Description of three new species of oak gallwasps of the genus Amphibolips Reinhard from Mexico (Hymenoptera, Cynipidae)

Abstract Three new species of oak gall wasps of the genus Amphibolips Reinhard, 1865 (Hymenoptera: Cynipidae: Cynipini) are described from Mexico: Amphibolips magnigalla Nieves-Aldrey & Castillejos-Lemus, Amphibolips kinseyi Nieves-Aldrey & Castillejos-Lemus and Amphibolips nigrialatus Nieves-Aldrey & Castillejos-Lemus. The specimens of the first two species were representative of sexual generations and come from the State of Oaxaca, while only a female, collected in the State of Veracruz, is described for A. nigrialatus. The new species induces galls on Quercus zempoaltepecana and Q. sapotifolia (Fagaceae, section Lobatae, red oaks). Descriptions of the diagnostic morphological characteristics of the three species and a key for their identification are provided. The taxonomic relationships of the new species with other species of Amphibolips are discussed; the three new species are closely allied amongst themselves and are related to A. dampfi Kinsey, 1937. With the three newly-described species, the number of Amphibolips in Mexico is increased to 23.


Introduction
Oak gall wasps (Cynipidae: Cynipini) include approximately 41 genera with circa 1,000 species (Liljeblad et al. 2008, Pénzes et al. 2018) distributed mainly in the Holarctic, Neotropical and Oriental regions (Ronquist et al. 2015). They represent the largest tribe of Cynipidae and are a monophyletic group of wasps that induce relatively more structurally complex and diverse galls of the known gall types (Cornell 1983, Stone and Cook 1998, Nieves-Aldrey 2001, Csöka et al. 2005. Cynipini species are predominantly associated with host species of Quercus (Liljeblad et al. 2008), but some genera of Cynipini use other hosts within the Fagaceae, such as Castanea, Castanopsis, Lithocarpus, Chrysolepis and Notholithocarpus (Stone et al. 2002. A particularity of the Cynipini is that most species exhibit life histories with alternating generations (e.g. asexual and sexual) (Stone et al. 2002).
The Nearctic region, particularly Mexico, is one of the centres of diversity of the oak gall wasps, which have been estimated to include more than 700 species (Stone et al. 2002, Liu et al. 2007). This diversity is directly related to the diversity of Quercus species, with more than 90 species recorded from the United States and Canada and 161 from Mexico (Valencia 2004, Liu andRonquist 2006). The most recent work on the number of Cynipidae species recorded from Mexico indicates the presence of 183 species in 16 genera (Pujade-Villar and Ferrer-Suay 2015) associated with approximately 35 Quercus species.
Amphibolips is exclusively associated with the Lobatae section of Quercus genus and is restricted to the American continent (Nieves-Aldrey et al. 2012). Fifty-three species of Amphibolips are recognised; the vast majority are found in the Nearctic region, three species are distributed in Panama, 19 species are endemic to Mexico and one is shared with the United States (Burks 1979, Melika and Abrahamson 2002, Medianero and Nieves-Aldrey 2010, Melika et al. 2011, Nieves-Aldrey et al. 2012, Pujade-Villar et al. 2018.
The morphological characteristics of adults and their galls are very uniform amongst the most well-known Amphibolips species. The galls induced by species of this genus develop mainly in buds, stems or leaves and are rarely found in acorns. They are usually globose or spindle-shaped and detachable, with a spongy parenchyma surrounding a central larval cell, sometimes supported by radiating filaments (Beutenmüller 1909, Kinsey 1937, Melika and Abrahamson 2002. The species of this genus can be easily recognised by the following diagnostic characteristics: antennae with 12 to 14 segments in females and 15 to 16 segments in males; body robust with strong coarse reticulate sculpture, notauli not well marked, mesoscutellum often emarginate posteriorly; metasomal tergites punctate posteriorly; metatarsal claws with a large secondary basal tooth; forewings usually more or less smoky and showing spots, bands or completely obscured; radial cell open; ventral spine of the hypopygium usually long and pointed apically, without setae forming an apical tuft (Melika and Abrahamson 2002, Medianero and Nieves-Aldrey 2010, Melika et al. 2011. Before 1937, only two species had been described in Mexico (A. palmeri Basset, 1890 and A. nigra Beutenmüller, 1911) (Bassett 1890;Beutenmüller 1911Beutenmüller , 1917. In 1937, Kinsey described nine species, six of which he grouped in the "niger" complex; the remaining three (A. dampfi Kinsey, 1937, A. nassa Kinsey, 1937 and A. fusus Kinsey, 1937) were not grouped. Melika et al. (2011) described two new species: A. zacatecaensis Pujade-Villar, 2011 andA. hidalgoensis Pujade-Villar &Melika, 2011. Parallel to the "niger" complex proposed by Kinsey (1937), a second group, the "nassa" species complex, was proposed and a species identification key was provided for A. palmeri, A. dampfi, A. nassa, A. hidalgoensis, A. zacatecaensis and A. fusus. Nieves-Aldrey et al. (2012) described seven new species outside of those in the "niger" group, raising the number of known species to 13. In the referenced paper, the "nassa" complex was criticised as useless, based on the assumption that it did not reflect the extant species diversity outside of the "niger" group, as the complex omitted the anterior wing colouration pattern, which was important for some of the species described by Kinsey, such as A. dampfi, A. fusus and A. nassa (Nieves-Aldrey et al. 2012). More recently, an additional species (A. cibriani Pujade-Villar, 2018) was described within the "nassa" group (Pujade-Villar et al. 2018), resulting in a total of 20 species of Amphibolips recorded from Mexico.
