Brazil, population of Lutzomyia longipalpis (Diptera: Psychodidae).

The phlebotomine sand fly, Lutzomyia longipalpis, is the vector of visceral leishmaniasis in the New World. Variability in its tergal spot morphology has led to conflicting interpretations of the species status of the various forms. An L. longipalpis field population from eastern Brazil was found with three co-occurring morphological variations--1-spot, 2-spot, and an intermediate form. Genetic profiles were established for each form. Fifteen isoenzyme loci provided the data matrix for comparison of genetic variation among the forms. Spot patterns and isoenzyme frequencies fit Hardy-Weinberg expectations, and no significant differences in isoenzyme frequencies were associated with morphological phenotype. The spot phenotype appears to be a polymorphic character not related to genetic isolation or differentiation at the species level.

Life cycle and fecundity analysis of Lutzomyia shannoni (Dyar) (Diptera: Psychodidae).

The life cycle of Lutzomyia shannoni (Dyar), was described for laboratory conditions with maximum daily temperature of 27-30 degree C, minimum daily temperatures of 22-27 degree C and relative humidity between 87-99%. Life cycle in each stage was as follows: egg 6-12 days (ave, 8.5 days); first stage larva 5-13 days (ave. 9.6 days); second stage larva 4-13 days (ave. 9.2 days); third stage larva 5-19 days (ave. 11.8 days); fourth stage larva 7-37 days (ave. 19.9 days); pupa 7-32 days (ave. 15.2 days). The life expectancy of adults ranged from 4 to 15 days (ave. 8.6 days). The entire egg to adult period ranged from 36 to 74 days (ave. 54.6 days). On average, each female oviposited 22.7 eggs; the average egg retention per female was 24.3 eggs.

Genetic structure of local populations of Lutzomyia longipalpis (Diptera: Psychodidae) in central Colombia.

Lutzomyia longipalpis (Lutz & Neiva), the sand fly vector of American visceral leishmaniasis in the New World tropics, has a broad but discontinuous geographical distribution from southern Mexico to Argentina. A baseline for population genetic structure and genetic variability for this species was obtained by analyzing 5 local, peridomestic populations at the approximate center of its distribution, the Magdalena River Valley of central Colombia. Three populations of L. longipalpis from El Callejón, a small rural community, were compared with 2 populations from neighboring areas 12 and 25 km distant for genetic variation at 15 isoenzyme loci. The mean heterozygosity ranged from 11 to 16%, with 1.2 to 2.3 alleles detected per locus. Nei's genetic distances among the populations were very low, ranging from 0.001 to 0.007. Gene flow estimates based on FST indicated high levels of gene flow among local L. longipalpis populations, with minimal population substructuring.

Genetic variability in biochemical characters of Brazilian field populations of the Leishmania vector, Lutzomyia longipalpis (Diptera: Psychodidae).

The phlebotomine sand fly Lutzomyia longipalpis is the insect vector of visceral leishmaniasis, a protozoan disease of increasing incidence and distribution in Central and South America. Electrophoretic allele frequencies of 15 enzyme loci were compared among the L. longipalpis populations selected across its distribution range in Brazil. The mean heterozygosity of two colonized geographic strains (one each from Colombia and Brazil) were 6% and 13% respectively, with 1.6-1.9 alleles detected per locus. In contrast, among the seven widely separated field populations, the mean heterozygosity ranged from 11% to 16% with 2.1-2.9 alleles per locus. No locus was recovered that was diagnostic for any of the field populations. Allelic frequency differences among five field strains from the Amazon basin and eastern coastal Brazil were very low, with Nei's genetic distances of less than 0.01 separating them. The two inland and southerly samples from Minas Gerais (Lapinha) and Bahia (Jacobina) states were more distinctive with genetic distances of 0.024-0.038 and 0.038-0.059, respectively, when compared with the five other samples. These differences were the consequence of several high frequency alleles (glycerol-3-phosphate dehydrogenase [Gpd1.69] and phosphoglucomutase [Pgm1.69]) relatively uncommon in other strains. The low genetic distances, absence of diagnostic loci, and the distribution of genes in geographic space indicate L. longipalpis of Brazil to be a single, but genetically heterogeneous, polymorphic species.

Patterns of genetic variability in colonized strains of Lutzomyia longipalpis (Diptera: Psychodidae) and its consequences.

The genetic qualities of laboratory colonies of phlebotomine sand flies have not been compared with field specimens despite 1) probable genetic shifts due to the colonization process and 2) the problems associated with the extrapolation of experimental data derived from colonized organisms to field populations. The present study compared the genetic profiles of five laboratory colonies of geographic strains of the New World sand fly Lutzomyia longipalpis, and contrasted them with field populations. The profiles were based on the variability exhibited with polyacrylamide gels at 14 enzyme loci. A general pattern of a loss of infrequent alleles and decreased heterozygosity emerged as an apparent consequence of colonization. The average number of alleles per locus ranged from 1.2 to 1.6, and the average heterozygosity ranged from 4% to 11%. The field collection from Lapinha Caves (near Belo Horizonte, Brazil) averaged 2.1 alleles with a heterozygosity of 16%. In contrast, the LAPINHA laboratory colony established from that site 24 years earlier showed very low values (1.2 alleles/locus and 4% heterozygosity) and fixation for alleles not present or rare in the field collection from the same site. The genetic differences between the other Brazilian colonies and the Lapinha Caves field samples were due to presence of both different alleles and highly diverged allelic frequencies. Biological inferences based on colonized sand flies must be tempered by recognizing that the colony may represent a highly skewed genetic subsample of the L. longipalpis field genome.

