An identification key to species in the mali complex of Aphelinus (Hymenoptera, Chalcidoidea) with descriptions of three new species

The Aphelinus mali complex consists of eleven described species. Monophyly of this complex is well supported by a combination of traits: (1) a single complete row of setae proximal to the linea calva of the fore wing, with a few additional setae in the angle between this row and the marginal vein; (2) linea calva open (no setae at its posterior edge); (3) head and body dark except for parts of the metasoma; (4) mesoand metacoxae dark; (5) metafemur pale, (6) metatibia dark. Species within the complex have been distinguished by color and shape of antennal segments (particularly the third funicular segment), color of legs and metasoma, and relative length of ovipositor versus mesotibia. We provide a key for identifying species in the mali complex, and describe three new species, Aphelinus glycinis sp. n., Aphelinus rhamni sp. n., and Aphelinus coreae sp. n. from material in laboratory cultures originally reared from soybean aphid in China and Korea as candidates for biological control of soybean aphid, Aphis glycines. JHR 26: 73–96 (2012) doi: 10.3897/JHR.26.2584 www.pensoft.net/journals/jhr Copyright Keith R. Hopper et al. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ReseARCH ARtiCle Keith R. Hopper et al. / Journal of Hymenoptera Research 26: 73–96 (2012) 74


introduction
The genus Aphelinus (Hymenoptera: Aphelinidae) comprises 84 recognized species (Noyes 2011), all of which are internal parasitoids of aphids.There are several complexes of closely related species in the genus, and identification of species within these complexes has been problematic (Heraty et al. 2007), leading to a confused literature on host specificity.The Aphelinus mali complex comprises 11 described species (Ashmead 1888; Evans et al. 1995;Gahan 1924;Girault 1913;Haldeman 1851;Hayat 1998;Prinsloo and Neser 1994;Timberlake 1924;Yasnosh 1963;Zehavi and Rosen 1988), and there are 6 other closely related species (Carver 1980;Hayat 1998;Howard 1917;Kurdjumov 1913;Walker 1839;Yasnosh 1963) that differ from the members of the complex in one or two traits (Table 1).The species within the complex have diverged little in morphology so the taxonomy within the complex has been confused, and many specimens have been identified as A. mali (Haldemann) or A. gossypii Timberlake that are likely different species based on differences in the aphid host species and geographical regions from which they were collected.We provide a key to identification of species in the A. mali complex based on 19 traits coded primarily from species descriptions.We describe three new species that were collected in China and Korea during exploration for natural enemies of the soybean aphid, Aphis glycines Matsumura, all of which are candidates for biological control of this important pest.
The genus Aphelinus consists of several complexes of cryptic species including the mali complex, the varipes complex (Heraty et al. 2007), the asychis complex (Kazmer et al. 1995(Kazmer et al. , 1996)), the perpallidus complex (unpublished data), and possibly others.Cryptic species are closely related species that differ little in the morphological features used for taxonomy, but differ critically in physiological, behavioral and ecological traits, such as climatic adaptation and host range (Darling and Werren 1990;DeBach 1969).Recent evidence from molecular studies suggests that cryptic species of hymenopteran parasitoids may be far more common than previously realized (Campbell et al. 1993;Clarke and Walter 1995;Darling and Werren 1990;Kankare et al. 2005a;Kankare et al. 2005b;Kazmer et al. 1996;Molbo et al. 2003;Rincon et al. 2006;Stouthamer et al. 2000;Stouthamer et al. 1999).The success of biological control programs depends on accurate species-level identifications of hosts and natural enemies, but choosing the best parasitoids for biological control programs is complicated by cryptic species (Rosen 1986;Wharton et al. 1990).Because cryptic species are difficult to recognize, studies on host ranges of parasitoids have often confounded more than one parasitoid species (Clarke and Walter 1995;Hopper et al. 1993), making analysis and prediction of host range difficult.Heightened concern about potential impacts of introduced parasitoids on non-target species makes accurate prediction of host range crucial to biological control introductions.