The objective of this study is to present a description of three new species of the Amphibolips genus in Mexico. One of these species represents the first record of Amphibolips for the State of Veracruz (A. nigrialatus Nieves-Aldrey & Castillejos-Lemus, new species) and the other two species were collected in the State of Oaxaca. One of the species from Oaxaca induces a strikingly-characteristic gall (A. magnigalla Nieves-Aldrey & Castillejos-Lemus, new species), while the other species from Oaxaca (A. kinseyi Nieves-Aldrey & Castillejos-Lemus, new species) shares characteristics with A. magnigalla and with another species previously described from Oaxaca (A. dampfi Kinsey, 1937). The species richness of this genus in Mexico is discussed, as well as the taxonomic problems existing within the group. An update to the identification key given in Nieves-Aldrey et al. (2012) is provided, including the new species described herein. This work is part of a larger study of the revision of the Amphibolips species of Mexico, which includes extensive sampling throughout most of Mexico. Rich materials of Amphibolips have been collected, including possible additional new species and are being studied with a phylogenetic approach, including genetic tools. The results will be published elsewhere.

Study material
Quercus species of the Lobatae section were sampled in Veracruz State in 2008 and in Oaxaca in 2018. The galls were collected directly from oak trees and stored in plastic containers with plastic or mesh lids until the emergence of the wasps. The emergence of the wasps occurred under laboratory conditions. The voucher specimens and their galls were deposited in the entomological collections of the Museo Nacional de Ciencias Naturales in Madrid, Spain and in the Colección Nacional de Insectos of the Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City, Mexico. The Quercus species were identified by Dr Susana Valencia-Ávalos at the Facultad de Ciencias of the Universidad Nacional Autónoma de México (UNAM). Voucher specimens were deposited in the Herbarium of the Facultad de Ciencias and in the Escuela Nacional de Estudios Superiores, Unidad Morelia (ENES-Morelia) of the UNAM. Observations on the habitats, distribution or affinities of the host Quercus species were mainly based on Valencia-Ávalos (2004), but other publications on Quercus species were also consulted.

Examination of types
The type specimen of Amphibolips dampfi Kinsey, 1937 was examined for comparison with the new species described. The male holotype of this species was borrowed from the American Museum of Natural History, New York (AMNH) (James Carpenter).

Specimen preparation
The images used for the morphological descriptions were taken with a FEI Quanta 200 (Oregon, EU) scanning electron microscope (SEM) in Madrid (Spain) and with a JEOL JSM-IT300 (Tokyo, Japan) SEM in Morelia (Mexico). For the SEM observations, two strategies were followed, depending on the number of individuals available for a given species. For the preservation of some unique specimens mounted in a conventional manner, a low vacuum technique was used without gold coating. When the number of specimens allowed it, some specimens were dissected in 99% alcohol and mounted in stubs to be coated with gold and observed with a high vacuum technique. The forewings were mounted on slides with euparal and examined with a Wild MZ8 and an Olympus SZX10 stereomicroscope. Images of the wings and adult habitus were acquired with a NIKON Coolpix 4500 digital camera attached to a Wild MZ8 light microscope, with the exception of some images taken with an Olympus SC100 camera with the help of CELLSENS STANDARD software. Measurements were made with a micrometric eyepiece calibrated to a Wild M5A stereomicroscope. Photographs of galls in the field and of gall dissections were taken with a Nikon D5300 camera.

Morphological terms
The terminology of the morphological structures and abbreviations follow that of Ronquist and Nordlander (1989), Nieves-Aldrey (2001) and Liljeblad et al. (2008). For wing venation, we follow Ronquist and Nordlander (1989) and for the terminology of the forewing cells, we follow Richards and Davies (1977). For sculpture terminology, we follow Harris (1979). The measurements of the structures were made according to Nieves-Aldrey (2001). The abbreviations used include F1-F12 for the antennal flagellomeres, POL (post-ocellar distance) for the distance between the inner margins of the posterior ocelli, OOL (ocellar-ocular distance) for the distance from the outer margin of a posterior ocellus to the inner margin of a compound eye and DOL (diameter of a lateral ocellus).  Pujade-Villar, 2018(Pujade-Villar et al. 2018). However, the forewing pattern of the new species is different from that of all the referenced species. The transversal clear band is larger and broader and extends to two-thirds of the radial cell in both sexes and the basal third of the wing is more heavily infuscate in the male (Fig. 4A, B).

