Zhang 2022 Anchored Phylogenomics and A Revised Classification of The Planidial Larva Clade of

Received: 16 September 2021 Accepted: 21 December 2021
DOI: 10.1111/syen.12533
ORIGINAL ARTICLE
Anchored phylogenomics and a revised classification

of the planidial larva clade of jewel wasps
(Hymenoptera: Chalcidoidea)
Junxia Zhang1 | John M. Heraty2 | Christopher Darling3,4 |

Robert L. Kresslein2 | Austin J. Baker2 | Javier Torréns5 |
6 7
Jean-Yves Rasplus | Alan Lemmon | Emily Moriarty Lemmon8
1
Key Laboratory of Zoological Systematics and Abstract
Application of Hebei Province, Institute of Life
Science and Green Development, College of Life Planidia are free-living, mobile first-instar larvae that are notable in their ability
Sciences, Hebei University, Baoding, Hebei, China to transition across different larval stages of their host, and for completing their
2
Department of Entomology, University of
development on the host prepupa as ectoparasitoids, effectively acting as larval-
California, Riverside, California, USA
3
Department of Natural History, Royal Ontario
pupal external koinobionts. Chalcidoid taxa with a planidium form a monophyletic
Museum, Toronto, Ontario, Canada group, the planidial larva clade (PLC). We conducted a phylogenomic study of the PLC
4
Department of Ecology and Evolutionary using anchored hybrid enrichment data. Phylogenetic analyses support the backbone
Biology, University of Toronto, Toronto,
Ontario, Canada relationship of PLC as: (Eutrichosomatinae, ((Philomidinae, Chrysolampinae),
5
CRILAR (CONICET), Anillaco, La Rioja, (Perilampinae, Eucharitidae))). Although excluded from the main analyses, the genus
Argentina
Jambiya, based on only 11 loci recovered, was placed as the sister of Chrysolampinae
6
INRAE, UMR 1062 CBGP CS30016,
Montferrier-sur-Lez, France + Philomidinae or Perilampinae + Eucharitidae. Our results demonstrate that Per-
7
Department of Scientific Computing, Florida ilampidae (Philomidinae, Chrysolampinae and Perilampinae) are paraphyletic. Diver-
State University, Dirac Science Library,
gence dating based on four node calibrations based on fossils suggests that the PLC
Tallahassee, Florida, USA
8
Department of Biological Science, Florida
arose approximately 111 Ma and the evolution of ant parasitism at least 64 Ma. Host
State University, Tallahassee, Florida, USA associations, direct versus indirect hyperparasitism, ability to attack a host within a
cocoon, soft versus hard planidial forms and mobility of the planidium were explored
Correspondence
John M. Heraty, Department of Entomology, using a likelihood-based ancestral state reconstruction method. A revised higher-
University of California, 900 University Ave,
level classification of the PLC is proposed, with Eutrichosomatinae elevated to
Riverside, CA 92521, USA.
Email: heraty@ucr.edu Eutrichosomatidae (stat. rev.), Chrysolampinae and Philomidinae placed in
Chrysolampidae (stat. rev.), Perilampidae (stat. rev.) restricted to what was referred
Funding information
Advanced Talents Incubation Program of the to as Perilampinae, and Eucharitidae maintained with four subfamilies, with
Hebei University, Grant/Award Number: Akapalinae (unknown biology) as sister group to the core Eucharitidae, all of which
521000981324; National Science Foundation,
Grant/Award Number: 1555808 are ant parasitoids. Jambiya is treated as an incertae sedis taxon within the planidial
clade.
KEYWORDS
anchored enrichment, Formicidae, immatures, parasitoids, phylogenomics, phylogeny
Systematic Entomology. 2022;47:329–353. wileyonlinelibrary.com/journal/syen © 2022 Royal Entomological Society. 329

13653113, 2022, 2, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/syen.12533 by CAPES, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
330 ZHANG ET AL.
proposed by Heraty et al. (2013). Monophyly of the PLC also has been
‘you can’t always get what you want…’ - Rolling Stones
supported by transcriptomic studies that utilized extensive data but
with limited taxon sampling (Peters et al., 2018; J. Zhang et al., 2020).
While the larvae within the PLC are similar in overall morphology and
I N T R O D U CT I O N behaviour, the adults have an astounding diversity of body forms
(Fig. 2), behaviours and host associations.
For parasitoids that lay eggs away from their hosts, a free-living Ectoparasitic first-instar larvae of some Chalcidoidea are known
first-instar larval stage is critical for host location. Smith (1912), with to have some degree of mobility in locating a feeding site on a host
reference to discussions with W.M. Wheeler, first used the term (i.e., Spalangia cameroni Perkins; Gerling & Legner, 1968) or even to
‘planidium’ in reference to the similar first-instar larva of Eucharitidae search a host to eliminate conspecific larvae (i.e., Nasonia vitripennis
and Perilampidae, deriving the term ‘from the Greek πλανήτης, a wan- [Walker], Krogmann & Abraham, 2003). Within the PLC, the initial
derer, and ίδιον, diminutive’ (Figure 1B–E). The homology of the mobility of the planidium is associated with egg placement and subse-
planidia of Eucharitidae and Perilampidae was first addressed by quent host location, then followed by the need to transition between
Heraty and Darling (1984), setting it apart from the planidiaform larval instars of the host to the pupa. Eutrichosoma Ashmead and
first-instar larvae found in other taxa such as Euceros Gravenhorst Chrysolampus Spinola deposit their eggs within the seed pod of the
(Ichneumonidae) (Tripp, 1961) and some families of Diptera host plant close to their weevil host (Baker & Heraty, 2020; Darling &
(e.g., Acroceridae, Nemestrinidae, Bombyliidae, Tachinidae, Asilidae) Miller, 1991). Philomidinae may lay their eggs into flowers to be trans-
(Clausen, 1940b). In Chalcidoidea, a planidiaform first-instar larva with ported by adult halictid bees to their ground-nesting larval hosts
distinct sclerotized terga is known only to occur in hyperparasitic (Heraty et al., 2019). Perilampinae deposit their eggs in the vicinity of
males of Coccophagus gurneyi Westwood (Aphelinidae) when eggs are their host with the first-instar fully responsible for host location
placed into a yet unparasitized scale host but they develop only (Clancy, 1946; Darling, 1999; Darling & Gibson, 2000; Darling &
as parasitoids of the larva of females of their same species (Flanders, Roberts, 1999; Perlman, 1995; Smith, 1912). Eucharitidae deposit
1937). However, planidia are better defined by how they transition their eggs at a far greater distance from their host and use a multitude
across different instars of their host as an external (Figure 1A,E) or of different behaviours to be transported into the ant nest and onto
internal parasitoid, and then develop as an external parasitoid of the their larval ant host (cf. Fig. 1D,E) (Clausen, 1940c, 1941;
host pupa (Baker et al., 2020; Clausen, 1940a; Darling, 1992, 1999; Heraty, 2000, 2002; Heraty et al., 2004). Current phylogenetic rela-
Darling & Miller, 1991). A critical distinction is that planidia are active tionships suggest that increasing mobility of the planidium is derived
larval parasitoids that develop only as external parasitoids of the host within the clade, and undergoes even further modification within the
pupa; with earlier descriptions instead focused on their highly sclero- Eucharitini, with planidia of some taxa able to actively jump large dis-
tized body and eggs being laid remote from their host. The planidial tances in their attempts to associate with an adult of their ant host for
larva clade (PLC), comprised of Eutrichosomatinae (Pteromalidae), Per- transport back to the nest (Torréns, 2013; Torréns et al., 2016).
ilampidae (Chrysolampinae, Perilampinae, Philomidinae and the Planidia are responsible for attacking the host, a process that can
unplaced genus Jambiya Heraty & Darling) and Eucharitidae be simple or complex. Many species are primary parasitoids attacking
(Akapalinae, Eucharitinae, Gollumiellinae and Oraseminae), was a specific host. In a simple case, the eggs of Eutrichosoma mirabile
F I G . 1 Immature stages within planidial larva clade. (A) Eutrichosoma mirabile Ashmead (Eutrichosomatidae), soft planidium attached to weevil
larva (modified from Baker & Heraty, 2020). (B) Perilampus sp. planidium dissected from larva of Anacamps wikeri Harrison (Gelechiidae); photo
credit Terry Harrison. (C–F) Kapala sp. (Eucharitidae) from Costa Rica: (C) fully developed hard planidia inside eggs in flower of Hamelia patens
Jacq.; (D) active planidium perched on base of Hamelia flower after corolla drop and next to nectar source used by ants; (E) planidium attached to
larva of Ectatomma ruidum (Formicidae: Ectatomminae); (F) fully developed female in host cocoon
ANCHORED PHYLOGENOMICS OF THE PLANIDIAL CLADE 331
F I G . 2 Planidial clade taxa. (A) Eutrichosomatidae: Eutrichosoma mirabile Ashmead (♀, UCRCENT00318910). (B–C) Chrysolampidae:
Chrysolampinae: (B), Chrysolampus schwarzi Crawford (♂, UCRCENT00456230); (C) Chrysomalla hesperis Darling (♀, UCRCENT0091180).
(D) planidial clade incertae sedis: Jambiya vanharteni Heraty & Darling (♀, UCRCENT00184056). (E–F) Chrysolampidae: Philomidinae: (E)
Aperilampus sp. (♂, CASENT 2022341); (F) Philomides frater Masi (♀, UCRCENT00422377). (G–J) Perilampidae: (g) Euperilampus tanyglossa
(♂, ROME174200); (H) Krombeinius sp. (♀, ROME176884); (I) Steffanolampus salicetum Steffan (♀, ROME188153); (J) Perilampus
(♀, ROME189000). (K–R) Eucharitidae: (K) Akapalinae: Akapala astriaticeps Girault (♀, UCRCENT00241748); (L) Gollumiellinae:
Anorasema pallidipes (♂, UCRCENT00241530); (M) Oraseminae: Orasema arimbome Dominguez et al. (♀, UCRCENT00425961); (N) Eucharitinae:
Psilocharitini: Neolosbanus palgravei (Girault) (♀, UCRCENT00241537); (O–R) Eucharitinae: Eucharitini: (O) Galearia latreillei
(♀, UCRCENT00161520); (P) Hydrorhoa stevensoni female (♀, UCRCENT00161471); (Q) Eucharis adscendens (♀, UCRCENT00161545);
(R) Lirata pustula Heraty (♂, UFES59788)
332 ZHANG ET AL.
Ashmead are deposited among the eggs of its host weevil within a segments (Fig. 1A) or strongly sclerotized (hard) with distinct darkened
seed pod of Acacia, and thus the planidium is already in close proxim- terga and a ventral membranous region (Fig. 1B,C,E), but even when
ity to its host (Baker & Heraty, 2020). In a more complex case, some weakly sclerotized there is usually some form of ornamentation on
species of Perilampus Latrielle are obligate indirect hyperparasitoids, the margin of the body segments (Baker & Heraty, 2020).
with planidia entering a secondary host, whether parasitized or not The presence of distinct terga appears correlated with increasing
and then attacking either a dipteran (Tachinidae) or hymenopteran degrees of mobility and likely environmental exposure. The terga of
(Ichneumonoidea) primary parasitoid; planidia do not develop further in some Eucharitini have an additional lateral longitudinal line of
unparasitized secondary hosts (Darling & Gibson, 2000; Smith, 1912). desclerotization (tergopleural line), that appears correlated with the
Even defining a planidium as an endoparasitoid or an ectoparasitoid can ability to jump as opposed to crawling in an inch-worm like fashion
be difficult (Heraty & Murray, 2013). Eutrichosoma and Chrysolampus (Torréns, 2013). A more weakly sclerotized larva without terga
are both ectoparasitic throughout their development on the host, (Chrysolampinae and Eutrichosomatinae) has been proposed as ances-
remaining attached to their larval host by their mandibles and undergo- tral within the clade, whereas the formation of well-sclerotized terga
ing limited feeding (Fig. 1A) (Baker & Heraty, 2020; Darling, 1992). In (Eucharitidae, Perilampinae and Philomidinae) and ultimately the ter-
Perilampus chrysopae Crawford, a primary parasitoid of Chrysopidae gopleural line are considered as derived (Baker & Heraty, 2020;
(Neuroptera), the planidium remains as an ectoparasitoid throughout Darling, 1992; Darling & Miller, 1991; Heraty & Darling, 1984). Once
development (Clancy, 1946). In those Perilampus species that are indi- located on the host pupa and feeding has begun, the first-instar can
rect hyperparasitoids, the planidium attacks and enters into the hemo- expand over a 1000-fold and the tergites appear only as minute dark
coel of its secondary host and, if that host is parasitized, the planidium bands (Heraty et al., 1993; Heraty & Barber, 1990; Smith, 1912).
enters the hemocoel of its primary parasitoid host larva, becoming Larvae within the PLC clade are hypermetamorphic, with the
external only at the start of histolysis during transformation of the pri- planidium attacking the host larva and the hymenopteriform later
mary host to a prepupa; if unparasitized, the planidia do not develop instar larvae developing in a ventral position on the host pupa
further (Bergold & Ripper, 1937; Darling & Gibson, 2000; Perlman, (Darling, 1992, 1999; Darling & Roberts, 1999; Heraty et al., 1993,
1995; Purrington, 1970; Smith, 1912). In Eucharitidae, planidia can be 2015). Later instars are usually external parasitoids, although Monacon
either always ectoparasitic on the host larva and attached by their Waterston (Perilampinae), for example, actively burrow into the host
mouthparts, or can burrow just under the cuticular surface of the host pupa during their third instar (Darling & Roberts, 1999).
while maintaining an external connection through the entrance hole in Host relationships are diverse across the PLC but can be either
the cuticle (Heraty et al., 2004; Heraty & Murray, 2013). In the latter conserved or highly variable within groups. Eutrichosomatinae attack
case, planidia become external at each larval moult. Where known, all seed-feeding weevils (Curculionidae) on Asteraceae or Fabaceae
members of the planidial clade complete their larval development as (Baker & Heraty, 2020; Charlet & Seiler, 1994). Chrysolampinae attack
ectoparasitoids feeding on the ventral region of the host prepupa. Nor- seed-feeding tychiine weevils or pollen-feeding Nitidulidae on a vari-
mally, only a single parasitoid completes development on a host. ety of host plants including Asteraceae and Fabaceae (Darling &
Planidia of Perilampus have been observed to search the prepupa of its Miller, 1991; Erdös, 1955; Graham, 1969; Nikol’skaya, 1952), whereas
primary host and kill other conspecific planidia (Clancy, 1946; Austrotoxeuma Girault from New Zealand have been associated with
Smith, 1912). This behaviour has not been documented in other taxa cerambycid beetles in dead twigs of Discaria Hook. (Rhamnaceae) and
but it may be more widespread. Only a few derived Eucharitini Coprosma J.R. Forst. & G. Forst. (Rubiaceae) (Bouček, 1988).
attacking large pupa sometimes support 2–3 parasitoids per host, but Aperilampus Walker (Philomidinae) have been reared from the pupa of
generally only a single parasitoid develops, even in those species in Halictus africanus Friese (Halictidae) (Darling, 1992) and Philomides
which multiple individuals per host have been documented Haliday has been observed on flower buds of Solanum torum
(Torréns, 2013; Torréns et al., 2008). Sw. (Solanaceae) (Heraty et al., 2019). Perilampinae have the broadest
Unfed planidia are generally minute, ranging from 0.07–0.40 mm taxonomic host range within the clade, and as primary parasitoids or
in total body length, although they can reach 1.2 mm in Aperilampus obligate indirect hyperparasitoids, they have been reared from eight
(Philomidinae) (Baker & Heraty, 2020; Darling, 1992, 1999; Darling & different insect orders. As primary parasitoids, they have been
Miller, 1991; Heraty et al., 2004; Heraty & Barber, 1990; Heraty & reported attacking Coleoptera (Bostrichidae and Ptinidae), Neuroptera
Darling, 1984). Morphology of the planidium is highly conserved (Chrysopidae) and Hymenoptera (Diprionidae, Tenthredinidae,
within groups, and although highly divergent in morphology between Eumenidae, Sphecidae, Chrysididae) (Abe, 1988; Bouček, 1977;
groups, there is some overall similarity. All planidia have a distinct cra- Clancy, 1946; Darling, 1983, 1992; Krombein, 1964; Principi, 1947;
nium and relatively large sickle-shaped mandibles. In the clades, Tripp, 1962). As indirect hyperparasitoids, they attack Hymenoptera
Eutrichosomatinae and Chrysolampinae, the planidia have distinct (Ichneumonoidea, Sclerogibbidae) and Diptera (Sarcophagidae,
antennae, positional homology of a number of distinct stout spines, Tachinidae) through secondary hosts in Lepidoptera, Orthoptera,
and 13 body segments after the head, with these proposed ancestral Phasmatodea and Rhaphidioptera (Aspöck, 2002; Bergold &
structures undergoing losses in more derived taxa (Baker & Ripper, 1937; Bouček, 1978; B. D. Burks, 1969; Darling, 1983;
Heraty, 2020; Darling, 1992; Heraty & Darling, 1984). Within the Laing & Heraty, 1982; Smith, 1912). The interactions are often not
PLC, the planidia may be weakly sclerotized (soft) with uniform body clear, and some records of Eumenidae and Sphecidae as direct
parasitoids may be the result of lepidopteran predation and ‘acciden- Eucharitidae), with Eucharitidae and Perilampinae monophyletic, has
tal hyperparasitism’ of the predator (cf. Perlman, 1995). The biology been supported in morphological analyses of the planidia by
of Akapalinae is unknown, but all other records within Eucharitidae Darling (1992) and Baker and Heraty (2020).
are as direct parasitoids of ants, although the use of prey items such Previous analyses of relationships within the core Eucharitidae
as immatures of Auchenorrhyncha or Thysanoptera as intermediates (excluding Akapalinae) support the monophyly of the three subfam-
to gain access to the ant colony are known (Das, 1963; ilies, with Gollumiellinae either paraphyletic or sister group to
Heraty, 1994a, 2000). Among their ant hosts, there is considerable Oraseminae + Eucharitinae (Fig. 4). Within Eucharitinae, Psilocharitini
fidelity for certain ant subfamilies or groups of ant genera (Murray can be monophyletic or paraphyletic, and Eucharitini are always
et al., 2013). monophyletic but with different relationships among the included cla-
Heraty and Darling (1984) provided a morphology-based phyloge- des. Monophyly of the Eucharitinae supports a single shift from
netic analysis of the planidia of Eucharitidae and Perilampidae. Their attacking ants that lack a cocoon to attacking those that pupate within
results proposed that Chrysolampinae were sister group to Peri- a cocoon (Heraty & Murray, 2013). Based on the relationships pro-
lampinae + Eucharitidae, and thus Perilampidae as defined at that posed by Murray et al. (2013), Pseudometagea Ashmead, a parasitoid
time were paraphyletic. Subsequent analyses of relationships across of Lasius Fabricius (Formicinae), was sister to the remaining
Chalcidoidea using either ribosomal DNA (18S and 28S) or a combina- Eucharitini, and supported a hypothesis of Formicinae as the ancestral
tion of ribosomal DNA and morphology recovered a PLC (Fig. 3) host association for the family, with a single shift to parasitism of
(Heraty et al., 2013; Munro et al., 2011). Idioporus (Pteromalidae) was poneromorph ants (PEM clade: Ponerinae, Ectatomminae,
included within the clade in Munro et al. (2011), but excluded in later Myrmeciinae) in the Chalcura, Schizaspidia and Kapala clades (green,
results; it has never been considered as related to the planidial clade Fig. 4). Morphological features of the pupa also support the mono-
(La Salle et al., 1997). The inclusion of Eutrichosomatinae was surpris- phyly and relationships of the PEM clade (Heraty et al., 2020). The
ing at the time, but was consistent with an earlier study demonstrat- grade of the Chalcura and Schizaspidia clades, both Palaeotropical or
ing morphological similarities of the labrum (Darling, 1988) and the Palaearctic in distribution, to the Kapala clade (Neotropical or Nearc-
later discovery of a planidial larva that was very similar to that of tic), suggested a single dispersal event to the New World at 23.3
Chrysolampinae (Baker & Heraty, 2020). An extensive analysis of rela- (16.8–30.9) Ma (Murray et al., 2013). Similar dates were proposed for
tionships within Eucharitidae by Murray et al. (2013) recovered similar a single dispersal event from the Old World to the New World within
planidial clade membership (Fig. 3), but the outgroup sampling was Oraseminae that was estimated at 19.6 (13.0–27.5) Ma by Murray
limited. Notably, the monophyly of each of Chrysolampinae, Peri- et al. (2013) and 24 (17–33) Ma by Baker et al. (2020).
lampinae and Eucharitidae were maintained across all analyses, while Phylogenetic hypotheses based on morphology of the planidia
the relationships between these groups differed considerably (Fig. 3). suggest that a hard planidium originated once and that the relation-
Taxa with a soft planidium were more basal and there was support, ships proposed are (Eutrichosomatinae, (Chrysolampinae,
albeit weak, for the monophyly of taxa with a hard planidium (Fig. 3). (Philomidinae, (Perilampinae + Eucharitinae)))), with larvae unknown
Monophyly of the hard planidia taxa (Philomidinae, Perilampinae, for Jambiya and Akapalinae (Baker & Heraty, 2020; Darling, 1992).
F I G . 3 Previous hypotheses of relationships within the planidial clade, with the groups with a soft planidium and hard planidium indicated.
Idioporus (Pteromalidae) is a whitefly parasitoid and not considered as a member of the clade. Jambiya is currently not placed into a higher-level
taxon (see text discussion)
334 ZHANG ET AL.
86
Gollumiellinae Gollumiellinae Gollumiellinae
100 100 100
Oraseminae Oraseminae Oraseminae
99
Psilocharitini 100 Psilocharitini
? ?
Psilocharis Psilocharis
100 Psilocharitini 100
Psilocharitini 100 Psilocharitini
Eucharitinae Neolosbanus Neolosbanus
59 100
79 Schizaspidia Pseudometagea 100 Pseudometagea
clade 91
61 Kapala
Kapala 54
Eucharis
Eucharitini clade clade
clade 100
Schizaspidia 96
Stilbula clade
86 Stilbula
clade Stilbula clade
Formicinae clade
100 Chalcura
Eucharis Eucharis clade
Myrmicinae clade clade 93
100 Schizaspidia
= >70% BS clade
Poneromorph 100
Pseudometagea
90
100 Kapala
clade
Munro et al. (2011) Heraty et al. (2013) Murray et al. (2013)
ribosomal 18S & 28S ribosomal (18S, 28S) & morphology ribosomal (18S, 28S) &
[48 Eucharitidae] [14 Eucharitidae] mitochondrial (COI, COII)
[195 Eucharitidae]
FIG. 4 Previous hypotheses of relationships within the core Eucharitidae (excluding Akapalinae) that employ molecular data
These same relationships were recovered by Heraty et al. (2013) using on their similar morphology to Chrysolampinae, and also for their use by
a combination of adult morphological and molecular data. Murray et al. (2013) and Baker et al. (2020). Spalangiinae and
We investigated the phylogeny of the PLC using an anchored Erotolepsiinae are monophyletic and sister to the planidial clade in
hybrid enrichment (AHE) phylogenomic approach with a restricted unpublished AHE and Ultraconserved Elements (UCE) phylogenies of
focus on just the planidial clade, as opposed to our recent studies Chalcidoidea. Pteromalinae were sister to the planidial clade in Heraty
across all Chalcidoidea, which allows us to reduce the homoplasy et al. (2013). Voucher specimens are mounted and tagged with both a
encountered and also for greater data inclusion through less trim- UCRCENT specimen identifier and a DNA code number (D####) and
ming of the sequence data. In contrast to an earlier AHE approach deposited in the University of California Entomology Research
to understanding Oraseminae (Baker et al., 2020), the AHE data in Museum. Table S1 provides details for species information. We
this study were analysed with an updated pipeline that enabled us excluded a second specimen of Philomidinae, Philomides hamooniae
to further divide the coding regions from adjacent flanking regions Heraty et al. (UCRCENT00491418), because of poor sequence capture
(mostly introns) and explore the effects of different loci and codon even though it did not affect the placement of our included philomidine
positions on phylogenetic reconstruction. Our results are used to genus Aperilampus in earlier analyses. Jambiya vanharteni Heraty & Dar-
map several traits of interest in the PLC, date the temporal diver- ling (UCRCENT00184056) also had poor capture, but because of its
gence of the PLC to better understand the evolution of planidia unique status, we included it in supplementary analyses (see below).
and host-shifts within this clade, and propose a revised classifica- Taxa were identified by coauthors Christopher Darling, John Her-
tion for the group. The proposed results are not always concordant aty, Javier Torréns, Jean-Yves Rasplus, Austin Baker and Roger Burks,
with earlier studies and in some cases require rethinking the evolu- primarily using Bouček (1978, 1988), Burks et al. (2017, 2018), Dar-
tion of host associations and patterns of geographic dispersal ling (1986) and Heraty (1994a, 1994b, 2002).
within these higher-level groups.
AHE probe sets

