Description and nesting biology of three new species of neotropical silk wasp (Hymenoptera: Apoidea: Pemphredoninae: Microstigmus)

ABSTRACT The neotropical apoid wasp genus Microstigmus is of particular interest biologically because it represents an origin of eusociality independent of vespid wasps and bees, and is part of the only eusocial lineage among the approximately 10,000 solitary species of apoid wasps. Females construct nests made of silk and the species exhibit an unusual diversity of nesting strategies. However, research is hampered because many species remain undescribed and the basic nesting biology of only a few species is known. I describe three new species from north-west Ecuador related to M. bicolor Richards, including diagnostic morphological characters, altitudinal ranges and molecular data as well as descriptions of their nests and nesting biologies. M. rosae sp. nov. is a mass provisioner that preys on nymphal Thysanoptera, while M. lydiae sp. nov. and M. mirandae sp. nov. are progressive provisioners that prey on nymphal leafhoppers (Cicadellidae). Nests of all three species can contain multiple adult females but more than half of nests contain only a single female. http://www.zoobank.org/urn:lsid:zoobank.org:pub:E46768B9-FD13-4370-8E31-8D1819B724F4


Introduction
Microstigmus is a genus of small neotropical apoid wasps in the subfamily Pemphredoninae (or Pemphredonidae sensu Sann et al. 2018Sann et al. , 2021. It is of special interest biologically because it represents an origin of eusociality independent of vespid wasps and bees (Matthews 1968), the only definite eusocial lineage among the large and diverse groups of apoid wasps (Melo 2020). Microstigmus has several unique features, notably the copious use of silk to build bag-like nests that can be found hanging beneath vegetation or human-built structures (Matthews 1991). Wasps live in groups of rarely more than 10 females, usually considerably fewer, and the genus is also unusual in including both mass provisioning and progressively provisioning species. Around 30 species have so far been described, but the genus is understudied in comparison with other eusocial Hymenoptera, with the biology of only a few species known in any detail (M. comes: Matthews 1968;Ross and Matthews 1989;Matthews 1991;M. myersi: Melo and de Campos 1993;M. thripoctenus : Asís 2003;M. nigrophthalmus: Lucas and Field 2011;Lucas et al. 2011aLucas et al. , 2011b. Here, I describe adults and nests of a further three new species from Ecuador closely related to M. bicolor Richards 1971 andM. nigrophthalmus Melo 1992, and provide diagnostic morphological characters and molecular data supporting their recognition. I also provide information about their nesting biologies.

Materials and methods
The three species described here are based on material collected by R. Bonifacii and J.P. Field during 2014-2017, principally from two areas in the north-west Ecuadorian Choco region on the edge of the Andes Mountain range: Mashpi Biodiversity Reserve (N 0.167°N, 78.872°W) and Santa Lucia Cloud Forest Reserve (0.118°N, 78.608°W). A few specimens from adjacent areas were also examined (e.g. Maquipucuna 0.125°N, 78.625°W). These sites include both primary and secondary forest and span an altitudinal range of 750-2300 masl. The three new species, while small-sized, are relatively large for the genus. They are closely related to each other and to M. bicolor Richards 1971 (see the monograph by Richards (1972) on the taxonomy of Microstigmus). Along with other species, the author was able to examine the holotype of M. bicolor in the British Museum (Natural History) (BMNH), and paratypes of M. nigrophthalmus Melo 1992, which is another species related to M. bicolor that has been described since Richards (1972). Specimens of M. pallidus Richards 1972 collected by the author in Ecuador were also examined, along with specimens from Costa Rica kindly provided by R.W. Matthews.

Morphology
Specimens of each species that had been collected in the field as adults were micropinned and viewed/photographed under a binocular microscope at 60 × or higher magnification, but all characters described are clearly visible at 60 ×. Additional specimens were imaged using a scanning electron microscope (SEM) at magnifications of 150-200 × in order to obtain images of microsculpturing and accurate morphological size ratios. Specimens had been stored in 95% ethanol until they were prepared for imaging. For SEM, each specimen was mounted on a JEOL aluminium stub (10 mm diameter × 10 mm high) using a PELCO™ carbon conductive sticky tab (10 mm diameter) and carbon coated using spectrographically pure carbon. Specimens were then imaged with a TESCAN Vega SEM in High Vacuum mode at 20.0kV with the scan mode set to Resolution.