Amphibolips magnigalla
Amphibolips magnigalla shares with Amphibolips dampfi, A. castroviejoi and the other two new species described herein, a mesoscutellum emarginate posteriorly. However, the emargination is comparatively less deep in A. magnigalla (Figs 1C,2C). Besides the main character of the forewing, the four species can be readily distinguished by the characters provided in the identification key in this paper.
Regarding the gall, the new species is easily distinguished by its large spindleshaped gall (approximately 10 cm in length × 2.5 cm in diameter), which is at least 2× larger than any other spindle-shaped gall described from Mexico. Amphibolips fusus and A. durangensis induce galls morphologically similar to the gall of the new species. However, besides the differences in size, the inner structure of the gall induced by the referenced species is different, being filled with a dense soft tissue, while the inner structure of the gall induced by the new species is often almost empty, with visible radiating filaments from the central larval cell.
Description. Body length: 5.8 mm (n = 1) for females; 5.2 mm (n = 3) for males. Female (Fig. 4C). Body almost entirely black; antennae, except two basal segments, mandibles, metasoma ventrally, hypopygium and parts of tibiae and tarsi, chestnut. Forewing predominantly black infuscate, except a wide clear transversal band that starts in the distal two thirds of radial cell and extends towards discoidal and Head, in dorsal view 2.3× wider than long. POL:OOL:DOL as 23:44:14. Head in anterior view (Fig. 1A) 1.2× wider than high, gena slightly broadened behind eye. Vertex, frons, lower face, gena and occiput with strong reticulate-rugose sculpture (Fig. 1B); two longitudinal carina present, extending from ventral margin of toruli to converge towards the anterior tentorial pits; irradiating carinae from clypeus absent; head moderately pubescent, except in vertex and frons. Clypeus more or less hexagonal, ventral margin strongly projecting over mandibles and slightly sinuate on anterior margin. Anterior tentorial pits well visible; epistomal sulcus and clypeo-pleurostomal lines slightly discernible. Malar space 0.7× height of compound eye. Toruli situated mid-height of compound eye; transfacial line 1.4× height of an eye; distance between antennal rim and compound eye slightly shorter than width of antennal socket including rim. Ocellar plate slightly raised.
Head posterior view (male) (Fig. 2B), heavily pubescent, with occiput coarsely rugose; dorsally the sculpture is transversely ribbed. Two carinae present, arising from dorsal part of the occipital foramen and ventrally continuing past posterior tentorial pits; posterior tentorial pits rounded; gular sulci united meeting at hypostoma. Posterodorsal margin of oral foramen not margined medially; hypostomal ridges well separated.
Mouthparts (male) ( Fig. 2A), mandibles strong, exposed; with dense setae in base, right mandible with three teeth; left with two teeth. Cardo of maxilla not visible, maxillary stipes 4.1× as long as wide. Maxillary palp with five segments. Labial palp with three segments; apical peg of last labial and maxillary segments present.
Mesonotum. Mesoscutum (Fig. 1C) barely pubescent and with strong reticulaterugose sculpture, the interspaces smooth and shining. Notauli somewhat obscured by the coarse sculpture, but visible; strongly convergent posteriorly; longitudinal median impression, not discernible. Anteroadmedian signa quite visible, extended back to near one half of mesoscutum; parapsidal signa distinct. Transscutal fissure narrow, sinuate. Mesoscutellum 1.2× as long as wide; about 0.7× as long as mesoscutum. Scutellar foveae ( Fig. 1D) rounded, elongated posteriorly, about 0.5× as long as mesoscutellum, separated medially by a groove, the foveae are deep, mostly smooth anteriorly and crossed posteriorly by irregular transversal rugae, the intervals smooth, posterior margins indistinct. Mesoscutellum strongly coarsely rugose, with a deep and broad median longitudinal impression which makes the mesoscutellum strongly emarginate posteriorly (Fig. 1D); the emargination reaches anteriorly the scutellar foveae. In lateral view, the posterior emargination of mesoscutellum is seen as two, slightly curved upwards, horn-like projections. Mesoscutellum in lateral view with the posterodorsal extension of body of subaxillular strip short, not reaching one half of mesoscutellar height. Mesopleuron coarsely reticulate rugose, the rugae not as strong as in mesoscutum (Fig. 1E).
