Revision of the neotropical genus Sendaphne Nixon (Hymenoptera, Braconidae, Microgastrinae)

The Neotropical genus of parasitoid wasps Sendaphne (Hymenoptera, Braconidae, Microgastrinae) is revised and the following six new species are described, all authored by Fernández-Triana and Whitfield: anitae, bennetti, broadi, dianariaspennae, penteadodiasae, and rogerblancoi. The greatest species richness is found in northern South America, but the genus extends north to 23° N in Mexico. Most species have been collected in rainforest below altitudes of 900 m, with only a few species found in cloud forests up to 1900 m. Nothing is known of the host caterpillars for these parasitoid wasps.


Introduction
The genus Sendaphne was described by Nixon (1965) to accommodate two Neotropical species with a number of distinctive features: a very long and bilobate glossa, slender body, extensive yellow coloration, mostly smooth mesosoma and metasoma, enlarged hypopygium, and very long and curved ovipositor.Penteado-Dias (1995) described two additional species, and Scatolini and Penteado-Dias (1999) added another one.Four of the five species described so far are from Brazil, with the exception being Sendaphne sulmo Nixon (1965), recorded from southeastern Mexico.No host is known for Sendaphne, although the related genus Promicrogaster Brues & Richardson, 1913, has been reared from hosts living in bracket fungi (Mason 1981), among other concealed hosts.
In spite of the rather unique morphological traits, the validity of Sendaphne as a distinct genus has been questioned even by the same author who described it (Nixon 1965: 204), and it might eventually be determined to be a synonym of the more abundant and diverse Promicrogaster.However, Mason (1981) considered the differences as sufficient to maintain them as two genera.That decision has been followed by all subsequent authors and, without a comprehensive phylogenetic study of Microgastrinae, we agree that is better to keep it that way at present.The morphological and molecular analysis of Whitfield et al. (2002), although not conclusive, also found those two genera to be closely related.An anticipated revision of Promicrogaster may help to elucidate the limits of these two genera in the future.
As part of comprehensive studies on the fauna of Microgastrinae from Area de Conservación Guanacaste (ACG), northwestern Costa Rica (e.g., Fernández-Triana et al. 2014 and references cited there) we found a new species from that area.The Canadian National Collection of Insects (CNC) in Ottawa, Canada and the NSF-funded "Insect Survey of a Hyperdiverse Country: Colombia" projects led by M. J. Sharkey, B. V. Brown and the Humboldt Institute (Colombia) also contained additional undescribed species from Central and South America.All these new species are described below, altogether with a key to all known, previously described species of Sendaphne.