Molecular data
In order to check the separation of the newly described taxa from each other, three genes were sequenced using adults or immature offspring from 3-13 individual nests of each species: mitochondrial cytochrome oxidase I (COI) gene, nuclear elongation factor-1α (EF-1α) and RNA Polymerase II (Pol II). Adult females had been identified using described differences in morphology and prey type (see below). Samples from both main study sites (Mashpi and Santa Lucia) were included for the two species that occurred at both sites, M. rosae sp. nov. and M. lydiae sp. nov. Single females of M. nigrophthalmus and M. brasiliensis Melo 1992 were also sequenced, using material collected from their type locality by the author and E. Lucas. DNA was extracted from abdomen or mesosoma using either an ammonium acetate method (Nicholls et al. 2000) or a DNeasy Tissue Kit (Qiagen). COI was amplified using the primers LCO1490 (Folmer et al. 1994) and H7005-mod1 (Donald et al. 2005). EF-1α was amplified using the primer pair HaF2For1 (Sipes and Wolf 2001;Patiny et al. 2008) and F2-rev1 (Danforth et al. 1999) and for Pol II, the polfor2a and polrev2a (Danforth et al. 2006) primers were used. The Polymerase Chain Reaction (PCR) profile was as follows with all three primers. Initial denaturation at 94°C (3 min), followed by 40 cycles of 94°C (30 s), annealing temperature (60 s) and 72°C (60 s), and a final extension phase at 72°C (4 min). Both COI and EF-1α were annealed at 45°C, and Pol II at 50°C. All PCR reactions were performed using MyFI mix (Bioline). Amplified PCR products were cleaned using Exosap-IT (Thermofischer) and sent for automated sequencing on an ABI Prism 3770 (Macrogen).
Sequences were aligned using Muscle in MEGA7 (Kumar et al. 2016), with slight alterations made by eye when necessary. The introns of EF-1α were removed due to ambiguity in alignment. The three sequences obtained for each individual were then concatenated (final length 2213 bp). After alignment, three M. rosae sp. nov. individuals were removed as they were identical to other individuals across all three genes. Using Geneious Prime 2020.1.1, a neighbour-joining tree was created for the remaining 19 individual Microstigmus plus a non-pemphredonine apoid outgroup with previously published sequences for the three genes, Isodontia mexicana . The genetic distance model used was Tamura-Nei and 100 bootstraps were performed. The tree was visualised using Treegraph2 (Stöver and Müller 2010). Sequences were deposited in GenBank, and the GenBank identifiers are listed in the Supplementary material.

Nesting biology
Nests were collected mainly at night, to ensure that all adult occupants were present. Adults and immatures were counted, and any parasite larvae or pupae recorded. Prey type was identified, along with whether provisioning was progressive or mass (Field 2005). Mass provisioning, where an egg is laid only once a full complement of prey has been placed in the cell, was identified when cells contained balls of numerous prey items with or without an immature. Progressive provisioning, where the egg is laid in the empty cell and the immature is provisioned gradually as it develops, was evident when cells contained eggs or part-grown larvae with few or no prey items.

Taxonomy
In the following descriptions, abbreviations for type specimen depositories are: NHM: The Natural History Museum, London NIBEQ: National Institute of Biodiversity of Ecuador, Quito Microstigmus rosae sp. nov.

Recognition
Unlike M. lydiae sp. nov. and M. mirandae sp. nov., this species has the female 3 rd antennal segment (i.e. the first flagellomere) almost square (Figure 3(a)), and runs to M. pallidus in Richards (1972). However, the overall colouration is darker than M. pallidus, with the head, including clypeus, mainly black (Figure 1(c)); the 3 rd antennal segment is slightly squarer than in M. pallidus; the sculpturing is coarser, e.g. on the mesopleurae; and the striations at the back of the scutum are longer and denser (Figure 2(a)). In addition, the centre of the posterior margin of tergite 7 in male M. rosae sp. nov. bears a pair of adjacent, upturned and incurved teeth.

