A new species of Nephus (Nephus) (Coleoptera, Coccinellidae) described from Reunion Island

Abstract We report here a new species belonging to Nephus (Nephus) Mulsant. Nephus (Nephus) apoloniasp. nov. was collected in the Reunion Island (Mascarene Archipelago, Indian Ocean). We describe this new species and redescribe and illustrate three other Nephus species already known from Reunion: Nephus (Nephus) oblongosignatus Mulsant, 1850, Nephus (Geminosipho) reunioni (Fürsch, 1974) and Nephus (Nephus) voeltzkowi Weise, 1910. Furthermore, we present a phylogenetic tree for these four species and calculate the genetic distances between them, using high-throughput DNA sequencing of the mitochondrial genome. The similar external morphology of N. apoloniasp. nov. and N. voeltzkowi very probably explains why individuals from the first species have been mistakenly identified as the latter and were not recognized as different until now. Other than external and genitalia traits, the present study provides molecular evidence confirming these are indeed two different species.


Introduction
The Coccinellidae is a diversified family composed of some 6000 species, and the largest of the superfamily Coccinelloidea (Robertson et al. 2015). Seago et al. (2011) formally recognised two subfamilies within the Coccinellidae, Microweisinae and Coccinellinae sensu Ślipiński (2007). This last subfamily includes most of the Coccinellidae tribes, among them the Coccidulini sensu Seago et al. (2011), to which Nephus belongs. Nephus was first considered by Mulsant (1846) as a subgenus of Scymnus but Mader (1924) and then eventually Pope (1957) treated it as a valid genus.
The following characters distinguish Nephus: antennae with nine or pseudo-11 antenomeres; prosternal process sub-quadrangular, as wide as long, with a shallow lateral depression, without carina; legs with tarsi trimerous; abdomen with six ventrites, with incomplete postcoxal line, recurved and not reaching posterior margin of first ventrite.

Material and methods
The specimens examined here were provided by the first author from a laboratory rearing (Laboratoire Evolution & Diversité Biologique, Université Toulouse III) initiated from field collected material: Nephus oblongosignatus and N. voeltzkowi were collected in Reunion Island in 2011, N. apolonia sp. nov. was collected in Reunion Island in 2013, and N. reunioni was collected in 2007 in Portugal, where the species had been introduced for biological control in the 1980's (Magro et al. 1999). The first author also observed specimens collected from 2006 to 2012 by the Insectarium of Reunion to investigate possible misidentifications of N. apolonia sp. nov. with N. voeltzkowi and to gather additional information on the geographical distribution of the new species on Reunion Island.
Photographs of the external morphology as well as male and female genitalia were taken using a Leica DMC 2900 Digital Camera attached to Leica M205C stereomicroscope using Leica Application Suite. Furthermore, specimens were examined with a JEOL JSM-6360LV scanning electron microscope in the Electronic Microscopy Center of Universidade Federal do Paraná. The length and width measurements of the species represent the average of the examined specimens.
The terminology used in the descriptions follows Ślipiński (2007). Labels of the type specimens are arranged in sequence from top to bottom, where the data for each label are within double quotes (""), a slash (/) separates the rows, and information between brackets ([]) provides additional details written on the labels.
We used the mitochondrial genome of Nephus species previously sequenced by Magro et al. (2020): N. reunioni, N. includens, N. voeltzkowi and N. apolonia sp. nov. (voucher number: NeSpa1), together with the mitogenome of N. oblongosignatus sequenced in the present study (accession numbers: see Table 1) following the same protocol (see details in Magro et al. 2020).
Molecular characterization and distance analyses were conducted on the cytochrome c oxidase I (COI) gene using MEGA v.7 (Kumar et al. 2016). Pairwise distances were estimated between specimens using the Kimura-2-parameters model (Kimura 1980). We reconstructed the phylogenetic relationships between the Nephus species based on the mitogenome sequences (all protein coding and tRNA genes, but we deleted the control region because of the high divergence between species and the presence of repeated sequences, leading to low quality alignments in this region), using as outgroup the available sequence of Cryptolaemus montrouzieri which belongs to the same tribe as Nephus (i.e., Coccidulini sensu Seago et al. 2011) together with other Coccinellidae species (accession numbers: see Table 1). Sequences were aligned using MAFFT default parameters (Katoh and Standley 2013). We inferred maximum likelihood trees and bootstrapping with RAxML 8.2.10 (Stamatakis 2014) under the best-fitting model of sequence evolution for the dataset (GTR+G model), selected using the Akaike information criterion (AIC) using jModelTest 2 (Darriba et al. 2012).