Methods
Among Microgastrinae, Sendaphne is one of the most rarely collected genera, and is poorly represented in collections.This study is based on 73 specimens from four sources: 38 Neotropical specimens deposited in the CNC, 18 specimens from Colombia (Humboldt Institute), 10 specimens from the ACG inventory, four specimens from Unité d'Entomologie fonctionnelle et évolutive, Gembloux Agro-Bio Tech, Université de Liège (FUSAGx) and three specimens from the Natural History Museum, London, England (BMNH).
All species previously described were deposited in the Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil (DCBU), Universidade Federal do Paraná, Curitiba, Paraná, Brazil (UFPR), or in the BMNH.We did not examine the holotypes of those species; however, their original descriptions and illustrations are sufficiently detailed to allow us to describe the new species with confidence.
Morphological terms and measurements of structures are mostly as used by Mason (1981), Huber and Sharkey (1993), Whitfield (1997), Karlsson andRonquist (2012), andFernández-Triana et al. (2014).Because the ovipositor in Sendaphne is strongly curved, its length was difficult to measure accurately; the ovipositor length measurements provided for each new species are only intended as an approximation.In any case, the ovipositor and its sheaths are some of the longest observed in any Microgastrinae genera; they are usually two times longer than the metatibia length.
Descriptions of the new species are based on the study of all available female specimens, so as to reflect intraspecific variation, but always include data from the holotype.As an exception, two new species only known from males are described below because they were sufficiently distinct to be distinguished from all others; the males of those two species may be identified by the key, but males of most other species may not be readily identified unless associated with females via rearing or molecular data.
The descriptions include 17 characters that are commonly used in describing Microgastrinae (e.g., body measurements such as length of body and fore wing, ovipositor sheath; and also color of particular body areas).Those descriptions are complemented with extensive color photos of every species.Geographic distribution is also provided in the key as supplementary information to aid the morphological identification of species, though we recognize that with time the current known geographic distribution may eventually become obsolete.
Photos were taken with a Keyence VHX-1000 Digital Microscope, using a lens with a range of 13-130×.Multiple images through the focal plane were taken of a structure and these were combined to produce a single in-focus image, using the software associated with the Keyence System.
Together with morphological studies, we also analyzed DNA barcodes (the 5' region of the cytochrome c oxidase I (CO1) gene, Hebert et al. 2003) whenever available.DNA barcodes for all ACG inventory Sendaphne specimens were obtained using DNA extracts prepared from single legs using a glass fibre protocol (Ivanova et al. 2006).Briefly, total genomic DNA was re-suspended in 30 μl of dH2O, and a 658-bp region near the 5' terminus of the CO1 gene was amplified using standard primers (LepF1-LepR1) following established protocols (Smith et al. 2006(Smith et al. , 2007(Smith et al. , 2008)).If the initial 658 bp amplification was unsuccessful, smaller sequences were generated using internal primers.If each amplification worked a composite sequence was generated, however in cases where only one read amplified, this shorter sequence was used.All information for the sequences associated with each individual specimen can be retrieved from the Barcode of Life Data System (BOLD) (Ratnasingham and Hebert 2007) using the following public DOI: http://dx.doi.org/10.5883/DS-SENDAPH.A Neighbor-Joining tree based on Kimura 2-parameter distances of all described species of Sendaphne with DNA barcodes available in BOLD was also generated (Suppl.material 2).

Results
Six new species of Sendaphne are described below, increasing the total known species from five to 11.We are aware of potential additional new species in the CNC collection, from Costa Rica (but not ACG), Ecuador, and Brazil.However, they are not described here because each is only represented by a single specimen and they are not sufficiently distinct to warrant description without further specimens and evidence.
Sendaphne is a Neotropical genus.To date it is most abundant and diverse in South America (eight species), while Central America has three species.It extends from 23° N in central Mexico (Durango) to 27° S in Paraguay (Pirapo) and southern Brazil (Santa Catarina).Most of the species have been collected in rain forests, at altitudes between 100 m and 900 m.However, a few species have been found only in cloud forests between 1,450 m and 1,900 m.The specimens collected at higher altitudes have darker coloration (especially on mesosoma and metasoma) than those found in the lowlands.
An interesting result of our morphological study was the relation between body and fore wing lengths.Body proportions in Microgastrinae have not been explored in detail, but in most genera and species with available data the fore wing length tends to be slightly longer than the body length (usually by 0.1-0.2mm).In the specimens of Sendaphne described here, the body length was longer than the fore wing length (usually by 0.2-0.4mm).The main reason is the long and slender body form (rather than exceptionally short wings), and an unusually enlarged and extended hypopygium.

Observations of COI barcodes for Sendaphne
Of 46 specimens sampled (Suppl.material 1), we recovered sequences for only 24 specimens.Only three sequences were over 600 base pairs, while the rest were mostly minibarcodes of 102-390 base pairs each.However, six out of the 11 known species now have some molecular data associated (Suppl.material 2).Many of the initial amplifications from ACG were characterised by unintended amplification of the endosymbiont bacteria Wolbachia that were amplified in the first round LepF1/LepR1.These sequences were not mistaken for the wasp DNA (Smith et al. 2012) and are retained on BOLD in the trace files.The standard strategy of amplifying two smaller and overlapping regions secondarily was then followed (as the first round was considered a 'failure').In this case the 5' amplification (LepF1/C_ANTMR1D) worked and the 3' amplification (RonMWASPdeg_t1 -LepR1) failed.Thus many of the ACG Sendaphne are ~260-280bp in length.Finally, regarding the CNC specimens, these were, on average, 35 years old.In those cases, greater success with smaller amplifications compared to larger amplifications is to be expected (Hajibabaei et al 2006).In 2010 and 2011 when these specimens were submitted to the Biodiversity Institute of Ontario (Guelph) for DNA barcoding, they only had the primers generating minibarcode (smaller amplicons) used for PCR.The longer amplicon generating primer pairs were not attempted on these specimens.

