A taxonomy review of Oreoderus Burmeister, 1842 from China with a geometric morphometric evaluation (Coleoptera, Scarabaeidae, Valgini)

Abstract The species of the genus Oreoderus are morphologically similar, and can be challenging to distinguish without dissecting the male genitalia. In this study, the Oreoderus species from China are reviewed. Three new species of Oreoderus are described: Oreoderus dasystibialis Li & Yang, sp. n., Oreoderus brevitarsus Li & Yang, sp. n. and Oreoderus oblongus Li & Yang, sp. n. A key of the male Oreoderus and a distribution map are provided. Oreoderus coomani Paulian, 1961 was found as a new record in China. The first description of the female of Oreoderus arrowi Ricchiardi, 2001 is provided. Oreoderus humeralis Gestro, 1891, Oreoderus quadricarinatus Arrow, 1944, Oreoderus crassipes Arrow, 1944, and Oreoderus momeitensis Arrow, 1910 are excluded from the Chinese fauna. Furthermore, we utilize geometric morphometric approaches (GM) to analyze the shape variation of four characters (pronotum, elytra, protibia and aedeagus) in Oreoderus. The morphological variations of Oreoderus and the taxonomic value of each character are discussed. The combined analysis of geometric morphometrics and comparative morphology support recognition of the three new species.


Introduction
. The materials used in the geometric morphometric analyses.

Taxonomic approaches
The description of morphological characters follows the terminology of Krikken (1984) and Ricchiardi (2001). Specimen length was measured from the anterior margin of the pronotum to the apex of the pygidium. Specimen width represents the maximum width of the elytra. Type specimens of the new species are deposited in the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZAS) and private collection of Enrico Ricchiardi, Turin, Italy (ERC). The images of female genitalia were drawn using Adobe Illustrator CS5, others were taken using a Nikon D5100 digital camera fitted to a Zeiss Stemi 2000-C stereomicroscope and processed in Helicon Focus 5.1 software and Adobe Photoshop CS5. The distribution map was made in ArcGis 10.0.

Character selection
Four characters (pronotum, protibia, elytra, and aedeagus) were examined and analyzed. There was negligible difference in the shape of pronotum and elytra for the male and female. However, sexual dimorphism often occurs in the protibia of cetoniines and other scarabs (Ricchiardi and Perissinotto 2014, McQuate and Jameson 2011, Holm 1993. Differences between both sexes include the number and acuteness of external teeth. Taking this into account, only male specimens were examined for protibia variation.

