A review of the Indonesian species of the family Signiphoridae (Hymenoptera, Chalcidoidea), with description of three new species

Abstract The Indonesian species of the family Signiphoridae (Hymenoptera, Chalcidoidea) are revised. Three species of Chartocerus are described as new (Chartocerus kartiniae Polaszek & Schmidt, sp. nov., C. sumatrensis Schmidt & Polaszek, sp. nov., and C. javensis Schmidt & Ubaidillah, sp. nov.) and four species of Signiphora, viz., S. bennetti Woolley & Dal Molin, S. flavella Girault, S. perpauca Girault and S. bifasciata Ashmead, are diagnosed.


Introduction
The Signiphoridae are a small family of Chalcidoidea with currently 88 described species in four genera (Noyes 2019). The family has a worldwide distribution, with the majority of species occurring in the tropics. Signiphorids are known primarily

Materials and methods
The specimens were collected during field training courses at the universities in Padang, Sumatra (Andalas University) and Malang, Java (Brawijaya University), as part of capacity building activities for young Indonesian entomologists, and as part of the Indonesian Biodiversity Discovery Project (Cancian de Araujo et al. 2017). The National Park has been recognised as one of the largest remaining tropical rain-forest ecosystems in Java, being designated as a National Park in 2003 with a present area of about 113,357 hectares. During the IndoBioSys Project, Malaise traps and Yellow Pan traps were employed in the Mount Halimun-Salak National Park in West Java. In addition, chalcid wasps were collected using a screen-sweep net (cf. Noyes 1982).
The descriptions of the three new species are based on specimens that were borrowed from the Museum Zoologicum Bogoriense, Research Center for Biology, Indonesian Institute of Sciences. 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 providing country of 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.

DNA sequencing
For DNA studies, whole specimens were sent to the Canadian Centre for DNA Barcoding (CCDB) in Guelph, Canada, for DNA extraction and barcode sequencing, and subsequent recovery of vouchers for preparation and morphological study. DNA extraction, PCR amplification, and sequencing were conducted at the CCDB using standardised high-throughput protocols (Ivanova et al. 2006, deWaard et al. 2008. The 658bp target region, starting from the 5' end of the mitochondrial cytochrome c oxidase I (COI) gene, includes the DNA barcode region of the animal kingdom (Hebert et al. 2003). All specimen data are accessible on BOLD through the following doi: dx.doi.org/10.5883/DS-INDOSIG. The specimen data include 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, primer information, and access to trace files. These data are also available through GenBank (Accession nos MH407234-MH407243).

Data analysis
Sequence divergence statistics were calculated using the Kimura two parameter model of sequence evolution (Kimura 1980), as it is commonly applied in the analysis of DNA barcode sequence data because it evaluates the substitution type (i.e., transitions vs transversions) in addition to the number of nucleotide substitutions between sequences. Barcode Index Numbers (BINs) were assigned by the BOLD system, representing globally unique identifiers for clusters of sequences that correspond closely to biological 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. Sequences were aligned using the BOLD Aligner (amino acid-based hidden Markov models). The analyses are based on sequences with a minimum length of 500 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.