MATERIAL AND METHODS
The AHE probe set initially applied was the Hym_Ich set originally
Taxon sampling developed for Ichneumonoidea (Hymenoptera) targeting for
541 anchored loci (212,392 bp) (Sharanowski et al., 2021). Later, the
A total of 105 terminal taxa were included that cover all of the known Hym_Cha probe set that was optimized for Chalcidoidea targeting for
families and subfamilies of the PLC: Eutrichosomatinae (3), 441 loci (421,012 bp) was utilized to improve the capture efficiency.
Chrysolampinae (5), Philomidinae (1), Perilampinae (18) and Eucharitidae For details about the procedures of probe design and optimization,
(2 Akapalinae, 3 Gollumiellinae, 24 Oraseminae, 37 Eucharitinae). In addi- see Baker et al. (2020). In total, 56 taxa were enriched with the
tion, 12 taxa of Pteromalidae (1 Pteromalinae, 4 Spalangiinae, Hym_Ich probe set and 42 taxa with the Hym_Cha probe set. For
2 Erotolepsiinae) and Torymidae (1 Chalcimerinae, 1 Glyphomerinae, seven taxa, the targeted AHE loci were extracted from the available
3 Toryminae) were included as outgroups. Torymidae were chosen based transcriptomes or genome (Peters et al., 2018; Table S1).
DNA extraction, AHE and sequencing Nasonia vitripennis, Gonatocerus morrilli, Aphelinus abdominalis,
Trichogramma evanescens, Tamarixia radiata, Sycophila biguttata as ref-
Genomic DNA was extracted using the QIAGEN DNeasy Blood & erences. For each exon, up to 6000 bp containing the best matching
Tissue Kit. Quality and quantity of DNA were checked using a region for each species were initially extracted using a customized
Qubit™ Fluorometer, and Bioanalyzer. AHE data were collected at java script (ExtractBestFromSingleScanResultsFile) and the excessive
the Centre for Anchored Phylogenomics (www.anchoredphylogeny. regions were later trimmed after ortholog alignment (see below for
com) following the library preparation and AHE protocols described in details).
Baker et al. (2020). Enriched library pools were sequenced at the
Translational Lab of the College of Medicine at Florida State Univer-
sity with either an Illumina 2500 sequencer (92 samples with single Orthology prediction and alignment
indexing) or an Illumina NovaSeq6000 sequencer (6 samples with dual
indexing). With the exception of 12 samples sequenced with a paired- Orthology was determined for each of the 1121 exon regions rather
end 200 bp protocol, all samples were sequenced with a paired-end than 441 loci. Orthology prediction in the standard AHE
150 bp protocol. Sequencing details are provided Table S1. The AHE phylogenomic pipeline first computes the pairwise distance between
data were then analysed following the pipeline depicted in Fig. S1. two sequences and then clusters the sequences using the distance
matrix with a Neighbour-Joining algorithm (see details in Hamilton
et al., 2016). However, highly divergent intron regions often inflate
Paired-read merging and assembly the pairwise distance and then place some orthologous sequences
into different clusters. To counter this artefact, we modified the
The sequenced reads were demultiplexed with no tolerance for any orthology prediction method in the standard AHE phylogenomic pipe-
mismatched indexes, and then the low-quality reads were removed line and only used the coding regions in the homologous sequences
using the Illumina Casava high-chastity filter. Paired reads were mer- for orthology determination. First, coding and non-coding regions
ged following Rokyta et al. (2012) to improve assembly efficiency. The were identified in homologous sequences using a customized java
assembly of merged and unmerged reads were conducted using a script (TranslateHomologs3) that translates each homologous
quasi-de-novo assembler (Hamilton et al., 2016) with the following sequence generated for each exon in all three reading frames, then
chalcidoid species as references: Nasonia vitripennis (Walker) selects the optimal (based on match to reference and minimizing stop
(Pteromalidae), Gonatocerus morrilli (Howard) (Mymaridae), Aphelinus codons) reading frame for each sequence. Reads were mapped to ref-
abdominalis (Dalman) (Aphelinidae), Trichogramma evanescens erence exon sequences and the assembly produced one or more
Westwood (Trichogrammatidae), Tamarixia radiata (Waterston) (homologous) sequences per reference exon sequence. For homo-
(Eulophidae), Orasema simulatrix Gahan (Fabricius) (Eucharitidae) and logues of each exon region, the nucleotide sequences of the coding
Sycophila biguttata (Swederus) (Eurytomidae). The targeted AHE loci regions (HomologsNT123), the first two codon positions of the coding
often contain multiple exons within one captured locus. In some cases, regions (HomologsNT12) and the sequences that contain both coding
the locus-based assembly approach failed to recover some of the adja- and non-coding regions (HomologsNTALL) were extracted. The
cent exons if the intermediate intronic regions were sequenced at pairwise distance matrix was then computed using the first two codon
lower coverage. In earlier analyses, we observed that this could result position sequences (NT12) because the explorative analyses
in a large amount of missing data. Thus, an exon-based assembly strat- suggested it resulted in a more complete dataset than that using all
egy was applied, in which the sequences of the targeted loci were split three codon positions (NT123) or complete (NTALL) sequences. Clus-
into exon regions identified during probe design for each reference spe- tering of the homologue sequences based on the pairwise distance
cies (in total 1121 exon regions) and then each split exon region was matrix followed Hamilton et al. (2016). The coding (NT123) and com-
assembled separately for the sequenced taxa. For each exon of each plete (NTALL; including both coding and non-coding) sequences of
individual, the number of reads mapping to each assembly cluster was the orthologs for each targeted exon region were then sorted sepa-
counted, and then the 5% quantile in the read counts for each exon rately for downstream analyses. Sequence clusters containing fewer
across all individuals and assembly clusters was computed. The assem- than 40 taxa were removed from the datasets, and in total
bly clusters with read counts greater than or equal to the 5% quantile 945 orthologous groups were retained for alignment.
value for corresponding exon were removed to filter out possible low- Both the NT123 and NTALL sets were aligned with Mafft
level contaminants. v.7.305b (Katoh & Standley, 2013) using the E-INS-i algorithm. The
initial multiple sequence alignments (MSAs) of the NTALL set were
processed in Geneious Prime v.2019.1.1 to remove the excessive
Extracting AHE loci from transcriptomes or genomes sequences in the transcriptome and genome taxa that often resulted
in difficulty of alignment. For the six transcriptome taxa, the extracted
For the six transcriptomes and one genome included in this dataset sequences that extended to adjacent exon regions were trimmed in
(Table S1), each was scanned for the 1121 target exon regions using a the MSAs. For the Nasonia vitripennis genome, the excessive regions
customized java script (QuickScan5) and the target exon sequences of at both ends that could not be aligned with the AHE taxa were
336 ZHANG ET AL.
trimmed from each MSA. Gaps were removed from the MSAs of analysis including all the AHE data generated for the Chalcidoidea
the NTALL set and a second round of alignment with Mafft v.7.305b phylogenomic project (over 600 taxa), since only checking for contam-
(E-INS-i algorithm) was conducted again. ination among the samples included in this study would not be able to
detect contaminants from samples processed together but not
included in this study. Exon-based assembly and orthology prediction
Overlap checking and identification of core coding and were performed as outlined above. The orthologs of the NT123 set
flanking regions were aligned using Mafft v.7.305b and E-INS-i algorithm, and a gene
tree was then built for each of the NT123 alignment using RAxML
The exon-based assembly strategy improves the assembly efficiency, v.8.2.8 (Stamatakis, 2014) with the GTRCAT model. Using a custom
but the assembled contigs often extend to the adjacent exon and intron Python script (J. Zhang et al., 2020), we flagged the taxa on each gene
regions, which cause the overlap of MSAs. Therefore, we checked for tree that may be subject to contamination errors (identical sequences
overlaps of all MSAs as follows. First, the sequence of Nasonia for unrelated taxa of different genera). Each gene tree and its
vitripennis (when it was absent in the MSA, the sequence of another corresponding sequence alignment were manually inspected to fur-
species was used instead) was extracted from each of the NT123 and ther determine contaminants and generate a list of sequences that
NTALL MSAs. All the gaps at both ends of the extracted representative needed to be removed due to contamination. When the source of
sequences were replaced with Ns, and then the representative contamination could not be distinguished, all flagged sequences were
sequences were mapped to the annotated genome of Nasonia removed from the dataset. From this list we extracted the contami-
vitripennis (GCF_000002325.3_Nvit_2.1) in Geneious Prime v.2019.1.1 nants that were included in this study and then removed them in the
with high/medium sensitivity and up to 10 iterations (other settings as corresponding NT123 and NTALL orthologous groups before MSA.
default). The mapped results were manually inspected for overlaps We also checked for potential alignment errors or paralogs in
which were marked and removed from corresponding NT123 and each of the coding and flanking MSAs. For each of the extracted cod-
NTALL MSAs. The MSAs that are redundant with others or not mapped ing and flanking MSAs, a gene tree was constructed using RAxML
to the annotated Nasonia vitripennis genome or very poorly aligned v.8.2.8 (Stamatakis, 2014) with the GTRGAMMA model. Gene trees
were deleted from downstream processes. were inspected using a customized Python script (J. Zhang
While conducting the overlap checking, we observed that some et al., 2020) to flag taxa with anomalous sequences that resulted in
of the NT123 MSAs contained a relatively larger proportion of missing abnormally long branches on the gene tree. These flagged sequences
data than the corresponding regions in the NTALL MSAs. This may be were then removed from the corresponding MSAs. The coding MSAs
due to the stringent translation algorithm during the orthology predic- were also manually checked in Geneious Prime v.2019.1.1. The gappy
tion. To further reduce the missing data in the datasets, we then iden- regions in the flanking MSAs were masked using Seqtools in the PASTA
tified and extracted the core (well-aligned) coding and flanking (intron package (Mirarab et al., 2015) with ‘masksites = 40’. We tried a more
and poorly aligned exon) regions in each of the NTALL MSAs after relaxed masking parameter (masksites = 20) or no masking of gappy
removing the overlaps. This procedure increased the number of regions in Seqtools and the explorative analyses resulted in almost
parsimony-informative sites (16,000 more) and decreased the miss- identical phylogenetic relationships. The coding and flanking MSAs with
ing/ambiguity proportion (15% less) in the concatenated matrix of less than 50 taxa were then excluded from the phylogenetic datasets,
coding sequences and thus were used for downstream analyses. The which resulted in 1332 sequence alignments (763 coding, 569 flanking
separation of well-aligned core coding sequences from flanking sequence alignments). For each of the 1332 alignments, statistics
regions allowed us to further investigate their signal in different phy- (e.g., number of taxa, alignment length, number of parsimony-
logenetic hypotheses. informative sites) were computed using AMAS (Borowiec, 2016).
The gaps in the extracted flanking MSAs inserted during align-
ment were removed and then the short sequences (<100 bp) were
trimmed. The resulted flanking regions with less than 40 taxa were Phylogenetic datasets
excluded from the dataset. The sequences of the remaining flanking
regions were then realigned to improve the alignment using Mafft The AHE data were mainly analysed with nucleotide sequences, and
v.7.305b and the E-INS-i algorithm. different concatenated datasets were generated to explore the phy-
logeny of PLC (see Table S3 for details of each dataset): (1) 763 cod-
ing (third codon RY recoded) + 569 flanking regions; (2) 763 coding
Error checking and alignment masking +569 flanking regions; (3) 569 flanking regions; (4) 763 coding
regions; (5) 763 coding regions with the third codon positions being
Contaminations may happen during DNA extraction, library prepara- RY recoded; (6) filtered 336 regions (300 coding regions with third
tion and sequence capture processes. Previous studies (J. Zhang codon RY recoded plus 36 flanking regions) with number of taxa
et al., 2020) have suggested that even small amounts of contamina- > = 80, alignment length ≥ 200, missing percentage ≤ 20%; (7) a
tion can lead to the wrong phylogenetic relationships. To remove the ‘decisive’ dataset extracted from the 763 coding regions with the
putative contaminants in the dataset, we conducted an independent third codon positions being RY recoded following Dell’Ampio
et al. (2014) and Misof et al. (2014), in which only data blocks that The 11 loci were identified by examining a separate set of gene trees
contain sequences with at least one representative of the predefined with Heimbra included; excluding any loci where the sequences were
taxonomic groups are retained (531 genes: 337 coding regions with identical and not part of the planidial clade taxa. Because of the extent
third codon RY recoded and 194 flanking regions). Concatenation of of contamination, we excluded Jambiya from our primary analyses for
sequence alignments was conducted using FASconCAT (Kück & discussion, but we did run separate analyses for (1) the entire set of
Meusemann, 2010). loci but for Jambiya loci with only their 11 loci included and (2) for all
of the taxa but with only the 11 loci included.
The orthology prediction of the 11 uncontaminated exon-only
Phylogenetic analyses loci was performed separately from the primary analysis, using the
same methods and parameters presented above. The resulting
Phylogenetic reconstruction was performed on each nucleotide sequence data were appended into correct orthologous clusters in the
dataset using the maximum likelihood (ML) criterion with IQ-TREE proofread exon-only dataset (dataset 4: 763 coding regions) produced
v.2.0.6 (Nguyen et al., 2015). The best-fitting model and optimized in the primary analysis. The 11 loci were realigned in Mafft v.7.305b
partition scheme were inferred for each supermatrix in IQ-TREE using with the E-INS-i algorithm and were concatenated and partitioned by
the option ‘-m MF + MERGE’. Each of the 763 coding and locus using FASconCAT v1.0 (Kück & Meusemann, 2010). Phyloge-
569 flanking regions was predefined as a single partition. For each netic reconstruction was performed using IQ-TREE 1.6.12 (Nguyen
dataset, we ran 40 independent ML tree searches (20 with random et al., 2015) on two datasets: (1) all 763 coding regions and (2) only
starting trees and 20 with parsimonious starting trees). Ultrafast boot- the 11 loci which contained uncontaminated sequence data from
strap (‘-B 2000 -bnni -wbt’) analyses were completed in IQ-TREE to Jambiya. Locus partitions were modelled using GTR + Γ + I (-m GTR
access node support. We also analysed the 763 coding regions + I + G). For each dataset 20 independent ML tree searches were run
(dataset 4) based on the translated amino acid sequences using the from random starting trees and node support was assessed using
mixture model (CAT+GTR) in PhyloBayes MPI v.1.8 (Lartillot standard non-parametric bootstrap analysis (-b 100).
et al., 2013). Two chains were conducted each with sampling fre-
quency as 1 and constant sites removed (-dc) to save computational
time. The convergence of the two chains were checked with bpcomp Partition support for phylogenetic hypotheses
(maxdiff = 0.096), and the first 2000 sampled trees were discarded as
burn-in. The consensus of the remaining 20,095 trees sampled from Partition support for different phylogenetic hypotheses was assessed
the two chains was conducted to compute the posterior probabilities on the ‘569 flanking’ (dataset 3), ‘763 cds’ (dataset 4) and ‘763 cds
of clades. (third codon RY recoded)’ (dataset 5) matrices respectively to evaluate
A coalescent-based species tree method that accounts for poten- the support of each partition for four alternative phylogenetic hypoth-
tial gene tree heterogeneity and discordance was applied to three eses (T1–T4, Figure 7) using the method in Shen et al. (2017). The