Forewing (Fig. 4A), about as long as body, radial cell 3.2× longer than wide; open along anterior margin; areolet obsolete, obscured by infuscation. M and Cu1 veins nearly straight, not reaching wing margin. Rs+M not reaching basalis. First abscissa of radius (2r) slightly angled, not projected. Cu1 vein not branched in two veins. Apical margin with very short or obsolete hair fringe. Metasoma (Fig. 3A, D), in dorsal view 1.6× as long as wide, in lateral view 1.2× as long as high. Second metasomal tergite covering about 0.7× length of metasoma. Anterior 2/3 smooth and shining; posterior one third with a band of micropunctures clearly visible; the punctate sculpture extended on subsequent tergites; ventral area of second metasomal tergite moderately pubescent. Projecting part of hypopygial spine moderately long (Fig. 3B); 4.6× as long as high in lateral view; laterally with long setae, longer than spine width, but not forming an apical patch.
Male ( Gall ( Fig. 5A-D). A large spindle-shaped gall with an elongated and narrow tip and base. The galls measure 100 × 25 mm on average. The surface of the gall is smooth, but some superficial longitudinal ridges are visible. The gall is monothalamic; the outer shell is thin, flexible and of fleshy consistency when it is fresh and becomes soft and light when it dries. They are light green without spots when they are fresh and light brown when they are dry. Internally (Fig. 5C), there is an oval larval cell in the centre of the gall (0.35 mm thick and 7 × 5 mm; n = 1). A spongy tissue occupies the entire space between the epidermis and the larval chamber, the outer shell is weakly attached to the internal spongy tissue when fresh and when the gall dries, the spongy tissue allows us to observe the radiant filaments, which extend from the larval chamber towards the internal walls of the galls (Fig. 5D). When it is dry, the gall is very fragile and can be easily crushed. At least half of the galls no longer showed spongy tissue when they were transferred to the laboratory. This caused the galls to collapse due to the fragility of the epidermis. Some of these collapsed galls presented internal modifications in the epidermis, probably caused by inquilines.
The galls develop on twigs of Quercus zempoaltepecana Trel. The gall closely resembles that of Amphibolips durangensis Nieves-Aldrey & Maldonado, 2012. However, the gall of A. magnigalla is distinguished by its larger size, which is at least 2× longer than that of A. durangensis and by its different internal structure, which is filled with less dense spongy tissue and radiant filaments (easily visible in the older galls). Distribution. A. magnigalla was found only in one site: Comaltepec (Oaxaca State, Mexico). The galls were relatively abundant on a single isolated tree, while we did not find galls on the nearby trees.
Biology. Sexual generation. The galls were collected at the end of April and the insects emerged shortly thereafter, in early May. It seems that it is normal for many insects to feed on the tissue of this species. A detached gall was observed in a field, relatively far from the tree, probably carried by a bird. Etymology. Named after Dr Alfred Kinsey, one of the most prominent cynipidologists and the pioneer of the study of Amphibolips in Mexico.

Amphibolips kinseyi
Diagnosis and comments. Amphibolips kinseyi is very similar to A. dampfi Kinsey, 1937. We collected the new species in sites near where collections by A. Dampf were made (near Ixtlán, Oaxaca), the material of which was later described by Kinsey (Kinsey 1937). Both species share a strongly emarginate mesoscutellum and have a similar forewing colour pattern. However, after a close comparison with the male holotype, we found some diagnostic differences that allowed us to describe our specimens as different and new species. The forewings of the males of A. dampfi and A. kinseyi are similar, being predominantly black infuscate and have a reduced clear transversal band; however, in A. dampfi, the first radial abscissa (2r vein) is strongly angled and projected into the radial cell (Fig. 9C), while it is weakly angled and not projected in A. kinseyi (Fig. 9D). The postero-lateral projections of the mesoscutellum are more or less pointed or acute in the A. dampfi males (Fig. 9A), but are more rounded and flatter in the case of the new species (Fig. 9B). Additional distinguishing characters are given in the identification key provided herein. The female forewing of A. dampfi was not available for description, as it was apparently lost in the only female collected. However, the female forewing of this closely-allied new species is described here. Consistent with other Amphibolips species from Mexico, the female forewing is different from the male forewing. In this case, the female forewing has a clear transversal band, which is larger and more extended than that of the male (Fig. 8C).
Description. Body length: 6.3 mm (n = 1) for females; 5.7 mm (n = 3) for males. Female. Body predominantly black (Fig. 8A); head, except the red mandibles and the mesosoma, black; metasoma reddish postero-ventrally; antennal flagellum reddish in distal half; legs reddish except black basal part of coxae. Forewing (Fig. 8C) predominantly black infuscate, but much less infuscate above the cubital veins and below the M+Cu1 vein. There is a wide, clear, transversal band, which starts in the apex of the radial cell and extends towards the discoidal and cubital cells to the Cu1a vein but does not reach the latero-ventral margin of the forewing.
