After the signature of a Memorandum of Understanding (MoU) between UFES and ZSM, a joint effort was settled by BCA, SS and MTT to accelerate the work process by using the ZSM expertise in molecular biology and LaBI-UFES expertise in Chalcididae taxonomy. BCA was responsible for the general coordination, samples sorting and genera identification (one week for sorting and identification), SS was in charge of the molecular coordination and MTT was in charge of the taxonomic coordination. MTT selected the specialists for each genus found during the sorting process. The specimens were illustrated following the specialist's request and the images sent via the ZSM server. Over one thousand images were prepared and sent in two weeks. Since Conura represented almost 40% of all specimens, TRAB was sent to ZSM to accelerate the identification process (one month). In order to avoid shipping a large amount of material, only a few specimens, not identified by the images, were sent from Germany to Brazil according to the specialist's request. Each specialist made the first identification of the material and selected the specimens with priority on the molecular pipeline. The material was plated in ZSM, sent to CCDB and the results analysed by SS, who communicated with the specialists and coordinated new rounds of molecular analysis. This process took around two months and was important to avoid sequencing the same species several times, thereby saving money and time. BCA, MTT and SS coordinated, together with the specialists, the interpretation of the morphological and molecular results and the definition of the species lists. The specialists prepared the results and discussion for each genus under MTT coordination (one month). BCA, MTT and PMS prepared the backbone of the manuscript while SS wrote the methodology. BCA, MTT and SS wrote the general discussion. In the end, all authors made changes and suggestions in the entire manuscript. All this coordination was performed by using e-mail and message apps, allowing real-time discussion. The entire process, from the beginning of the samples sorting until the paper submission, including delays, took ten months.

The taxonomic data compilation for the named specimens was based mainly on Noyes (2018). The data for each species is listed in Table 1.

Specimens were sorted to genus level at ZSM and then forwarded to taxonomic specialists at LaBI-UFES. Specimens for DNA barcoding were selected by the specialists based on morphological examination, aiming to obtain a set of specimens that reflected the species diversity. Long series of the same species were excluded to reduce cost and effort for the molecular analyses. Whenever possible, the specimens were identified to species level. Putative new species were flagged at this step. All specimens were imaged using a Nikon V1 camera attached to a Leica Z16 APO objective. Most specimens became part of revisional studies conducted as masters or doctoral theses at LaBI-UFES. The new species that were discovered will be formally described as part of the resulting taxonomic treatments of Neotropical and world generic revisions.

Whole specimens were submitted to the Canadian Centre for DNA Barcoding (CCDB) in Guelph, Canada, for DNA extraction and sequencing. The voucher specimens were submitted to non-destructive DNA extraction and sequencing for subsequent preparation and morphological study. DNA extraction, PCR amplification and sequencing were conducted at the CCDB using standardised protocols (Ivanova et al. 2006, deWaard et al. 2008, Ratnasingham and Hebert 2007 http://ccdb.ca/). The 658 bp target region, starting from the 5’ end of the mitochondrial cytochrome c oxidase subunit I (COI) gene, includes the DNA barcode region for the animal kingdom (Hebert et al. 2003). The DNA extracts are stored at the CCDB. All specimen data are available through the Barcode of Life Database (BOLD) as a single, citable dataset (dx.doi.org/10.5883/DS-PECHAL1). The dataset includes collecting locality, geographic coordinates, elevation, collector, one or more digital images, identifier and voucher depository. Sequence data can be obtained through BOLD and include a detailed LIMS report, including chromatograms, primer information and access to trace files. The sequences are also available on GenBank (Accession nos. MK202011-MK202148). 

Data on genetic material contained in this paper and the Barcode of Life Database (BOLD) are published for non-commercial use only, according to the agreements with the country providing the analysed samples. Use by third parties for purposes other than non-commercial scientific research may infringe the conditions under which the genetic resources were originally accessed and should not be undertaken without obtaining consent from the corresponding author of the paper and/or obtaining permission from the original providers of the genetic material.

Sequences were aligned using the BOLD Aligner (amino acid-based hidden Markov models). The analyses are based on sequences with a minimum length of 300 bp and < 1% ambiguous bases. Genetic distances and summary statistics were calculated using analytical tools in BOLD and are given as mean and maximum pairwise distances for intraspecific variation and as minimum pairwise distances for interspecific variations.

Sequence divergence statistics were calculated using the Kimura two-parameter model of sequence evolution (Kimura 1980). The Barcode Index Numbers (BINs) that are assigned by the BOLD system represent globally unique identifiers for clusters of sequences that correspond closely to putative species (Ratnasingham and Hebert 2013). For BIN assignment, a minimum sequence length of 500 bp is required and sequences between 300 and 500 bp can join an existing BIN, but will not create or split BINs. BINs provide an interim taxonomic system and a way to determine Molecular Operational Taxonomic Units (MOTUs), prior to detailed taxonomic studies including morphology.