Methods
Three new species in the A. mali complex were collected from Aphis glycines in the Peoples Republic of China near Beijing and Xiuyan (Liaoning Province) and in the Republic of South Korea near Miryang (Gyeongsangnam Province) and maintained as laboratory cultures at the Beneficial Insects Introduction Research Unit, USDA-ARS, Newark, DE.All of the specimens described below were taken from lab cultures, killed in 95% ethanol, and most were critical-point-dried and card-mounted.Selected specimens were then slide-mounted in Canada balsam.Specimens photographed for coloration (15)(16)(17)(18)(19)(20)(29)(30)(31)(32)(33)(34) were killed in ethanol and photographed as soon as possible, by placing specimens on a layer of KY® jelly in a small watch glass, submerging the specimen in ethanol, and photographing using a Leica MZ 16 stereomicroscope, fiber optic illumination, a Zeiss Axiomat MRc5 camera, and Helicon Pro image-stacking software.Slide-mounted specimens were photographed using differential interference contrast optics (DIC) with an Olympus BH2 compound microscope, and the same camera and software.Final modifications to images were made using Adobe Photoshop, Adobe Lightroom, and Adobe InDe- Walker 1839 prociphili 3  Carver 1980 sharpae 3  Hayat 1998 brunneus 4  Yasnosh 1963 daucicola 4  Kurdjumov 1913 lapisligni 4  Howard 1917 1 new species described in this paper 2 insufficient description to be included in tree or key 3 difference from mali complex: more than 1 line of setae in delta region 4 difference from mali complex: posterior femur dark sign.Type material and other specimens examined have been deposited as indicated in the species descriptions.We tabulated and coded 19 traits for species in the A. mali complex, using the original species descriptions for the most part.These traits included color of scape, pedicel, club, coxae, femora, tibiae, tarsi, and metasoma, as well as shape of third funicle and club (length:width) and length of ovipositor relative to mesotibia.For some traits, males and females differed (e.g., F3 shape, procoxae color) and the values were scored separately.When trait data were lacking from original descriptions, we used data from later descriptions.Trait values for the new species in the complex were taken from specimens freshly killed in ethanol and slide-mounted specimens.These traits were used to construct an on-line, interactive, multiple entry identification key to the mali complex which is available on request.Of the 19 traits, 12 proved to be most consistent and useful in distinguishing species, and these are presented in Table 2.
Table 3 is a list of anatomical terms used in the paper followed by URI values (uniform resource identifiers), that will link the terms to precise definitions and illustrations in the Hymenoptera Anatomy Ontology project (see http://portal.hymao.organd http://hymao.orgfor more information on this initiative).Additional information on morphological terminology in Chalcidoidea is available in Gibson (1997) and http://www.canacoll.org/Hym/Staff/Gibson/apss/chglintr.htm.
The ventral surface of the antennal scape refers to the surface that is ventral when the antennae are deployed, or anterior when the antennae are folded on the face.F1, F2 and F3 refer to the first, second and third segments of the funicle of the antennal flagellum, respectively.T1, T2 etc. refer to metasomal terga.We use the term ovipositor to refer to the anatomical cluster consisting of the first valvula, second valvula, third valvula, first valvifer and second valvifer.Length of the ovipositor is the measurement (generally of a slide-mounted specimen) from the anterior margin of the second valvifer to the posterior (distal) end of the third valvula.