Head, in dorsal view 2.1× as wide as long; 0.8× as wide as mesosoma. POL 0.7× the OOL; lateral ocelli separated from inner margin of an eye for a distance of 3× the diameter of a lateral ocellus. Head in anterior view (Fig. 6A) 1.2× wider than high, gena slightly broadened behind eye. Vertex, frons, lower face and gena, with strong coarsely-rugose sculpture. A medial frontal pit visible followed by a sulcus addressed to the median ocellus. Face with two longitudinal carinae visible, extending from ventral margin of toruli to converge towards the space between the anterior tentorial pits; irradiating carinae from clypeus virtually absent; head moderately pubescent, except in vertex and frons. Clypeus more or less hexagonal, ventral margin strongly projecting over mandibles and weakly sinuate on anterior margin. Anterior tentorial pits well visible; epistomal sulcus and clypeo-pleurostomal lines visible. Gena slightly depressed basally and projected over the mandibles. Malar space 0.7× height of a compound eye. Toruli situated mid-height of compound eye; transfacial line 1.7× height of an eye; distance between antennal rim and compound eye 0.8× width of antennal socket including rim. Ocellar plate slightly raised.
Mouthparts (Fig. 6A), mandibles strong, exposed; with dense setae in base, right mandible with three teeth; left with two teeth. Antenna (Fig. 6E), about one half as long as body length; with 13 antennomeres; 12 and 13 incompletely separated ventrally. Flagellum not broadening towards apex; with relatively long, erect setae. Relative length/width of antennal segments as: 0. . Scape slightly longer than wide, flattened and smooth ventrally. Pedicel, short, small, as long as wide, 0.5× as long as scape; F1 1.3× as long as F2, F11 2.3 times as long as wide, 2× as long as F10. Placodeal sensilla present on flagellomeres F3-F11, disposed in dense rows of 6-8 sensilla, only in half dorsal area of each flagellomere. Coeloconic and trichoidea sensilla are also present and visible (Fig. 6F).
Mesosoma in lateral view 1.3× as long as high. Pronotum, moderately pubescent; lateral surface of pronotum with strong irregular reticulate rugose sculpture (Fig. 6D). Pronotum medially short; ratio of length of pronotum medially/laterally = 0.2. Pronotal plate indistinct dorsally. Mesonotum. Mesoscutum (Fig. 6C) barely pubescent and with strong coarse reticulate sculpture, the interspaces smooth and shining. Notauli shallow and crossed by the general sculpture, but well visible and almost complete; longitudinal median impression not visible. Anteroadmedian signa well marked, extended back to near one half of mesoscutum; parapsidal signa distinct. Transscutal fissure very narrow, sinuate. Mesoscutellum 1.3× as long as wide; about 0.5× as long as mesoscutum. Scutellar foveae ovoid elongated posteriorly, about 0.5× as long as mesoscutellum, separated medially by a deep groove, the foveae are deep, crossed by irregular transversal rugae, the intervals smooth, posterior margins shallowly indicated. Mesoscutellum strongly coarsely rugose, with a deep and broad median longitudinal impression which makes the mesoscutellum strongly emarginate posteriorly (Fig. 6C); the emargination reaches anteriorly the scutellar foveae. In lateral view, the posterior emargination of mesoscutellum appears as two rounded apically and slightly flat, curved upwards projections. In lateral view, the space between the mesoscutellar projections and the posterior limit of mesoscutellum is high. Axillula large, deep, heavily pubescent, with distinct margins. Mesoscutellum in lateral view with the posterodorsal extension of body of subaxillular strip long, nearly reaching upper margin of mesoscutellum. Mesopleuron coarsely reticulate rugose, the rugae not as strong as in mesoscutum.
Forewing (Fig. 8C), about 1.2× as long as body, radial cell 3.7× as long as wide; open along anterior margin; areolet very small, but visible. All veins heavily infuscate. M and Cu1 veins nearly straight, not reaching wing margin. Rs+M reaching basalis, well-marked. First abscissa of radius (2r) slightly angled, not projected. Cu1 vein not branched in two veins. Apical margin with very short or obsolete hair fringe. Metasoma (Fig. 7D), in lateral view 1.3× as long as high. Second metasomal tergite covering about 0.6× length of metasoma. Anterior 2/3 smooth and shining; posterior one third with a band of micropunctures clearly visible; the punctate sculpture extended on subsequent tergites; ventral area of second metasomal tergite moderately pubescent, with a relatively dense patch of setae. Projecting part of hypopygial spine moderately long (Fig. 7D); about 5× as long as high in lateral view; laterally with long setae, longer than spine width, but not forming an apical tuft.