Morphology and imaging
Morphological terminology and the format for species descriptions follow Hayat (2009). Photographs were made using a Leica Ortholux compound microscope with Nomarski Differential Interference Contrast (DIC) illumination and a Leica DM 5000 B with DIC. Images were processed using the stacking software AutoMontage (Synoptics, Cambridge, UK) and Helicon Focus (version 7.0), and further edited using Adobe Photoshop CC 2019. Plates were compiled with Adobe Illustrator CS6. Diagnosis. Body dark brown or black, often with metallic luster. Fore tibial spur simple, without comb of setae (cf. Fig. 3H). Propodeum posteriorly without lamelliform process (except in C. kartiniae with a suggestion of a lamelliform process on the median sclerite). Female antenna with 4 anelli (except in C. kartiniae with 2 anelli), male with 3 anelli. Seta M6 on fore wing marginal vein present, and with additional seta between M2 and M3 (M2b, Fig. 4E). Occipital margin rounded or acute. Mesofemur with 3 or 4 long spines (cf. Fig. 4D). Diagnosis. Head and body dark brown (Fig. 1A, B), fore wing behind marginal vein with infuscation except hyaline area near posterior margin (Fig. 1F). Antenna (in female) with 4 anelli (Fig. 1H). Clava elongate, about 6.7 times as long as broad and 1.5 times as long as scape length (Fig. 1E). Fore wing marginal fringe slightly longer than half the length of the wing disc (Fig. 1F). Midtibial spur subequal in length to corresponding basitarsus (Fig. 1C).
Morphology. Head 1.78 times as broad as frontovertex width (Fig. 1B), frontovertex width slightly less than length of clava (0.84), distance from mouth margin to facial  ridge 0.61 times head height. Antenna with 4 anelli, F4 longer than each of the preceding segments, clava 6.7 times as long as broad and 1.51 times as long as scape length, pedicel 0.45 times as long as scape length (Fig. 1E). Midlobe of mesoscutum with 4 setae in anterior half and 6 setae in posterior half, scutellum with 7 setae along posterior margin. Fore wing 2.7 times as long as broad (Fig. 1F), longest setae of marginal fringe 0.56 times as long as width of disc, seta M1 absent, M2b present. Mesofemur ventrally with 3 long spines (Fig. 4D). Midtibial spur subequal in length to corresponding basitarsus (Fig. 1C), the latter 0.55 times as long as midtibia. Ovipositor nearly twice as long as midtibia (1.82) and 1.29 times as long as hind tibia.
Male. Colour and structure similar to female ( Fig. 2A, B), but antenna ( Fig. 2D) with 3 anelli and longer, clava 7.28 times as long as broad and 2.76 times as long as scape (Fig.  2D). Setae of fore wing (Fig. 2E) stouter than in female. Genitalia as in Fig. 2G, phallobase with a pair of setae, digitus about 3 times as long as broad, strongly curved at apex.
Host. Unknown. Distribution. Only known from the type locality near Malang, East Java, Indonesia. Etymology. The species is named after the island of Java. DNA barcode data. The species exhibits an intraspecific variation of 0.2% and a distance of 12.2% to the nearest neighbour species, C. sumatrensis sp. nov. (Suppl. material 1, Genbank Accession Numbers: MH407241, MH407242).
Remarks. The species is similar to Chartocerus kerrichi (Agarwal) and C. sumatrensis sp. nov. but can be distinguished from the former by the longer clava (6.7 times as long as broad, compared to 4.5 times in kerrichi), the longer fore wing marginal fringe (0.56 times as long as width of disc compared to 0.36-0.40 times in kerrichi), and the longer midtibial spur (subequal in length to corresponding basitarsus whereas distinctly shorter in kerrichi). From C. sumatrensis sp. nov. it can be separated by the shorter ovipositor (less than 2.0 times as long as midtibia, compared to 2.9 times in sumatrensis) and the longer midtibial spur (subequal in length to corresponding basitarsus, compared to 0.7 times in sumatrensis). Diagnosis. Chartocerus kartiniae sp. nov. is unique among Signiphoridae and is provisionally placed in Chartocerus on a balance of genus-level characters. The female can be quickly diagnosed among all Chartocerus species by the following combination of characters: metanotum pale (Fig. 3A); fore wing with discal seta (Fig. 3C); antennal funicle 2-segmented (Fig. 3I). Description (female holotype). Colour. Head and body dark brown (Fig. 3A, B), with metanotum distinctly pale in contrast, legs pale brown with fore tibiae and all tarsi pale yellow (Fig. 3F), antenna yellow-brown with radicle and scape paler (Fig. 3E). Fore wing with brown infuscation centrally and on marginal vein (Fig. 3C).