Results and discussion
The species of Nephus present the following characteristics: antennae with nine or pseudo-11 antenomeres (  Each elytron black with one big oval yellowish elongated spot, reaching middle of elytron; spermatheca with sharp base and truncated apex (Fig. 4)........

Diagnosis.
Nephus oblongosignatus is similar to N. voeltzkowi and N. apolonia sp. nov. but differs in the body shape, size and shape of the spots and the pattern of genitalia.
Remarks. It should be noted that Fürsch (2007), in his remarks about N. reunioni, mentions "The species is referred from various authors from South Africa (det. Fürsch), and even from Israel and Portugal. These specimens are breeded for pest control, but they seem to be misidentifications and in fact N. derroni." In what concerns the Portugal population, we do not agree with Fürsch's statement. Indeed, the specimens collected in Portugal and analyzed in the present study correspond to the original N. reunioni description by Fürsch presented in Chazeau et al. (1974). Raimundo (1992), who first described N. reunioni for Portugal, also illustrated the external morphology and genitalia corresponding to the original description by Fürsch in Chazeau et al. (1974). In both cases, the observations show that the specimens from the Portuguese population are distinct from N. derroni, first described from S. Tomé and presented in Fürsch (1974). Weise, 1910 Figs 1, 4 Nephus (Nephus) voeltzkowi Weise, 1910: 512 (original description);Fürsch 2007: 6 (systematics). Nephus seychellensis Sicard, 1912: 362 (original description); Chazeau et al. 1974: 272 (synonymy). Scymnus (Nephus) voeltzkowi: Korschefsky 1931: 153 (catalog) ;Fürsch 1966: 181 (systematics). Nephus (Nephus) voeltzkowi: Poussereau et al. 2018: 128 (systematics) Diagnosis. Nephus voeltzkowi resembles N. oblongosignatus and N. apolonia in the color of the integument and spots but differs in the shape and size of the spots and the female genitalia.

Remarks.
It should be noted that only female specimens of N. voeltzkowi were observed here. Furthermore, although Magro et al. (2020) performed a large sampling campaign on Reunion, they never found N. voeltzkowi males, and eventually demonstrated that Reunion females are parthenogenetic. As indicated by Magro et al. (2020), the presence of N. voeltzkowi was reported before by Chazeau et al. (1974) in their fauna of ladybirds from Reunion, but the sex of the specimens was not mentioned: although Chazeau et al. (1974) presented an illustration of the genitalia of a N. voeltzkowi male, the possibility that the drawing was based on a Madagascar specimen was not discounted (Chazeau pers. com.). In the absence of the original material, we cannot confirm this information. Diagnosis. Nephus apolonia sp. nov. is similar to N. voeltzkowi and N. oblongosignatus but differs by the size and shape of the spots and the pattern of genitalia.
Female. Length 1.88 mm, width 1.30 mm. Similar to male. Genitalia with coxites longer than wide, subtriangular, 3.0× longer than wide; stylus mamiliform with long bristles (Fig. 5L). Spermatheca heavily sclerotized, slightly arched, not very striated, and marked by one strong constriction in the middle, with sharp base and rounded apex (Fig. 5K).
Etymology. This species is named after an early name of Reunion Island, mentioned as "Santa Apolonia" on the Portolan charts (nautical charts) from the XVI th century (GENUNG, 2017).
Type locality. Reunion Island: from a laboratory rearing (Laboratoire Evolution & Diversité Biologique, Université Toulouse III) initiated from field material collected in December 2013 in Manapany-les-Bains and Étang-Salé.
Molecular analysis. The mitochondrial genome of one specimen of Nephus apolonia is deposited in Genbank under accession number MN164644. Genetic distances based on the COI sequences between N. apolonia and other Nephus species confirm that N. apolonia is different from the other species, as all distances are within the same range (i.e., 0.13-0.17) ( Table 2). In the phylogenetic tree reconstructed from 14,867 pb of aligned mitochondrial genomes (Fig. 6), most nodes, including N. apolonia, were supported by high bootstrap values.