Intraspecific variation in key morphological characters of Culiseta melanura (Diptera:Culicidae).

Culiseta melanura (Coq.), the enzootic vector of eastern equine encephalitis in North America, is polymorphic for a trait used as a key diagnostic character. The absence of white abdominal bands distinguishes this species in several prominent keys to North American mosquitoes. However, this is an environmentally induced, nongenetic trait that cannot be used as a key character for diagnosing Cs. melanura. In light trap collections, banded specimens occur in early spring and summer, and nonbanded adults appear in late summer-autumn. Larvae reared in laboratory conditions produce nonbanded adults. Progeny reared from banded mothers are uniformly nonbanded. Biochemical genetic results indicate that banding is not correlated with a distinctive genotype or presence of cryptic species. In 18 enzyme loci screened, neither diagnostic alleles nor large differences in allele frequencies were detected between field-collected representatives of the two forms. Genetic variability was relatively low in the 28-year-old laboratory colony (average heterozygosity = 7%; average number of alleles per locus = 1.4), whereas in field samples, the variability was typical of field populations (average heterozygosity = 12-19%; average number of alleles per locus = 1.6-1.8), with the presence of both polymorphic and private alleles. The population genetic profile and comparisons among geographically distinct populations represent the first such presentation for any species in the genus Culiseta.

Systematics of mosquito disease vectors (Diptera, Culicidae): impact of molecular biology and cladistic analysis.

The field of medical entomology, by nature of its association with problems of human health, has been conservative in its application of molecular and computer technologies to systematic research. Recently, however, these methods have opened new interpretations for systematics of disease vectors. Medically important insects, particularly mosquitoes, are among those more thoroughly described by conventional taxonomy, and thereby provide a secure framework for testing congruencies with molecular data. In turn, molecular investigations have provided a stimulus to vector systematics in the discovery and delineation of cryptic species complexes, as well as providing new perspectives on relationships at higher taxonomic divisions. In this review, examples involving cladistic analysis, cytogenetics--in situ hybridization, isoenzymes, DNA sequencing, and restriction fragment polymorphism are drawn from the following taxa: Aedes communis; Aedes (Ochlerotatus) group G; Aedes (Stegomyia) species including A. aegypti, A. albopictus, and A. scutellaris group; Anopheles albitarsis, Anopheles dirus, Anopheles gambiae, Anopheles nuneztovari, Anopheles pseudopunctipennis, and Anopheles punctulatus groups; Culex pipiens and the Culex subgenus Melanoconion; and the tribe Sabethini.

CHROMOSOMAL REPATTERNING AND LINKAGE GROUP CONSERVATION IN MOSQUITO KARYOTYPIC EVOLUTION.

Chromosome number and morphology in mosquitoes is remarkably uniform: virtually all mosquitoes have a diploid chromosome number of six (2N = 6), and their chromosomes are invariably metacentric or submetacentric. Numerical changes obviously have not been important in mosquito chromosomal evolution, and because of the morphological similarity of their chromosomes, it appears that structural changes have played little or no role in mosquito karyotypic evolution. The goal of the present study was to identify the types and relative numbers of chromosomal changes in mosquito evolution and to extend the comparison where possible to the higher diptera. To do this, we compared the enzyme linkage maps of six species of Aedes to each other and to enzyme maps of seven other mosquito species and to Drosophila melanogaster. Our results indicate that Aedes chromosomes have been modified by inversions, most which were paracentric, and by translocations, most which were Robertsonian. Intrageneric comparison of Aedes enzyme maps also revealed groups of linked enzyme loci whose integrity has been maintained throughout Aedes evolution (conserved linkages/syntenies). Intergeneric comparisons of Aedes enzyme maps with those of species in the genera Culex, Anopheles, and Toxorhynchites disclosed conserved associations of enzyme loci between mosquito genera. These findings lead us to postulate that the ancestral mosquito karyotype consisted of six chromosomal elements which, other than being combined in different ways in various mosquito groups, have remained essentially intact during mosquito evolution. Furthermore, the identification of groups of linked enzyme loci common to mosquitoes and to D. melanogaster indicates that linkage group conservation may characterize the karyotypic evolution of all dipteran insects.

Population genetic evidence for species A, B, C and D of the Anopheles dirus complex in Thailand and enzyme electromorphs for their identification.

Mixtures of chromosomal forms A, B, C and D in natural populations of Anopheles dirus Peyton & Harrison sensu lato in Thailand show significant positive values of Wright's fixation index for six enzyme-electromorph loci. The mean value of FIS over all loci was found to be +0.28 (SD 0.02), with a range of +0.57 (Odh) to +0.10 (Idh-2). Partitioning electromorph data for the chromosomal forms reduces the mean FIS to 0.03 (SD 0.01), which suggests that positive assortative mating is a characteristic of each form. This supports the hypothesis that the chromosomal/electrophoretic forms A, B, C and D represent four distinct biological species within the An. dirus complex. An example is given of the use of enzyme electromorphs as a means of vector identification during a malaria entomological field study involving a mixture of An. dirus species A and D. Electromorph identifications of 323 sp. A and 161 sp. D were more than 98% correct when cross-referenced to specific DNA probes.