Chartocerus kartiniae
Morphology. Head 1.52 times as broad as frontovertex width (Fig. 3B), frontovertex width 0.87 times length of clava. Antenna with only 5 antennomeres (excluding radicle); antenna with 2 anelli, clava 5.5 times as long as broad and 1.7 times as long as scape length, pedicel about 0.5 times as long as scape length (Fig. 3E). Midlobe of mesoscutum with 7 setae in posterior half, scutellum with 7 setae along posterior margin. Propodeum with a suggestion of a lamelliform process on the median sclerite. Fore wing 2.8 times as long as broad (Fig. 3C), longest setae of marginal fringe 0.78 times as long as width of disc, all marginal vein setae present, stigmal seta long; discal seta present. Foretibial spur bifid, without a comb (Fig. 3I). Midtibial spur 0.9 times as long as corresponding basitarsus (Fig. 3F), the latter 0.57 times as long as mid tibia. Mesofemur with four long spines anteroventrally. Ovipositor 2.0 times as long as mid tibia and 1.5 times as long as hind tibia.
Host. Unknown. Distribution. Known only from the type locality at Mount Halimun, West Java, Indonesia.
Etymology. The species is named after Raden Adjeng Kartini. DNA barcode data. No sequence data were available for analysis.
Remarks. This species is unique for the genus, having only two funicle antennomeres, and a prominent discal seta on the fore wing. The pale metanotum is unusual (or possibly unique) among known species of Chartocerus (J.B. Woolley, pers. comm.). The foretibial spur is bifid and without a comb, supporting placement of the species in the genus Chartocerus. A full DNA barcode sequence could not be obtained for the single specimen, but analysis of an available DNA fragment suggests membership of the Chartocerus lineage (distant from Signiphora and Thysanus Walker), and a possible sister-group relationship to remaining Chartocerus for which DNA data are available. Diagnosis. Head and body dark brown (Fig. 4A, B), fore wing basally with brown infuscation (Fig. 4E). Antenna (in female) with 4 anelli (Fig. 4H). Clava elongate, about 6 times as long as broad and 1.5 times as long as scape length (Fig. 4G). Fore wing 2.9 times as long as broad, anteriorly with 4 setae, posterior margin of hind wing disc slightly rounded.

