﻿Description of Chilearinus Sharkey gen. nov. and status of Nearctic Earinus Wesmael, 1837 (Braconidae, Agathidinae) with the description of new species

﻿Abstract The Neotropical members formerly included in Earinus Wesmael, 1837 are transferred to a new genus, Chilearinus Sharkey gen. nov. Presently three Nearctic species of Earinus are recognized, i.e., Earinuserythropoda Cameron, 1887, Earinuslimitaris Say,1835, and Earinuszeirapherae Walley, 1935, and these are retained in Earinus. Earinuschubuquensis Berta, 2000 and Earinusscitus Enderlein, 1920 are transferred to Chilearinus, i.e., C.chubuquensis, and C.scitus, comb. nov. One other species is transferred to Chilearinus, i.e., Microgasterrubricollis Spinola, 1851, Chilearinusrubricollis, comb. nov. Two other Neotropical species, Earinushubrechtae Braet, 2002 and Earinusbourguignoni Braet, 2002 were described under the genus Earinus but are here transferred to Lytopylus, L.hubrechtae, and L.bourguignonicomb. nov. Two new species of Chilearinus are described, C.covidchronos and C.janbertspp. nov. The status of Agathislaevithorax Spinola,1851, Agathisrubricata Spinola,1851, and Agathisareolata Spinola, 1851 is discussed. A neotype is designated for Earinuslimitaris (Say, 1835) and diagnosed with a COI barcode. Earinusaustinbakeri and Earinuswalleyispp. nov. are described. The status of both Earinus and Chilearinus in the Americas is discussed. A revised key to the genera of Agathidinae of the Americas is presented.


DNA extraction and sequencing
Molecular work was carried out at the CBG using standard protocols. A leg from each frozen-then-oven-dried specimen was destructively sampled for DNA extraction using a glass fiber protocol (Ivanova et al. 2006). Extracted DNA was amplified for a 658 bp region near the 5′ terminus of the cytochrome c oxidase subunit I (COI) gene using standard insect primers LepF1 (5′-ATTCAACCAATCATAAAGATATTGG-3′) and LepR1 (5′-TAAACTTCTGGATGTCCAAAAAATCA-3′) (Ivanova and Grainger 2007). If initial amplification failed, additional PCRs were conducted following established protocols using internal primer pairs: LepF1-C113R (130 bp) or LepF1-C_ANTMR1D (307 bp) and MLepF1-LepR1 (407 bp) to generate shorter overlapping sequences. Most amplicons were Sanger sequenced, but some recent specimens were analyzed on SEQUEL.
The BOLD database can be used to identify specimens using the following steps: (1) navigate to the identification tab of the BOLD Systems database (http://www.boldsystems.org/index.php/IDS_OpenIdEngine); (2) paste the COI sequence of the query organism (in forward orientation) into the query box and search against the appropriate library (e.g., All Barcode Records on BOLD, Species Level Barcode Records, etc.); (3) the search results page shows the top hits based on percentage similarity starting with the closest matches (This page also provides additional information to help verify the identity of a match, such as links to the BIN where specimen data, including images, can be found, a distribution map, and a tree-based identification tool); (4) use the Tree-Based Identification button to generate a neighbor-joining tree and find the query taxon (name in red). This allows you to visualize how distant the query sequence is from the closest matches.