Male (Figs 6B, 7A, E, 8B, D). Differs from the female as follows: smaller size, length 5.7 mm on average (n = 3). Body and wings almost completely black, except the mandibles, metasoma ventrally, tarsomeres of legs and half of the apical flagellomeres of antennae which are more or less reddish (Fig. 8B). Antennae, legs and wings relatively longer. Antenna with 14 segments (Fig. 7A as wide as high; apical part of gena slightly expanded. Frontal pit distinct, prolonged by a groove towards median ocellus. Pair of frontal longitudinal carinae more convergent towards epistomal line (Fig. 6B). Projection of anterior margin of clypeus more incised. Forewing (Fig. 8D) relatively longer 1.3× as long as body. Almost completely black, except the distal transversal clear band that is much smaller and less extended. Gall (Fig. 8E, F). The gall is similar to the gall of Amphibolips dampfi described by Kinsey (1937). A moderate to large "oak apple", irregularly spherical gall with spongy inner consistency. Some are slightly elongated towards the apex. The surface is slightly rough when intact, but may have more pronounced irregularities, which cause deformations on the surface or in the general shape. Monothalamic. They are light green without spots when they are fresh and light brown when they are dry. The epidermis is thin, at 0.4 mm thick; firmly attached to the internal spongy tissue when fresh; firm and brittle when dry. The consistency is relatively hard and fleshy when green and brittle when dry. Internally, the spongy tissue occupies the entire space between the epidermis and the larval chamber (Fig. 8F). Diameter of 30 mm and height of 31 mm on average (diameter of 16 to 44 mm and height of 18 to 51 mm; n = 18). Rigid and oval larval cell, 0.4 mm thick and 6.5 mm long × 5 mm in diameter on average (n = 2). Galls are formed on the twigs of Quercus zempoaltepecana Trel. Galls are relatively common in the study area.
Distribution. Known only from the type locality along the route from Ixtlán to Tepanzacoalcos (Oaxaca State, Mexico).
Biology. Sexual generation. The galls were collected in late April and the insects emerged shortly thereafter, in early May. It is normal to find galls deformed and/or attacked by inquilines and parasitoids; the deformed or attacked galls are usually relatively small. Etymology. Named after the smoky black forewing. Kinsey, 1937 and the new species Amphibolips kinseyi. Despite being based on a single female holotype, we found distinctive diagnostic characters that let us describe the specimen as belonging to a new species. The strongly-emarginate mesoscutellum relates the new species to A. kinseyi and A. dampfi, but in A. nigrialatus, the posterolateral projections of the scutellum are pointed apically and curved upwards. Moreover, the scutellar foveae are very large in the new species, extending approximately one half of the length of the mesoscutellum, medially confluent and not separated by a carina or groove, while they are well separated by the mesoscutellar impression in the other two species. The forewing colour of A. nigrialatus is the darkest we have seen in females of Amphibolips species from Mexico and the black smoky colouration even extends to the costal and basal cells and below the cubital vein. In this last character, it resembles A. castroviejoi from Panama, but in this late species, the forewing area anterior to the transversal band is even darker (Fig. 15F), besides other distinguishing characters given in the identification key (transversal clear band larger, smooth scutellar foveae and visible notauli). The clear transversal band in the discoidal cell of forewing is very short and narrow in A. nigrialatus, measuring not more than one-fifth of the length of the radial cell (Fig. 13A), while in A. kinseyi, it is wider, measuring at least one-half the length of the radial cell (Fig. 8C). Additionally, the green spherical gall of the new species (A. nigrialatus) on Quercus sapotifolia is distinguishable from the galls of A. dampfi and A. kinseyi on Q. ocoteifolia Liebm. and Q. zempoaltepecana Trel., respectively.

Diagnosis and comments. Amphibolips nigrialatus is closely allied to A. dampfi
Description. Body length: 6.6 mm (n = 1) for the female. Female. (Fig. 13B). Body almost completely black with the exception of the mandibles, the antennae apically, the metasoma ventrally, especially the hypopygium and parts of the legs, including the tarsi, which are reddish. Forewing predominantly black infuscate, except a large basal area delimited by the medial and cubital veins and another smaller area above the medial vein. A small clear transversal band, that starts below the radial cell and extends towards the medial vein, but does not reach the cubital vein, is present (Fig. 13A).