datasets: (1) 1332 trees from 763 coding and 569 flanking regions hypothesis recovered from the unconstrained concatenated analysis
(dataset 1); (2) 336 trees from filtered 336 regions (dataset 6); (3) 531 on the ‘763 cds (third codon RY recoded) + 569 flanking’ dataset
trees from the decisive dataset (dataset 7). First, the ML gene tree (dataset 1) is T1. The T2 hypothesis was recovered by some concate-
and 100 non-parametric bootstrap replicates were constructed for nation and coalescent analyses, which involves a rearrangement of
each alignment in IQ-TREE v.2.0.6 using the best-fitting model Eutrichosomatinae as the sister to the clade with Perilampinae and
selected by ModelFinder (Kalyaanamoorthy et al., 2017). For each Eucharitidae. The T3 hypothesis represents a planidial larval transition
gene tree, the average bootstrap support and average branch length from soft to hard, with Philomidinae sister to Perilampidae
were calculated using an R script (available at https://github.com/ + Eucharitidae, as proposed by Baker and Heraty (2020). The T4
marekborowiec/good_genes/blob/master/tree_props.R). Branches in hypothesis shows the result from Murray et al. (2013) for Eucharitini,
gene trees with bootstrap scores ≤10 were collapsed using Newick in which the PEM (including the Chalcura clade, Schizaspidia clade
Utilities v1.6.0 (Junier & Zdobnov, 2010) to account for branches on and Kapala clade) is monophyletic and Pseudometagea is sister to
gene trees with little or no support (C. Zhang et al., 2017), and then remaining Eucharitini. The tree that matches each of these alternative
the species trees were estimated using the Accurate Species Tree hypotheses was reconstructed with a constrained topology in IQ-
Algorithm (ASTRAL-III v.5.6.1; Mirarab et al., 2014; C. Zhang TREE v.2.0.6. The site-wise log-likelihood for each of the alternative
et al., 2017). Branch support for the species trees were estimated hypotheses was estimated using RAxML (v.8.2.12) with the option ‘-f
using 100 bootstrap replicates of each gene tree. G’, and then parsed for each locus using a Perl script to obtain the
gene-wise log-likelihood scores (GLS; Shen et al., 2017). The differ-
ence in gene-wise log-likelihood scores (ΔGLS values) for the different
Phylogenetic analyses including Jambiya hypotheses was calculated, and following the method in J. Zhang
et al. (2020) a cutoff of 2 was used to determine if the locus strongly
Our sequences of Jambiya vanharteni were contaminated by Heimbra supported the unconstrained hypothesis (T1, ΔGLS >2) or the con-
sp. (Eurytomidae) (UCRCENT00161500) for all but 11 exon-only loci. strained hypotheses (T2, T3 or T4, ΔGLS < 2).
338 ZHANG ET AL.
Divergence dating analyses We also estimated node ages using all of the AHE data (dataset 2:
763 cds + 569 flanking) in MCMCtree (PAML package v 4.9j;
Four fossil records of chalcidoid wasps were used to calibrate the Yang, 2007). The best ML tree topology from dataset 1 (763 cds with
nodes for the divergence time estimation. †Torymus bruesi (Huber) third codon RY recoded + 569 flanking) was also applied in the
(Torymidae: Toryminae) from the Eocene–Oligocene boundary of Flo- MCMCtree analyses with the maximum age of the root being con-
rissant shales in Colorado (33.7–23.8 Ma) was used to constrain the strained to 200 Ma and the 95% probability range of ages for the four
age of the most recent common ancestor of Didactyliocerus Masi and fossil calibrations applied at corresponding nodes. Two independent
Torymus Dalman (Calibration 1; Brues, 1910 as †Torymus sackeni Brues). MCMCtree runs were conducted (the posterior mean for all the nodes
The prior of this node was set to a lognormal distribution at M = 10, from each run were plotted to check for convergence following the
S = 1.2, offset = 23.8 (in real space) with a 95% probability range of MCMCtree manual), each with 10,000,000 generations (sampling
24.5–58.8 Ma and the median prior probability at 28.7 Ma. Three fos- every 100 generations) and the first 2,000,000 generations discarded
sils are from Baltic amber (44.1 1.1 Ma; Ritkowski, 1997): as burn-in.
(1) †Brachyelatus marthae Burks, Krogmann & Heraty (Perilampidae:
Chrysolampinae; R. A. Burks et al., 2019) to constrain the age of the
most recent common ancestor of Brachyelatus Hoffer & Novicky and Ancestral states reconstruction
Chrysolampus (Calibration 2); (2) †Perilampus pisticus Heraty & Darling
(Perilampidae: Perilampinae; Heraty & Darling, 2009) to constrain the Mesquite v.3.61 (Maddison & Maddison, 2019) was used to trace nine
age of the most recent common ancestor of Perilampus and Krombeinius trait histories (Suppl. nexus file 1). For likelihood mapping, polymorphic
Bouček (Calibration 3); and (3) †Palaeocharis rex Heraty & Darling states were coded as new states (see discussion below). We used an
(Eucharitidae: Psilocharitini; Heraty & Darling, 2009) to constrain the alternative Mesquite dataset for mapping traits using parsimony with
age of the most recent common ancestor of Oraseminae and variable states as polymorphic (Suppl. nexus file 2). Ancestral state recon-
Eucharitinae (Calibration 4) since the monophyly of Psilocharitini is not struction (ASR) likelihood probabilities are reported in Table S6. All states
supported by the current analyses. Following Murray et al. (2013), the were treated as unordered. We assessed trait evolution on five different
priors for these three nodes were all set to a lognormal distribution at hypotheses: T1, the tree obtained from our likelihood analyses with
M = 8.08, S = 1.0, offset = 39.2 (in real space), with a 95% probability Eutrichosomatidae sister to the remaining groups, which was variably
range of 40.1–64.6 Ma and the median prior probability at 44.1 Ma. supported in most analyses (Fig. 5); T2, the alternative hypothesis to T1
Divergence dating analyses conducted in BEAST v2.6.3 (Bouckaert with Chrysolampidae (Chrysolampinae + Philomidinae) sister to the
et al., 2019) applied the relaxed lognormal molecular clock model on two remaining groups; T3, Philomidinae sister to Perilampinae + Eucharitidae,
subsets of the AHE data each with 50 loci, because the concatenated which favours a single hard planidium origin; T4, in which the hypotheses
matrix is too large for MCMC sampling in BEAST. One subset included of T1 are retained, but within Eucharitini, Pseudometagea are sister to the
50 loci with medium evolutionary rate (one flanking and 49 coding remaining lineages and the poneromorph (PEM) parasitoids are monophy-
regions), and the average branch lengths of each gene tree was used as letic following the results of Murray et al. (2013); and the BEAST result.
an approximation for its evolutionary rate with the lowest average
branch length indicating the slowest evolutionary rate. The other subset 1. Host order: 0, Coleoptera; 1, Diptera; 2, Hymenoptera; 3,
contained 50 loci randomly selected from 569 flanking regions. BEAUti Neuroptera; 4, Lepidoptera, 5, mixed hosts (multiple orders). This
v2.6.3 (Drummond & Rambaut, 2007) was used to generate BEAST XML was the most conservative assignment of the actual host being
files. BEAST analyses were conducted using a fixed tree topology (best consumed by the parasitoid.
ML tree from dataset 1: 763 cds with third codon RY recoded + 569 2. Host family: 0, Curculionidae; 1, Ptinidae; 2, Schizophora
flanking) with branch length transformed to be ultrametric in Mesquite (Chloropidae/Sarcophagidae/Tachinidae; including Cecidiomyiidae
v3.6.1 (Maddison & Maddison, 2019). Two independent BEAST runs for Spalangia); 3, Ichneumonoidea; 4, Formicidae; 5, Aculeata
were completed for each dataset, each for 150,000,000 generations excluding Formicidae; 6, Cynipidae; 7, Chrysopidae; 8, mixed
(trees sampled at every 5000 generations). Tracer v1.7.1 (Rambaut orders (sawflies [Diprionidae/Tenthredinidae], Diptera and
et al., 2018) was used to check when the MCMC chains had reached a Ichneumonoidea through an alternate host, grasshoppers, acule-
stationary distribution by visual inspection of plotted posterior esti- ates, phasmids, chrysomelids; only within Perilampus). The host
mates, and to check that the ESS (Effective Sample Size) was greater than diversity is very broad and complicated by the obligate indirect
200 for most parameters. Using LogCombiner v2.6.3 (Drummond & hyperparasitism in some Perilampidae (=Perilampinae). The group-
Rambaut, 2007), trees sampled during the first 30,000,000 generations ing of multiple orders allowed for single state coding which is
(20%) in each run were removed as burn-in and the remaining trees were required for the likelihood analysis, but variable states were coded
combined for annotation. The resulting 24,002 trees were summarized as polymorphic in the parsimony analysis.
in TreeAnnotator v2.6.3 (Drummond & Rambaut, 2007) using the ‘user 3. Ant host subfamily: 0, no ant host; 1, Formicinae; 2, Myrmicinae; 3,
target tree’ and ‘mean age’ options, and then displayed in FigTree v1.4.4 poneromorph/PEM (Ponerinae, Ectatomminae, Myrmeciinae). This
(Rambaut, 2009). The 95% highest probability density (95% HPD) values character focused only on Eucharitidae. Poneromorphs are not a
were summarized for each analysis. monophyletic group, but instead an assemblage of subfamilies with
F I G . 5 Summary of phylogenomic analyses. The best tree from maximum likelihood (ML) analysis on the full dataset of 1332 regions (with
third base of codons RY coded). Boxes show the ML bootstrap support from the concatenation analyses and whether the clade is recovered in
the best ML tree and ASTRAL analyses of different datasets. The family (and subfamily) composition and designation of clades are indicated on
the right side of the tree. The scale bar represents substitutions per position. Taxon names include information on sex (female, male, unknown)
and geographic region. AT, afrotropical; AU, Australian, IM, Indomalaysian; NA, nearctic; NT, neotropical; PA, palearctic
similar morphology and behavioural patterns (Bolton, 2003). Multi- 4. Primary parasitoid or obligate indirect hyperparasitoid: 0, primary;
ple subfamilies of hosts within the PEM are generally shared within 1, obligate indirect hyperparasitoid; 2, either primary or hyper-
clades of Eucharitidae, and thus merging of the subfamilies into a parasitoid depending on host (polymorphic coding). States 1 and
single group is justified (Murray et al., 2013). 2 occur only in the Perilampidae (=Perilampinae). An example of
340 ZHANG ET AL.
state 2 is found in the Perilampus hyalinus species group, which are codon positions RY coded, the coding dataset shows dramatically
primary parasitoids of sawfly hosts but also can be obligate indirect increased ultrafast bootstrap support for the clade with
hyperparasitoids of ichneumonids or tachinids through lepidop- Chrysolampidae (Philomidinae, Chrysolampinae), Perilampidae and
teran secondary hosts. We scored Krombeinius (Perilampinae) as a Eucharitidae (93% vs. 65%), but decreased support for some other
primary parasitoid in the matrix, but also tested the impact of scor- nodes, such as the node including the Kapala clade, Stibula clade,
ing it as an obligate indirect hyperparasitoid because of the uncer- Pseudometagea and Eucharis clade (94% vs. 100%). The filtered-
tainty of this association. 336-region-dataset shows increased support over the full dataset for
5. Planidium attachment to host larva: 0, external; 1, partially internal the clade with Chrysolampinae + Philomidinae, Perilampinae and
(burrowed just below cuticle); 3, fully internal in either secondary Eucharitidae (91% vs. 65%), but decreased support for some other
or primary host (hyperparasitoid). nodes, for example, the Stibula clade (98% vs. 100%) and the node
6. Attacking host with a cocoon: 0, cocoon absent (includes Diptera with Pseudometagea and the Eucharis clade (68% vs. 91%). The deci-
puparium); 1, cocoon present (Hymenoptera, Neuroptera). All sive dataset recovers a similar topology to the full dataset with some
members of the planidial clade complete their development on the minor rearrangements within Perilampinae, Oraseminae clade and
prepupa, whereas the outgroups may develop on larvae or pupae. Kapala clade, but shows decreased support for certain nodes, for
Cocoon absence or presence is only relevant within the planidial example, the clade with Philomidinae, Chrysolampinae, Perilampinae
clade. and Eucharitidae (72% vs. 80%), the Stibula clade (92% vs. 100%) and
7. Planidium: 0, none (hymenopteriform); 1, present. Planidium is the node with Pseudometagea and Eucharis clade (79% vs. 91%). The
defined as a mobile first-instar larva that is an external larval-pupal analysis on the amino acid sequences resulted in a tree that is largely
koinobiont with features such as setation or mandibular structure congruent with the nucleotide tree (third codon positions RY coded).
that is characteristic of members of the planidial clade (Baker & One noticeable difference is the placement of Pseudometagea as sister
Heraty, 2020). to the Stibula clade plus Eucharis clade, but this relationship is poorly
8. First-instar larva type: 0, hymenopteriform and not a larval-pupal supported (posterior probability as 0.71; see Supplementary Material).
koinobiont; 1, soft planidium; 2, hard planidium. We coded the Results from partition support analyses are shown in Fig. 7 and
planidium in two different ways (7 above) to investigate the impact Table S4.
of dividing the character without ordering. Divergence dating on the 50 loci with medium evolutionary rates
9. Tergopleural line: 0, absent; 1, present. This feature was first using BEAST is shown in Fig. 6, and the age estimates for the major
defined by Heraty and Darling (1984) and is likely associated with clades from different analyses are summarized in Table S5. The
the ability of the planidium to jump (Torréns, 2013). BEAST runs on the two different datasets resulted in similar age esti-
mates for most nodes, but the 50-flanking-dataset estimated younger
ages than the 50-medium-dataset for the PLC (node 1; 99.52
vs. 111.37 Ma) and the Chrysolampidae + Perilampidae +
RESULTS Eucharitidae clade (node 3; 97.91 vs. 106.35 Ma), and an older age for
the Gollumiellinae clade (node 9; 56.49 vs, 48.64 Ma). The MCMCtree
Summary statistics for the 1332 loci (not RY coded for the coding analyses based on all AHE data provided similar age estimates for
regions) are provided in Table S2. Concatenation of the loci most nodes but usually with wider 95% HPD. For the major clades
(or subsets of loci) resulted in seven matrices each with 105 taxa. The within Eucharitidae (node 10: Oraseminae + Eucharitinae; node 11:
total number of nucleotide sites in these supermatrices ranged from Oraseminae; node 12: Eucharitinae; node 13: Eucharitini), the
93,939 to 327,879 and the number of parsimony-informative sites MCMCtree estimated much older ages than the BEAST analyses. In
ranged from 21,350 to 177,377 (Table S3). The flanking dataset con- the following discussion, we mainly focus on the BEAST result from
tained more missing data (gaps + ambiguites% in the matrix) than the the 50 loci with medium evolutionary rate unless otherwise noted.
coding dataset (36.35% vs. 23.68%) (see details in Table S3). The sum- ASR probabilities for trait evolution on the five different hypothe-
mary of results from phylogenetic analyses of the different datasets is ses (T1–T4, Figure 6) are presented in Table S6, with characters plot-
shown in Fig. 5. The log-likelihood of the best ML tree found from ted on the five hypotheses available in the Mesquite nexus file posted
each dataset and average bootstrap support on the best ML tree are at Dryad (see data deposition). Reconstructions are discussed below.
given in Table S3. The ML trees from the concatenation analyses of
nucleotide sequences, including analyses with Jambiya, and the spe-
cies trees estimated from ASTRAL analyses are provided in Supple- DI SCU SSION
mentary Material. The concatenated and coalescent analyses of
nucleotide sequences resulted in similar phylogenetic relationships Our AHE results all strongly support monophyly of the PLC,
(Fig. 5). The flanking dataset shows lower ultrafast bootstrap support including Jambiya, and suggest a sister relationship of the PLC with a
for certain nodes than the coding dataset, for example, the planidial clade of ‘Erotolepsiinae + Spalangiinae’ (Pteromalidae) (Figs 5 and 6;
larval clade and its sister (99% vs. 100%), the Stilbula clade (98% Figs S2–S12 and S14; paraphyletic in S13). As with previous studies
vs. 100%) and the Eucharis clade (95% vs. 100%). With the third (Baker et al., 2020; Darling, 1992; Heraty et al., 2013;
F I G . 6 Chronogram of the planidial larva clade divergence. Times shown are the mean age estimates from the BEAST analysis on the 50 loci
with medium evolutionary rate (95% highest probability density for major clades indicated as grey bars). Numbers for major clades are consistent