Key to Sendaphne species
[This key is intended for female specimens, although two species are only known from males, and in those cases the key accommodates them.Generally, males tend to have darker coloration than the females, especially on the metasoma].
Distribution.Only known from the type locality in Mexico.Molecular data.No DNA could be recovered from the specimen sampled.
Comments.Even though only one male specimen is known, it is sufficiently distinctive to warrant description.This species is the northernmost known distribution of the genus Sendaphne.
Etymology.Named after Dr. Andrew Bennett of the Canadian National Collection, Ottawa, and Canadian expert on Ichneumonidae, in appreciation for his support and encouragement to study braconid wasps.Diagnosis.This species is morphologically similar to Sendaphne penteadodiasae but it has a slightly different color pattern, fore wing vein 1Cu-a much shorter than vein 1Cu-b (subequal in penteadodiasae), much longer and narrow T1, and slightly longer metacoxa.

Sendaphne brasilianus
Distribution.Only known from the type locality in Brazil.Molecular data.No specimen is known to have been sampled for DNA.
Comments.We could not study a specimen of this species, but the original description is sufficiently detailed for recognition, including several line drawings of the metasoma, fore wing, tip of antenna and hind leg (Penteado-Dias 1995).Diagnosis.This species is morphologically similar to Sendaphne bennetti from Mexico, but S. broadi has a narrower mediotergite 1 (length 6.0 × its width at posterior margin vs 4.0 × in bennetti), a more transverse first discal cell in the fore wing (2.0 × as wide as high vs 1.3 ×), and a geographical distribution far apart.
Distribution.Ecuador.Molecular data.No DNA could be recovered from the three specimens sampled.
Comments.Even though only male specimens of this species are known, they are sufficiently distinctive to warrant description.
Distribution.Brazil and Colombia.Molecular data.Four of the paratypes from Brazil (DNA Voucher codes: CNCHYM 07023, CNCHYM 07024 and CNCHYM 07040) as well as the holotype were sampled for DNA.Only one of the paratypes (CNCHYM 07040) rendered a minibarcode of 103 base pairs.
Comments.The Brazilian specimens of S. dianariaspennae were collected between January and March, while the Colombian specimens were collected between May and August.

Sendaphne jatai
Diagnosis.This is the only species where female specimens have the body entirely yellow.Distribution.Brazil, Ecuador, French Guiana.Molecular data.The two specimens from Ecuador rendered minibarcodes of 102 base pairs (CNCHYM 07034) and 164 base pairs (CNCHYM 07035).
Distribution.Brazil, French Guiana, Peru.Molecular data.The CNC specimen from the type locality in Brazil (CNCHYM 07019) rendered a minibarcode of 164 base pairs.
Comments.We could not see the holotype of this species, but the original description is adequately detailed for discrimination.We examined one female from the type locality (collected 30 years after the holotype female), the only known male specimens (from French Guiana), which were discussed in Braet (2006), and the first known specimen from Peru.Other material mentioned in the original description.Almost 70 specimens (females and males) from Brazil, Paraná, Telêmaco Borba, collecting dates between viii.1986 andiii.1987.