Geometric morphometric approaches
The morphology of the four characters (pronotum, protibia, elytra, and aedeagus) was represented by curves. Each curve was based on homologous or corresponding criteria. The pronotum was represented by 5 curves. Curve 1 represented the outline of the pronotum, which resampled into 50 semi-landmarks (SLM). Curve 2 and Curve 3 represented the outline of the carinae, which resampled into 15 SLM. Curve 4 and Curve 5 represented the outline of lateral carinae, which resampled into 10 SLM. The pronotum, elytra and aedeagus were each represented by a single curve, which resampled into 50 SLM (Fig. 1A-D).
These curves were digitized with tps-DIG 2.05 (Rohlf 2006) and all semi-landmarks were converted to landmarks. Landmark configurations were scaled, translated and rotated against the consensus configuration using the GLS Procrustes superimposition method (Bookstein 1991). The principal component analysis (PCA) and canonical variate analysis (CVA) were analyzed in MorphoJ 1.06c (Klingenberg 2011). Because shape differences among species were studied in the PCA analysis, the average or consensus configuration of landmarks for each species was computed. Minimum spanning trees (MST), based on Euclidean distance of the original data points, was computed in PAST 2.04 (Hammer et al. 2001). Curve selection of four characters. A the red curve (Curve 1) is the outline of pronotum, which resampled into 50 semi-landmarks (SLM); the two green curves (Curve 2, 3) are outline of the carinae, which resampled into 15 SLM; two blue curves (Curve 4,5) are the outline of lateral carinae, which resampled into 10 SLM B the curve is the outline of elytra, which resampled into 50 SLM C the curve is the outline of protibia, which resampled into 50 SLM D the curve is the outline of the left paramere, which resampled into 50 SLM.
The Procrustes distances and Mahalanobis distances computed from canonical variate analysis (CVA) can be used to explain the differences and modes of evolution. Procrustes distance is a measure of the absolute magnitude of the shape deviation and indicates how big the differences are between the average group shape. Mahalanobis distance provides an indication of how different an individual is relative to the others in the sample, and how distinctly groups are separated from one another.
Diagnosis. Oreoderus can be distinguished from all other genera within the tribe Valgini by the following characters: a) protibia with only two or three external teeth; b) the first joint of the hind tarsus shorter than the second one; c) pronotum elongated with four carinae; d) visible sternite V twice longer than sternite IV.
Diagnosis. Based on the morphological comparison and PCA analysis of four characters (see below), this new species is close to O. dasystibialis and O. bidentatus, but differs from O. bidentatus by the shape of the carinae of the pronotum and can be separated from O. dasystibialis by the sharp teeth on the protibia and the absence of a thick brush on meso-and metatibia in the male. Finally, the aedeagi are very distinc- Description of the holotype, male. Length 8.2 mm; width 3.9 mm. Color: light brown to brown. Head: clypeus anteriorly rounded, lateral margin extended, with erected setae. Frons densely covered with testaceous scales. Ocular canthus short and broad, covered with same scales as clypeus. Antenna with 10 segments, clubs much longer than antennomeres 2-7. Pronotum: widest at the base, lateral margins sinuate. Surface covered with oblong lied-down scales. Carinae sharp, highly prominent, ending over 1/2 of the pronotum length; lateral small carinae short, sharp, not reaching the lateral margin of the pronotum. Scutellum: triangular, rounded at the apex, covered with shorter oval scales than pronotum. Elytra: with rows of punctate lines, covered with similar scales as scutellum. Propygidium: anteriorly glabrous and slightly punctate, posterior densely punctate and covered with scales. Spiracles moderately elevated. Pygidium: with thick, lied-down scales and one central scale tuft at the hind margin. Venter: coarsely and densely punctate. Visible sternite V smooth and bald in the middle.
Visible sternite V twice longer than sternite IV. Legs: slender, femora and tibia covered with testaceous scales except for protibia. Outer margin of mesometatibia covered with more dense scales. Protibia short and bidentate, external teeth sharp. Tarsomeres with short setae. Parameres: short, lateral margin sinuate, the apex covered with yellow setae.
Description of female. Length 7.5-10.4 mm; width 3.5-4.7. Pronotum broader than the male one; carinae much shorter, reaching about 1/3 of the disc. Propygidium much longer, and pygidium with a sharp stylus. Visible sternite V much broader. Protibia short and with thick brush in the inner margin; tooth slightly blunt, mesometatibia with same brush on the inner and outer margin.
Etymology. The new species is named for the short tarsi of protibia. Diagnosis. based on the morphological comparison and PCA analysis of four characters (see below), this new species is close to O. brevitarsus and O. bidentatus, but differs from O. bidentatus by the smaller lateral carinae of the pronotum and can be separated from O. brevitarsus by the blunt teeth on protibia and the appearance of a thick brush on meso-and metatibia in the male. The aedeagi are very distinctive among these three species. The apex of the parameres in the new species is much wider than in the other two.
Description of the holotype, male. length 8.5 mm; Width 4.3 mm. Color: light brown to brown. Head: clypeus short, anteriorly straight, with erected setae. Frons densely punctate, covered with testaceous scales. Ocular canthus short, covered with same scales. Antenna with 10 segments, clubs much longer than antennomeres 2-7. Pronotum: widest at the base, lateral margins sinuate. Surface densely punctate, covered with thick testaceous scales. Carinae sharp, highly prominent, ending at 2/3 of the pronotum length; lateral small carinae long, sharp, reaching the lateral margins of the pronotum. Scutellum: triangular, rounded at the apex, covered with testaceous scales. Elytra: with rows of punctate line, covered with same scales as scutellum. Propygidium: covered with lied down scales. Spiracles moderately elevated. Pygidium: triangular form, coarsely punctate, covered with thick lied down oval scales. Venter: coarsely and densely punctate with each point bearing a testaceous scale. A longitudinal groove is visible in the middle of visible sternites I-IV; visible sternite V longer twice than the sternite IV. Legs: slender, covered with testaceous scales except protibia. Protibia extended, bidentate, teeth blunt. Meso-and metatibia with thick brush on the outer margins. Tarsomeres with short setae. Parameres: relatively longer and much broader, the apex is the widest part.
Etymology. the new species is named according to its thick brush on meso-and metatibia.
Distribution. China: Hainan. Remarks. only three males were collected in Hainan Island, two of them on Mt. Jianfengling.  Diagnosis. Based on the morphological comparison and PCA analysis of four characters (see below), this new species is close to O. maculipennis, but lacks a spine on the outer margin of meso-and metatibia and elytra without white patch scales in the new species. The parameres are very short in O. oblongus.

