Research Article |
Corresponding author: Jeremy D. Blaschke ( jblaschke@uu.edu ) Academic editor: Michael Ohl
© 2024 Allaina L. Armstrong, Jayme E. Sones, Volker Lohrmann, Paul D. N. Hebert, Dan H. Janzen, Winnie Hallwachs, Jeremy D. Blaschke.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Armstrong AL, Sones JE, Lohrmann V, Hebert PDN, Janzen DH, Hallwachs W, Blaschke JD (2024) Six in one: cryptic species and a new host record for Olixon Cameron (Rhopalosomatidae, Hymenoptera) revealed by DNA barcoding. Journal of Hymenoptera Research 97: 363-378. https://doi.org/10.3897/jhr.97.116726
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Olixon testaceum is a widely distributed species of brachypterous parasitoid wasp (Vespoidea: Rhopalosomatidae) occurring in Meso- and South America, but little is known of its biology. Here, the first known host of O. ? testaceum is identified as the cricket Anaxipha sp. (Grylloidea: Trigonidiidae) through DNA barcoding of six Olixon larvae and their hosts. Barcoding results also indicated substantial genetic diversity within nominal O. testaceum specimens. The number of species and statistical significance of these groups were tested using Maximum Likelihood phylogenies, distance-based cluster analyses, and coalescence models. All analyses revealed at least six distinct lineages, which suggests six or more cryptic species within O. ? testaceum. Combined with what is currently known about Rhopalosoma host use, these results indicate that rhopalosomatids may be generalist rather than specialist parasitoids, and further confirm the benefits of open global collaboration and DNA barcoding in advancing taxonomic knowledge.
Cricket-assassin wasp, integrative taxonomy
In recent decades, new discoveries have greatly increased our knowledge of the diversity, systematics, and behavior of Olixon Cameron, 1887–an historically understudied genus of cricket-assassin wasps (Vespoidea: Rhopalosomatidae) (
Of the 74 currently described species of extant Rhopalosomatidae (
Despite their brachypterous wings which would seem to limit long-range dispersal, Olixon testaceum, as currently understood, is among the most widespread of all rhopalosomatids, occurring throughout Meso- and South America, from Argentina to Arizona (
An initial search of publicly available rhopalosomatid records revealed 221 sequences from specimens identified as O. cf. testaceum using the key to species in
Specimens from each sequence cluster in this study were identified to genus using
The publicly available records (n = 221) were combined with additional private sequences of rhopalosomatids made available by JS, PH, DJ, and WH to ensure maximum coverage, including another two larvae (for a total of six). Specimens were predominantly collected via weekly Malaise trap samples (n = 398) between 2012 and 2020. All but 10 specimens (sourced from GenBank) were sequenced at the Centre for Biodiversity Genomics, and most sequences (n = 309) were generated by a Sequel (Pacific Biosciences) high-throughput sequencer, while 95 were analyzed using Sanger sequencing (https://ccdb.ca/resources/). All sequences, specimen images, and collection data are available in the dataset “DS-RHOP” on BOLD.
An initial Maximum Likelihood (ML) phylogeny of Rhopalosomatidae was created using RAxML (v.8.2.12) (
To narrow the focus to only larvae and potential cryptic species of O. testaceum, a new ML phylogeny was created using all six larvae, specimens from each apparent O. testaceum clade, all other Olixon specimens available (five in total), and four outgroup specimens (two each for Liosphex Townes, 1977 and Rhopalosoma Cresson, 1865). Sequences were aligned using MAFFT v.7.450 (
Specimens used in phylogenetic and statistical analyses. Process and Voucher IDs from BOLD: www.boldsystems.org.