Sequences between 100 and 499 bp that did not meet the requirements for BIN assignment were submitted to a cluster analysis that included all sequences with a length of at least 100 bp. The analysis tool is provided by the BOLD system and generates OTUs independent of the BIN registry, using the REfined Single Linkage algorithm (RESL – Ratnasingham and Hebert 2013). In the tables presenting Sample IDs and BINs, "N/A" was used for missing data.

Conura was the most abundant and richest genus sampled with a total of 166 specimens sorted into 113 species, a trend found in other checklist papers of Chalcididae (Arias and Delvare 2003, Tavares and de Araujo 2007). It was the second most common and diverse genus in the fogging samples, with six specimens representing six species, (one formally described and five new species). Conura currently includes 301 valid species (Noyes 2018), with 8 recorded in Peru so far (Table 1). This study recovered two of those species, Co. attacta and Co. immaculata and added another 22, totalling 30 described species for the country (Fig. 2). At least 37 species obtained in this study are new species, which are being described in papers in preparation at LaBI-UFES. One species (Co. sp90) was represented by 11 specimens and another two (Co. sp3 and Co. sp35) by six specimens; however, 77% of the sampled species were singletons. Despite being the second most dominant genus in the fogging samples, only six species singletons were obtained.

Figure 2.  

Diversity of Conura subgenera and species groups (Delvare 1992) and respective BINs. All specimens are females, except F and K: A. Co. (Ceratosmicra) camescens (immaculata group; BOLD:ADI0819); B. Co. (Ce.) sp77 (onorei group); C. Co. (Ce.) dorsimaculata (side group); D. Co. (Conura) dares (dares group); E. Co. (Co.) nigrifrons (maculata group; BOLD:ADE9829); F. Co. (Co.) sp90 (vau group); G. Co. (Spilochalcis) sp76 (picta group); H. Co. (Sp.) sp04 (chrysomera group; BOLD:ADI0628); I. Co. (Sp.) sp23 (surumuae group); J. Co. (Sp.) vesicula (vesicula group); K. Co. (Sp.) sp29 (annulipes group); L. Co. (Sp.) sp45 (femorata group); M. Co. (Sp.) amoena (flava group; BOLD:ADI0816); N. Co. (Sp.) sp63 (pygmaea group); O. Co. (Sp.) sp16 (aequalis group; BOLD:ADE9956); P. Co. (Sp.) sp17 (arcuaspina group; BOLD:ADE9958); Q. Co. (Sp.) sp68 (contributa group); R. Co. (Sp.) santaremensis (discolor group); S. Co. (Sp.) adela (maculipennis group; BOLD:ADI1049); T. Co. (Sp.) sp62 (rasplusi group); U. Co. (Sp.) sp44 (rufodorsalis group); V. Co. (Sp.) sp89 (xanthostigma group; BOLD:ADH8745) ; X. Co. (Sp.) attacta (femorata group); Z. Co. (Ce.) immaculata (immaculata group).

Conura systematics is rather complicated, with three subgenera, three species complexes and 63 species groups (Delvare 1992). All subgenera and species complexes were represented in the samples, as well as 24 of the 63 species groups (Suppl. material 1). A total of 135 specimens, representing the majority of sampled species of Conura, were chosen for the molecular pipeline. Of those, 80 specimens produced sequences of at least 200 bp and were included in the cluster analysis (Table 3). The clusters presented an average of 13.51% nearest neighbour (NN) distance for the genus Conura. Comparing the subgenera separately, the average nearest neighbour (NN) distance is 8.32% for the subgenus Ceratosmicra, 24.63% for subgenus Conura and 12.48% for subgenus Spilochalcis. Thirty-seven BINs were associated with the specimens sequenced, all new to BOLD (Table 3).

Some species had specimens recovered in different BINs, but were morphologically indistinct at first analysis, such as Co. sp3, Co. sp6, Co. sp9, Co. sp12 and Co. sp18 (Fig. 3). Further morphological analyses did reveal differences within Co. sp9 and Co. sp12, but no significant differences were found amongst Co. sp3, Co. sp6 and Co. sp18. Co. sp9 and Co. sp12 were then divided into Co. sp9 and Co. sp89 and Co. sp12 and Co. sp90, respectively. Co. sp9 and Co. sp89 differ in number and size of metafemur teeth, the first one with 13 and the second with 18 smaller, very close together and the gaster of C. sp9 is higher than in C. sp89. Conura sp12 and C. sp90 were more similar and harder to split, the only clear difference between these species was the lower face strigate in C. sp12 and shallow fovea in C. sp90. This suggests that the DNA barcode may be a tool to refine taxonomy and even for detecting potential cryptic species of Chalcididae. The other three species remained morphologically indistinct, in spite of their different BINs. Perhaps further studies, including aspects of internal morphology, genitalia, biology and additional developmental data could help elucidating those cases.

