Journal of Natural History

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Description and nesting biology of three new

species of neotropical silk wasp (Hymenoptera:
Apoidea: Pemphredoninae: Microstigmus)

Jeremy Field

To cite this article: Jeremy Field (2023) Description and nesting biology of three new species
of neotropical silk wasp (Hymenoptera: Apoidea: Pemphredoninae: Microstigmus), Journal of
Natural History, 57:1-4, 1-18, DOI: 10.1080/00222933.2022.2157345

To link to this article: https://doi.org/10.1080/00222933.2022.2157345

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UK Limited, trading as Taylor & Francis
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 JOURNAL OF NATURAL HISTORY
2022, VOL. 57, NO. 01–04, 1–18
https://doi.org/10.1080/00222933.2022.2157345

Description and nesting biology of three new species of

neotropical silk wasp (Hymenoptera: Apoidea:
Pemphredoninae: Microstigmus)
Jeremy Field
Centre for Ecology and Conservation, University of Exeter, Penryn, UK

ABSTRACT ARTICLE HISTORY

The neotropical apoid wasp genus Microstigmus is of particular Received 24 January 2022
interest biologically because it represents an origin of eusociality Accepted 5 December 2022
independent of vespid wasps and bees, and is part of the only KEYWORDS
eusocial lineage among the approximately 10,000 solitary species wasps; progressive
of apoid wasps. Females construct nests made of silk and the provisioning; neotropics
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 descrip­
tions 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. 2018, 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:

CONTACT Jeremy Field j.p.field@exeter.ac.uk

Geolocation information: study area N 00°07 W 078°36
Supplemental data for this article can be accessed online at https://doi.org/10.1080/00222933.2022.2157345
© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Published online 27 Jan 2023

2 J. FIELD

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.
2011a, 2011b). Here, I describe adults and nests of a further three new species from
Ecuador closely related to M. bicolor Richards 1971 and M. nigrophthalmus Melo 1992, and
provide diagnostic morphological characters and molecular data supporting their recog­
nition. 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 micro-
pinned 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
 JOURNAL OF NATURAL HISTORY 3

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 denatura­
tion 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. indivi­
duals 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 (Field
et al. 2011). 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.
In addition, single adult females from each of eight nests of M. lydiae sp. nov. and
16 of M. mirandae sp. nov., the two morphologically most similar species, were
genotyped at 29 of the microsatellite loci developed previously for
M. nigrophthalmus (Lucas et al. 2009: MN loci nos. 1, 2, 4, 5, 7–10, 12, 14, 18–22,
24–26, 28–30, 34, 35, 37, 40, 41, 43, 49, 50). Loci were amplified using multiplex PCR,
carried out in 6 uL reactions using Qiagen PCR master mix (Quiagen Inc.). PCR
products were diluted and added to formamide before samples were sequenced
using a MiSeq Benchtop Sequencer (Illumina) at the University of Exeter, Penryn.
Allele sizes were scored using the software Geneious 10.0.9.

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.
4 J. FIELD

Results and discussion

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 3rd 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 3rd 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

Figure 1. (a) Microstigmus mirandae sp. nov., dorsal view of female with (inset) whole body; (b)
M. lydiae sp. nov., dorsal mesosoma of female; faces of females: (c) M. rosae sp. nov., (d) M. lydiae sp.
nov. with arrow indicating dark line along top edge of clypeus, (e) M. mirandae sp. nov.; (f) face of male
M. mirandae sp. nov. Scale bars: 0.5 mm.
 JOURNAL OF NATURAL HISTORY 5

Figure 2. Electron micrographs showing dorsal view of mesosoma and propodeum of female (a)
Microstigmus rosae sp. nov., (b) M. lydiae sp. nov., (c) M. mirandae sp. nov.; (d) lateral mesosoma of
M. mirandae sp. nov. female. White arrows in a–c indicate striations along posterior edge of scutum.
Scale bars: 0.3 mm.

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 speci­
mens, 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,
6 J. FIELD

Figure 3. Electron micrographs showing antenna of female (a) Microstigmus rosae sp. nov., (b)
M. lydiae sp. nov., (c) M. mirandae sp. nov. White brackets indicate antennal segment 3 (i.e. the first
flagellomere). Scale bars: 0.2 mm.

sometimes including a whiteish mark at the base of the 6th sternite. Legs pale yellow-
orange 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; 3rd 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 mar­
gin 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 postero-
dorsally. Omaular sulcus and hypersternaulus well developed, the former unusually
 JOURNAL OF NATURAL HISTORY 7

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 abdo­
men 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, 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.

Type material
Holotype female. ‘ECUADOR: Pichincha Province: Santa Lucia nr Nanegal (1900 m).
Cabana N 0.11843 W 78.61186. 27/8-3/9/2014 J.P.Field’ with ‘12’ additionally pencilled
onto the label (NHM).
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.
8 J. FIELD

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 man-
made 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

Figure 4. Nests of (a) Microstigmus rosae sp. nov., (b) M. lydiae sp. nov., (c) M. mirandae sp. nov. Arrows
indicate nest entrances. (a) and (b) are in situ beneath leaves of Xanthosoma sagittifolium. The scale in
(c) is millimetres. Photo credits - (a) and (b): R. Bonifacii; (c) C. Savill and J. Field.
 JOURNAL OF NATURAL HISTORY 9

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 (1971, 1972) descriptions of M. eberhardi and M. soror, they can be separated
from other described species that run to couplet 7 as follows.