Description
Female. Length 3.5 mm. Colouration: head black except sides of lower face dark orange or pale orange, these lateral orange areas not fully meeting centrally (Figure 1(c)). A narrow line adjacent to the inside of the eyes and a strip running around the back of the head, connecting the back of the eyes to the occipital carina, also orange. Clypeus black with orange lower margin. Mandibles dark yellow or pale yellow, similar in colour to antennal scape, with dark red-edged tips. Antennae blackish brown, somewhat paler/yellowish ventrally, darker towards the tips. Pronotum orange with anterior dark mark centrally, and black areas on underside of pronotum in most specimens. Scutum varying in colour from bright orange to a dirty orange-brown. Scutum coloured homogeneously in most specimens, but a few have a hint of the pattern seen in M. lydiae sp. nov. (Figure 1(b)), with a central paler strip running antero-posteriorly and slightly darker areas laterally. Scutellum and metanotum orange laterally with central black area. Mesopleurae partly brown or black, with a small orange area postero-centrally. Underside of mesothorax black. Propodeum and abdomen black/dark brown, with a small dark orange area on each side at the base of the second tergite in some specimens. Variable pale marks on sternites in some specimens, sometimes including a whiteish mark at the base of the 6th sternite. Legs pale yelloworange except: coxae and trochanters more yellowy-white; hind femora dark brown or black dorsally; and hind tibiae more or less brown-marked.
Maximum width of face across the eyes approximately 1.1 times the distance from top of the head to apex of clypeus when head is viewed face-on. Inner margins of compound eyes approximately parallel on face; minimum interorbital distance (across middle of face) approximately 1.1 times interorbital distance near top of the eyes. Ocelli in a not quite equilateral triangle, with the posterior side shorter and the two posterior ocelli separated by a distance approximately equal to their diameters. Mandibles with upper tooth shorter than lower tooth. Malar space short, approximately equal to the width of antennal segment 3. Clypeus raised, anterior margin weakly convex with a more or less weak central tooth often present. Basal flagellar segments of antennae short and relatively square: fifth antennal segment 1.25 times longer than wide; 3 rd segment 1.4 times longer than wide and viewed from above, third segment clearly shorter than pedicel (Figure 3(a)). Integument predominantly shining. Frons finely reticulate with fairly dense, shallow punctures. Scutum impunctate, finely reticulate, with striations along the posterior margin visible at 60 × (Figure 2(a)), these striations longer (length 20% of length of scutum) and denser than in M. lydiae sp. nov. and M. mirandae sp. nov. Pronotum with a transverse carina centrally. Scutellum weakly convex when viewed laterally, and moderately raised centrally. Posterior half of the raised area with a raised longitudinal keel centrally with weaker carinae each side. Metanotum with a sharp, raised longitudinal keel centrally. Mesopleurae mostly coarsely sculptured, especially ventrally, but smooth posterodorsally. Omaular sulcus and hypersternaulus well developed, the former unusually wide, wider than in M. lydiae sp. nov. and M. mirandae sp. nov. Propodeum shining and very coarsely sculptured, more so than in M. lydiae sp. nov. and M. mirandae sp. nov.
Pilosity: compound eye bare, top of head and dorsal mesosoma with dense, short setae. In perfect specimens there are two longer, erect setae in the ocellar area that are slightly longer than the distance between the outer edges of the two posterior ocelli. Clypeus and lower edge of mandibles with longer setae. Antennae covered with dense, short setae, especially towards the tips; setae so dense that junctions between basal antennal segments hard to discern in some specimens. Dorsal area of propodeum bare, but posterior face moderately setose, setae longer than on head and mesosoma. Mesopleurae with short, dense setae especially towards the venter and beneath. First two abdominal tergites almost bare, with short setae becoming denser towards the tip of the abdomen, including a few longer setae at the posterior edges of tergites and sternites.
Male. Similar to female except somewhat paler colouration: entire underside of head and pronotum, and the posterior half of tergite 7, pale; and orange area on posterior mesopleurae more extensive than in female, running from wing base to nearly the underside of the mesosoma. Malar space noticeably longer than in the female, approximately 1.5 times the length of the antennal pedicel. Lower face of male is snowy white, starting half-way down the eyes and including the clypeus, and anterior (ventral) side of scape and mandibles pale yellow, contrasting with the white lower face. Male posterior tergites bear central depressions. When the abdomen is in its natural position (segments telescoped), these appear as small, groovelike depressions in the centres of tergites 3-7, the overall impression being of a central flattening of the entire posterior dorsal abdomen, with the black posterior tergal margins appearing emarginate. When the abdominal segments are pulled out, it can be seen that these depressions cover only the apices of tergites 3-5, where they are semi-circular; but cover the entire lengths of the centres of tergites 6-7, where they are oval-shaped. The surface of tergite 2 is smooth, without depressions or grooves. Tergite 7 bears a pair of adjacent, upturned and incurved teeth at the centre of its posterior margin. Sternite 7 narrows towards its bluntly rounded, upturned tip.
A further 4 female and 2 male paratypes also deposited at NHM, including 2 females and 2 males with the same data as the holotype except with pencil numbers 9, 10 and 13 (females, the latter specimen has the head micro-pinned separately onto the same piece of plastozoate as the body) or '12' (one of the two males). The second male, also from Santa Lucia, has similar data to the holotype but: '(1600 m). N 0.11843 Under W 78.61186 a sign. 3/9/2014 J.P.Field' with '25' additionally pencilled onto the label. The final female paratype is from the Mashpi site and has the abdomen missing, with data: 'ECUADOR: Pichincha, Mashpi Reserve nr Pacto (1050 m). N 00°09.771' W 078°52.271, 18-24/9/2014 R. Bonifacii' with '104' additionally pencilled onto the label.
Of two additional female paratypes, both with the abdomen missing, one has data: 'ECUADOR: Pichincha, Mashpi Reserve nr Pacto (1050 m). N 00°09.771' W 078°52.271, 6-7/ 9/2014 J.Field, 34'. The second female has the same data except that the date, collector and pencil label are: 18-24/9/2014 R.Bonifacii, 101. These last two paratypes are currently in the author's collection but the intention is to deposit them at NIBEQ once a visit to Ecuador is possible, post-Covid pandemic. An additional 2 female and 2 male paratypes from the Mashpi site have already been deposited at NIBEQ, and these have similar data to the Mashpi paratypes above except dates in September 2017.
Etymology. This species is named after Rosa Bonifacii in recognition of her excellent PhD work focussing on its biology.