Species | Location | Date Collected | Elevation (M) | Stage | Process ID | Sample ID | BIN |
---|---|---|---|---|---|---|---|
O. ? testaceum 1 | Cortes, HND | 7/2/2014 | 1219 | Adult | GMHJK402-15 | BIOUG18597-F10 | BOLD:ACE2345 |
O. ? testaceum 1 | Cortes, HND | 7/27/2012 | 1219 | Adult | GMHDO003-13 | BIOUG04583-G09 | BOLD:ACE2345 |
O. ? testaceum 1 | Cortes, HND | 6/18/2015 | 1219 | Adult | GMHMQ600-15 | BIOUG26862-E11 | BOLD:ACE2345 |
O. ? testaceum 1 | Cortes, HND | 7/24/2014 | 1196 | Adult | GMHKP138-15 | BIOUG19409-D02 | BOLD:ACE2345 |
O. ? testaceum 1 | Cortes, HND | 7/16/2015 | 1219 | Adult | GMHMU283-16 | BIOUG28324-G04 | BOLD:ACE2345 |
O. ? testaceum 2 | ACG, CRI | 5/25/2020 | 1366 | Adult | CRALC14238-21 | BIOUG72979-B05 | BOLD:AEO2513 |
O. ? testaceum 3 | ACG, CRI | 3/13/2014 | 853 | Adult | JICFX017-16 | BIOUG29019-H01 | BOLD:ACZ7577 |
O. ? testaceum 3 | ACG, CRI | 1/9/2014 | 831 | Adult | PLEAI182-19 | BIOUG48962-D09 | BOLD:ACZ7577 |
O. ? testaceum 3 | ACG, CRI | 5/12/2014 | 575 | Adult | GMAAT178-16 | BIOUG27868-D08 | BOLD:ACZ7577 |
O. ? testaceum 3 | ACG, CRI | 8/10/2015 | 575 | Adult | GMADY103-16 | BIOUG28200-H03 | BOLD:ACZ7577 |
O. ? testaceum 3 | ACG, CRI | 4/14/2014 | 575 | Adult | GMAAR037-16 | BIOUG28246-C12 | BOLD:ACZ7577 |
O. ? testaceum 3 | ACG, CRI | 1/26/2017 | 828 | Adult | PLVAK389-20 | BIOUG55894-F08 | BOLD:ACZ7577 |
O. ? testaceum 4 | ACG, CRI | 1/28/2020 | 15 | Adult | CROAC13695-21 | BIOUG68837-C03 | BOLD:AEM2374 |
O. ? testaceum 4 | ACG, CRI | 3/3/2022 | 62 | Adult | CROCA33528-21 | BIOUG68316-G10 | BOLD:AEM2374 |
O. ? testaceum 5 | Paramaribo, SUR | 10/2/2017 | n/a | Larva | GMSPA14567-21 | BIOUG70270-H11 | BOLD:AEK9228 |
O. ? testaceum 6 | ACG, CRI | 8/5/2012 | 300 | Adult | GMCRH028-13 | BIOUG05414-E09 | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 1/18/2018 | 828 | Adult | PLBCJ264-20 | BIOUG57554-G01 | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 8/30/2018 | 811 | Adult | PLEFA082-21 | BIOUG64629-H07 | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 5/26/2020 | 15 | Adult | CROAD12739-22 | BIOUG80688-H11 | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 6/16/2020 | 15 | Adult | CROAD18508-22 | BIOUG81047-E07 | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 1/19/2017 | 828 | Larva | PLVAJ397-22 | BIOUG55891-B08_parasite | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 8/20/2020 | 791 | Larva | PLDFN085-21 | BIOUG63752-H08 | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 8/27/2020 | 811 | Larva | PLEFO304-21 | BIOUG59841-H04 | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 1/18/2018 | 809 | Larva | PLKCJ206-20 | BIOUG59151-H08 | BOLD:ACG4885 |
O. ? testaceum 6 | ACG, CRI | 8/30/2018 | 809 | Larva | PLKDP220-20 | BIOUG58943-C02 | BOLD:ACG4885 |
O. banksii | IO, USA | 22/8/2009 | – | Larva | Olixon_Larva_IO | – | |
O. banksii | TX, USA | 7/6/2011 | 81 | Adult | BBHYA2946-12 | BIOUG02644-C10 | BOLD:ACA7258 |
O. banksii | VA, USA | 9/28/1993 | – | Adult | SICOD002-19 | CCDB-34061-A02 | BOLD:AEA2163 |
O. banksii | OK, USA | 6/19/2011 | – | Adult | BBHYA2958-12 | BIOUG02644-D10 | BOLD:ACA7139 |
Olixon sp. | WA, AUS | 11/21/2014 | – | Adult | GMCWM011-15 | BIOUG23860-E06 | BOLD:ACZ3980 |
Liosphex sp. | ACG, CRI | 4/28/2014 | 575 | Adult | GMAAS028-16 | BIOUG28345-C04 | BOLD:ADA1369 |
Liosphex sp. | ACG, CRI | 6/11/2015 | 1220 | Adult | GMCCI021-17 | BIOUG36436-H10 | BOLD:ADL6377 |