Chartocerus sumatrensis
Description (female holotype). Colour. Head dark brown (Fig. 4A, B), legs brown with tarsi pale yellow (Fig. 4C), antenna brown with radicle and basal 3 funicle antennomeres pale yellow (Fig. 4G). Fore wing basally with brown infuscation, and a darker patch behind distal part of submarginal vein and proximal part of marginal vein, and longitudinal dark patch along basal part of hind margin (Fig. 4E).
Morphology. Head 1.69 times as broad as frontovertex width (Fig. 4B), frontovertex width subequal to length of clava, distance from mouth margin to facial ridge 0.66. Antenna with 4 anelli, increasing in size towards apex (Fig. 4H), clava about 6 times as long as broad and 1.5 times as long as scape length, pedicel about 0.5 times as long as scape (Fig. 4G). Midlobe of mesoscutum with 11 setae in posterior half, scutellum with 8 setae along posterior margin. Fore wing 2.9 times as long as broad (Fig. 4E), longest setae of marginal fringe 0.38 times as long as width of disc, seta M1 absent, M2b present (Fig. 4E). Hind margin of hind wing disc slightly rounded and nearly parallel to anterior margin (Fig. 4F). Mesofemur ventrally with 3 long spines (Fig. 4D). Midtibial spur 0.7 times as long as corresponding basitarsus (Fig. 4C), the latter 0.53 times as long as midtibia. Ovipositor 2.9 times as long as midtibia and 2.0 times as long as hind tibia.
Host. Unknown. Distribution. Only known from the type locality near Padang in West Sumatra. Etymology. The species is named after the island of Sumatra. DNA barcode data. A single, full-length barcode sequence of the species is separated from its nearest neighbour species, C. javensis sp. nov., by 12.2% (Suppl. material 1, Genbank Accession Number: MH407238).
Remarks. The species is similar to Chartocerus kerrichi and C. javensis sp. nov., but can be distinguished from the former by the longer ovipositor (2.9 times as long as midtibia, compared to 2 times in kerrichi) and the more slender clava (about 6 times as long as broad, compared to 4.5 times in kerrichi). From C. javensis sp. nov. it can be separated by the longer ovipositor (2.9 times as long as midtibia, compared to less than 2 times in javensis) and the shorter midtibial spur (0.7 times as long as corresponding basitarsus, compared to subequal in javensis).
Morphology. Head 1.5 times as broad as frontovertex width (Fig. 5B), frontovertex width 1.04 times length of clava. Antenna with 6 antennomeres and with 3 anelli, clava 4.6 times as long as broad and 1.7 times as long as scape length, pedicel 0.6 times as long as scape length (Fig. 5E). Midlobe of mesoscutum with 2 fine setae in posterior half, scutellum with 4 fine setae along posterior margin. Fore wing 3.5 times as long as broad (Fig. 5C), longest setae of marginal fringe 1.52 times as long as width of disc, marginal vein seta M1 absent, M5 absent in some paratypes; stigmal seta long (Fig.  5C), vestigial seta base/socket present between M5 and M6 on lower edge of marginal vein, discal seta absent (Fig. 5C). Midtibial spur 0.84 times as long as corresponding basitarsus (Fig. 5F), the latter 0.46 times as long as midtibia. Ovipositor 1.8 times as long as midtibia and 1.4 times as long as hind tibia.
Distribution. Very widespread in the New World (Woolley and Dal Molin 2017). DNA barcode data. The species exhibits an intraspecific variation of 0.2% and a distance of 12.5% to the nearest neighbour species, S. flavella (Suppl. material 1, Genbank Accession Numbers: MN520843, MN520844).
Remarks. The species belongs to the Signiphora flavopalliata Ashmead species group and can be separated from other species in the group by the central dorsal pale band extending from the distal mesoscutum to the proximal propodeal triangle (female), absence of M1 from the marginal vein, and the presence of a minute anterior seta on the marginal vein distal to M2 ( fig. 38 in Woolley and Dal Molin 2017), though this seta is not present in every specimen.  6A) with variable extent of brown markings, clava only apically or completely dusky brown (Fig. 6D). Discal seta on fore wing missing, seta M1 on marginal vein present (rarely absent) (Fig. 6E). Mt8 in female transverse, without a medial emargination.
Description (female). Colour. Body yellow (Fig. 6A), head yellow except occiput with curved brown band along occipital carina (Fig. 6B). Antenna with scape yellow and flagellum brown, apical half of clava darker brown (Fig. 6D). Fore wing with dark band behind marginal vein that is proximally and distally diffusely delimited (Fig. 6E). Third valvula brown.
Morphology. Head 1.76 times as broad as frontovertex width (Fig. 6B), frontovertex width 0.77 times length of clava, distance from mouth margin to facial ridge 0.57. Antenna with 3 anelli, F1 0.4 times as long as broad, F2 0.8 times as long as broad, F3 subquadrate, clava about 6 times as long as broad and 1.7 times as long as scape length (Fig. 6D), pedicel 0.77 times as long as scape length. Midlobe of mesoscutum with 2 setae on disc and anteriorly with 6 setae, scutellum with 6 setae along posterior margin. Fore wing 2.9 times as long as broad (Fig. 6E), longest setae of marginal fringe 1.4 times as long as width of disc, seta M1 present, M2b absent. Midtibial spur subequal in length to corresponding basitarsus (Fig. 6C), the latter 0.56 times as long as midtibia. Ovipositor 2.1 times as long as midtibia and 1.4 times as long as hind tibia.
Male. The species reproduces primarily parthenogenetically; males are very rare (Woolley and Dal Molin 2017) and were not recorded in the study area.
Host. Polyphagous on many species of Diaspididae (Hemiptera). For a full list of host records see Woolley and Dal Molin (2017).
Distribution. Cosmopolitan and, apart from Indonesia, occurring in the following countries (after Woolley and Dal Molin 2017): Algeria, Argentina, Australia, Brazil, Chile, Honduras, Honduras, Greece, India, Israel, Mexico, Morocco, New Zealand, Peru, Puerto Rico, South Africa, Spain, Trinidad and Tobago, USA, Venezuela. DNA barcode data. The sequence data did not reveal any intraspecific variation and a distance of 9.7% to the nearest neighbour species, S. perpauca (Suppl. material 1, Genbank Accession Numbers: MH407235, MH407237, MH407239, MH407240, MH407243). Remarks. The species exhibits variation in colour patterns and several similar looking species that had been described mainly based on colour differences were synonymised with S. flavella by Woolley and Dal Molin (2017). It resembles S. perpauca (see below) but lacks the discal seta in the fore wing.  Description (female). Colour. Body yellow (Fig. 7A), head yellow except occiput with curved brown band along occipital carina (Fig. 7B). Antenna with scape yellow and flagellum brown, apical half of clava distinctly darker (Fig. 7D). Fore wing with dark band behind marginal vein that is proximally and distally diffusely delimited (Fig.  7E), discal seta present. Mt8 in female transverse, without a medial incision.