with those of Table S5. Asterisks refer to calibration nodes. The family (and subfamily) composition and designation of clades are indicated on the
right side of the tree as in Figure 5. Insets refer to distribution of hard and soft planidium type across planidial clade, and ant hosts for core
Eucharitidae (excluding Akapalinae)
Munro et al., 2011), Perilampidae s.l., comprised three subfamilies Based on our results (Fig. 5), we propose a new classification for
(Chrysolampinae, Philomidinae and Perilampinae), are always the PLC. There is no easily recognizable morphological support for the
paraphyletic to Eucharitidae (Figs 5 and 6; Figs S2–S14). planidial clade, although it is monophyletic in morphological, molecular
342 ZHANG ET AL.
and combined analyses (Heraty et al., 2013; Murray et al., 2013). The labrum in Philomidinae); mandibular dentition 3:2 (likely
planidia, a defining feature, are similar, but they lack concrete plesiomorphic, but also with equal dentition [2 teeth] in
apomorphies to support the clade (Baker & Heraty, 2020). Instead, it Chrysolampinae and Philomidinae); axillae not strongly advanced
is the behaviour of the planidium as an external larval-pupal (likely plesiomorphic but also strongly advanced in
koinobiont that defines their monophyly. The morphological diversity Eutrichosomatidae); stigmal vein of fore wing relatively long (although
of the group (Fig. 2) would make it difficult to readily assign a member variable in many included taxa); hyaline break of fore wing absent
to the clade without their recognition as part of one of the higher- (likely plesiomorphic and present in most Chrysolampinae); spur vein
level subfamily groups. We argue that the behavioural and host diver- of hind wing absent (present in some Chrysolampus). Based only on
sity across the various taxa would lose informativeness if treated as a Perilampus (Perilampinae) and Schizaspidia Westwood (Eucharitidae),
single family. We thus propose four families: Eutrichosomatidae Krogmann and Vilhelmsen (2006) proposed support based on pres-
Peck, 1951 (stat. rev.), Chrysolampinae and Philomidinae (previously ence of a profurcal bridge in the mesosoma. Although based on a
in Perilampidae) placed together in the family Chrysolampidae Dalla number of new dissections, this appears to only be a feature of
Torre, 1898 (stat. rev.), Perilampidae Förster, 1856 (stat. rev.) being Aperilampus (Philomidinae), Perilampus and Eucharitini (John
restricted to what was referred to as Perilampinae, and Eucharitidae M. Heraty). Using planidial morphology, Baker and Heraty (2020) pro-
Walker, 1846 being maintained with four subfamilies, including posed ‘ectoparasitic koinobiont’ (their character 23) as a synapomor-
Akapalinae (unknown biology) as sister group to the core Eucharitidae, phy of the clade.
which are all ant parasitoids. Jambiya is treated as an incertae sedis
taxon within the planidial clade.
Eutrichosomatidae had variable placement as either sister group Host associations
to the remaining planidial clade (Figs 5 and 6; Figs S2, S5–S8, S11 and
S14), sister to Perilampidae and Eucharitidae (Figs S3, S4, S10, S12 When treated at the ordinal level (character 1), Hymenoptera were
and S13) or unresolved (Fig. S9). Chrysolampidae, including supported as the ancestral host (p. 0.61–0.65 for T1–T4, 0.81 for
Chrysolampinae and Philomidinae, were always monophyletic and BEAST) (Table S6). As this may be outgroup dependent, and with
strongly supported. Perilampidae was monophyletic and sister to some of our outgroups parasitizing a very different hymenopteran
Eucharitidae. Eucharitidae always had the same subfamily and tribal (Cynipidae), we looked at the ASR with the host of the cynipid
relationships with Akapalinae sister to (Gollumiellinae, (Oraseminae parasitoids treated as missing, and although lower, the ASR of Coleop-
+ Eucharitinae)), and with Psilocharitini, and Psilocharis Heraty, always tera (p. 0.55) was now favoured over Diptera (p. 0.240) and Hymenop-
paraphyletic to Eucharitini. Jambiya was placed as either the sister tera (p. 0.19). When treated as families (character 2), Curculionidae
group of Perilampidae and Eucharitidae (Figure S12: all loci) or as sis- (p. 0.38–0.39 for T1–T4) were equivocal to Schizophora (p. 0.39–0.41
ter to Chrysolampidae (Fig. S13: 11 loci). The placement of Jambiya for T1–T4). For the BEAST hypothesis, Curculionidae (p. 0.51) were
outside of any of the other higher-level groups, and the relationships more strongly favoured over Diptera (p. 0.22). Schizophora were not
proposed within Perilampidae, with Euperilampus sister to the scored for any of the ingroup taxa, but instead were included as mixed
remaining genera and Perilampus paraphyletic, are the same as the orders for hyperparasitic Perilampus. We are unclear why Hymenop-
results from earlier studies (Heraty et al., 2013; Munro et al., 2011; tera were favoured using likelihood mapping for host order, whereas
Murray et al., 2013). For Oraseminae, even with a much reduced Curculionidae were very slightly favoured for host family. Using parsi-
taxon sampling and with a different AHE assembly, the relationships mony mapping (with polymorphic coding), the ancestral host was
among genera and species were the same as those proposed in Baker unresolved (equivocal) for the host order, but Curculionidae were the
et al. (2020). The relationships among groups and genera also were unambiguous ancestral state for host family. Overall, with beetles as
the same in our more extensive unpublished analyses of all the hosts of Eutrichosomatidae, Chrysolampinae and four of the six
Chalcidoidea using AHE and UCE, thus there was no impact of using genera of Perilampinae, it makes more intuitive sense for Coleoptera
more localized taxa in a more restricted framework; the results are to be the ancestral host.
the same.
Planidium
Planidial larva clade
Our ASR of a planidium as a two-state character (character 7) has the
Diagnosis presence of a planidium as the ancestral state for the PLC supported
across all analyses (p. 0.97–1.0) (Table S6). When treated as three
Morphological support for the PLC (as Clade 1, Fig. 6) proposed in states (character 8), there was slight support for a hard planidium as
Heraty et al. (2013) included antenna without basiconic sensilla ancestral (p. 0.39–0.62) over a soft planidium (p. 023–0.32). Even
(although found in some Perilampinae); male scape with ventral pores when the three hard planidia taxa, Philomidinae, Perilampidae and
(lost in several taxa and likely plesiomorphic); labrum with marginal Eucharitidae, were all treated as monophyletic (T3), the probability of
digits (absent in some Chrysolampus and a very different plate-like a hard planidium as ancestral (p. 0.39) was still slightly higher than a
soft planidium (p. 0.23). When Chrysolampidae were sister to the of the rather high mutation rate in the flanking regions and the satura-
remaining (T2), there was slightly higher support for a hard planidium tion of third codon positions in the coding regions.
as ancestral (p. 0.62). As expected, for character 7, our parsimony
reconstruction had unequivocal support for presence of a planidium
as ancestral, but was unresolved when separated into a hard and soft Diagnosis
planidum.
Adults of Eutrichosomatidae are recognized primarily by the axillae
strongly advanced and widely separated medially; antenna with
Eutrichosomatidae (stat. rev.) 12 flagellomeres; frenum with no dorsal projection; propodeal spiracle
reniform (Bouček, 1975).
Eutrichosomatidae had variable placement as either sister group to
the remaining planidial clade or sister to Perilampidae and Eucharitidae,
but as a family, they were monophyletic in all analyses (Fig. 5). Host associations
Eutrichosomatidae, currently with three genera, Eutrichosoma,
Peckianus and Collessina (Bouček, 1975), has been placed within Eutrichosoma mirabile have been reared as parasitoids of seed-feeding
Cleonyminae (Cleonymidae) (Ashmead, 1899), Tanaostigmini weevils (Baker & Heraty, 2020; Bouček, 1975).
(Encyrtidae) (Ashmead, 1904), Pteromalidae Bouček (1975), or treated
as its own family, Eutrichosomatidae (Peck, 1951). The labral morphol-
ogy of adult Eutrichosoma mirabile (Ashmead) is similar to that of Planidium
Eucharitidae and Perilampidae but this was initially considered as inde-
pendently derived (Darling, 1988). Its placement within the PLC was The morphology of the soft planidium of E. mirabile was described in
first proposed by Heraty et al. (2013) based on combined morphological Baker and Heraty (2020). This is the only planidium with seta on ter-
and Sanger-sequencing data, and later supported by first-instar larval gum X and spiracles on terga II, IV, V and VI.
morphology and behaviour (Baker & Heraty, 2020). Results from most
of the concatenated and ASTRAL analyses show Eutrichosomatidae as
sister to the rest of PLC (T1) as shown in Fig. 5: (Eutrichosomatidae, Chrysolampidae (stat. rev.)
(Philomidinae + Chrysolampinae), (remaining planidial clade)), although
the node with ‘Philomidinae + Chrysolampinae’ and the remaining PLC Despite extreme morphological differences in the adults (Fig. 2) and
(node 3, Fig. 5) is not strongly supported (ultrafast bootstrap ranging their larvae (hard versus soft planidium, Fig. 3), Chrysolampidae, includ-
from 65% to 93%). This placement of Eutrichosomatidae is consistent ing Chrysolampinae and Philomidinae, were always monophyletic and
with the phylogeny based on planidial morphology in which all except strongly supported (Fig. 5). Monophyly of these two subfamilies has
Eutrichosomatidae share the synapomorphy of absence of seta on ter- not been supported in any of the prior analyses, whether based on
gum X (Baker & Heraty, 2020). However, the concatenated analyses on molecular data (Munro et al., 2011; Murray et al., 2013), combined data
the full dataset (with third codon positions not RY coded) and the (Heraty et al., 2013) or morphology of the planidium (Baker &
flanking dataset, as well as the ASTRAL analysis on the 531 loci, Heraty, 2020; Darling, 1992). Except for Munro et al. (2011), the other
recover a different relationship (T2): (Philomidinae + Chrysolampinae, studies support monophyly of taxa with a hard planidium (Philomidinae,
(Eutrichosomatidae, remaining planidial clade)) in which the node with Perilampidae sens. nov. and Eucharitidae), although with different rela-
Eutrichosomatidae and the remaining PLC is again not strongly tionships among them. Three larval characters that were optimized as
supported (ultrafast bootstrap as 65% and 93% respectively; Figs S9 synapomorphies for the clade with Philomidinae, Perilampidae sens.
and S10). Exploring the partition support for either of these hypotheses nov. and Eucharitidae include the presence of paired spiracles on TII
(T1 vs. T2) shows that there are more regions in the 569-flanking- (although absent in Eucharitidae), reduced antenna and incomplete
dataset strongly supporting T2 (ΔGLS < 2; 16 regions) over T1 terga (Baker & Heraty, 2020). Our AHE data propose a different rela-
(ΔGLS > 2; 9 regions); more regions of the 763-coding-dataset (not RY tionship: ((Chrysolampinae + Philomidinae), (Perilampidae sens. nov.
coded) strongly support T1 (54 regions) over T2 (45 regions); and more + Eucharitidae)) (T1), which is strongly supported by the concatenated
regions of the 763-coding-dataset (RY coded) strongly support T1 analyses (Fig. 7; Figs S2–S8, S12–S13; Chrysolampidae not resolved in
(30 regions) over T2 (20 regions) (Fig. 7; Table S4). Studies have coaelescent analyses). The results of the partition support analyses
suggested that saturated third codon positions may result in misleading show that there are far more loci that support T1 over T3: 55 regions
phylogenetic relationships (Breinholt & Kawahara, 2013). With the third of the 569-flanking-dataset strongly supported T1 and 13 regions
codon positions being RY coded, the coding dataset shows dramatically strongly supported T3; 165 regions of the 763-coding-dataset (not RY
increased ultrafast bootstrap support for the clade with Philomidinae, coded) strongly supported T1 and 24 regions strongly supported T3;
Chrysolampinae, Perilampidae and Eucharitidae (93% vs. 65%), and thus 96 regions of the 763-coding-dataset (RY coded) strongly supported T1
increased support for the T1 hypothesis. Therefore, the placement of and 13 regions strongly supported T3 (Fig. 7; Table S4). Strong support
Eutrichosomatidae as shown in T2 in some analyses may be an artefact for a monophyletic Chrysolampidae (Chrysolampinae + Philomidinae),
344 ZHANG ET AL.
(third RY)
(third RY)
(third RY)
F I G . 7 Partition support analyses testing locus support for competing hypotheses. Four alternative topological hypotheses were tested in the
PL analyses: T1, maximum likelihood (ML) tree recovered by most analyses; T2, ML tree constrained to recover different placement of
Eutrichosomatinae as shown in some analyses; T3, ML tree constrained to recover different placement of Chrysolampinae and Philomidinae as in
Heraty et al. (2013); T4, ML tree constrained to recover different relationships within the core Eucharitidae as in Murray et al. (2013).
Distributions of phylogenetic signal for alternative hypotheses in the flanking-region or the coding-region (third codons RY or not RY coded)
datasets. Δ gene-wise log-likelihood scores (ΔGLS) values (y axis) were calculated by measuring the difference in gene-wise log-likelihood scores
for T1 versus T2 or T1 versus T3 or T1 versus T4. Blue bars denote genes supporting T1, whereas red bars denote genes supporting T2 or T3
or T4
including an additional philomidine genus, Philomides, was also recov- with Philomidinae as sister to Perilampidae + Eucharitidae (T3, at the
ered in our unpublished AHE and UCE analyses of Chalcidoidea. chrysolampine node) had little impact on the higher probability for a
hard planidum as the ancestral state (p. 0.57–0.70; lowest for T3) over
a soft planidium (p. 0.21–0.31) at node 3 (Fig. 6). Given the high
Diagnoses phylogenomic support for a monophyletic Chrysolampidae that
includes Philomidinae (T1, T2, T4, BEAST), this suggests that for the
The only morphological support for monophyly in adults is the pres- PLC at node 1 a hard planidium was ancestral, with a reversal to a soft
ence of bidentate symmetrical mandibles, with both teeth long and planidium in Chrysolampinae (p. 0.39–0.62), as compared to a single
medially directed (cf. Darling, 1986; Heraty et al., 2019). Males also evolution of a hard planidium (T3) (p. 0.39) (Table S4).
have pores on the ventral surface of the scape, but this is likely
plesiomorphic. There is no support for monophyly of the two subfam-
ilies based on features of the planidia (Baker & Heraty, 2020). Perilampidae + Eucharitidae
Chrysolampinae are composed of six genera, Australotoxeuma Girault,
Brachyelatus Hoffer & Novicky, Chrysolampus Spinola, Chrysomalla Förster, A sister group relationship between Perilampidae and Eucharitidae was
Elatomorpha Zerova and Parelatus Girault. Adults have a more typical highly supported in all of our results (node 5, Figs 5 and 6; Figs S2–S13).
pteromaloid body form and are recognized by a triangular prepectus that is The same results were recovered using combined data (Heraty
lateral and freely articulating with the pronotum, a weakly sclerotized flap- et al., 2013) and in studies of planidial morphology (Baker &
like labrum usually with marginal digits, and the male scape with pores Heraty, 2020; Darling, 1992; Heraty & Darling, 1984). Jambiya was not
located in distinct pits (Bouček, 1988; Darling, 1986). The planidium is included within this clade, but it was the sister group of Perilampidae +
weakly sclerotized and without terga, lacks spiracles and has distinct anten- Eucharitidae in the analysis that included all AHE loci and only the 11 loci
nae (Baker & Heraty, 2020; Darling & Miller, 1991). Host associations are for Jambiya, but without bootstrap support (Fig. S12). The probability of
all with Coleoptera in the families Curculionidae, Nitidulidae and possibly a hard planidium for this node (5) was 1.0 across all analyses. None of the
Cerambycidae (Askew, 1980; Bouček, 1988; Darling & Miller, 1991). groups basal to this node attack hosts that pupate in a cocoon (unknown
Philomidinae are composed of three genera, Aperilampus Walker, for Jambiya), but both Perilampidae and Eucharitidae have at least some
Philomides Haliday and Vidlinus Heraty et al. (Heraty et al., 2019). taxa attacking hosts within a cocoon (Character 6). For this node, across
Adults are distinctive, having a large shoulder-like prepectus that all hypotheses (T1–T4, BEAST), it was more likely that hosts being
overlaps the pronotum, a strongly sclerotized flap-like labrum, attacked would not have a cocoon (p. 0.98–1.0).
scattered minute ventral pores on the male scape and numerous other
distinctive features (Bouček, 1988; Heraty et al., 2019). The only host
record for Philomidinae is from the pupa of a ground-nesting halictid Perilampidae (stat. rev.)