List of anatomical terms and links to URI locations in the Hymenoptera Anatomy Ontology portal.leg The anatomical cluster that is composed of the coxa and all distal leg segments and is connected to the pectus. http://purl.obolibrary.org/obo/HAO_0000494

Results and discussion
Following the work of Zehavi and Rosen (1988), we consider the following traits to be diagnostic for the A. mali complex: (1) head and body dark except for parts of the metasoma; (2) metafemur pale, (3) a single complete row of setae proximal to the linea calva of the fore wing, with a few additional setae in the angle between this row and the marginal vein; ( 4) linea calva open (no setae at its posterior edge); (5) mesoand metacoxae dark; (6) metatibia more or less dark.The A. mali complex consists of eleven described species, and there are six similar species with either a dark metafemur or more than one line of setae proximal to the linea calva (Table 1).Species within the complex have been distinguished by color and shape of antennal segments (particularly the third funicular segment), color of legs and metasoma, and relative length of ovipositor versus mesotibia (Ashmead 1888; Evans et al. 1995;Gahan 1924;Girault 1913;Haldeman 1851;Hayat 1998;Prinsloo and Neser 1994;Timberlake 1924;Yasnosh 1963;Zehavi and Rosen 1988).
Key to species in the Aphelinus mali complex 1 Female: procoxa white or yellowish-white, male: procoxa yellowish-white or grey, both sexes: meso-and metacoxae dark .Diagnosis.Female.Head and mesosoma dark brown to black; legs with procoxa yellowish white, meso-and metacoxae dark brown to black, femora yellowish white, protibia yellowish white, mesotibia yellowish white with center greyish, metatibia dark grey to black with base pale; metasoma with base, apex, and venter yellow, remainder brown; antenna white to yellowish white; F3 1.3-1.7 times as long as broad; club 3.2 times as long as broad.Male similar except procoxa grey; pro-and mesofemur sometimes with darkened center; metasoma brown with base and apex yellow; scape dark greyish brown with greyish yellow distal tip, swollen in center, maximum width 3× distal end width, with three to five volcano-shaped secretory pores in a single line on ventral surface, pedicel greyish yellow, third funicle more than 2 times as long as broad, club 3.9 times as long as broad.
Head. (Figs 2, 8) Head 1.3× as broad as high in frontal view, about as broad as mesosoma; frontovertex width 0.4× of head width, 1.2× as long as broad, and 0.8× as broad as scape length; posterior ocelli 1.0× their diameter from eye margin, 3× their diameter from one another, and 0.33× their diameter from occipital margin; mandible with 2 acute teeth and a broad truncate surface below the teeth, antenna as in Fig. 8 with scape 4.8× as long as broad, pedicel 2.2× as long as broad, F1 anneliform, 1.7× as wide as long, F2 1.2× as broad as long, F3 1.6-1.7×as long as broad, club 3.2× as long as broad, 2.7× times as long as F3, and with 6-7 longitudinal sensilla.
Mesosoma.(Figs 4,6,13) Mesoscutum and scutellum with fine reticulate sculpture, longest diameter of reticulations approximately twice the diameter of the scutellar sensilla, interior of reticulations with fine, granulate surface (visible only in slidemounts under high magnification); mid-lobe of mesoscutum with 2 pairs of long setae and about 40 short setae, side lobes each with 2 long and 1 short setae; scutellum with 2 pairs of long setae and two sensilla directly posterior to the anterior pair of setae, scutellar sensilla slightly posterior to middle of scutellum; mesotibial spur 0.8× mesobasitarsus; metatibial spur 0.6× metabasitarsus.
Fore wing.(Fig. 10) 2.3× as long as broad; costal cell with 1 complete row of dorsal setae and 2 rows of ventral setae, the posterior row extending from under the proximal end of the marginal vein almost to stigma, costal cell 1.2× longer than marginal vein; submarginal vein with 5-6 setae; marginal vein with 10 setae along the margin; stigmal vein short with stigma rounded; delta region proximal to linea calva with one complete line of 13-15 setae and 2-6 additional setae in angle with marginal vein, linea calva with no dorsal setae at its posterior edge; wing distal to linea calva with dense, evenly spaced, dorsal setae and much smaller ventral setae.