Rhopalosoma sp. | ACG, CRI | 7/12/2018 | 809 | Adult | PLKDI021-20 | BIOUG58153-D12 | BOLD:ADC7061 |
Rhopalosoma sp. | ACG, CRI | 5/14/2012 | 300 | Adult | GMCGG056-14 | BIOUG17755-D09 | BOLD:ACG8319 |
To test for potential cryptic species diversity within O. testaceum, both distance-based cluster analyses and phylogenetically informed tests of species delimitation were employed. Assemble Species by Automatic Partitioning (ASAP) (
Additional distance-based cluster analyses were carried out in R (
Phylogenetically informed species delimitation methods were also used. The Multi-rate Poisson Tree Process (mPTP) introduced by
High throughput sequencing (HTS) has the advantage of generating sequences of biota associated with the target specimen. This property enables the identification of potential host-parasitoid interactions, predator-prey relationships, pathogen infections, etc. If a specimen yields more than one sequence contig, the additional contig(s) can be examined for biologically relevant associations. To identify the host for each larva, we compared any additional sequence information generated by the HTS to BOLD and generated a new barcode record when a potential orthopteran host sequence was found.
Thirty-four sequences were used to generate the ML tree (Fig.
Maximum Likelihood phylogeny of Olixon testaceum. Bootstrap support >75 is shown. Putative cryptic species of O. testaceum are labeled. Vertical bars indicate statistically significant species groups identified by Hierarchical clustering (HCL), Assemble Species by Automatic Partitioning (ASAP), Multi-rate Poisson Tree Process (mPTP), and Bayesian Poisson Tree Process (bPTP). Light orange bars for bPTP are not significant.
All novel host associations of Olixon. Sample IDs from BOLD: www.boldsystems.org.
Species | BIN | Location | Larva Sample ID | Host Sample ID | Host Species | Host BIN | Status |
---|---|---|---|---|---|---|---|
O. ? testaceum 5 | BOLD:AEK9228 | Paramaribo, SUR | BIOUG70270-H11 | – | – | – | |
O. ? testaceum 6 | BOLD:ACG4885 | ACG, CRI | BIOUG55891-B08_parasite | BIOUG55891-B08 | Anaxipha sp. | BOLD:ACO0556 | Known |
O. ? testaceum 6 | BOLD:ACG4885 | ACG, CRI | BIOUG63752-H08 | BIOUG63752-H08.NTS | Anaxipha sp. | BOLD:ACO0556 | Known |
O. ? testaceum 6 | BOLD:ACG4885 | ACG, CRI | BIOUG59841-H04 | BIOUG59841-H05 | Trigonidiidae | BOLD:ACG0099 | Associated |
O. ? testaceum 6 | BOLD:ACG4885 | ACG, CRI | BIOUG59151-H08 | BIOUG59151-H08.NTS | Anaxipha sp. | BOLD:ACO0556 | Associated? |
O. ? testaceum 6 | BOLD:ACG4885 | ACG, CRI | BIOUG58943-C02 | BIOUG58943-C02.NTS | Trigonidiidae | Associated? |
One larva (BIOU70270-H11) was collected in Suriname. It was found without a host and no associated specimens are confirmed at this time. The other five larvae were collected in ACG, Costa Rica. Two (BIOUG55891-B08_parasite and BIOUG63752-H08) were still attached to their host and barcodes for all four specimens were recovered. Both hosts were identified as Anaxipha sp. (Trigonidiidae). Three specimens (BIOUG59841-H04, BIOUG59151-H08, and BIOUG58943-C02) were not attached to a host, likely reflecting their detachment upon exposure to the ethanol in the Malaise trap. Potential orthopteran hosts associated with these samples (i.e., those collected from the same site, date, and trap) were all identified via barcodes as trigonidiid crickets.