Figure 3.  

Species of Conura with morphologically indistinct specimens and two BINs, females: A and B. Conura (Spilochalcis) sp3 (femorata group; A. BOLD:ADE9827; B. BOLD:ADE9681); C & D. Co. (Conura) sp6 (maculata group; C. BOLD:ADI0818; D. BOLD:ADE9680); E & F. Co. (Sp.) sp18 (discolor group; E. BOLD:ADE9682; F. BOLD:ADE9822).

Figure 4 shows a neighbour-joining distance tree (Fig. 4) with sequences longer than 200 bp and the subgenera of Conura highlighted with different colours. All subgenera, species complexes and several species groups were recovered as distinct groups. However, these results may suggest that the relationships within Conura do not fully correspond to the hypotheses previously proposed by Delvare 1992. Some morphology-based phylogenetic studies, developed at LaBI-UFES, also refute Delvare 1992. However, it remains to be demonstrated to what extent the fast-evolving COI gene is suitable for assessing higher level phylogenetic relationships within Chalcididae.

Figure 4.  

A neighbour-joining distance tree of the Conura specimens with sequences longer than 200 bp. The colours represent subgenera. Available BINs are presented between brackets before the OTUs.

Melanosmicra is the only genus of the subfamily Chalcidinae found as new to Peru in this work. The number of specimens obtained with Malaise trap sampling (64 specimens or 15% of the total) was expressive (Table 4). Those specimens belong to 11 species, with three new species (Fig. 5). From this total, 21 specimens, representing all species found, were submitted to the molecular pipeline, producing 15 sequences longer than 100 bp, which corresponded to four different BINs. The molecular findings were congruent with the morphological results. The numbers here reported put Peru as the second country in terms of number of species of Melanosmicra, with eight described species and three species new species, after Brazil, with 13 species recorded (Navarro-Tavares and Tavares 2008).

Figure 5.  

Diversity of Melanosmicra. A. M. areta, female (BOLD:ADF1337); B. M. carenata, female; C. M. flavicollis, female (BOLD:ADE9942); D. M. gracilis, male; E. M. immaculata, male (BOLD:ADF0815); F. M. nigra, male; G. M. rugosa, female; H. M. tricolor, male; I. M. sp1, female (BOLD:ADF1769); J. M. sp2, female; K. M. sp3, male. 

Brachymeria was represented by 67 specimens belonging to 14 species, 11 new species and three described (Table 5). Only the singleton B. sp 1 was collected in the fogging samples; the remainder were captured by Malaise trap. Brachymeria mnestor and B. pandora are frequently collected in the Neotropics. B. caudigera is very uncommon and it had been recorded so far only from the type locality (Jataí, State of Goiás, Brazil) (Fig. 6). Only four Brachymeria species had been previously recorded from Peru (Table 1). Therefore, with the records in this study, this number is increased to 18.

Figure 6.  

Diversity of the Brachymeria described species: A. B. caudigera, female; B. B. mnestor, male; C. B. pandora, male.

Seventeen specimens, representing eleven species, were selected to be submitted to molecular pipeline and produced five sequences larger than 500 bp, resulting in four different BINs. Molecular and morphological results were congruent.

Brachymeria is distributed worldwide and includes about 307 described species (Noyes 2018). There are 46 described species in the Neotropical region (Noyes 2018) and at least 60 species awaiting description (unpublished data, MTT). Therefore, the Brachymeria fauna, sampled in Panguana, represents almost 10% of the Neotropical fauna.

Ceyxia was represented by five species and six specimens collected only with the Malaise trap (Table 6). Six species had previously been recorded for Peru: Ce. acutigaster, Ce. atuberculata, Ce. concitator, Ce. decreta, Ce. flaviscapus and Ce. villosa (Andrade and Tavares 2009). We recovered Ce. acutigaster and Ce. villosa and another three species: Ce. amazonica, Ce. bellissima and Ce. sp1. Thus, eight species are now known for the genus in Peru. Specimens of Ce. acutigaster, Ce. amazonica and Ce. bellissima were submitted to the molecular pipeline, but no sequences were obtained (Fig. 7).

Figure 7.  

Diversity of the Ceyxia described species, females: A. Ce. acutigaster; B. Ce. amazonica; C. Ce. bellissima.