Sendaphne paranaensis
Diagnosis.S. paranaensis is one of only three Sendaphne species with head entirely yellow (or yellow-orange).It differs from the other two species (S. jatai and S. olearus) in having dark brown areas on the anteromesoscutum and mesopleuron, and mediotergites 4+ entirely black.
Distribution.Brazil, Paraguay.Molecular data.Of the four specimens in the CNC sampled for DNA, only one (CNCHYM 07037) rendered a minibarcode of 164 base pairs.
Comments.The male specimens from Paraguay are much darker in coloration, but similar variation is mentioned in the original description for the male paratypes from Brazil (Scatolini and Penteado-Dias 1999: 53).Diagnosis.This species is morphologically similar to Sendaphne brasilianus but it has a slightly different color pattern, fore wing vein 1Cu-a subequal to vein 1Cu-b (much shorter in brasilianus), shorter and wider T1, and slightly shorter metacoxa.
Distribution.Only known from the type locality in Brazil.Molecular data.No DNA could be recovered from the two specimens sampled.
Comments.The specimens of this species (housed in the CNC) were previously identified by W.R.M. Mason as "Sendaphne sulmo".However, the morphological differences from the original description of Sendaphne sulmo (see key and diagnosis above), and the disparate geographical distribution allows us to consider the Brazilian and Mexican specimens as separate species.
Etymology.Named after Dr. Angélica Maria Penteado Martins-Dias (Brazil), in recognition of her career studying Braconidae, and also for her work describing most of the previously known species of Sendaphne.Diagnosis.This is the most distinctive species of Sendaphne based on coloration (head, mesosoma, metasoma, and metacoxa black), shape of first discal cell, and narrow mediotergite 1.
Distribution.Only the summit cloud forest at 1,450 m on Volcán Cacao, northwestern Costa Rica.
Molecular data.In BOLD there are data for 16 specimens of this species (the holotype, the paratypes and other specimens that we could not examine) which rendered partial barcodes, most of them from 260 to 390 base pairs.Only the holotype (DNA Voucher code: DHJPAR0031465) had a longer barcode (633 base pairs).
Etymology.This unique species, the only Sendaphne known from Costa Rica so far, is named after Sr. Roger Blanco Segura, of Area de Conservación Guanacaste (ACG), northwestern Costa Rica, in recognition of his 3+ decades of intense care and management of ACG in an enormous variety of circumstances and for a very large array of purposes.Sendaphne sulmo Nixon, 1965: 204. Figs 8-14, 76-78 Holotype.Female, BMNH.MEXICO, Tabasco, Teapa (not examined).
Diagnosis.This is the only known species of Sendaphne with a higher ocellar triangle (i.e., anatomical line tangent to posterior margin of anterior ocellus crosses far above anterior margin of posterior ocelli).The distance between anatomical line tangent to posterior margin of anterior ocellus and anterior margin of posterior ocelli is 0.5 × the diameter of anterior ocelli (Fig. 77), while for all other known species of Sendaphne it is usually 0.1-0.3×.
Comments.We could only study some photos of the holotype (Figs 76-78) and the original description which included a line drawing of the metasoma (Nixon 1965: 207, Figure 255).The drawing shows a T1 slightly narrower medially than the photos of the actual holotype reveal, but the rest of the original description is in agreement with the photos we examined.The males mentioned above are, however, different from the female holotype in having a darker anteromesoscutum, scutellar disc, and metasoma , and also the ocellar triangle is not as elevated.Lacking more specimens to examine (especially females), we have refrained from considering those male specimens as a different species because the two localities are not too far apart and males are known to be darker in other species of Sendaphne.If more material becomes available for study in the future, the status of those specimens may be clarified.
Molecular data.The three male specimens sampled for DNA rendered minibarcodes of 103 base pairs each.ICI-03), and by BOLD/iBOL of the Biodiversity Institute of Ontario and University of Guelph.JFT thanks Yves Braet (Belgium) for making specimens from French Guiana available for this study and Gavin Broad (BMNH) for sending pictures of the specimens housed in London.The suggestions from two anonymous reviewers and the editor considerably improved the final version of this paper.