Seven Sycoryctine Fig Wasp Species (Chalcidoidea: Pteromalidae) Associated with Dioecious Ficus hirta Inhabiting South China and Southeast Asia

Simple Summary The non-pollinating fig wasps are essential components of fig wasp communities, negatively impacting mutualism. However, this group of fig wasps has received less taxonomic attention than pollinating fig wasps. This study presents seven new non-pollinating fig wasp species associated with Ficus hirta fig trees inhabiting South China and Southeast Asia. The presence of a long ovipositor sheath characterizes this group of fig wasps. An identification key is provided to distinguish between them, and the relationships with their host fig trees are discussed. The type specimens and examined materials are deposited in the South China Botanical Garden, Chinese Academy of Sciences, China. Abstract Even though non-pollinating fig wasps are essential components in tropical and subtropical habitats, yet they are poorly described in the Oriental communities. This study presented seven new sycoryctine fig wasp species associated with Ficus hirta fig trees inhabiting South China and Southeast Asia. These new sycoryctine species belong to the genera Philotrypesis, Sycoryctes, and Sycoscapter. They can be easily distinguished by their adaptive morphologies such as face sculpture, body-color, and ovipositors. An identification key is provided to differentiate between them, and the relationships with their host fig trees are also discussed. The holotypes and paratypes are both deposited in the South China Botanical Garden, Chinese Academy of Sciences, China.


Introduction
The taxonomy of sycoryctines has changed over the past ten years, and their phylogeny is currently more defined due to the advancement of molecular techniques [5,6]. These fig wasps belong to the subfamily Sycoryctinae and are highly diversified and geographically widespread [5]. Sycoryctines are associated with all six subgenera and at least 15 sections in Ficus [7]. Surprisingly, an estimated 826 species (2.7 species per Ficus) are waiting to be discovered in the Old World [5]. Two sycoryctine species have been described Biology 2022, 11, 801 2 of 12 recently in Taiwan [8] and India [9], and hopefully more Oriental sycoryctines are going to be described.
Their complicated trophic relationships make sycoryctines the ideal study species for the population dynamics in tropical and sub-tropical habitats [13]. A recent molecular study showed eight allopatric sycoryctine species associated with Ficus hirta Vahl in South China and Southeast Asia. The marked barcoding gaps ranged from 7.2% to 15.7% for the Cytb gene sequence in the same genus [14]. However, the morphological traits of these species remain unknown. This study compared their morphology and reported seven new species belonging to the genera Philotrypesis, Sycoryctes, and Sycoscapter. An identification key is provided to distinguish between them, and the relationships with their host

Materials and Methods
The specimens of sycoryctine fig wasps associated with F. hirta were collected in 23 sampling sites for Philotrypesis, 15 sites for Sycoscapter, and two sites for Sycoryctes during the years 2010 to 2018 are distributed from South China to Java (Table 1). The distribution of the species in each genus is allopatric except for two sites with two species in each genus co-occurred, QMS in Thailand and XI in China (Table 1). When figs close to ripening were dissected, the wasps emitted therein were collected. Two to twenty-four wasps in each site with one wasp exited from one natal fig were collected and stored in 75% ethanol. Later, the wasps were dehydrated through an ethanol series (80%, 90%, and 100%) and critical-point dried (LEICA EM CPD300, Leica Microsystems GmbH, Wetzlar, Germany) before being mounted on cards following Noyes (1982) [15]. Each photo was taken using a digital camera connected to a stereomicroscope (LEICA M205 FA, Leica Microsystem GmbH, Wetzlar, Germany). The images were processed using LAS X 3.08.19082 software to create a stacked image with increased focal depth [16]. Physical characteristics were measured using ImageJ 1.8.0_172 software (National Institutes of Health, Bethesda, MD, USA). Specimen measurements were taken with an accuracy of 0.001 mm and rounded to the nearest 0.01 mm.
Specimens were mounted on brass stubs and sputter-coated with gold (LEICA EM ACE600, Leica Microsystem GmbH, Germany) before the observation and photographed using SEM (JEOL JSM-6360LV, JEOL Ltd., Tokyo, Japan). Morphological terminology follows Gibson (1997) and the Hymenoptera Anatomy Ontology (HAO) Portal [17,18]. The holotypes and a group of paratypes are deposited in the Plant Science Center, South China Botanical Garden, Chinese Academy of Sciences (23 • 10 48" N; 113 • 21 8" E). Diagnosis: The female of this genus can be recognized by its lengthened seventh and eighth urotergites and its subquadrate pronotum.        Male. Unknown. Etymology: Named after the Java Island of Indonesia.
Head. Width 0.2-0.5 mm. Eye longer than gena. Antenna inserted above the bottom line of compound eye. Toruli approach, distance between toruli smaller than diameter of one torulus. Funicular segments slightly longer than wide. Face sculpture smooth. Epistomal margin slightly protruded.
Head. Width 0.3-0.5 mm. Eye longer than gena. Antenna inserted at the bottom line of compound eye. Toruli approach, distance between toruli smaller than diameter of one torulus. Funicular segments slightly longer than wide. Face sculpture smooth. Epistomal margin slightly protruded.