Biology.
Hundreds of nests of this species were located at the two principal study sites, particularly at Mashpi. Nests (Figure 4(a)) were found throughout the 750-2000 masl altitudinal range investigated, and were commoner than nests of the other two species. Nests were found hanging under the leaves of a variety of plants, less often from manmade structures. At Mashpi, they were particularly common beneath the large leaves of Xanthosoma sagittifolium (Araceae), with more than one nest commonly found on the same plant and sometimes hanging under the same leaf of X. sagittifolium. Each nest was suspended by a thin, normally straight, petiole. Nests were typically brown in colour with a rough external appearance, apparently constructed from tiny pieces of plant material (often with the appearance of wood fragments) interwoven with strands of silk with occasional fragments of arthropod exoskeleton embedded. The nest entrance is located at the apex, where the petiole meets the main nest structure, and the lower part is divided into between 1 and 9 vertically orientated cells, visible externally as bumps on the surface of the nest. The nest entrance diameter was 1.5-2 mm and the nest petiole was usually straight but occasionally slightly or strongly curved, shorter (5-10 mm, mean 7.25) than in M. mirandae sp. nov. Each nest cell contained a single offspring or was empty. Approximately 80% of nests contained just a single adult female, but there could be up to four females and three males. Each offspring was mass provisioned with between 30 and 80 nymphal thrips (Thysanoptera). Cells sometimes contained pupae of braconid wasps (Heterospilus: Marsh and Melo 1999) or chalcidid wasps, and parasite larvae were observed feeding externally on Microstigmus larvae and prepupae.
The following two new species, Microstigmus lydiae sp. nov. and M. mirandae sp. nov., run to M. bicolor (couplet 7) in Richards (1972). Based on the material examined and Richards (1971Richards ( , 1972 descriptions of M. eberhardi and M. soror, they can be separated from other described species that run to couplet 7 as follows.

Recognition
This species has the female 3 rd antennal segment (i.e. the first flagellomere) relatively long and thin, like M. mirandae sp. nov., but differs from M. mirandae sp. nov. in: (1) darker overall colouration of head and thorax, with a thick black line along top edge of clypeus (Figure 1(d)); (2) mesopleurae lacking striations latero-ventrally; (3) presence of short striations at back of scutum at high power (Figure 2(b)). (4) Anterior face of male antennal scape pale yellow, contrasting with white lower face and mandibles; (5) surface of male second tergite smooth, without depressions or grooves.

