DNA Barcoding of the parasitoid wasp subfamily Doryctinae (Hymenoptera: Braconidae) from Chamela, Mexico

Abstract Background and aims. The Doryctinae is a considerably diverse, poorly studied group of parasitoid wasps and one of the most diverse subfamilies within Braconidae. Taxonomic knowledge of this group remains highly incomplete, specially in the tropics. In Mexico, it has been reported as the subfamily with the highest number of recorded genera. A preliminary Barcoding study carried out in the Chamela region, located near the Mexican pacific coast in Jalisco, identified 185 barcoding species of Dorytinae assigned to 19 identified doryctine genera. This work updates the later study, representing a three years effort to assess the species richness of this subfamily for the Chamela region. Materials and methods. Ten collecting field trips of 5 to 10 days each were carried out from June 2009 to May 2011. A 2% divergence criterion using the BIN system implemented in BOLD was followed in order to establish species boundaries among the specimens that were collected. Results and conclusions. A total of 961 specimens were collected, from which 883 COI sequences were obtained. The sequences generated corresponded to 289 barcoding species and 30 identified genera. The most speciose genera were Heterospilus Haliday (170 spp.), Ecphylus Förster (19 spp.), Allorhogas Gahan (15 spp.) and Callihormius Ashmead (14 spp.). Addition of previously collected material increased the diversity of the subfamily in the region to 34 genera and 290 species. Paraphyly of Heterospilus with respect to Neoheterospilus and Heterospathius was again recovered. Twenty new species and two new genera (Sabinita Belokobylskij, Zaldívar-Riverón et Martínez, Ficobolus Martínez, Belokobylskij et Zaldívar-Riverón) have been described so far from the material collected in this work.


Introduction
Biodiversity inventories represent an integral component for the adequate management of natural resources of any country. In the case of faunistic studies, however, these rarely include arthropod taxa due to their considerably high diversity and scarce taxonomic knowledge. In this context, DNA Barcoding (Hebert et al. 2003a) represents a fast, valuable approach to built species inventories of highly diverse, poorly known invertebrate groups.
With over 1,300 described and almost 3,000 estimated species, Doryctinae is one the four most diverse subfamilies of braconid parasitoid wasps together with the Braconinae, Microgastrinae and Opiinae (Jones et al. 2009, Yu et al. 2012. Doryctine wasps are mainly known to be idiobiont ectoparasitoids of xylophagous and bark boring coleopteran larvae, though other host groups (e.g. Lepidoptera, Diptera, Hymenoptera) and biologies (e.g. phytophagy, endoparasitoidism) have also been reported (Belokobylskij 1992, Belokobylskij et al. 2004. Several doryctine species are known to be parasitoids of insect pests (LaSalle and Gauld 1993), therefore they play an important role for maintaining the balance of terrestrial ecosystems (Hawkins and Hochberg 1994).
The taxonomic knowledge of the Doryctinae is still far from complete, especially in the tropics and subtropics, where most of its species richness is known to occur (Belokobylskij 1992). In the Mexican territory, this was recently reported as the subfamily with the highest number of recorded genera (63 genera; Coronado-Blanco and Zaldívar-Riverón 2014). However, this number contrasts with its scarce number of described species that have been reported for the country. Recently,  showed the preliminary results of a DNA Barcoding survey of the Doryctinae from Chamela Biological Station (CBS) in Mexico, which located near the Pacific coast in the state of Jalisco and is mainly composed of tropical dry forest (Noguera et al. 2002).  not only revealed and extraordinary, mostly undescribed species richness for this group (185 Barcoding and 20 recognized genera), but also showed some previously undetected taxonimic problems.
Here we show the final list of the DNA Barcoding species inventory of the Doryctinae from the CBS, which was carried out during three years of collecting effort. We updated the list of genera that occur in the region and show their number of DNA Barcoding species based on the barcoding index criterion (BIN; Ratnasingham and Hebert 2013). This faunistic study highlights the extraordinary, neglected species richness of this parasitoid group in the Mexican territory, and is also serving as a basis for the subsequent description of several new taxa.