Color.(Figs 2,4,6) Head and mesosoma dark brown to black; legs with procoxa yellowish white, meso-and metacoxae dark brown to black, femora yellowish white, protibia yellowish white, mesotibia yellowish white with center greyish, metatibia dark grey to black base pale, tarsi pale brown with tips and metabasitarsus greyish brown; metasoma with base (T1 and T2), apex, and venter yellow, remainder brown; antennae yellowish white; compound eyes burgundy, and ocelli red in life, both silvercolored in dried specimens.
Hosts.In the field, Aphis glycines is the only known host.In laboratory experiments, A. glycinis parasitizes A. glycines and closely related species in the genus Aphis.
Etymology.This species is named for the host from which it was collected.The species epithet is a noun in genitive case.
Relationships.Aphelinus glycinis is closest to A. engaeus and A. ficusae Prinsloo and Neser based on our matrix of traits (Table 2).Aphelinus glycinis differs from A. engaeus in having elongated third funicle segments in males and females, and it differs from A. ficusae in having an ovipositor more than 1.2× as long as the mesotibia and grey procoxa in males.It also differs from these species in its aphid hosts and geographical distribution.Aphelinus glycinis is a specialist on Aphis species close to Aphis glycines, but A. engaeus is reported from Schizaphis graminum (Rondani) and Sitobion ochnearum (Eastop) and A. ficusae was reared from an undetermined aphid on Ficus sycomorus (Prinsloo and Neser 1994).Furthermore, Aphelinus glycinis was collected in northeastern China, whereas A. engaeus and A. ficusae have been reported only from South Africa.

Aphelinus rhamni
Hopper & Woolley, sp.n. urn:lsid:zoobank.org:act:1132B1E4-8F2E-4FF3-9E6B-30FAFD497EA1http://species-id.net/Aphelinus_rhamniFigs 15-28 Diagnosis.Females.Head and mesosoma dark brown to black; legs with coxae dark brown to black, profemur dark grey with pale apex, mesofemur dark grey to black, metafemur white, protibia white with pale greyish base, mesotibia dark grey to black with pale base and apex, and metatibia dark grey to black with pale base; metasoma yellowish brown with base and apex yellow; antennae yellow with basal half of scape and pedicel sometimes greyish; F3 quadrate; club 2.8 times as long as broad.Males similar except scape swollen in middle, 3× broader in middle than at distal end, with 2 or 3 volcano-shaped secretory pores; scape dark yellowish grey, pedicel pale greyish yellow; club 3.3 times as long as broad.
Head. (Figs 16,22) Head 1.2× as broad as high in frontal view, about as broad as mesosoma; frontovertex 0.4× head width and as broad as scape length; posterior ocelli sensilla, interior of reticulations with fine, granulate surface (visible only in slidemounts under high magnification); mid-lobe of mesoscutum with 2 pairs of long setae and 35-40 short setae, side lobes each with 2 long and 1 short seta; scutellum with 2 pairs of long setae, pair of scutellar sensilla directly posterior to the anterior pair of setae and slightly posterior to middle of scutellum; mesotibial spur equal in length to mesobasitarsus; metatibial spur 0.5× metabasitarsus.
Fore wing.(Fig. 24) 2.2× as long as broad ; costal cell with 1 row of dorsal setae and two rows of ventral setae, the posterior row extending from under the proximal end of the marginal vein almost to stigma, costal cell 1.1× as long as marginal vein; submarginal vein with 5 setae, marginal vein with 10 setae along the anterior margin; stigmal vein short with stigma rounded; delta region proximal to linea calva with one complete line of 13-15 setae and 2-6 additional setae in angle with marginal vein, linea calva with no setae at its posterior edge; wing distal to linea calva with evenly spaced, dense dorsal setae and much smaller ventral setae.