Description
Female. Length 4 mm. Colouration: head black except lower face across most of its width, and a narrow line adjacent to the inside of the eyes, pale orange (Figure 1(d)). Dark orange strip running around back of head, connecting back of eyes to occipital carina, present in some specimens. Clypeus pale orange but always with a thick, black line along upper edge (Figure 1(d)). Mandibles creamy white, noticeably whiter than yellowy scape, with dark red-edged tips. Antennae yellow beneath, brown above, darker towards the tips. Pronotum orange with anterior dark mark centrally and dark brown areas on underside of pronotum in some specimens. Scutum with thick central orange-yellow strip running antero-posteriorly, approximately one-third the width of the scutum. Laterally, each side of this strip dark brown/black (Figure 1(b)). Scutellum and metanotum pale orange, each with central black area, the two areas merged, the black areas less extensive laterally than in M. mirandae sp. nov. or M. rosae sp. nov. Mesopleurae pale brown with small yellow-orange area postero-centrally. Underside of mesothorax dark brown/black. Propodeum (including sides) and abdomen black/dark brown, with a pale mark on each side at the base of the second tergite. Variable pale marks on sternites in some specimens. Legs pale yellow-orange except coxae and trochanters yellowy-white beneath, and hind femora with variable amounts of dark brown, especially dorsally. Maximum width of face across the eyes approximately 1.1 times the distance from top of the head to apex of clypeus when head is viewed face-on. Inner margins of compound eyes approximately parallel on face; minimum interorbital distance (across middle of face) approximately 1.1 times interorbital distance near top of the eyes. Ocelli in a not quite equilateral triangle, with the posterior side shorter and the two posterior ocelli separated by a distance approximately equal to their diameters. Mandibles with upper tooth shorter than lower tooth. Malar space longer than in M. rosae sp. nov., 1.3 times the width of antennal segment 3. Clypeus raised, anterior margin weakly convex. Basal flagellar segments of antennae longer and thinner: fifth antennal segment 1.5 times longer than wide; 3 rd segment 2.1 times longer than wide and viewed from above, third segment similar in length to pedicel (Figure 3(b)). Integument predominantly shining. Frons finely reticulate with sparse, shallow punctures. Scutum impunctate, finely reticulate, with striations along the posterior margin visible at high power, these striations short (length less than 10% of the length of the scutum) and sparse (Figure 2(b)). Pronotum with a transverse carina centrally. Scutellum moderately raised centrally. Raised area with a very slight depression centrally and a short, raised longitudinal keel centrally in the posterior half of the raised area; weaker carinae each side. Metanotum with a sharp, raised longitudinal keel centrally. Mesopleurae smooth and shining, lacking latero-ventral striations posteriorly, or with at most one or two weak striations. Omaular sulcus and hypersternaulus well developed, the former wide. Propodeum shining and coarsely sculptured.
Pilosity: compound eye bare, top of head and dorsal mesosoma with dense, short setae visible at high power. In perfect specimens there are two longer, erect setae in the ocellar area, these being relatively shorter than in M. rosae sp. nov., approximately the same length as the distance between the centres of the posterior ocelli. Clypeus and lower edge of mandibles with longer setae. Antennae covered with dense, short setae, especially towards the tips. Dorsal area of propodeum bare, but posterior face with moderately dense setae, these longer than on head and mesosoma. Mesopleurae with short setae, these becoming denser and longer towards the venter and beneath. First two abdominal tergites almost bare, with short setae becoming denser towards the tip of the abdomen, including a few longer setae at the posterior edges of tergites and sternites.
Male. Similar to female except somewhat paler colouration: top of head dark brown/ black, but underside of head pale, with orange strip running around back of head, connecting back of eyes to occipital carina. Underside of mesothorax and mesopleurae paler brown than in female, and yellow-orange central area of mesopleurae larger than in female. Lower face of male is snowy white, starting half way down the eyes and including the clypeus and mandibles (except edges of mandibular tips dark red). Anterior (ventral) side of scape yellow, contrasting with the white lower face and mandibles. Malar space noticeably longer than in the female, nearly twice as long as the antennal pedicel. Male posterior tergites bear central depressions. When the abdomen is in its natural position (segments telescoped), these appear as small, groove-like depressions in the centres of tergites 3-7, the overall impression being of a central flattening of the entire posterior dorsal abdomen. The surface of tergite 2 is smooth, without depressions or grooves. Tergite 7 is black, concolorous with the other tergites, and has a small, triangular, nick-like emargination at the centre of its posterior margin. Sternite 7 narrowing towards its triangular, upturned tip. One female and one male paratype also deposited at NHM: the male has the same data as the holotype except date is 23/8/2014, altitude 1700 m, 'Under sign', pencil label '2'. The female is 'ECUADOR: Pichincha, Mashpi Reserve nr Pacto (1337 m). N 00°09.771' W 078°52.271, 18-24/9/2014 R.Bonifacii', pencil label '106'.
Two further female paratypes have the same data as the holotype except different date/pencilled numbers (27/8-3/9/2014, numbers 7 and 11), and a further male paratype is: 'ECUADOR: Pichincha, Mashpi Reserve nr Pacto (1337 m). N 00°09.771' W 078°52.271, 24/3/2017 R.Bonifacii MS 966'. These last three paratypes are currently in the author's collection but the intention is to deposit them at NIBEQ once a visit to Ecuador is possible, post-Covid pandemic.
Etymology. This species is named after Lydia Field, whose enthusiasm for the rainforests of Ecuador has been infectious.