Taxonomic account
Chilearinus Sharkey, gen. nov. http://zoobank.org//82CEAEE1-8CDB-48DD-B79F-1B59F8CF74A1 Type species. Chilearinus janbert Sharkey, sp. nov. Etymology. A conjunction of Chile, where 90% of the species are likely to be found, and Earinus, a reference to the probable sister group of the species, based on preliminary analyses. The genus is masculine.
Diagnosis. Notauli absent; hind coxal cavities open; tarsal claws with basal lobes; second submarginal cell quadrate, never petiolate; foretibia lacking sclerotized spines/ pegs; hind wing Cub strong and emanating from an angle on the basal cell. Most similar morphologically to Earinus and Lytopylus. Earinus and Chilearinus do not have overlapping distributions. The former is restricted to the Nearctic and the latter to the Neotropics; therefore, there is little chance of confusing the two. Nonetheless, the lack of pegs on the foretibia of members of Chilearinus and the morphological characters given in the key (below) can also be employed to differentiate them. Members of Lytopylus differ most significantly in that they lack vein Cub in the hind wing. See couplet 25 in the key below.
Description. Head. Lateral carina on frons (as found in members of Alabagrus) absent; interantennal space slightly raised above antennal sockets; gena not extended ventroposteriorly into sharp prominence; mandible dorsoventrally flattened (twisted); labial palpus with 4 segments, third segment slightly more than ½ length of apical segment. Mesosoma. Propleuron lacking a sharp bump; notauli absent; mesoscutum smooth with a median pit (presumably a remnant of notauli), postscutellar depression absent; propodeum mostly smooth, sometimes with weak smooth sculpture medially; sclerite between hind coxal cavities and metasomal foramen absent. Precoxal groove absent or smooth and weakly impressed. Legs. Foretibia lacking dull pegs (unlike Earinus); mid-and hind tibia with blunt apical or preapical pegs; all tarsal claws with a rounded basal lobe. Wings. Forewing RS+Ma vein mostly present but not usually completely tubular; second submarginal cell large, quadrate and usually (perhaps always) higher than long; RS of forewing complete to wing margin; hind wing r and r-m cross veins absent; hind wing vein Cub strong and emanating from an angle on the basal cell. Metasoma. First median tergite smooth, longer than apical width, lateral longitudinal carina absent or weak and short; remaining terga smooth; ovipositor ranging from as long as the body to twice the length of the body, but this is based on small sample of a few dozen species.
Biology. Unknown. Diversity and distribution. This is a species-rich genus with hundreds of species, based on specimens identified by MS. It is widespread in Chile and southern Argentina. A few species are found at high altitudes as far north as Ecuador and Colombia.
Notes. Sharkey (1997) included members of what are now Chilearinus in a broader concept of Earinus. Spinola (1851) described three species of Agathidinae from Chile. Since members of Chilearinus are by far the most species-rich of Chilean agathidines, and since his descriptions do not contradict membership in the genus, these species are probably members of Chilearinus, i.e., Agathis laevithorax, Agathis rubricata, and Agathis areolata. They certainly are not members of Agathis since this genus does not extend into the southern regions of South America. These specimens should be in the Hymenoptera collection of Maximilian Spinola whose collection is housed in the Museo Regionale di Scienze Naturali (MRSN) in Turin (Torino). One of us (MS) could not locate these specimens during a visit to MSRN in 1985, but a specimen of Chilearinus, Microgaster rubricollis Spinola, 1851, was present. Microgaster may seem an odd place for placement of what we now consider an agathidine, but such was the classification at the time. It is clear from the following that Spinola knew the species was closely related to Earinus, "Este Microgastro habria pertenecido á [sic] la primera seccion del G. Microdus, N. V. Es., y al sub-género Earinus Wesm." (Spinola 1851: 34).
It is almost pointless to present a morphological key to the five recognized species of Chilearinus as they represent just five species out of hundreds. Many undescribed species will undoubtedly key to these named species. The only way to handle speciesrich undocumented genera such as Chilearinus is to include COI barcode data in the diagnoses. We know this diagnostic is sufficient to differentiate all but a few species of Agathidinae (Sharkey et al. 2018). Nonetheless, despite the absurdity, a key is presented below to mollify critics (e.g., Zamani et al. 2020).
Key to the few described species of Chilearinus  Diagnosis. COI barcode. BOLD sample ID H12114. BOLD BIN: Paratypes. None. Etymology. A conjunction of Paul Hebert and Dan Janzen in recognition of their enormous contributions towards the conservation of nature.

Earinus Wesmael, 1837
Note.In the Americas, there are three previously recognized species of Earinus, i.e., E. erythropoda Cameron, 1887, E. limitaris (Say, 1835), and E. zeirapherae Walley, 1935, and here we describe two more, Earinus austinbakeri sp. nov. and Earinus walleyi sp. nov. In the Nearctic, Earinus is common and widespread with the southernmost record being the sole recognized specimen of E. erythropoda from northern Sonora state, Mexico. Earinus differs from Chilearinus in the possession of pegs/spines in the foretibia and the characters given in the key.
Based on the collection in the Hymenoptera Institute (MS's personal collection, which will eventually be deposited in the CNC) and borrowed specimens, there are probably between eight and 12 species in the Nearctic region. They are extremely similar in color, but there are obvious differences among specimens in body dimensions, degree of punctation, color of the hind coxae, ocellar configuration, ovipositor length, length and density of setae on the ovipositor sheath, and dimensions of the first metasomal tergum. Unfortunately, these are not sufficient to allow confident delineation of species limits. For example, the differences in the key between E. limitaris and E. erythropoda are trivial. There are numerous specimens scattered over the Nearctic region that will key to E. erythropoda, but they might all be E. limitaris, or the two nominal species may be conspecific, or there may be multiple cryptic species. Likewise, there are probably a number of undescribed Nearctic species that will key to either E. zeirapherae or E. austinbakeri. In other words, the key is sufficient to discriminate among the barcoded species and E. zeirapherae but not among these and the undescribed species. The key is presented in part to satisfy the code of Zoological Nomenclature to act as a diagnosis for E. austinbakeri and E. walleyi. Only dense sampling of COI barcodes and perhaps other genes will supply the information necessary to delimit Nearctic Earinus species.
Key to the species of Earinus of North America Morphological diagnosis. Very similar to E. austinbakeri but differing by the characters given in the key as well as having the ovipositor sheath more setose. The COI barcodes of the two species differ by 6.29% (p-distance), reinforcing the conclusion that they are different species.
Distribution. The holotype and paratypes were found at two localities just north and northeast of Lake Ontario. This species may be widespread throughout the eastern USA as far south as the Carolinas.