1. Scutum black. Prey on thrips............................................................ M. eberhardi and M. soror

- Scutum entirely orange or with large orange areas both anteriorly and posteriorly
(Figure 1). Prey on Homoptera (Cicadellidae) (prey of M. bicolor unknown) . . . . . . . . . 2

2. Head (and mesosoma plus the dorsal surface of the petiole in M. nigrophthalmus)
entirely orange, except the male lower face is white, and sometimes the tiny area
between the ocelli is black. Larger species (female length 4.5–5.2 mm) with more
spherical nests where cells are not visible from the outside....................................................
....................................................................................................... M. bicolor and M. nigrophthalmus
- Dark patches on the head (Figure 1) as well as the mesosoma, least so in pale
specimens of M. mirandae sp. nov. (Figure 1(a)). Dorsal surface of petiole black/
brown or with black/brown marks. Smaller species (female length 4 mm) with less
spherical nests that have cells visible as bumps on the outside (Figure 4).................. 3

3. Colouration of female head and mesosoma darker, with a thick black line along
top edge of clypeus (Figure 1(d)). Mesopleurae lacking striations latero-ventrally.
Short striations present at the back of the scutum at high power (Figure 2(b)).
Anterior face of male antennal scape pale yellow, contrasting with white lower
face and mandibles. Surface of male second tergite smooth, without depressions
or grooves.................................................... .................................................... M. lydiae sp. nov.
- Colouration of female head and mesosoma paler, with the clypeus entirely pale,
lacking a thick black line along its top edge (Figure 1(e)). Mesopleurae with clear
striations latero-ventrally (Figure 2(d)). Striations absent or very weak at the back of
the scutum, even at high power (Figure 2(c)). Anterior face of male antennal scape
white (except yellowish distally at the junction with segment 2), concolorous with
lower face and mandibles. Male second tergite with a central, narrow, groove-like
depression running along the posterior three-quarters or more of the segment..........
............................................................................................................................... M. mirandae sp. nov.

Microstigmus lydiae sp. nov.

Recognition
This species has the female 3rd 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
10 J. FIELD

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 under­
side 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 seg­
ments of antennae longer and thinner: fifth antennal segment 1.5 times longer than wide;
3rd 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
 JOURNAL OF NATURAL HISTORY 11

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.

Type material
Holotype female. ‘ECUADOR: Pichincha Province: Santa Lucia nr Nanegal (1900 m).
Cabana N 0.11843 W 78.61186. 29/8/2014 J.P.Field’ with ‘8’ additionally pencilled onto
the label (NHM).
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
12 J. FIELD

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).

Microstigmus mirandae sp. nov.

Recognition
This species has the female 3rd 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 through­
out 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 meso­
soma, 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
 JOURNAL OF NATURAL HISTORY 13

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; 3rd
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 reticu­
late. 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 shin­
ing 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 meso­
pleural 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 depres­
sions in the centres of tergites 3–7, the overall impression being of a central flatten­
ing 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
14 J. FIELD

emargination at the centre of its posterior margin. Sternite 7 narrowing towards its
bluntly rounded, upturned tip.

Type material
Holotype female. ‘ECUADOR: Pichincha, Mashpi Reserve nr Pacto (1050 m). N 00°09.956’
W 078°53.288, 8/12/2016 J.Field’. Second label: ‘Nest under butterfly house’ with ‘502’
written in pencil (NHM).
Three female and one male paratypes are also deposited at NHM, from the same
Mashpi site (all ‘8/12/2016 J.Field’, and all with second label: ‘Nest under butterfly house’
and pencil numbers 500, 501, 503 [females] and 500 [male]).
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 altitu­
dinal 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
 JOURNAL OF NATURAL HISTORY 15

Figure 5. Neighbour-joining tree for individuals of the three newly described species together with
two other Microstigmus species and outgroup Isodontia mexicana. Letters M or SL after a species name
indicate the collection site (Mashpi or Santa Lucia). Numbers at nodes indicate percentage bootstrap
support. The scale is substitutions per site.

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, 1991;
Ross and Matthews 1989). In M. nigrophthalmus, however, almost all females in multi-
female 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. 2011a, 2011b)).
16 J. FIELD

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. 2011, 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.

Acknowledgements
Rosa Bonifacii kindly provided information and numerous specimens, and carried out the micro­
satellite genotyping. She and Mika Peck were excellent company during collecting trips. Paul John
Parsons carried out the sequencing and produced Figure 5. Charlotte Savill carried out the SEM
imaging, and Miranda Sealy helped to prepare the figures. I thank the staff at Mashpi and Santa
Lucia for their hospitality, and W.A. Foster for advice about etymology.

Disclosure statement
No potential conflict of interest was reported by the author.

Funding
The author reported there is no funding associated with the work featured in this article.
 JOURNAL OF NATURAL HISTORY 17

ORCID
Jeremy Field http://orcid.org/0000-0003-0663-4031

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