Biology.
Approximately 30 nests of this species were located. Nests (Figure 4(b)) were found only at higher altitudes (1000-2000 masl), principally at Santa Lucia and only rarely at Mashpi and Maquipucuna. Nests were found hanging under the leaves of a variety of plants and from man-made structures. Nests were typically brown in colour with a rough external appearance, apparently constructed from tiny pieces of plant material (such as wood fragments) interwoven with strands of silk. The nest entrance is located at the apex, where the petiole meets the main nest structure, and the lower part is divided into between 1 and 15 vertically orientated cells, visible externally as bumps on the surface of the nest. The nest entrance diameter was 2-2.5 mm and the nest petiole was straight or occasionally slightly curved, and shorter (7-8 mm, mean 7.5) than in M. mirandae sp. nov. Each nest cell contained a single offspring or was empty. Approximately 50% of nests contained more than one adult female, with up to six females and six males per nest. Offspring were provisioned progressively with leafhopper nymphs (Cicadellidae). Cells sometimes contained pupae of braconid wasps (Heterospilus: Marsh and Melo 1999).

Recognition
This species has the female 3 rd antennal segment (i.e. the first flagellomere) relatively long and thin, like M. lydiae sp. nov. It differs from M. lydiae sp. nov. in: (1) paler overall colouration of head and mesosoma, with the clypeus entirely pale, lacking a thick black line along its top edge (Figure 1(e)); (2) mesopleurae with clear striations latero-ventrally (Figure 2(d)); (3) striations at back of scutum absent or very weak, even at high power (Figure 2(c)); (4) anterior face of male antennal scape white (except yellowish distally at the junction with segment 2), concolourous with lower face and mandibles; (5) male second tergite with a central, narrow, groove-like depression running along the posterior three-quarters or more of the segment.