A total of 18 specimens of Stypiura were obtained, representing 12 species (Table 7). According to Bouček (1992), amongst the genus of Phasgonophorini, only Stypiura had already been recorded from Peru. However, Bouček did not provide information on which species were found in Peru. In the present work, almost all recorded species are new. The only species found that had been previously described was S. batesii, which, so far, had not been recorded from Peru. Thus, 12 species are hereby recorded for that country: S. batesii, S. sp1, S. sp2, S. sp3, S. sp4, S. sp5, S. sp6, S. sp7, S. sp8, S. sp9, S. sp10 and S. sp11 (Fig. 8).

Figure 8.  

Diversity of Stypiura, females. A. S. batesii; B. S. sp1; C. S. sp2; D. S. sp3; E. S. sp4; F. S. sp5; G. S. sp6 (BOLD:ADF0812); H. S. sp7; I. S. sp8; J. S. sp9; K. S. sp10 ; L. S. sp11. 

The most abundant species were S. sp6 and S. sp8, with 3 and 4 specimens, respectively. The species S. batesii, S. sp2, S. sp6 and S. sp7 were chosen for the molecular pipeline. Despite the cluster being congruent with the morphological delimitation of the species, the only species that received a BIN was S. sp6.

The current classification of Dirhinus presents three subgenera: Dirhinus, Hontalia and Pareniaca, all found in the New World; the second is exclusively neotropical. Although it is quite diverse for the New World (16 species), the only species known from Peru is D. (Dirhinus) giffardii, which was not found in the studied samples. After morphological analysis of the 28 specimens obtained, we were able to recognise nine species (Fig. 9). Eight species belong to subgenus Pareniaca (six new to science) and one species belongs to the subgenus Hontalia. We submitted 18 specimens, representing all species found of Dirhinus, to the molecular pipeline. Ten produced sequences longer than 100 bp and were included in a cluster analysis (Table 8). The clusters presented an average of 6.05% of nearest neighbour distance (NN), with the clustering fully corresponding with the results of the morphological analysis. Seven BINs were associated with the successfully sequenced samples, all new to BOLD (Table 8). These seven new species correspond to an increase of 43.7% in the Dirhinus diversity known from the New World.

Figure 9.  

Diversity of Dirhinus, females, except F and H, males. A. D. cameroni; B. D. buscki (BOLD: ADF1234); C. D. kirbyi; D. D. sp1; E. D. sp2; F. D. sp3; G. D. sp4; H. D. sp5; I. D. sp6 (BOLD: ADE9797)

Notaspidium is the only genus of Haltichellinae previously recorded from Peru. It was known from two species (N. apantelis and N. giganteum). Only N. apantelis was obtained in the present study. Notaspidium was the dominant genus in the fogging samples, with 11 specimens and 8 species (Table 9). A total of 37 specimens and 12 species were found in the morphological analysis (Fig. 10), four of them new species. Eighteen specimens and 12 species were selected to the molecular pipeline. Eleven specimens produced sequences longer than 100 bp and were included in a cluster analysis (Table 2). The clusters presented an average of 11.26% nearest neighbour (NN) distance, fully matching the results of the morphological analysis. The four BINs found are new to BOLD (Table 9).

Figure 10.  

Diversity of the Notaspidium described species: A. N. acutum, female; B. N. apantelis, female; C. N. boharti, male; D. N. braziliensis; E. N. burdicki; F. N. minutum; G. N. truncatum; H. N. villegsasi

This is the first time that the genus Aspirrhina has been recorded from Peru. Three species (A. remotor, A. bifurca and A. dubitator) were identified, based on 12 specimens (Fig. 11). Aspirrhina bifurca was the most common species, with 8 specimens. Eight specimens representing all putative taxa found were submitted to the molecular pipeline, but no sequences were obtained.

Figure 11.  

Diversity of Aspirrhina. A. A. remotor, male; B. A. bifurca, male; C. A. dubitator, female.

Although it is one of the most common genera obtained in neotropical Malaise trap samples, along with Conura and Brachymeria, this is the first time Haltichella has been recorded from Peru. Twenty-seven specimens were collected corresponding to four new species (Fig. 12). One species (Ha. sp3) was represented by 24 specimens and all others were singletons. Fourteen specimens were submitted to the molecular pipeline (Table 10). Only two specimens (representing Ha. sp3 and Ha. sp4) had the COI-5P fragment successfully amplified, but the sequences generated did not meet the criteria for BIN assignment. These specimens formed two separate clusters, which were congruent with the morphological analysis.

Figure 12.  

Diversity of Haltichella. A. Ha. sp1, female; B. Ha. sp2, female; C. Ha. sp3, male; D. Ha. sp4, male.

Hockeria is here recorded for the first time from Peru and was represented by two specimens of the same species (Fig. 13). Those two males do not seem to correspond to any known neotropical species. Both specimens were submitted to the molecular pipeline, but no sequences were obtained.

Figure 13.  

Hockeria sp1, male.