bee (Darling, 1992). In a field photograph, a female appears to be
ovipositing into a flower bud of Solanum torum (Solanaceae) (Heraty Perilampidae are composed of six genera, Burskilampus Bouček, Eup-
et al., 2019), with additional images on inaturalist.org of Aperilampus erilampus Walker, Krombeinius Bouček, Monacon Waterson, Perilampus
and Philomides suggesting oviposition into flower buds (see especially Latreille and Steffanolampus Peck, with Perilampus being the most diverse
https://www.inaturalist.org/observations/11153148). The planidium genus with over 160 species worldwide (Noyes, 2019). Perilampidae was
has sclerotized terga, spiracles on the second body segment and has monophyletic in all of our results as well as those of earlier studies
the antennae highly reduced (Darling & Roberts, 1999). (Heraty et al., 2013; Munro et al., 2011; Murray et al., 2013).
Euperilampus were monophyletic and sister group to the rest of
Perilampidae, with Krombeinius sister to a paraphyletic Perilampus that
Host associations included Burksilampus, Monacon and Steffanolampus. The same relation-
ships were recovered in earlier studies (Heraty et al., 2013; Munro
ASR predictions for the ancestral host for Chrysolampidae as sister to et al., 2011). In the restricted sense, Perilampidae are easily
Perilampidae + Eucharitidae (node 3, Fig. 6; T1, T4), for the planidial circumscribed both for adults (Bouček, 1978, 1988; Darling, 1996) and
clade with Chrysolampidae sister to all members (T2), or for Philomidinae first-instar larvae (Heraty & Darling, 1984; Smith, 1912).
as sister to Perilampidae and Eucharitidae were similar to the planidial
clade results, with either Curculionidae (p. 0.38–0.51) or Schizophora
(p. 0.24–0.41) as the ancestral host (Table S6). There was higher support Diagnosis
for Curculionidae (p. 0.51) over Schizophora (p. 0.24) on the BEAST tree.
Adults with large mandibles, right with three teeth, left with two and
overlapping when closed; clypeus usually differentiated by sutures, apex
Planidium usually entire and weakly emarginate or rounded; labrum usually flat-
tened and flap-like with marginal digits and tapered or spatulate setae
Having Chrysolampidae monophyletic and sister to the rest of the that can be extended beyond the clypeus, usually with aboral digits or
taxa (node 3, Fig. 6; T1, T2, T4, BEAST) or forced to be paraphyletic setae; mesosoma compact, short and high in lateral view, mesoscutellum
346 ZHANG ET AL.
usually vaulted over metascutellum and propodeum; pronotum always may represent modified antennae, 12 strongly sclerotized tergites,
visible in dorsal view; prepectus closely associated and on the same one pair of spiracles situated between TI and TII, setae present on ter-
plane as lateral panel of pronotum and usually fused to pronotum; gites I-IX, long cerci on TXII surrounded by cuticle, ventral membra-
metasoma sessile or petiolate, usually with a raised scale; petiole with nous region usually with ornamentations including sharp spicules,
posterior ventral margin overlapping first gastral sternum by a thin trans- tubercles, or plates and a terminal membranous region that in at least
verse lamina; second gastral tergum with a broad antecostal sulcus; gas- some species functions as a caudal sucker facilitating movement
ter usually triquetrous, short and triangular in profile, comprised mainly (Heraty & Darling, 1984). All of these are regarded as plesiomorphic
the second and third gastral terga, with lateral areas differentiated from features relative to Eucharitidae (Baker & Heraty, 2020). Modifica-
dorsal area; ovipositor aciculate, thin and needle-like. Where known, tions within the family include the fusion of the first and second terga
Perilampidae have a hard planidium with one pair of spiracles and a in Steffanolampus, Monacon and Krombeinius (also fused within some
series of sharp spicules on the ventral membranous region. Eucharitidae), variation in the length of setae that may be related to
mode of parasitism, and absence of ventral ornamentation in Per-
ilampus chrysopae (Clancy, 1946; Darling, 1995, 1999; Darling &
Host associations Roberts, 1999). The planidium has not been described for Eup-
erilampus, which is unfortunate because of its proposed sister group
ASR predicted Hymenoptera (character 1; p. 0.58–0.90) and mixed position in the family.
orders (character 2, p. 0.43–0.56) as the ancestral hosts. Mixed orders
are based on the variety of hosts in Euperilampus and some Perilampus
which can be primary or indirect parasitoids of Diptera and Hymenop- Interpretation of results
tera. Under parsimony and polymorphic states, Hymenoptera are the
ancestral host order, but the host family choice is equivocal. This would The results of our study agree with prior analyses, with the consistent
suggest that associations with Coleoptera in Burskilampus, Monacon, result being that Euperilampus is sister group to the other genera of
Steffanolampus and some Perilampus would be considered as derived. Perilampidae. The other four genera are imbedded in a paraphyletic
Euperilampus has been reared from tachinid puparia and ichneumonoid Perilampus, and the only intuitive but weakly supported result is the
pupae (Bouček, 1978; Darling, 1983; D. Christopher Darling apparent monophyly of the New World hyalinus and platigaster spe-
unpublished museum records) and is regarded as an obligate secondary cies groups (Fig. 5, apical three species). These results must be reg-
hyperparasite through Lepidoptera. Krombeinius was reared from a arded as highly provisional because very few species of Perilampidae
eumenid wasp (Krombein, 1978) and Perilampus have been reared from have been included and the results generally are not compatible with
Sphecidae (Krombein, 1960; Krombein et al., 1999), and these could be morphology and host associations. Euperilampus was regarded as the
the result of accidental secondary parasitism through a secondary lepi- most derived genus of Perilampidae based on an analysis of 22 mor-
dopteran prey item. Species in the Perilampus fulvicornis species group phological characters (Darling, 1983), and also by a comparative analy-
(UCRCENT12639, 12640) and the carinate species of Perilampus sis of the labrum (Darling, 1995). Euperilampus is a hyperparasitoid,
(Darling, 1996) (16360, 12636, 12658) are both derived and monophy- whereas the intuitive ancestral host association in Perilampidae is pri-
letic in our results, and both are known to be obligate secondary mary parasitism of xylophagous beetles. Steffanolampus has this asso-
hyperparasitoids of Ichneumonoidea through Lepidoptera, with the ciation, and also the plesiomorphic configuration of the prepectus in
hyalinus group also known as hyperparasitoids of Tachinidae and as pri- the family (Darling, 1996), but in our analysis it is imbedded in a
mary parasitoids of sawflies (Tripp, 1962). Perilampus are also primary paraphyletic Perilampus, as are the other genera. The important result
parasitoids of Chrysopidae (Clancy, 1946) and Raphidioptera of this analysis is unequivocal support for a monophyletic Per-
(Aspöck, 2002). We scored Perilampus salieus in our analysis as a para- ilampidae and generic relationships are best considered as unresolved.
sitoid of a pyralid moth, Acropolitus, based on a likely incorrect record
made by Riek (1966), but this did not have an impact on our ASR
results. Within Perilampidae, our ASR results for hyperparasitism (char- Eucharitidae
acter 4) suggest that primary parasitism is ancestral (p. 0.97–0.98); scor-
ing Krombeinius as a hyperparasitoid rather than a primary parasitoid Eucharitidae are a diverse assemblage of 65 genera and 484 species
only slightly lowers the probability of primary parasitism as ancestral. classified into four subfamilies (Heraty, 2019). The AHE data reveal
The ASR for attacking a host in a cocoon (as a hyperparasitoid) was similar relationships (Figs 5 and 6; Figs S2–S13) for the four recog-
considered to be ancestral for Perilampidae (p. 0.91–0.94). nized subfamilies, Akapalinae, Gollumiellinae, Oraseminae and
Eucharitinae, to results from previous studies (Baker et al., 2020;
Heraty et al., 2013; Munro et al., 2011; Murray et al., 2013). Three
Planidium taxa of Gollumiellinae (two Anorasema and one Gollumiella) were
included in this study. They are recovered as one clade (100% ultra-
Perilampidae have a hard planidium that has a sclerotized cranium fast bootstrap) by all analyses except the concatenated flanking-
with hooks, spines or setae and sensory structures of which some region ML analysis, which places Gollumiella as the sister to the clade
with Oraseminae and Eucharitinae, but this relationship is not well 26 regions strongly supported T4 (Fig. 7; Table S6). Thus, based on
supported (ultrafast bootstrap = 86%). Even with a much reduced the AHE data, the hypothesis of Pseudometagea apical and the PEM
taxon sampling within Oraseminae and with a different AHE assembly, parasitoids paraphyletic is the most strongly supported scenario.
the relationships among genera and species were the same as those
proposed in Baker et al. (2020). Within Eucharitinae, neither the tribe
Psilocharitini nor the genus Psilocharis is recovered as monophyletic, Adult diagnosis
and the genus Neolosbanus is strongly supported as the sister to
Eucharitini (Fig. 5). Psilocharitini have been monophyletic in Adults with mandibles usually falcate (sickle-shaped) and overlapping
morphology-only analyses (Heraty, 1994b, 2002) or combined ana- when closed; labrum flattened and usually with 4–16 elongate mar-
lyses (Heraty et al., 2013), but paraphyletic with Neolosbanus sister to ginal digits and no aboral digits or vestigial sockets; pronotum usually
the Eucharitini in molecular-only analyses (Munro et al., 2011; Murray reduced and ventral to mesoscutum, except Akapalinae which have a
et al., 2013) (Fig. 4). Very few taxa in the Psilocharitini have ever been narrow transverse pronotum visible dorsally; prepectus either freely
included in a phylogenetic analysis, and increased taxon for the tribe articulated along anterior margin or fused with pronotum; metasoma
could address the problem of monophyly. petiolate; gaster often swollen and rounded in females; first gastral
Our results within the Eucharitini are consistent across all ana- sternum overlapping posterior ventral margin of petiole; ovipositor
lyses and propose that the PEM parasitoids (green clades, Fig. 6 inset) aciculate or broadly expanded and toothed.
are a grade from which the remaining members of the Eucharitini and
Pseudometagea (blue clade) are derived. These are similar to the
results of earlier analyses (Fig. 4) (Heraty et al., 2013; Munro Host associations
et al., 2011). The results of Murray et al. (2013) were the product of a
comprehensive analysis of 195 taxa with four gene regions (Fig. 4). Our AHE results suggest that the ancestral host of the core
They recovered Pseudometagea (blue) as sister to the remaining Eucharitidae (node 8; Gollumiellinae, Oraseminae, Eucharitinae) is
Eucharitini, and the clade of PEM parasitoids were monophyletic slightly favoured as being poneromorph parasitoids (T1–T3; p. 0.32).
suggesting a biogeographic shift from the Old World to the New The BEAST results with similar relationships suggest that having no
World (our T4 topology). Pseudometagea is the only member of the ant host is favoured (p. 0.42) over either formicine (p. 0.16) or a
Eucharitinae with a partially internal planidium, similar to poneromorph host (p. 0.25). For Eucharitini (node 13), with a hypothe-
Gollumiellinae and Oraseminae, which supports its sister relationship sis of Pseudometagea as sister to the rest of the tribe (T4), as proposed
within the remaining tribe (Heraty & Murray, 2013). However, ASR of by Murray et al. (2013), Formicinae is clearly the favoured ancestral
whether the planidium remains external or becomes partially internal host (p. 0.91). The alternative hypotheses (T1–T3, BEAST) strongly
(character 5) supports a shift from external to internal under all favour poneromorphs as a grade to the remaining (p. 1.0).
hypotheses (T1–T4, BEAST), so there is no argument for why Pseudo- For core Eucharitidae, external parasitism is favoured as the
metagea as sister to the remaining Eucharitini based on this trait. We ancestral state for Eucharitidae (p. 0.75–0.77) across all analyses,
have two competing topologies that revolve around the placement of despite internal parasitism by the planidium in both Gollumiellinae and
Pseudometagea as apical (our T1–T3, BEAST hypotheses) or Pseudo- Oraseminae. There was a single irreversible shift to attacking a host
metagea as sister (T4) following Murray et al. (2013). The sister group with a cocoon (character 6) in Eucharitinae (Neolosbanus + Eucharitini;
relationship was also proposed in a molecular study with two ribo- p. 0.83–0.93) that was unaffected by the different hypotheses. This
somal genes (18S and 28S) by Heraty et al. (2004). Having the may be a trait of the entire subfamily, but the host of Psilocharis
poneromorph parasitoids both sister and paraphyletic to the remains unknown.
remaining Eucharitini, including Pseudometagea does not make sense
in terms of host relationships, morphology or biogeographic patterns
of dispersal and establishment as proposed by Murray et al. (2013). Planidium
Additional support for monophyly of the poneromorph parasitoids
also comes from synapomorphic features of their planidia (Heraty The eucharitid planidium has the antennae lacking, at most 12 distinct
et al., 2015) and their pupae (Heraty et al., 2020). darkened and strongly sclerotized tergites (reduced number in only a
We tried to examine the conflicting hypotheses for Eucharitini by few taxa), setae found only on tergites I–VII and TXII (cerci), ventral
looking at gene conflict in our AHE data. Partition support analyses membranous area smooth without any spines or ornamentation and
indicate that an overwhelming number of loci in the AHE dataset sup- the spiracles lacking (Heraty et al., 2004; Heraty & Darling, 1984).
port the T1 hypothesis (Pseudometagea apical) over the T4 hypothesis Modifications within the family include fusion of the first and second
(Pseudometagea sister) (Fig. 7): 382 regions of the 569-flanking- terga in Gollumiellinae and Eucharitinae and the presence of a lateral
dataset strongly supported T1 and 29 regions strongly supported T4; tergopleural line in Gollumiellinae and most Eucharitinae, with a partial
470 regions of the 763-coding-dataset (not RY coded) strongly tergopleural line in Orasema tolteca Mann (Heraty, 2000; Heraty
supported T1 and 24 regions strongly supported T4; 275 regions of et al., 2004; Heraty & Barber, 1990). The presence of a tergopleural
the 763-coding-dataset (RY coded) strongly supported T1 and line, which is an indication of mobility and the ability to jump
348 ZHANG ET AL.
(character 9) was considered to be absent in the ancestor of core to the 86 (63–110) Ma proposed by Murray et al. (2013). Our esti-
Eucharitidae (p. 0.84), whereas it is favoured as being present in mates for this divergence are comparable based on the flanking
ancestral Eucharitinae (p. 1.0), which was a result not affected by dif- regions (90 Ma) and our MCMCtree results (89 Ma) (Table S5). Based
ferent hypotheses. on transcriptome data but only three taxa (Perilampus, Orasema,
Eucharis), Peters et al. (2018) had a much younger estimate of the diver-
gence of Eucharitidae + Perilampidae at 49 Ma. Notably, we have
Interpretation of results representatives of Chrysolampidae (Brachyelatus), Perilampidae (Per-
ilampus) and Eucharitidae (Palaeocharis Heraty & Darling, Eucharitinae)
The conflict in relationships between our study and prior analyses in Baltic amber that have been dated at 44 Ma (R. A. Burks
affects our interpretation of the ancestral ant host for both core et al., 2019; Heraty & Darling, 2009). Fossils of higher Chalcidoidea
Eucharitidae and also for Eucharitini, but has no effect on the other (other than Mymaridae and Rotoitidae) prior to the Late Cretaceous
traits of interest. It also has drastic effects on our interpretation of boundary are rare and usually lack the derived features of modern fami-
biogeographic patterns. The evolution of pupal morphology within lies (Hass et al., 2018), and thus our estimates placing the divergence of
Eucharitini also supports monophyly of the poneromorph parasitoids the PLC and the Eucharitidae + Perilampidae prior to this time period
(Heraty et al., 2020). For the observed conflict in Eucharitini, we could are likely overestimates. Estimates of a divergence during the Pal-
propose that a less dense taxon sampling of the Eucharitinae clade in aeogene or early Eocene of these groups and the establishment of both
the AHE datasets may account for the discordance between T1 and a hard and soft planidium during this period are more likely.
T4. However, we observed identical relationships in Oraseminae Our estimated dates for crown Perilampidae range from 68 (51–