Color.(Figs 16,18,20) Head and mesosoma dark brown to black; legs with coxae dark brown to black, profemur dark grey with pale apex, mesofemur dark grey to black, metafemur white, protibia white with pale greyish base, mesotibia dark grey to black with pale base and apex, and metatibia dark grey to black with pale base; metasoma yellowish brown with venter of T1 and apex yellow; antennae yellow sometimes with basal half of scape and pedicel greyish yellow; compound eyes dark burgundy and ocelli red in life, both silver-colored in dried specimens.
Host.In the field, Aphis glycines is the only known host.In laboratory experiments, A. rhamni parasitizes A. glycines and closely related species in the genus Aphis, and rarely Rhopalosiphum padi L. and Schizaphis graminum.
Etymology.This species is named for the primary host plant of the aphid species from which it was collected.The species epithet is a noun in genitive case.
Relationships.Aphelinus campestris and Aphelinus gossypii are the closest described species to A. rhamni based on our matrix of traits (Table 2).Aphelinus rhamni differs from both species in having a more elongate club and in coloration of the metatibia.Aphelinus rhamni has a much narrower host range than A. gossypii, which is reported from at least 18 species of aphids in 10 genera and two tribes, including species which A. rhamni does not parasitize in laboratory experiments.
Aphelinus coreae sp.n. urn:lsid:zoobank.org:act:F4B3A880-2136-474C-815C-13406F2A48A0http://species-id.net/Aphelinus_coreaeFigs 29-42 Diagnosis.Females.Head and thorax dark brown to black; legs with coxae dark brown to black, profemur dark grey with distal half pale, mesofemur dark grey to black, metafemur pale yellowish white, protibia pale yellowish white to somewhat fuscous, mesotibia dark grey to black with distal half pale, and metatibia dark grey to black with pale base; metasoma dark brown with base and apex yellow; antennae yellow; F3 quadrate.Males similar except scape swollen in middle, 2.0× as broad in middle than at distal end, with two or occasionally three circular secretory pores in the middle of a shallow depression on ventral surface, scape dark yellowish grey with distal half yellow, pedicel greyish yellow.
Head. (Figs 30,36) Head 1.3× as broad as high in frontal view, about as broad as mesosoma; frontovertex 0.4× head width and as broad as scape length; posterior ocelli approximately their own diameter from eye margin, 5× their diameter from one another, and 0.5× their diameter from occipital margin; mandible with two acute teeth and a broad truncate surface below the teeth, ventral tooth sometimes not distinct; antennae as in Figs 30 and 36 with scape 4.0× as long as broad, pedicel 1.6× as long as broad, F1 anneliform, F2 1.4× as broad as long, F3 subquadrate or very slightly broader than long, club 3.75× as long as broad and 3.5× longer than F3, with 7-8 linear sensilla.
Mesosoma.(Figs 32,34,41) Mesosoma and scutellum with fine reticulate sculpture, longest diameter of reticulations approximately 2-3× diameter of scutellar sensilla; interior of reticulations with fine, granulate surface (visible only in slide-mounts under high magnification), mid-lobe of mesoscutum with 2 pairs of long setae and about 40-60  and mesotibia.Like A. rhamni, A. coreae has a much narrower host range than A. gossypii.Aphelinus coreae is very close to A. rhamni, but male A. coreae have shorter clubs and, as noted in the key, the two species differ in coloration of scape and mesotibia.Although difficult to distinguish, these species are reproductively isolated in laboratory crosses.Their DNA differs by 2130 fixed substitutions and 293 indels across 1.8 megabases of homologous DNA sequence.They also differ in host specificity: A. coreae parasitizes species of Aphis, e.g. A. nerii Boyer de Fonscolombe and A. rumicis L., not parasitized by A. rhamni in laboratory experiments.

table 1 .
Species in Aphelinus mali complex and related species The label data for each specimen has been digitized and all specimens bear individual accession numbers for Texas A&M University Insect Collection (e.g.TAMU x0616203), as well as a machine-readable bar-code.

table 2 .
Traits coded for species in mali complex of Aphelinus.