All statistical analyses confirmed the presence of at least six distinct lineages within this dataset (Fig.
Mean pairwise intra- and interspecific genetic distances (K2P) between sampled Olixon testaceum specimens. Bold values = intraspecific genetic distance. “n/a” = not applicable due to single taxon.
O. ?testaceum 1 | 0.000 | |||||
O. ?testaceum 2 | 0.138 | n/a | ||||
O. ?testaceum 3 | 0.149 | 0.072 | 0.003 | |||
O. ?testaceum 4 | 0.144 | 0.071 | 0.074 | 0.010 | ||
O. ?testaceum 5 | 0.159 | 0.107 | 0.113 | 0.104 | n/a | |
O. ?testaceum 6 | 0.149 | 0.079 | 0.063 | 0.061 | 0.103 | 0.006 |
Our analyses indicate several genetic lineages fall within the morphological variation of “Olixon testaceum.” However, since the holotype has not been barcoded, we are unable to link any particular genetic lineage with the “real” O. testaceum. To indicate this uncertainty and acknowledge that future morphological work is needed to formally describe these cryptic species, we refer to the species group collectively as “O. ? testaceum.”
Although O. ? testaceum is one of the most widespread morphospecies of Rhopalosomatidae, very little is known about its biology (
Olixon is now known to parasitize species within two subfamilies of crickets: Trigonidiinae (herein,
Furthermore, the identification of Anaxipha as a host of O. ?testaceum is quite remarkable as Anaxipha are also among the known hosts of the distantly related R. ?nearcticum (
Generalized phylogeny of Rhopalosomatidae showing currently confirmed host associations. The tree topology is based on
While a new host record and discovery of cryptic species is significant, there is still much work to be done. Future efforts relating to O. ? testaceum should investigate morphological or ecological differences that might further distinguish clades from one another. A previous study in the ACG (
Such integrative taxonomic research may reveal more cryptic species within O. testaceum beyond those discovered here. In their revision of the New World Olixon,
Rhopalosomatidae appears to be a hotspot for cryptic species diversity. Our ML tree included four specimens of nominal O. banksii specimens from Iowa, Texas, Virginia, and Oklahoma. Three specimens from IO, TX, and VA showed low intra-group variation (patristic distance = 0.032), while the average nearest-neighbor distance between these three and a specimen from OK was significantly higher (patristic distance = 0.234). Another closely related species, O. melinsula, is currently known only from Texas to Florida (along the Gulf of Mexico) and southern Paraguay. This distribution pattern may well represent “two sibling species, so similar as to be indistinguishable at the moment” (
The utility of DNA barcoding to identify and reveal cryptic species complexes is well established across a wide range of taxa and biomes (e.g.,
The taxonomic impediment is a significant and well-known problem in entomology (e.g., Agnarsson and Kunter 2007;
This research was funded in part by an undergraduate research grant from Union University (Jackson, TN) received by AA and JDB. A special thanks to the taxonomists and parataxonomists in Costa Rica for collecting specimens, to the Centre for Biodiversity Genomics (CBG) for sequencing specimens, to BioAlfa for providing images of specimens, to the government of Costa Rica for allowing use of the data, and to Barcode of Life Data Systems for informatics support and metadata. All ACG specimens were collected, exported and DNA barcoded under Costa Rican government permits issued to BioAlfa (Janzen and Hallwachs 2019) (R-054-2022-OT-CONAGEBIO; R-019-2019-CONAGEBIO; National Published Decree #41767), JICA-SAPI #0328497 (2014) and DHJ and WH (ACG-PI-036-2013; R-SINAC-ACG-PI-061-2021; Resolución N°001-2004 SINAC; PI-028-2021). Awards from the Walder Foundation, the Canada Foundation for Innovation, the New Frontiers in Research Fund, and Genome Canada through Ontario Genomics to PDNH aided the CBG’s capacity to gather sequence data and sustain BOLD. We thank Paul Bertner for his permission to use his photo of Olixon (Fig.