whether taxon sampling was rich (Baker et al., 2020) or limited (this 87) Ma (BEAST) to 66 Ma (MCMCtree) (Table S5). We were able to
study). If the T1 hypothesis indeed represents the true evolutionary sample Euperilampus from both North America and Australia, with the
history of Eucharitinae, this indicates a rather complicated shift of ant known distribution of the genus also in South America, Madagascar
hosts and biogeographic patterns within this group. and Southeast Asia (Bouček, 1978; Darling, 1983). While the esti-
mated divergence between the Euperilampus species is only 26–
30 Ma (Fig. 6), their divergence with other Perilampidae would corre-
Jambiya incertae sedis late with a Gondwanan pattern of divergence, and also suggests the
early evolution of obligate indirect hyperparasitism.
Jambiya includes a single species, Jambiya vanharteni. The adults have Core Eucharitidae (unknown biology for Akapalinae) are parasitoids
features which are characteristic of both Perilampidae and of ants in the subfamilies Ectatomminae, Formicinae, Myrmeciinae,
Chrysolampinae that ‘confuse the putative synapomorphies of Myrmicinae and Ponerinae (Heraty, 2002). The most recent estimate for
Chrysolampinae and Perilampinae’ (Heraty & Darling, 2007). In contrast the median crown age of the ants is 112 (103–123) Ma, with median
to Chrysolampinae, they have an asymmetrical 3/2 mandibular formula ages for the five subfamilies ranging between 45 and 61 Ma (Borowiec
and do not have pores located in distinct pits on the male scape. As well, et al., 2019). Earlier studies of ant relationships generally suggested older
they have a distinctive scimitar-shaped ovipositor that suggests oviposi- divergence times ranging from 111–169 Ma (Brady et al., 2006; Moreau
tion into plant tissue versus an acicular ovipositor found in et al., 2006; Moreau & Bell, 2013; Schmidt, 2013). For core Eucharitidae
Chrysolampinae and Perilampidae (Heraty & Darling, 2007). However, (Gollumiellinae, Oraseminae, Eucharitinae), our study suggests a median
basal Eucharitidae (Akapalinae, Gollumiellinae, Oraseminae) all have age of 65 (48–82) Ma, about 47 Ma after their ant hosts, which is consis-
expanded ovipositor suited for plant oviposition (Heraty et al., 2004). tent with the finding by Murray et al. (2013). However, if we consider
Jambiya was placed as either the sister group of Perilampidae and that the ancestral host of core Eucharitidae is Formicinae, as proposed
Eucharitidae (Fig. S12, all loci) or as sister to Chrysolampidae (Fig. S13, by Murray et al. (2013), then the difference is much less, with Borowiec
11 loci) but always with poor support. As in prior analyses, it does not et al. (2019) proposing a median age of 60 (51–71) Ma for Formicinae. If,
group within any of the major clades. Until we can get better data on the as proposed by this study, parasitism of poneromorph parasitoids is
group, we consider Jambiya as incertae sedis within the planidial clade. ancestral, albeit only with a slightly higher probability over Formicinae,
then the gap is much greater. Ponerinae, the host of Neolosbanus
(Eucharitinae, Psilocharitini) are estimated at 73 (61–84) Ma (Borowiec
Divergence times and evolution of the PLC et al., 2019). Although the MCMCtree analyses based on all AHE data
estimated older ages for Oraseminae (55 Ma) and Eucharitini (52 Ma)
Our BEAST dating analyses estimated the initial divergence of the than the BEAST analyses and some recent studies (Baker et al., 2020;
PLC likely occurred in the Lower Cretaceous (median node age Murray et al., 2013), they still postdated the diversification of their
111 Ma, 95% highest posterior density interval 83–141 Ma; Fig. 6, corresponding ant host lineages. Our study supports the conclusion by
Table S5). This is concordant with the proposal of just over 100 Ma Murray et al. (2013) that the diversification of Eucharitidae arose when
by Murray et al. (2013), which used the same Baltic amber calibration the major ant lineages were already established and diversifying.
fossils as this study. Similarly, our estimate for the divergence of In terms of biogeography, two significant events have been pro-
Eucharitidae + Perilampidae was 96 (72–122) Ma, which was similar posed within Eucharitidae. Oraseminae were proposed to have dispersed
to the New World in a single event dated at either 20 (13–27) Ma based those of previous studies. The use of phylogenomic data in the plan-
on ribosomal and mitochondrial data (Murray et al., 2013) or 24 (17–33) idial clade has helped to confirm and support some of our questions
Ma based on AHE data (Baker et al., 2020), with only the genus Orasema but has also raised some important contradictory results that can
occurring in the New World. Our BEAST results also suggest a single dis- hopefully be addressed in future studies.
persal event and comparable median age of Orasema at 20 (13–27)
Ma. Our MCMCtree results suggest a much older age of 48 (36–62) Ma, AC KNOW LEDG EME NT S
which is little different from the crown age of Oraseminae (Table S5). This work was funded by the Advanced Talents Incubation Program of
However, all of the younger MCMCtree divergences within Oraseminae the Hebei University to Junxia Zhang (Grant No. 521000981324); NSF
and Eucharitinae are much older than as proposed by our BEAST results DEB-1555808 to John Heraty, James B. Woolley and Matt J. Yoder,
(Table S5), and incongruent with the earlier studies. Therefore, we are and a UCR Hatch project fund to John Heraty. We would like to
more comfortable with the BEAST results in these derived clades. Within express our gratitude to Scott Heacox and Chrysalyn Dominguez for
Eucharitinae, members of the proposed PEM parasitoids were suggested help with sequencing efforts. The Supercomputer Centre at Hebei Uni-
to have originated in the Old World at 38 (28–49) Ma and underwent a versity provided computational resources for part of the analyses.
single dispersal event to the New World, forming the Kapala clade, at
23 (16–31) Ma (Murray et al., 2013). Our BEAST results, with the PEM CONFLIC T OF INT ER E ST
clade sister to the remainder (T1) and equivalent to the age of Eucharitini, The authors declare that there is no conflict of interest.
suggest a median age of origin at about 28 (21–35) Ma, with the
MCMCtree results again much older at 52 (42–63) Ma. The mean age AUTHOR CONTRIBU TIONS
estimate of the Kapala clade from the BEAST analyses is about 12.5 Ma, Junxia Zhang, John M. Heraty, D. Christopher Darling and Robert
and thus would also support a single recent New World dispersal event. L. Kresslein contributed to the design of the project and analyses.
A similar aged dispersal event for Orasema and the Kapala clade from a Austin J. Baker, Javier Torréns and Jean-Yves Rasplus contributed
paraphyletic Old World ancestral host suggests a Beringian Land Bridge critical specimens for the analysis. Alan Lemmon and Emily Moriarty
dispersal event (Baker et al., 2020; Murray et al., 2013). As proposed by Lemmon contributed to initial data acquisition and bioinformatics. All
this study, a sister group relationship between the Kapala clade and the authors contributed to manuscript preparation.
other Eucharitini (Pseudometagea, Eucharis clade, Stilbula clade) would
suggest that the entire sister group to the Kapala clade was widespread DATA AVAILABILITY STAT EMEN T
around the world, which overall we consider to be an untenable hypoth- The matrices, partition files, scripts, trees in ‘.phylip’ format and the
esis, although it is highly supported across the majority of loci (Fig. 7). Mesquite nexus files for trait evolution over five trees are openly
available in Dryad (doi: https://doi.org/10.5061/dryad.j9kd51cdq),
with raw read data available in the NCBI Sequence Read Archive
C O N CL U S I O N (SRA) as project PRJNA787274.
Our results strongly support the monophyly of the PLC, including ORCID
Jambiya, and we present a revised classification of the group. A Junxia Zhang https://orcid.org/0000-0003-2179-3954
planidium, which is a highly mobile external larval-pupal koinobiont, John M. Heraty https://orcid.org/0000-0002-9246-5651
evolved once, with either parallel evolution of a hard planidium Christopher Darling https://orcid.org/0000-0002-1207-7967
(with terga) or independent loss of the hard planidium in Robert L. Kresslein https://orcid.org/0000-0002-6606-7860
Chrysolampinae. Obligate indirect hyperparasitism appears to have Austin J. Baker https://orcid.org/0000-0002-4728-726X
evolved multiple times within Perilampidae. Ant parasitism evolved Javier Torréns https://orcid.org/0000-0002-3608-132X
once in Eucharitidae, as did the ability to attack ants within a cocoon Jean-Yves Rasplus https://orcid.org/0000-0001-8614-6665
in Eucharitinae. Our results suggest that the ability to attack the Emily Moriarty Lemmon https://orcid.org/0000-0001-5911-6102
immature stages of PEM ants was ancestral for Eucharitidae, con-
tradicting the proposal of Formicinae as the ancestral hosts by Murray RE FE RE NCE S
et al. (2013). Despite morphological evidence to support the mono- Abe, M. (1988) A biosystematics study of the genus Athalia Leach of Japan
phyly of the PEM parasitoids, our results strongly support these as a (Hymenoptera: Tenthredinidae). Esakia, 26, 91–131.
Ashmead, W.H. (1899) On the genera of the Cleonymidae. Proceedings of
grade to the remaining lineages in the Eucharitini, and not a derived
the Entomological Society of Washington, 4, 200–206.
lineage within the tribe as proposed by Murray et al. (2013). The con- Ashmead, W.H. (1904) Classification of the chalcid flies, or the superfamily
tradictory results for Eucharitini may be a product of incomplete taxon Chalcidoidea, with descriptions of new species in the Carnegie
sampling, although reduced taxon sampling for Oraseminae provided Museum, collected in South America by Herbert H. Smith. Memoirs
of the Carnegie Museum, 1, 225–551.
identical results to the more extensive sampling of Baker et al. (2020).
Askew, R.R. (1980) The biology and larval morphology of Chrysolampus
Despite the rearrangements of the relationships of some taxa, our thenae (Walker) (Hym., Pteromalidae). Entomologist’s Monthly Maga-
divergence dating of events across the entire clade are similar to zine, 115, 155–159.
350 ZHANG ET AL.
Aspöck, H. (2002) The biology of Rhaphidioptera: a review of present (Helianthus) in the Northern Great Plains. Annals of the Entomological
knowledge. Acta Zoologica Academiae Scientiarum Hungaricae, 48- Society of America, 87, 831–835.
(Supplement 2), 35–50. Clancy, D.W. (1946) The insect parasites of Chrysopidae (Neuroptera).
Baker, A.J. & Heraty, J.M. (2020) Larval morphology and life history of University of California Publications in Entomology, 7, 403–496.
Eutrichosoma mirabile Ashmead and description of a new species of Clausen, C.P. (1940a) The immature stages of the Eucharidae. Proceedings
Eutrichosoma (Hymenoptera, Chalcidoidea). Journal of Hymenoptera of the Entomological Society of Washington, 42, 161–170.
Research, 75, 67–85. Clausen, C.P. (1940b) Entomophagous insects. New York and London:
Baker, A.J., Heraty, J.M., Mottern, J., Zhang, J., Hines, H.M. et al. (2020) McGraw-Hill Book Company, Inc.
Inverse dispersal patterns in a group of ant parasitoids Clausen, C.P. (1940c) The oviposition habits of the Eucharidae
(Hymenoptera: Eucharitidae: Oraseminae) and their ant hosts. Sys- (Hymenoptera). Journal of the Washington Academy of Sciences, 30,
tematic Entomology, 45, 1–19. 504–516.
Bergold, G. & Ripper, W. (1937) Perilampus tristis Mayr as a hyperparasite Clausen, C.P. (1941) The habits of the Eucharidae. Psyche, 48, 57–69.
of the pine shoot tortricid R. buoliana Schiff. Zeitschrift für Para- Darling, D.C. (1983) A review of the New World species of Euperilampus
sitenkunde, Berlin, 9, 394–417. (Hymenoptera; Chalcidoidea), with notes about host associations
Bolton, B. (2003) Synopsis and classification of Formicidae. Memoirs of the and phylogenetic relationships. Quaestiones Entomologicae, 19, 1–40.
American Entomological Institute, 71, 1–370. Darling, D.C. (1986) Revision of the New World Chrysolampinae
Borowiec, M.L. (2016) AMAS: a fast tool for alignment manipulation and (Hymenoptera: Chalcidoidea). The Canadian Entomologist, 118,
computing of summary statistics. PeerJ, 4, e1660. 913–940.
Borowiec, M.L., Rabeling, C., Brady, S.G., Fisher, B.L., Schultz, T.R. & Darling, D.C. (1988) Comparative morphology of the labrum in Hymenop-
Ward, P.S. (2019) Compositional heterogeneity and outgroup choice tera: the digitate labrum of Perilampidae and Eucharitidae
influence the internal phylogeny of the ants. Molecular Phylogenetics (Chalcidoidea). Canadian Journal of Zoology, 66, 2811–2835.
and Evolution, 134, 111–121. Darling, D.C. (1992) The life history and larval morphology of Aperilampus
Bouček, Z. (1975) The pteromalid subfamily Eutrichosomatinae (Hymenoptera: Chalcidoidea: Philomidinae) with a discussion of the
(Hymenoptera: Chalcidoidea). Journal of Entomology (B), 43, 129–138. phylogenetic affinities of the Philomidinae. Systematic Entomology,
Bouček, Z. (1977) A faunistic review of the Yugoslavian Chalcidoidea 17, 331–339.
(Parasitic Hymenoptera). Acta Entomologica Jugoslavica, 13(Supple- Darling, D.C. (1995) New species of Krombeinius (Hymenoptera:
ment), 1–145. Chalcidoidea: Perliampidae) from Indonesia, and the first description
Bouček, Z. (1978) A generic key to Perilampinae (Hymenoptera, of first-instar larva for the genus. Zoologische Mededelingen, Leiden,
Chalcidoidea) with a revision of Krombeinius n.gen. and Euperilampus 69, 209–229.
Walker. Entomologica Scandinavica, 9, 299–307. Darling, D.C. (1996) Generic concepts in the Perilampidae (Hymenoptera:
Bouček, Z. (1988) Australasian Chalcidoidea (Hymenoptera). A biosystematic Chalcidoidea): an assessment of recently proposed genera. Journal of
revision of genera of fourteen families, with a reclassification of species. Hymenoptera Research, 5, 100–130.
Wallingford, United Kingdom: CAB International, p. 832. Darling, D.C. (1999) Life history and immature stages of Steffanolampus
Bouckaert, R., Vaughan, T.G., Barido-Sottani, J., Duchêne, S., salicetum (Hymenoptera: Chalcidoidea: Perilampidae). Proceedings of
Fourment, M., Gavryushkina, A. et al. (2019) BEAST 2.5: an advanced the Entomological Society of Ontario, 130, 3–14.
software platform for Bayesian evolutionary analysis. PLoS Computa- Darling, D.C. & Gibson, C. (2000) Life and death on the Leslie Street spit.
tional Biology, 15, e1006650. Rotunda, 25, 1–6.
Brady, S.G., Schultz, T.R., Fisher, B.L. & Ward, P.S. (2006) Evaluating alter- Darling, D.C. & Miller, T.D. (1991) Life history and larval morphology of
native hypotheses for the early evolution and diversification of ants. Chrysolampus (Hymenoptera: Chalcidoidea: Chrysolampinae)
Proceedings of the National Academy of Sciences of the United States in western North America. Canadian Journal of Zoology, 69,
of America, 103, 18172–18177. 2168–2177.
Breinholt, J.W., & Kawahara, A.Y. (2013) Phylotranscriptomics: Saturated Darling, D.C. & Roberts, H. (1999) Life history and larval morphology of
third codon positions radically influence the estimation of trees Monacon (Hymenoptera: Perilampidae), parasitoids of ambrosia bee-
based on Next-Gen data. Genome Biology and Evolution, 5(11), 2082– tles (Coleoptera: Platypodidae). Canadian Journal of Zoology, 77,
2092. https://doi.org/10.1093/gbe/evt157 1768–1782.
Brues, C.T. (1910) The parasitic Hymenoptera of Florissant, Colorado. Bulle- Das, G.M. (1963) Preliminary studies on the biology of Orasema assectator
tin of the Museum of Comparative Zoology, Harvard College, 54, 1–125. Kerrich (Hym., Eucharitidae), parasitic on Pheidole and causing dam-
Burks, B.D. (1969) New Perilampidae (Hymenoptera: Chalcidoidea). Pro- age to leaves of tea in Assam. Bulletin of Entomological Research, 54,
ceedings of the Entomological Society of Washington, 71, 73–81. 373–378.
Burks, R.A., Heraty, J.M., Dominguez, C., & Mottern, J.L. (2018) Complex Dell’Ampio, E., Meusemann, K., Szucsich, N.U., Peters, R.S., Meyer, B. et al.
diversity in a mainly tropical group of ant parasitoids: Revision of the (2014) Decisive data sets in phylogenomics: lessons from studies on
Orasema stramineipes species group (Hymenoptera: Chalcidoidea: the phylogenetic relationships of primarily wingless insects. Molecular
Eucharitidae). Zootaxa, 4401(1), 1. https://doi.org/10.11646/ Biology and Evolution, 31, 239–249.
zootaxa.4401.1.1 Drummond, A.J. & Rambaut, A. (2007) BEAST: Bayesian evolutionary anal-
Burks, R.A., Heraty, J.M., Mottern, J., Dominguez, C., & Heacox, S. (2017) ysis by sampling trees. BMC Evolutionary Biology, 7, 214.
Biting the bullet: revisionary notes on the Oraseminae of the Old Erdös, J. (1955) Magyarország Allatvilága. XII. Kötet. Hymenoptera
World (Hymenoptera, Chalcidoidea, Eucharitidae). Journal of Hyme- II. 2. Füzet. Fémfürkészek I. Chalcidoidea I. Fauna Hungariae, 2,