Head, in dorsal view (Fig. 10A) 2.2× as wide as long; narrower than mesosoma. OOL 1.4× POL 0.7× DOL; posterior ocelli separated from internal orbit of an eye by 2.2× its diameter. Head in anterior view (Fig. 11A) 1.3× as broad as high; gena slightly broadened behind eyes. Vertex, frons, lower face and gena, with coarse reticulate-rugose sculpture. Face with two longitudinal carinae, extending from ventral margin of toruli to converge towards the anterior tentorial pits; irradiating carinae from clypeus indistinct or absent; head moderately pubescent, except in vertex and frons. Clypeus more or less hexagonal, ventral margin strongly projecting over mandibles and moderately incised or sinuate on anterior margin. Anterior tentorial pits well visible; epistomal sulcus and clypeo-pleurostomal lines also distinct. Malar space 0.8× height of a compound eye. Toruli situated about mid-height of compound eye; transfacial line 1.7× height of an eye; distance between antennal rim and compound eye almost equal to the width of antennal socket including rim. Ocellar plate slightly raised. Mouthparts (Fig. 11A), mandibles strong, exposed, with dense setae in base, right mandible with three teeth, left with two teeth. Antenna (Fig. 10B), 0.6× as long as body length; with 13 antennomeres; 12 and 13 incompletely separated (Fig. 10C) (0.16). Pedicel, short, 0.4× as long as scape and slightly broader than long. F1 1.6× as long as F2; F8-F10 as long as wide, F11 2.1× times as long as wide, 1.9× as long as F10. Placodeal sensilla present on flagellomeres F3-F11, disposed in dense rows of 8-10 sensilla, only in half dorsal area of each flagellomere.
Mesonotum. Mesoscutum (Fig. 10A) barely pubescent and with strong coarse reticulate sculpture, the interspaces smooth and shining. Notauli indistinct, obscured by the coarse sculpture; more so in anterior one third of mesoscutum; longitudinal median impression indistinct. Anteroadmedian signa and parapsidal signa distinct. Transscutal fissure very narrow, sinuate. Mesoscutellum as long as wide; about 0.5× as long as mesoscutum. Scutellar foveae rounded transverse, about 0.5× as long as mesoscutellum, the scutellar foveae are confluent and not separated medially by a carina or groove; some transverse strong rugae visible with smooth and shining intervals. Mesoscutellum strongly coarsely rugose, with a deep and broad median longitudinal impression which makes the mesoscutellum strongly emarginate posteriorly (Fig. 10A); the emargination not reaching anteriorly the scutellar foveae. Postero-lateral projections of scutellum pointed apically and curved upwards (Fig. 11C, D). Mesopleuron coarsely reticulate rugose, the rugae not as strong as in mesoscutum (Fig. 11B). Mesoscutellum in lateral view with the posterodorsal extension of body of subaxillular strip short, not reaching one half of mesoscutellar upper margin.
Forewing (Fig. 13A), about 1.1× as long as body, radial cell 3.9× as long as wide; open along anterior margin; areolet absent. All veins heavily infuscate. M and Cu1 veins nearly straight, not reaching wing margin. Rs+M complete, reaching basalis. First abscissa of radius (2r) slightly angled, not projected into the radial cell. The two branches of the cubitalis vein are not interrupted by a gap. Apical margin with very short hair fringe. Metasoma (Fig. 12A) in dorsal view 1.5× as long as wide; in lateral view 1.2× as long as high (Fig. 11F). Second metasomal tergite covering about 0.64× the length of metasoma. In dorsal view, anterior half of T2 smooth and somewhat shining, posterior half with two types of microsculpture clearly visible, first a series of slightly hexagonal cells and in the back micropunctures, both microstructures occupying the same proportion (Fig. 12B). In lateral view, anterior 2/3 smooth and somewhat shining; posterior one third with a band of micro- punctures clearly visible. The punctate sculpture extended on subsequent tergites. Ventral area of second metasomal tergite moderately pubescent, with a relatively dense patch of setae. Projecting part of hypopygial spine long (Fig. 12D); about 6.3× as long as high in lateral view; laterally with long setae, longer than spine width, but not forming an apical tuft.
Male. Unknown. Gall (Fig. 13C). A regular, spherical, moderately-sized gall with a green colour when it is fresh. When dry, the gall acquires a rough and slightly elongated appearance and turns brown in colour. The galls measure on average 16.5 × 21.5 mm (diameter of 14 to 19 mm and length of 17 to 25 mm; n = 4). The gall is monothalamic. The outer shell is thin, flexible and of fleshy consistency when fresh and becomes rigid and hardly detachable from the parenchyma when dry. Internally, there is a spherical larval cell in the centre of the gall (5 × 5 mm; n = 1); a spongy tissue occupies the entire space between the epidermis and the larval chamber and is hardly separable from the larval chamber. When it is dry, the gall is moderately fragile.
On twigs of Quercus sapotifolia Liebm. Closely resembles that of Amphibolips oaxacae Nieves-Aldrey & Pascual, 2012, A. michoacaensis Nieves-Aldrey y Maldonado, 2012, A. trizonata Ashmead, 1896 and Amphibolips kinseyi sp. nov. However, the gall of A. nigrialatus differs in its size, which is approximately half that of the other species. The gall is similar to that of A. murata Weld, 1957, but not as rough when dry and to that of A. quercusfuliginosa Ashmead, 1885, from which it is impossible to differentiate according to the original description of the gall. Nonetheless, the adults are completely different.