Description
Female. Length 4 mm. Colouration: head (including underside, face and clypeus; Figure 1(e)) and mesosoma predominantly pale orange-yellow; abdomen black ( Figure 1(a)). On head, the small area between the ocelli is black in darker specimens. Vertex more or less diffusely brown, with the brown extending down onto face in darker specimens, which also have the underside of the head and mesosoma dark brown/black. In paler specimens, head (including underside and clypeus) pale yellow-orange throughout except vertex diffusely pale brown. Mandibles pale yellowy-white with dark red tips. Antenna pale yellow, more or less brown towards the tips, especially dorsally. On mesosoma, pronotum with anterior dark mark centrally. Scutum, especially at sides, more or less pale brown. Scutellum and metanotum each with central black area, the two areas merged. Mesopleurae pale brown. Underside of mesothorax usually pale or dark brown. Propodeum and abdomen black/dark brown, with a pale mark each side at the base of the second abdominal tergite. Variable pale marks on sternites in some specimens. Coxae and trochanters pale-white, femora partly or entirely (hind femur) dark brown; hind tibiae with a brown mark in some specimens. Legs otherwise pale orange/yellow.
Maximum width of face across the eyes approximately 1.1 times the distance from top of the head to apex of clypeus when head is viewed face-on. Inner margins of compound eyes approximately parallel on face; minimum interorbital distance (across middle of face) approximately 1.1 times interorbital distance near top of the eyes. Ocelli in a not quite equilateral triangle, with the posterior side shorter and the two posterior ocelli separated by slightly less than their diameters. Mandibles with upper tooth shorter than lower tooth. Malar space (Figure 1) longer than in M. rosae sp. nov., 1.5 times the width of antennal segment 3. Clypeus raised, anterior margin weakly convex. Basal flagellar segments of antennae relatively long and thin: fifth antennal segment 1.6 times longer than wide; 3 rd segment 2.5 times longer than wide and, when viewed from above, similar in length to or slightly longer than pedicel (Figure 3(c)). Integument predominantly shining. Frons finely reticulate with sparse, shallow punctures. Scutum shining, impunctate and finely reticulate. A few short striations along the posterior margin of scutum visible on electron micrographs (Figure 2(c)), but these striations short and sparse, not clearly discernable with a binocular microscope at 60 ×. Pronotum with a transverse carina centrally. Scutellum moderately raised centrally. Raised area with a very slight depression centrally and a raised longitudinal keel centrally in the posterior half of the raised area; weaker carinae each side. Metanotum with a sharp, raised longitudinal keel centrally. Mesopleurae with weak sculpturing dorsally, but posteriorly with some some stronger striae latero-ventrally continuing onto the underside of the mesosoma (Figure 2(d)). Omaular sulcus and hypersternaulus well developed, the former wide. Propodeum shining and coarsely sculptured.
Pilosity: compound eye bare, top of head and dorsal mesosoma with dense, short setae visible at high power. In perfect specimens there are two longer, erect setae in the ocellar area, these being approximately the same length as the distance between the centres of the posterior ocelli and less noticeable than in M. lydiae sp. nov. and M. rosae sp. nov. Clypeus and lower edge of mandibles with longer setae. Antennae covered with dense, short setae, especially towards the tips. Dorsal area of propodeum bare, but posterior face with dense setae, these longer than on head and mesosoma. Mesopleurae with short setae, these becoming denser and longer towards the venter and beneath. First two abdominal tergites almost bare, with short setae becoming denser towards the tip of the abdomen, including a few longer setae at the posterior edges of tergites and sternites.
Male. Similar to female except somewhat paler colouration: posterior face and dorsal area of propodeum black or with brown patches but sides of propodeum, scutum, mesopleurae and underside of mesothorax pale yellow/orange. Underside of abdomen largely pale yellow. Antennal flagellar segments are an exception, being almost entirely brown, darker towards the tips. Malar space noticeably longer than in the female (Figure 1), nearly twice as long as the antennal pedicel. Ventral mesopleural striations less discernible in male. Lower face of male is snowy white, starting half-way down the eyes and including the clypeus and mandibles (Figure 1(f)), except edges of tips of mandibles dark red. Anterior (ventral) side of scape also white. Male posterior tergites bear central depressions. When the abdomen is in its natural position (segments telescoped), these appear as small, groove-like depressions in the centres of tergites 3-7, the overall impression being of a central flattening of the entire posterior dorsal abdomen. In contrast with the male of M. lydiae sp. nov., the surface of tergite 2 also has a central narrow, groove-like depression covering the posterior three-quarters or more of the length of the tergite, this depression being deeper towards the apex of the segment. Tergite 7 is black and concolorous with the other tergites, and has a small, triangular, nick-like emargination at the centre of its posterior margin. Sternite 7 narrowing towards its bluntly rounded, upturned tip. Two further female and one further male paratypes are from the same Mashpi site with similar data but different dates/collectors and no second label (females: '6-7/9/2014 J. Field 32' and '1/4/2017 R.Bonifacii MS1056'; the first of these two females has the head micropinned separately onto the same piece of plastozoate as the body; male '12/3/2017 R.Bonifacii MS 886'). These last three paratypes are currently in the author's collection but the intention is to deposit them at NIBEQ once a visit to Ecuador is possible, post-Covid pandemic.
Etymology. This species is named after Miranda Debenham, who has been an inspiration throughout this work.

