A DNA Barcode Library for North American Pyraustinae (Lepidoptera: Pyraloidea: Crambidae)

Although members of the crambid subfamily Pyraustinae are frequently important crop pests, their identification is often difficult because many species lack conspicuous diagnostic morphological characters. DNA barcoding employs sequence diversity in a short standardized gene region to facilitate specimen identifications and species discovery. This study provides a DNA barcode reference library for North American pyraustines based upon the analysis of 1589 sequences recovered from 137 nominal species, 87% of the fauna. Data from 125 species were barcode compliant (>500bp, <1% n), and 99 of these taxa formed a distinct cluster that was assigned to a single BIN. The other 26 species were assigned to 56 BINs, reflecting frequent cases of deep intraspecific sequence divergence and a few instances of barcode sharing, creating a total of 155 BINs. Two systems for OTU designation, ABGD and BIN, were examined to check the correspondence between current taxonomy and sequence clusters. The BIN system performed better than ABGD in delimiting closely related species, while OTU counts with ABGD were influenced by the value employed for relative gap width. Different species with low or no interspecific divergence may represent cases of unrecognized synonymy, whereas those with high intraspecific divergence require further taxonomic scrutiny as they may involve cryptic diversity. The barcode library developed in this study will also help to advance understanding of relationships among species of Pyraustinae.

antisocialis can be easily distinguished from those of P. fodinalis in having the uncus more slender and provided with a lateral point on either side at the tip in male genitalia. Therefore, we suspect that very recent speciation or extensive introgressive hybridization may have occurred leading to this rare case of barcode sharing. In addition, the status of three subspecies of Pyrausta fodinalis should be reconsidered according to our analysis based on our morphological and molecular data. This species is an economic pest feeding on rhizomes of peppermint and other mints and occurs from Nova Scotia to British Columbia and Washington and south to Florida, eastern Texas and Utah. Munroe (1976)  Nascia acutellus and its sibling species Nascia cilialis (Hübner), 1796 (type locality: Oesterreich) are Holarctic species. As mentioned in Munroe (1976), the former is confined to the eastern part of North America, whereas the latter ranges from Europe to Japan with some geographical variation. Munroe (1976)  Pyrausta grotei. Three distinct clusters were displayed and the genetic distance was higher than 2.0% between each other. One includes two records from California, another two clusters includes one from California and another from Texas and Arizona. Our results suggest that augustalis may be a distinct, valid species, but more detailed study is needed.  Poole (1996) and listed three subspecies of Pyrausta insequalis, namely P. i. plagalis, P. i. petaluma and P. i. fascetalis. In our analyses, four distinct clusters of Pyrausta insequalis and one cluster of Pyrausta borealis (Alberta, British Columbia and Quebec, Canada) were displayed. Interestingly, the genetic distance was lower than among three clusters of Pyrausta insequalis. One was identified as P. i. plagalis and another P. i. petaluma including specimens from Western of North America. The third one was an undetermined subspecies from Mexico. However, P. i. plagalis mostly sampled from eastern North America, including Quebec, Oklahoma and Mississippi (with one exception from Arizona) diverged from the other three clusters of Pyrausta insequalis but clustered together with Pyrausta borealis. The genetic distance was lower than 2.0% between eastern P. i. plagalis and Pyrausta borealis but higher than 2.0% between eastern P. i. plagalis and Western P. i. plagalis as well between eastern P. i. plagalis and P. i. petaluma. By contrast, the genetic distance was less than 2.0% between Pyrausta borealis and western P. i. plagalis as well between Pyrausta borealis and P. i. petaluma. Our findings suggest that the genetic diversity of P. insequalis is most likely related to geographical variation and that historical introgression may have occurred between eastern P. i. plagalis and Pyrausta borealis, but more investigations are desirable. Our morphological study showed that differential diagnosis in genital structure is subtle based on the females from Arizona and California, but it is difficult to separate them with the limited material at hand. Further study is desirable. Sierra tend to be large, and those from the eastern part of the range have more fulvous than those from the West. In our analysis, four distinct clusters were displayed for Pyrausta nexalis. However, the genetic distance was lower than 2.0% between two clusters, and examination of their male genitalia indicated that they were all Pyrausta nexalis. One cluster includes three records from Washington and Arizona and another includes three samples from Texas and Arizona. Three records from California and Texas and one record from California formed two additional distinct clusters. Our results suggest the presence of overlooked cryptic species. But more material and investigation are needed to assess the significance of the morphological variation of Pyrausta nexalis related to genetic divergence. 10. Pyrausta nicalis (Grote, 1878) Type locality: USA, California, Sierra Nevada. [BMNH] This species is similar in general aspect to Pyrausta grotei (Munroe, 1976 out that all three names were based on material from the same region and that there was some morphological variation. Interestingly, our analyses revealed three distinct clusters. One should be the real Pyrausta nicalis based on our morphological study, including two records sampled from Utah and Nevada, United States. Another two clusters from Western Canada may represent two distinct species that have been overlooked. In addition, the taxonomic status of uxorculalis and subnicalis should be re-examined to assess whether they may deserve valid species status. and P. p. perrubralis (Packard), 1873 (type locality: USA, California). In our analysis, four distinct clusters of Pyrausta perrubralis were displayed. However, one comprising four records including the allotype of P. p. shastanalis formed a distinct cluster more than 2.0% distant from the other three clusters. By contrast, the genetic distance was lower than 2.0% among another three clusters. Our findings suggest that P. p. shastanalis should be elevated to full species level. We retain its subspecies rank at present pending a detailed investigation of genital morphology.

Pyrausta scurralis (Hulst, 1886) Type locality: USA, Arizona. [AMNH]
This species is similar and formerly was considered to be an inland race of However, the genetic distance was lower than 2.0% from each other. The morphological variation of the Pyrausta signatalis seems common in the eastern part of the range.  Munroe (1976)

indicated that
Pyrausta zonalis is most similar to Pyrausta napaealis in appearance but smaller. This species not uncommon in the drier parts of southern California and occurs from Apple Valley, San Bernardino County, to Painted Gorge, Imperial County, and east to Ivanpah and Palo Verde Mountains. In our analyses, two distinct clusters of Pyrausta zonalis were displayed. One incudes only one record from Oso Flaco Lake, San Luis Obispo County, California, another includes remaining records San Benito County and San Diego County, California. The genetic distance was higher than 2.0% between them. Our results implied that the single specimen may represent an overlooked species.

Sitochroa chortalis (Grote, 1873) Type locality: not fixed
This species is similar to its allies and often seen along with Crambinae and other moths in summer meadows and pastures by day. It has been reported from Nova Scotia to southern British Columbia and south to New Jersey, Arizona and north California. Munroe (1976) treated Sitochroa chortalis as a new combination transferred from the genus Eurycreon. But he was not able to designate a lectotype from an unknown number of syntypes described respectively in New York, Massachusetts and Alabama. In our analyses, three distinct clusters were displayed.
One includes two records from New Brunswick and the second includes five records from New Brunswick and Ontario. However, the genetic distance was lower than 2.0% between them. The third cluster identified as Sitochroa chortalis includes a few records from Wisconsin, New Mexico, Washington, Arizona, Newfoundland and Labrador, Ontario, Saskatchewan. The genetic distances was higher than 2.0% between it and the other two clusters. This suggests that a possible cryptic species has been overlooked in eastern Canada.