Diagnoses of Female Sycoryctine Species Associated with Ficus Hirta
The female of Philotrypesis guangdongensis is morphologically similar to P. yunnanensis and P. fujianensis; however, its toruli located slightly below the bottom line of compound eyes. The mouthpart of P. fujianensis is longer than F. guangdongensis and F. yunnanensis and it extended just below the central of the compound eyes. The black band of P. yunnanensis on scutellum is indistinct compared to P. guangdongensis and P. fujianensis, P. yunnanensis also has a non-bifurcated line on its mesoscutum.
Both the females belong to the genus Sycoryctes and Sycoscapter have metallic green body color and a relatively long ovispositor; however, the knob of Sycoryctes on stigmal vein does not produce downward. Sycoscapter chinensis does not has deep face sculpture and an acute epistomal margin projection compared to S. thaiensis and S. singaporensis.

Discussion
This study confirmed that the dioecious F. hirta inhabiting Southeast Asia is associated with at least seven morphologically distinct sycoryctine fig wasp species. The seven sycoryctine species associated with F. hirta can be distinguished morphologically by antennae, epistomal margin, face sculpture, body-color, and ovipositors. This study shows that the number of non-pollinating species on a dioecious fig tree across many geographical areas is higher than previously thought. The limited number of non-pollinating species is either due to the less sampling effort or may be due to the low dispersal ability of fig wasps in the  dioecious fig community, which may promote the diversification of these sycoryctine fig wasps [14].
F. hirta is a shrub widely distributed in the tropics and subtropics from Java in the south to China in the north and westwards into northeast India [19]. It was initially thought to be symbiosis with one pollinating species and two non-pollinating species [20]. However, through our extensive geographical sampling and molecular sequencing analysis, the nonpollinating fig wasps in the genus of Philotrypesis and Sycoscapter, which have initially been considered one species, are divided into four and three species, respectively [14]. These non-pollinators are mainly allopatric distributed. The differences in barcode gaps among them in the same genus are no more than 15.7%. Compared with the same genus in other fig species [5], these species are closely related. Some cases have been found in other broadly distributed fig species, such as F. pumila [21], F. racemosa [22], and F. septica [23]. Those results suggest that fig wasps are more likely to differentiate into new species due to their relatively short generation time than their host figs.
Although we have found more related species using molecular sequencing in both pollinating and non-  [25]. For example, the fig wall thickness of F. hirta at the northern limit of China is thicker than that of south China. Accordingly, the ovipositor length of Philotrypesis fujianensis distributed there is also significantly longer.
Exploring the speciation or host switching in the conservative sycoryctine phylogeny is pivotal to understanding the biological variability of non-pollinating fig wasps in the Old World. It is noteworthy that maritime Southeast Asia comprises thousands of tropical, segregated islands. The species richness across these habitats is consistently underestimated [26]. F. hirta is also distributed on some surrounding islands, such as Kalimantan. We may find more species if we identified the samples of non-pollinating fig wasps from more islands. Hence, additional sampling is necessary to establish a solid reference for further comparative studies, especially within mainland and maritime Southeast Asia.