Earinus erythropoda Cameron, 1887
Holotype. ♀, "N. Sonora, Mexico, Morrison" (British Museum Natural BM3c893, viewed). Notes. The sole identified specimen is the holotype. It differs little from many specimens that are widespread in the United States. It could be that they all belong to E. limitaris, or several more species may have similar morphologies. COI barcode data are needed. Several line drawings, modified from Berta (2000), are included in the key and others are in Berta's (2000) treatment. ( Diagnosis. Consensus COI barcode based on 9 specimens.
Biology. The following are listed as hosts of E. limitaris by Yu et al. (2016); all belong to Noctuidae: Egira dolosa, Enargia decolor, Homoglaea hircina, Ipimorpha pleonectusa, and Orthosia hibisci. Because there are probably a number of cryptic species in E. limitaris, these records need confirmation.
Notes. There are 15 specimens from one locality in Quebec that are in a different BIN (BOLD:ADF5580) which differs by only 2.54% (p-distance) from E. limitaris (Earinus sp. in Fig. 3). Because of the small distance between these two BINs, we refrain from describing this BIN as a new species but suggest that it may be a distinct species. Broader geographic sampling is required to clarify the significance of this barcode split.
Like many of Say's types, the type of B. limitaris is lost (Muesebeck 1927 Body black: palpi white: thorax longitudinally indented behind the middle: wings nearly hyaline, at base yellowish; nervures fuscous; stigma large; first cubital cell complete; second rather large, quadrangular: radial cellule also rather large: feet honeyyellow; posterior pair of tibiae whitish, their tips and annulus near the base black; posterior pair of tarsi black. Length seven twentieths of an inch. Var. a. Maxillary palpi, first joint black. ♀ Oviduct hairy, decurved, somewhat robust." Except for the body length, this description is consistent with all of the estimated 8-12 Nearctic species of Earinus. We have a number of specimens of what we believe to be E. limitaris. The neotype was selected because it is geographically closest to the two specimens included in Say's (1835) original description, despite the fact that it is a male.
Distribution. Unknown, except for barcoded specimens (West Virginia, southern Ontario), as well as either Missouri or Indiana, or both. It is unknown if Say's (1835) two specimens are conspecific. Based on specimens that one of us (MS) recently viewed, this species is probably widespread across southern Canada and northern United States, extending south as far as southern New Mexico (presumably at high altitudes) in the west and South Carolina in the east. The holotype of E. erythropoda may also belong here, which would extend the distribution into northern Sonora state, Mexico.

GAATAATAAATAT TAAAAT TGATAAAATGTCT T TAT TAAT T TGAT-CAATTTTAATTACTGCTATTTTATTATTATTRTCTTTACCAGTTT-TAGCAGGAGCTATTACTATATTATTAACAGATCGTAATTTAAATA-CAAGATTTTTTGATCCTTCYGGAGGGGGTGACCCAATTTTATAT-CAACATTTATTT
Morphological diagnosis. Very similar to E. zeirapherae, differing by the characters given in the key as well as having the ovipositor sheath less setose. The COI barcodes of the two species differ by 6.29% (p-distance) all but ensuring that they are different species.
Paratypes. All are from the same locality as the holotype, 07PROBE-23096, 07PROBE-23097, 09PROBE-A0304. These are specimen IDs; more data on the specimens can be found by searching for these codes on BOLD (http://www.boldsystems.org).
Distribution. Unknown but likely widespread in Alaska and northern and midlatitudinal areas of Canada. Some or all records in Yu et al. (2016) for E. zeirapherae occurring from Alaska, Nunavut, and the Yukon may belong to this species.

Earinus zeirapherae
Biology. The following are all reported as hosts by Yu et al. (2016). All belong to Tortricidae: Acleris hudsoniana, Choristoneura rosaceana, Rhyacionia adana, Zeiraphera canadensis, Zeiraphera griseana, and Zeiraphera ratzeburgiana. Since there are many species, including E. austinbakeri and E. walleyi, that are morphologically similar to E. zeirapherae, all hosts that do not belong to the genus Zeiraphera need confirmation. Notes. The holotype (Fig. 10) is from Nova Scotia, as is the male in Figure 11; both were reared from Zeiraphera ratzburgiana. Contrary to the image of the holotype in Figure 10, the original description by Walley (1935) states that the fore and mid coxae and hind coxa are basally blackish, "front and middle coxae mostly, all trochanters faintly, hind coxae basally … blackish." (Walley 1935: 56). It seems likely that over time the coxae of the holotype have faded. There are other specimens in the Canadian National Collection that have similar coloration but that are not likely to be conspecific based on other characters, e.g., one specimen from New Mexico. This serves as a reminder that the key will only function to separate the described species from each other.