Biology.
Approximately 20 nests of this species were located, all at Mashpi. Nests (Figure 4(c)) were therefore found only at the lower end (600-1086 masl) of the altitudinal range we principally investigated. Nests were found hanging under the leaves of a variety of plants and man-made structures. Nests were typically dark brown in colour with a rough external appearance, apparently constructed from small pieces of plant material (such as wood fragments) interwoven with strands of silk. The nest entrance is located at the apex, where the petiole meets the main nest structure, and the lower part is divided into between 1 and 7 vertically orientated cells, visible externally as bumps on the surface of the nest. Each nest cell contained a single offspring or was empty. Nests were larger (depths typically 13-14 mm, entrance diameter 2.25-2.5 mm) than nests of M. rosae sp. nov. and M. lydiae sp. nov., and had a longer petiole (13-18 mm, mean 15.1) that was always strongly bent at some point between the main nest structure and the substrate (Figure 4(c)). Most nests contained just a single adult female, but there could be up to four females and two males. Offspring were provisioned progressively with leafhopper nymphs (Cicadellidae) and no parasites were observed. Molecular data. The molecular data were consistent with the species delineations above. First, in the neighbour-joining tree ( Figure 5), individuals of each species identified using the morphological characters above formed a cluster with each other, including individuals from the two main collection sites and across each species' observed altitudinal range. Interestingly, the two progressive provisoners that prey on leafhopper nymphs, M. lydiae sp. nov. and M. mirandae sp. nov., clustered together, along with M. nigrophthalmus, the only other progressive provisioner in the tree, which again preys on leafhopper nymphs. Second, the two most similar species, M. lydiae sp. nov. and M. mirandae sp. nov., had alleles in common at only nine of the 29 microsatellite loci scored, at four of which they were both monomorphic for the same allele. Among the remaining 20 loci where there were no alleles common to both species, each species was monomorphic for a different allele at eight loci, while one (n = 10 loci) or both (n = 2) species were polymorphic at the other 12 loci. The microsatellite data will be presented fully elsewhere.

Conclusion
In this paper, I describe three new species in the apoid wasp genus Microstigmus that are related to M. bicolor. I provide molecular as well as morphological data: molecular data are likely to prove useful for species identification given the very small size and relatively homogeneous appearance of different Microstigmus species. Nest and prey type are also useful characters.
Microstigmus and related pemphredonine genera represent the only lineage of apoid wasps known to have evolved eusociality, and are unique in that adults produce silk for nest construction (Matthews 1991). The form of sociality exhibited by the three species described here awaits elucidation. In M. comes, groups typically comprise a mother and her daughters, with only one female having developed ovaries (Matthews 1968(Matthews , 1991Ross and Matthews 1989). In M. nigrophthalmus, however, almost all females in multifemale nests are mated and have developed ovaries, and multiple females can have brood developing simultaneously, with breeders as well as non-breeders foraging to provision offspring (Lucas and Field 2011;Lucas et al. 2011aLucas et al. , 2011b). Although nests of the species described here can contain up to 4-6 adult females and 2-6 males, at least half of the nests of each species contain just a single female (see also Asís 2003 in M. thripoctenus). In part, this is likely to reflect the fact that nests are often founded by single females and become social only once the first female offspring reach adulthood. In addition, however, it seems likely that random adult mortality results in group size fluctuations so that social nests might sometimes revert to being solitary. Even in M. comes, where nests contain up to eight females, nests are often founded by groups and eusociality is well documented, half of nests contain only one female (Matthews 1968;Ross and Matthews 1989).
As well as group size and the form of sociality, prey type and provisioning mode vary between Microstigmus species. Different species prey exclusively on Collembola, thrips (Thysanoptera) or Homoptera. Species such as M. comes and M. rosae sp. nov. are mass provisioners, laying the egg only once a full complement of prey has been collected for the offspring. In contrast, other species such as M. nigrophthalmus, M. lydiae sp. nov. and M. mirandae sp. nov. feed immature offspring gradually as they grow (progressive provisioning). While the three progressive provisioners in Figure 5 all prey on Homoptera, M. eberhardi, which is a progressive provisioner also in the M. bicolor group, preys on thrips (Richards 1972).
Variation in mode of provisioning within a single genus is unusual in aculeates. The three progressively provisioning Microstigmus species are clustered together in Figure 5. This contrasts with ammophilines, another apoid wasp lineage, where the four progressive provisioners included in a recent phylogeny represent four independent origins of the trait (Field et al. , 2020. However, at least one more distantly related Microstigmus species not in the M. bicolor group appears to practise a form of progressive provisioning (Melo and de Campos 1993). The evolutionary history of traits such as prey type, provisioning mode and social phenotype within Microstigmus, and more widely among pemphredonines, is of great interest and awaits a detailed phylogeny including a larger number of species.