Study area
Specimens belonging to the subfamily Doryctinae were collected in the Chamela biological station (Fig. 1), owned by the Instituto de Biología, Universidad Nacional Autónoma de México (UNAM). This station is located within the Chamela-Cuixmala Biosphere Reserve (CCBR), near the Pacific coast in the state of Jalisco(19°29' N, 105° 02' W). The prevailing climate in the area is tropical sub-humid (mean annual temperature 24.6 °C, annual precipitation 788 mm; García 1988). There is a strong seasonality in the region, with alternating rainy (Jul-Oct) and dry (Nov-Jun) seasons. Altitude ranges from 0 to 500 meters above sea level. The dominant type of vegetation is tropical dry forest, which is characteristic of the Mexican Pacífic coast, though patches of tropical evergreen forest, coastal dune, xeric shrubland and mangrove are also present (Rzedowski 1978, Noguera et al. 2002. The relevance of this area lies in the variety of habitats it supports, as well as in its high endemic component of plants, vertebrates and insects (Noguera et al. 2002).

Specimen sampling
A total of 10 collecting trips of 5 to 10 days each were carried out from June 2009 to May 2011. Collects were performed both during the rainy and dry seasons in order to have a complete representation of the examined group through the year. Specimens were collected in 24 sites within the CBS boundaries (Table 1), using for this: 1) malaise traps, 2) light traps, 3) yellow pan traps, as well as 5) sweep nets for at least 4 h per day (Marshall 1994). The collected materials were preserved in 96% ethanol and kept at -20 °C . All specimens were sorted out at subfamily level following the taxonomic key of New World genera of Braconidae (Wharton et al. 1997). Doryctine specimens were then identified to genus level using the relevant literature (Marsh 1997, Marsh 2002 and subsequent descriptions of genera (e.g.   (Hebert et al. 2003b) and LCO1490/HCO2198 (Folmer et al. 1994) primers. Sequences were edited with Sequencher 4.0.5 (Gene Codes Corp.) and aligned manually based on their translated amino acids. All of the COI sequences generated are deposited in GenBank (accession numbers GU715182-288, HM420734-5, HM434309-544, HM882254, HQ200960-201008, HQ201239-54). All sequences and their specimen information are available in the project file "Parasitoid Wasps (Braconidae: Doryctinae) of Chamela-Cuixmala Biosphere Reserve" (ASDORproject) in the projects section of the Barcode of Life Data Systems (www.barcodinglife.com)

Species boundaries
Species boundaries were established following a 2% divergence criterion (Hebert et al. 2003a). This criterion has been shown to represent a fast, generally reliable tool for exploring species richness in different animal taxa (Ratnasingham and Hebert 2013). This established genetic distance is based on the assumption that COI divergences usually do not exceed a 2% divergence within a recognized species, whereas different species generally show a higher divergence (Hebert et al. 2003a). Sequences divergence of the sequenced specimens were obtained using the K2P distance model (Kimura 1980). A neighbourjoining (NJ) tree and an accumulation curve were reconstructed as implemented in the BOLD system (Barcode of Life Data System, www.boldsystems.org). BINs were obtained for each specimen from the BOLD system, and this was employed to establish the number of barcoding species.

Species boundaries
A total of 961 specimens were collected during the ten field trips, of which 883 COI sequences were generated. Fifteen of these sequences had a length lower than 500 bp and thus did not receive a BIN, though they were included in the analysis. The sequences generated belonged to 289 barcoding species and 30 identified genera, though four barcoding species could not be assigned to any genus ( The reconstructed NJ tree recovered the megadiverse genus Heterospilus as nonmonophyletic with respect to Heterospathius and Neotherospilus (Fig. 2). The accumulation curve estimations of species richness (Fig. 3) indicates that the number of species that occur in the region has not been reached. Neighbour-joining tree obtained from BOLD that includes 883 nucleotide sequences belonging to doryctine specimens. The distance model used was the Kimura 2 Parameter, the marker was COI-5P, and the 1st, 2nd and 3rd codon positions were included. The sequences had a lengrh greater than 200bp. See Suppl. material 1.  DNA barcoding species accumulation curve for the Doryctinae from the CBS (Suppl. material 1). Table 3.
List of the new species described from specimens collected in this study discriminated remain undescribed, 20 of them and two new genera (Sabinita and Ficobolus) were already described by AZR and collaborators based on the material collected in this work. Most of these new described species belong to small, poorly known genera. The published records for the Chamela region indicate that the species richness of the Doryctinae is considerably higher than the those observed for Microgastrinae (103 spp.;Fernández-Flores et al. 2013), considered to be the second largest subfamily of Braconidae (Jones et al. 2009), and Rogadinae (27 spp.; Aguilar-Velasco 2013).