noptera Research, 55, 139–188. https://doi.org/10.3897/jhr.55. 1–48.
11482 Flanders, S.E. (1937) Ovipositional instincts and developmental sex differ-
Burks, R.A., Krogmann, L. & Heraty, J.M. (2019) Chrysolampine wasps ences in the genus Coccophagus. University of California Publications
(Chalcidoidea: Perilampidae sensu lato) from Baltic amber. Insect Sys- in Entomology, 6, 403–422.
tematics and Diversity, 3, 1–5. Gerling, D. & Legner, E.F. (1968) Developmental history and reproduction
Charlet, L.D. & Seiler, G.J. (1994) Sunflower seed weevils (Coleoptera: of Spalangia cameroni, parasite of synanthropic flies. Annals of the
Curculionidae) and their parasitoids from native sunflowers Entomological Society of America, 61, 1436–1443.
Graham, M.W.R.d.V. (1969) The Pteromalidae of north-western Europe Junier, T. & Zdobnov, E.M. (2010) The Newick utilities: high-throughput
(Hymenoptera: Chalcidoidea). Bulletin of the British Museum (Natural phylogenetic tree processing in the Unix shell. Bioinformatics, 26,
History) (Entomology), Supplement 16, 1–908. 1669–1670.
Hamilton, C.A., Lemmon, A.R., Lemmon, E.M. & Bond, J.E. (2016) Expanding Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haeseler, A. &
anchored hybrid enrichment to resolve both deep and shallow rela- Jermiin, L.S. (2017) ModelFinder: fast model selection for accurate
tionships within the spider tree of life. BMC Evolutionary Biology, 16 phylogenetic estimates. Nature Methods, 14, 587–589.
(212), 1–20. Katoh, K. & Standley, D.M. (2013) MAFFT multiple sequence alignment
Hass, M., Burks, R.A. & Krogmann, L. (2018) A new lineage of Cretaceous software version 7: improvements in performance and usability.
jewel wasps (Chalcidoidea: Diversinitidae). PeerJ, 6, e4633. Molecular Biology and Evolution, 30, 772–780.
Heraty, J.M. (1994a) Biology and importance of two eucharitid parasites Krogmann, L. & Abraham, R. (2003) The first instar larva of Nasonia
of Wasmannia and Solenopsis. In: Williams, D.F. (Ed.) Exotic ants: biol- vitripennis (Walker, 1836) (Hymenoptera: Chalcidoidea:
ogy, impact and control of introduced species. Boulder, CO: Westview Pteromalidae) with a pair of appendages on the labrum and closed
Press, pp. 104–120. spiracles. Entomologische Mitteilungen aus dem Zoologischen Museum
Heraty, J.M. (1994b) Classification and evolution of the Oraseminae in the Hamburg, 14, 97–103.
Old World, with revisions of two closely related genera of Krogmann, L. & Vilhelmsen, L. (2006) Phylogenetic implications of the
Eucharitinae (Hymenoptera: Eucharitidae). Life Sciences Contributions, mesosomal skeleton in Chalcidoidea (Hymenoptera, Apocrita) – tree
Royal Ontario Museum, 157, 1–174. searches in a jungle of homoplasy. Insect Systematics, 20, 615–674.
Heraty, J.M. (2000) Phylogenetic relationships of Oraseminae Krombein, K.V. (1960) Biological notes on some Hymenoptera that nest in
(Hymenoptera: Eucharitidae). Annals of the Entomological Society of Sumach pith. Entomological News, 71(29–36), 63–69.
America, 93, 374–390. Krombein, K.V. (1964) Natural history of Plummers Island, Maryland XVIII.
Heraty, J.M. (2002) A revision of the genera of Eucharitidae The hibiscus wasp, an abundant rarity and its associates
(Hymenoptera: Chalcidoidea) of the world. Memoirs of the American (Hymenoptera: Sphecidae). Proceedings of the Biological Society of
Entomological Institute, 68, 1–367. Washington, 77, 73–112.
Heraty, J.M. (2019) World catalog of Eucharitidae. Available at: http:// Krombein, K.V. (1978) Biosystematic studies of Ceylonese wasps. III. Life
www.hymenoptera.ucr.edu/eucharitidae [accessed February 2021] history, nest and associates of Paraleptomenes mephitis (Cameron)
Heraty, J.M. & Barber, K.N. (1990) Biology of Obeza floridana (Ashmead) (Hymenoptera: Eumenidae). Journal of the Kansas Entomological Soci-
and Pseudochalcura gibbosa (Provancher) (Hymenoptera: Eucharitidae). ety, 51, 721–734.
Proceedings of the Entomological Society of Washington, 92, 248–258. Krombein, K.V., Norden, B.B., Rickson, M.M. & Rickson, F.R. (1999) Biodi-
Heraty, J.M. & Darling, D.C. (1984) Comparative morphology of the plan- versity of the domatia occupants (ants, wasps, bees, and others) of
idial larvae of Eucharitidae and Perilampidae (Hymenoptera: the Sri Lankan myrmecophyte Humboldtia laurifolia Vahl (Fabaceae).
Chalcidoidea). Systematic Entomology, 9, 309–328. Smithsonian Contributions to Zoology, 603, 1–34.
Heraty, J.M. & Darling, D.C. (2007) A new genus and species of Per- Kück, P. & Meusemann, K. (2010) FASconCAT: convenient handling of
ilampidae (Hymenoptera: Chalcidoidea) with uncertain placement in data matrices. Molecular Phylogenetics and Evolution, 56, 1115–1118.
the family. Journal of the Entomological Society of Ontario, 138, La Salle, J., Polaszek, A., Noyes, J.S. & Zolnerowich, G. (1997) A new whitefly
33–47. parasitoid (Hymenoptera: Pteromalidae: Eunotinae) with comments on
Heraty, J.M. & Darling, D.C. (2009) Fossil Eucharitidae and Perilampidae its placement, an implications for classification of Chalcidoidea with par-
(Hymenoptera: Chalcidoidea) from Baltic amber. Zootaxa, 2306, ticular reference to the Eriaporinae (Hymenoptera: Aphelinidae). System-
1–16. atic Entomology, 22, 131–150.
Heraty, J.M. & Murray, E. (2013) The life history of Pseudometagea Laing, J.E. & Heraty, J.M. (1982) The parasite complex of the over-
schwarzii with a discussion of the evolution of endoparasitism and wintering population of Epiblema scudderiana (Lepidoptera:
koinobiosis in Eucharitidae and Perilampidae (Chalcidoidea). Journal Olethreutidae) in southern Ontario. Proceedings of the Entomological
of Hymenoptera Research, 35, 1–15. Society of Ontario, 112, 59–66.
Heraty, J.M., Wojcik, D.P. & Jouvenaz, D.P. (1993) Species of Orasema par- Lartillot, N., Rodrigue, N., Stubbs, D. & Richer, J. (2013) PhyloBayes MPI:
asitic on the Solenopsis saevissima-complex in South America phylogenetic reconstruction with infinite mixtures of profiles in a
(Hymenoptera: Eucharitidae, Formicidae). Journal of Hymenoptera parallel environment. Systematic Biology, 62, 611–615.
Research, 2, 169–182. Maddison, W.P. & Maddison, D.R. (2019) Mesquite: a modular system for
Heraty, J.M., Hawks, D., Kostecki, J.S. & Carmichael, A. (2004) Phylogeny evolutionary analysis. Version 3.6.1. Available at: http://
and behaviour of the Gollumiellinae, a new subfamily of the ant- mesquiteproject.org [Accessed February 2021].
parasitic Eucharitidae (Hymenoptera: Chalcidoidea). Systematic Ento- Mirarab, S., Reaz, R., Bayzid, M.S., Zimmermann, T., Swenson, M.S. &
mology, 29, 544–559. Warnow, T. (2014) ASTRAL: genome-scale coalescent-based species
Heraty, J.M., Burks, R.A., Cruaud, A., Gibson, G.A., Liljeblad, J. et al. (2013) tree estimation. Bioinformatics, 30, 541–548.
A phylogenetic analysis of the megadiverse Chalcidoidea Mirarab, S., Nguyen, N. & Tandy, W. (2015) PASTA: ultra-large multiple
(Hymenoptera). Cladistics, 29, 466–542. sequence alignment. In: Sharan, R. (Ed.) Research in computational
Heraty, J.M., Mottern, J. & Peeters, C. (2015) A new species of molecular biology. RECOMB 2014. Lecture notes in computer science,
Schizaspidia, with discussion of the phylogenetic utility of immature Vol. 8394. Cham: Springer.
stages for assessing relationships among eucharitid parasitoids of Misof, B., Liu, S., Meusemann, K., Peters, R.S., Donath, A., Mayer, C. et al.
ants. Annals of the Entomological Society of America, 108, 865–874. (2014) Phylogenomics resolves the timing and pattern of insect evo-
Heraty, J.M., Derafshan, H.A. & Ghafouri Moghaddam, M. (2019) Review lution. Science, 346, 763–767.
of the Philomidinae Ruschka (Hymenoptera: Chalcidoidea: Per- Moreau, C.S. & Bell, C.D. (2013) Testing themuseumversus cradle tropical
ilampidae), with description of three new species. Arthropod System- biological diversity hypothesis: phylogeny, diversification, and ances-
atics & Phylogeny, 77, 39–56. tral biogeographic range evolution of the ants. Evolution, 67, 2240–
Heraty, J.M., Mbanyana, N. & Van Noort, S. (2020) A new species of 2257.
Eucharissa Westwood (Eucharitidae) from South Africa, with an eval- Moreau, C.S., Bell, C.D., Vila, R., Archibald, S.B. & Pierce, N.E. (2006) Phy-
uation of the importance of pupae for assessing relationships in logeny of the ants: diversification in the age of angiosperms. Science,
these ant parasitoids. Journal of Hymenoptera Research, 79, 43–55. 312, 101–104.
352 ZHANG ET AL.
Munro, J.B., Heraty, J.M., Burks, R.A., Hawks, D., Mottern, J., Cruaud, A. Torréns, J. (2013) A review of the biology of Eucharitidae (Hymenoptera:
et al. (2011) A molecular phylogeny of the Chalcidoidea Chalcidoidea) from Argentina. Psyche: A Journal of Entomology,
(Hymenoptera). PLoS One, 6, e27023. 926572, 1–14.
Murray, E.A., Carmichael, A.E. & Heraty, J.M. (2013) Ancient host shifts Torréns, J., Heraty, J.M. & Fidalgo, P. (2008) Biology and description of a
followed by host conservatism in a group of ant parasitoids. Proceed- new species of Lophyrocera Cameron (Hymenoptera: Eucharitidae)
ings of the Royal Society B: Biological Sciences, 280, 20130495. from Argentina. Zootaxa, 1871, 56–62.
Nguyen, L.T., Schmidt, H.A., Von Haeseler, A. & Minh, B.Q. (2015) IQ- Torréns, J., Heraty, J.M., Murray, E. & Fidalgo, P. (2016) Biology and phylo-
TREE: a fast and effective stochastic algorithm for estimating genetic placement of a new species of Lasiokapala Ashmead from
maximum-likelihood phylogenies. Molecular Biology and Evolution, 32, Argentina (Hymenoptera: Eucharitidae). Systematic Entomology, 41,
268–274. 596–606.
Nikol’skaya, M. (1952) Khaltsidy Fauny SSSR English translation: the Chalcid Tripp, H.A. (1961) The biology of a hyperparasite, Euceros frigidus Cress.
Fauna of the USSR, Israel Prog. Sci. Transl., Jerusalem, 1963, Vol. 44. (Ichneumonidae) and description of the planidial stage. The Canadian
Moscow and Lenningrad: Zoologicheskim Institutom Akademii Nauk Entomologist, 93, 40–58.
SSSR, p. 575. Tripp, H.A. (1962) The biology of Perilampus hyalinus Say
Noyes, J.S. (2019) Universal Chalcidoidea database. World Wide Web Elec- (Hymenoptera: Perilampidae), a primary parasite of Neodiprion
tronic Publication. Available at: http://www.nhm.ac.uk/chalcidoids swainei Midd. (Hymenoptera: Diprionidae) in Quebec, with
[accessed June 2021]. descriptions of the egg and larval stages. The Canadian Entomolo-
Peck, O. (1951) Superfamily Chalcidoidea. In: Muesebeck, C.F.W., gist, 94, 1250–1270.
Krombein, K.V. & Townes, H.K. (Eds.) Hymenoptera of America north Yang, Z. (2007) PAML 4: a program package for phylogenetic analysis
of Mexico - synoptic catalog, agriculture monograph no. 2. Washington, by maximum likelihood. Molecular Biology and Evolution, 24,
DC: USDA. 1586–1591.
Perlman, S.J. (1995) The Evolution of Indirect Hyperparasitism, with special Zhang, C., Sayyari, E. & Mirarab, S. (2017) ASTRAL-III: increased scalability
reference to the family Perilampidae (Hymenoptera: Chalcidoidea). Mas- and impacts of contracting low support branches. In: Meidanis, J. and
ter’s thesis. University of Toronto. Nakhleh, L. (Eds.) Comparative Genomics: 15th International Work-
Peters, R.S., Niehuis, O., Gunkel, S., Bläser, M., Mayer, C., Podsiadlowski, L. shop, RECOMB CG. Springer, pp. 53–75.
et al. (2018) Transcriptome sequence-based phylogeny of chalcidoid Zhang, J., Lindsey, A.R.I., Peters, R.S., Heraty, J.M., Hopper, K.R.,
wasps (Hymenoptera: Chalcidoidea) reveals a history of rapid radia- Werren, J.H. et al. (2020) Conflicting signal in transcriptomic markers
tions, convergence, and evolutionary success. Molecular Phylogenetics leads to a poorly resolved backbone phylogeny of chalcidoid wasps.
and Evolution, 120, 286–196. Systematic Entomology, 45, 783–802.
Principi, M.M. (1947) Contributo allo studio dei Neurotteri italiani.
V. Bollettino dell’Istituto di Entomologia della R. Università degli studi di
SUPPORTING INF ORMATION
Bologna, 16, 134–175.
Purrington, F.F. (1970) Ecology of Metzneria lappella (Lep., Gelechiidae) Additional supporting information may be found in the online version
and its hymenopterous parasites in eastern North Dakota. Annals of of the article at the publisher’s website.
the Entomological Society of America, 63, 942–945.
Rambaut, A. (2009) FigTree, version 1.4.3. Available at: http://tree.bio.ed. Figure S1. Flowchart of the AHE data-processing pipeline.
ac.uk/software/figtree [accessed January 2020].
Rambaut, A., Drummond, A.J., Xie, D., Baele, G. & Suchard, M.A. (2018) Figure S2. The best tree from ML analysis on the full-1332-region
Posterior summarisation in Bayesian phylogenetics using Tracer 1.7.
dataset (third codons RY coded). The numbers along the branches are
Systematic Biology, 67, 901–904.
Riek, E.F. (1966) Australian Hymenoptera Chalcidoidea, family Pteromalidae, ML bootstrap supports. The scale bar is in substitutions per position.
subfamily Perilampinae. Australian Journal of Zoology, 14, 1207–1236.
Ritkowski, S. (1997) K-Ar Altersbestimmungen der bernsteinführenden Figure S3. The best tree from ML analysis on the full-1332-region
Sedimente des Samlandes (Paläogen, Bezirk Kaliningrad), pp. 19–24.
dataset (third codons not RY coded). The numbers along the branches
In Metalla, Sonderheft 66 zum Symposium Neue Erkenntnisse zum
are ML bootstrap supports. The scale bar is in substitutions per
Bernstein (eds Ganzelewski, M., Rehren, T.H. & Slotta, R.). Bochum,
Germany: Deutsches Bergbau Museum. position.
Rokyta, D.R., Lemmon, A.R., Margres, M.J. & Aronow, K. (2012) The
venom-gland transcriptome of the eastern diamondback rattlesnake Figure S4. The best tree from ML analysis on the 569-flanking-region
(Crotalus adamanteus). BMC Genomics, 13(312), 312.
dataset. The numbers along the branches are ML bootstrap supports.
Schmidt, C.A. (2013) Molecular phylogenetics of ponerine ants
(Hymenoptera: Formicidae: Ponerinae). Zootaxa, 3647, 201–250. The scale bar is in substitutions per position.
Sharanowski, B., Ridenbauch, R.D., Piekarski, P.K., Broad, G., Burke, G.R. et al.
(2021) Phylogenomics of Ichneumonoidea (Hymenoptera) and implica- Figure S5. The best tree from ML analysis on the 763-coding-region
tions for evolution of mode of parasitism and viral endogenization.
dataset (third codons not RY coded). The numbers along the branches
Molecular Phylogenetics and Evolution, 156(107023), 1–19.
Shen, X.-X., Hittinger, C.T. & Rokas, A. (2017) Contentious relationships in are ML bootstrap supports. The scale bar is in substitutions per
phylogenomic studies can be driven by a handful of genes. Nature position.
Ecology and Evolution, 1(0126), 1–10.
Smith, H.S. (1912) IV. The chalcidoid genus Perilampus and its relations to
Figure S6. The best tree from ML analysis on the 763-coding-region
the problem of parasite introduction. U.S. Department of Agriculture,
Technical Series, 19, 33–69. dataset (third codons RY coded). The numbers along the branches
Stamatakis, A. (2014) RAxML version 8: a tool for phylogenetic analysis and are ML bootstrap supports. The scale bar is in substitutions per
post-analysis of large phylogenies. Bioinformatics, 30, 1312–1313. position
Figure S7. The best tree from ML analysis on the filtered Table S1. Specimen information and AHE assembly data.
336-region dataset (third codons RY coded). The numbers along the
branches are ML bootstrap supports. The scale bar is in substitu- Table S2. Summary statistics for each locus and corresponding gene
tions per position. tree (gene trees generated from the RY coded sequences for the cod-
ing regions).
Figure S8. The best tree from ML analysis on the ‘decisive’
531-region dataset (third codons RY coded). The numbers along the Table S3. Summary statistics for the datasets in the concatenation
branches are ML bootstrap supports. The scale bar is in substitutions analyses.
per position.
Table S4. Partition support summary.
Figure S9. Species tree estimated from all 1332 gene trees using
ASTRAL. The numbers along the branches are bootstrap supports. Table S5. Estimates of divergence times (in millions of years) for nodes
The scale bar is in coalescent units. in Figure 7 from BEAST and MCMCtree analyses.
Figure S10. Species tree estimated from the ‘decisive’ 531 gene trees Table S6. ASR probabilities for certain transition. For full range of
using ASTRAL. The numbers along the branches are bootstrap sup- values, see supplementary Mesquite file.
ports. The scale bar is in coalescent units.
Table S7. Character analysis background and literature.
Figure S11. Species tree estimated from the filtered 336 gene trees
using ASTRAL. The numbers along the branches are bootstrap sup- Suppl. Nexus File 1. planidial_likelihood_mesquite_final_Sept_12_
ports. The scale bar is in coalescent units. 2021.nex (in dryad).
Figure S12. Concatenated IQ-TREE analysis of all loci and including Suppl. Nexus File 2. planidial_mesquite_parsimony_final_Sept_12_
the 11 informative loci for Jambiya. 2021.nex (in dryad).
Figure S13. Concatenated IQ-TREE analysis of only the 11 informative