Signiphora perpauca Girault
Morphology. Head 1.76 times as broad as frontovertex width (Fig. 7B), frontovertex width 0.77 times length of clava, distance from mouth margin to facial ridge 0.57 times head height. Antenna with 3 anelli (Fig. 4D), F1 0.4 times as long as broad, F2 0.8 times as long as broad, F3 subquadrate, clava about 6 times as long as broad and 1.7 times as long as scape length (Fig. 7D), pedicel 0.77 times as long as scape length. Midlobe of mesoscutum with 2 setae on disc and anteriorly with 6 setae, scutellum with 6 setae along posterior margin. Fore wing 2.9 times as long as broad (Fig. 4E), longest setae of marginal fringe 1.4 times as long as width of disc, seta M1 present, M2b absent. Midtibial spur subequal in length to corresponding basitarsus (Fig. 4C), the latter 0.56 times as long as midtibia. Ovipositor 2.1 times as long as midtibia and 1.4 times as long as hind tibia.
DNA barcode data. The sequence of the single examined specimen is separated by 9.7% from the nearest neighbour species, S. flavella (Suppl. material 1, Genbank Accession Number: MH407234).
Remarks. The species belongs to the Signiphora flavopalliata species group and can be separated from other species in the group, among other characters, by its light colour (female) and the presence of a discal seta in the fore wing ( fig. 306 in Woolley and Dal Molin 2017).
Diagnosis and remarks. The Neotropical species was recorded as a parasitoid of the cycad aulacaspis scale, Aulacaspis yasumatsui Takagi (Hemiptera, Diaspididae) in the Bogor Botanic Gardens in 2011 (Muniappan et al. 2012). This represents the first record of the family Signiphoridae from Indonesia. The species can be separated from other Signiphora species by the following characters (see Muniappan et al. 2012 and figures therein): discal seta present on both fore wing and hind wing, fore wing infuscate from the wing base to the distal end of the stigmal vein, submarginal vein with 2 setae, marginal vein of the hind wing with 1 seta, female antenna with 2-3 annelli, marginal fringe of fore wing very long, nearly half as long as width of disc, mesosoma brown except scutellum and metanotum which are pale tan, yellow or white, mesoscutum with about 16 setae, scutellum with 6 setae.
with special thanks to Ari Jamsari (Andalas University, Padang) and Fatchiyah Fatah (Brawijaya University, Malang) for course organisation and management, and Hagus