Distribution. Known only from the type locality in Veracruz State, Mexico. Biology. Presumably, a sexual generation. The gall was collected in late April and the insect emerged shortly afterwards. Forewing with a heavily-infuscate spot on the basal area of radial cell; remainder of the forewing, hyaline to only slightly infuscate (Fig. 15D)  Basal and first cubital cells colourless or only weakly infuscate prior to the heavily-infuscate basal half of the radial cell (Fig. 15A, B). Mesoscutellum weakly emarginate posteriorly (Fig. 14C) Mesoscutellum only slightly emarginate posteriorly (Fig. 14B). Clear transversal band relatively smaller and narrow (Fig. 15G)  Mesoscutellum more deeply emarginate posteriorly; postero-lateral projections of mesoscutellum moderately expanded (Fig. 1D). Non-infuscate clear forewing transversal band large and broad, almost reaching ventral margin of forewing; medially measuring approximately three-quarters of the length of the radial cell (Fig. 4A)  7 Scutellar foveae smooth. Notauli visible (Fig. 14A). Basal area anteriorly the transversal clear band of forewing completely black; Costal cell infuscate; without less infuscate areas above the cubital veins and below the M+Cu1 vein (Fig. 15F). Transversal clear band relatively large, reaching ventrally the posterior margin of forewing (Fig. 15F)  Transversal clear band large; dorsally extended on 2/3 of apical area of radial cell and extended posteriorly to reach margin of the wing (Fig. 4B)  Posterolateral projections of the mesoscutellum acute pointed (Fig. 9A). Notauli hardly visible. Radial cell with first radial abscissa projected into the radial cell at tip of angle (Fig. 9C).

Discussion
The current study increases the number of Amphibolips in Mexico from 20 to 23. However, this number may rapidly increase since fieldwork is revealing new species that are still being studied and will eventually be published elsewhere.
The three species described herein present the typical diagnostic features of the Amphibolips species out of the "niger" group (Kinsey 1937, Melika et al. 2011, Nieves-Aldrey et al. 2012). We observed one morphological character, which seems to be shared only by Amphibolips species, namely, the absence of a gap or space between the two branches of the cubital vein of the forewing. A second not previously noticed character, present in the species from Mexico, is a pair of longitudinal carinae running from the ventral margin of the toruli to the anterior tentorial pits or the epistomal sulcus. However, this feature needs to be checked in all the Amphibolips species.
Considering that the complete life cycle of most species is unknown, it is difficult to propose a pattern of morphologies or phenologies for these species. In some cases, mistakes could be made, for example, some descriptions may pertain to different generations of currently-recognised species. Nieves-Aldrey et al. (2012) mentioned the difficulties in separating Amphibolips species (excluding those of the "niger" complex) based on external morphology of their galls, considering that some species are very similar (e.g. A. nigrialatus new species and A. kinseyi new species). In the same study, the question arises about the variability in a single Amphibolips species and the need to use new tools (specifically, molecular markers) that allow the clarification of the boundaries between species.
The new species described herein are very similar and share a set of similar morphological characteristics. The forewings are very dark and heavily black infuscate, but with a clear transversal band that is more or less extended in both sexes; the mesoscutellum is very deeply emarginate posteriorly, with the emargination reaching the scutellar foveae and almost dividing the mesoscutellum into two parts. Another shared feature of the three new species is that all are distributed in the States of Oaxaca and Veracruz in southern Mexico and none exceeds the Trans-Mexican volcanic belt. Their host oak species (Quercus zempoaltepecana and Q. sapotifolia) are similar in their affinity for slopes in humid climates near the Gulf of Mexico and their occurrence within tropical communities with many Quercus species. After studying large collections of Amphibolips collected across extensive areas of Mexico, we observed a distinct morphological pattern within the geographic distribution of the species, which is also confirmed in the species from the United States (unpublished observations). The more southern a species is distributed, the stronger and deeper its mesoscutellar emargination appears. These two patterns are consistent within a north-south distribution in Mexico.
The presence of four described species from the State of Oaxaca (Amphibolips oaxacae Nieves-Aldrey & Pascual, A. dampfi Kinsey, A. magnigalla sp. nov. and A. kinseyi sp. nov.) and three species from Panama (A. castroviejoi Medianero & Nieves-Aldrey, A. aliciae Medianero & Nieves-Aldrey and A. salicifoliae Medianero & Nieves-Aldrey) allows us to propose that Amphibolips must be present throughout Central America. Although they have not been cited from Chiapas, Mexico to Costa Rica, its presence in those geographic areas is likely, given that most host Quercus species recorded from south of Mexico and Panama are also present in Mesoamerica (Burger 1977, Breedlove 2001, Valencia 2004. Currently, broader work is being carried out to understand the relationships between the Mexican and Panamanian species of Amphibolips.