How to cite this article: Zhang, J., Heraty, J.M., Darling, C.,
loci for Jambiya.
Kresslein, R.L., Baker, A.J., Torréns, J. et al. (2022) Anchored
phylogenomics and a revised classification of the planidial
Figure S14. Phylogenetic reconstruction based on the amino acid
larva clade of jewel wasps (Hymenoptera: Chalcidoidea).
sequences of 763 coding regions using CAT+GTR model in Phy-
Systematic Entomology, 47(2), 329–353. Available from:
loBayes. The numbers along the branches are posterior probabilities
https://doi.org/10.1111/syen.12533
(only values >0.7 are shown). The scale bar is in substitutions per
position.

Zhang 2022 Anchored Phylogenomics and A Revised Classification of The Planidial Larva Clade of

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Zhang 2022 Anchored Phylogenomics and A Revised Classification of The Planidial Larva Clade of

Uploaded by

Copyright:

Available Formats

Received: 16 September 2021 Accepted: 21 December 2021

Anchored phylogenomics and a revised classification

Junxia Zhang1 | John M. Heraty2 | Christopher Darling3,4 |

Systematic Entomology. 2022;47:329–353. wileyonlinelibrary.com/journal/syen © 2022 Royal Entomological Society. 329

AHE probe sets

Figure S13. Concatenated IQ-TREE analysis of only the 11 informative

You might also like