Oreoderus oblongus
Description of the holotype, male. Length 8.4 mm; width 4.0 mm. Color: light brown to brown. Head: clypeus anteriorly rounded, with erect setae on the anterior margin. Frons covered with testaceous scales. Ocular canthus short, covered with same scales. Antenna with 10 segments, clubs not much longer than antennomeres 2-7. Pronotum: widest at base, lateral margins sinuate. Surface densely punctate, covered with lied down scales. Carinae sharp, highly prominent, ending around 2/3 of the pronotum length; Lateral small carinae short, sharp, not joining the lateral margin of the pronotum. Scutellum: triangular, rounded at the apex, covered with testaceous scales. Elytra: with rows of punctate line, densely covered with oval scales. Propygidium: broad, punctate, covered with lied down scales. Spiracles moderately elevated. Pygidium: triangular, punctuation rounded, with thick lied down scales. Venter: coarsely and densely punctate with testaceous scales. Visible sternite V twice longer than sternite IV. Legs: slender, covered with oval scales except for protibia. Protibia tridentate; the third tooth small, far from the first two teeth. Meso-and metatibia covered with only sparse scales. Tarsomeres with short setae. Parameres: perpendicular to phallobase, the apex is sharp.
Description of female. Length 9.7 mm; width 4.5 mm. Pronotum a little broader than the male; carinae slightly shorter. Propygidium much longer, and pygidium more highly prominent. Visible sternite V much broader. External tooth of protibia apparently blunt. Tarsomeres more robust.
Etymology. the new species is named for the oblong shape of the body. Distribution. China: Yunnan.
Remarks. This species was previously known from Vietnam and Laos. This is the first record for Yunnan, China. Description of female. Length 8.4 mm; width 2.8mm. Color: light brown to brown. Head: clypeus anteriorly rounded, sharp in the apex, with erected setae. Frons covered with testaceous scales. Ocular canthus short and broad, covered with same scales. Antenna with 10 segments, club much longer than antennomeres 2-7. Pronotum: widest at base, lateral margin sinuate. Surface densely punctate, covered with testaceous scales. Carinae and lateral carinae sharp, highly prominent, ending before middle of pronotum. Scutellum: triangular, rounded at apex, covered with testaceous scales. Elytra: coarsely punctate, covered with testaceous scales. Propygidium: apparently longer than in male, hind margin rounded. Propygidial spiracles moderately el- evated. Pygidium: narrower than in male, with thick lied down scales. Venter: coarsely and densely punctate with testaceous scale. Sternite V twice longer than Sternite IV; Sternite VI much narrower than male. Legs: slender, covered with testaceous scales except protibia. Protibia tridentate, tooth blunter than in male; meso-and metatibia with a spine on the outer margin. Tarsomeres much shorter than in male, covered with short setae.

Oreoderus bidentatus Ricchiardi, 2001
Oreoderus bidentatus Ricchiardi, 2001: 526. Remarks. Oreoderus bidentatus was described from three males from Assam (2 paratypes) and Meghalaya (holotype) and one female from Yunnan (Ricchiardi 2001). One of the authors (Ricchiardi 2001) decided to determine the female specimen from Yunnan as belonging to this species because its two-teethed protibia and other characters similar to the males of O. bidentatus (shape of pronotum and carinae; anterior margin of clypeus, etc.). The second female specimen from the same Chinese Province was identical to the paratype. We hope that the finding of a male specimen from Yunnan will definitively confirm the distribution of this species. The absence of findings of O. bidentatus in Myanmar is probably only due to the lack of research in that country.

Morphological variations of Oreoderus
Oreoderus is the largest genus in the tribe Valgini and easily distinguished by its covering of scales. The clypeus is usually rounded in front except for O. clypealis (the front margin straight and recurved), O. gestroi, O. gravis and O. waterhousei (with a process on the front margin). The pronotum is nearly trapezoidal, longer than wide. There are two pairs of carinae on the pronotum, the length of carinae varies among species. The middle carinae are moderately prominent except O. insularis, O. argillaceus (highly prominent, forming two tubercles) and O. coomani (only slightly prominent). The elytra are short and broad, similar morphologically and sometimes decorated with patches of scales (O. bidentatus, O. maculipennis and O. birmanus etc.). The pygidium is nearly triangular, covered with thick scales. Females of some species have a stylus on the hind margin (O. bidentatus, O. brevitasus).
The morphological variation of four characters (pronotum, protibia, elytra and aedeagus) was investigated based on 34 species (84 specimens) using geometric morphometrics. The shape information was extracted from the landmark data using the Procrustes fit. To see the variations, we used the principal component analysis (PCA). The first two PCs together accounted for 77.11%, 89.14%, 60.50% and 55.96% of the total variance in the analysis of pronotum, elytron, protibia and aedeagus, respectively. The main shape change of the pronotum was observed in the length/width ratio of the pronotum and the carinae (Fig. 7A). The main shape change of the elytra was observed in the length/width ratio (Fig.  7B). The main shape change of the protibia was observed in the length/width ratio, while a secondary shape change can be observed in the curvature of the second teeth on protibia (Fig. 7C). The main shape change of the paramera was in their base, while a secondary shape change can be observed in the length/width ratio of the external part of parameres (Fig. 7D). Morphological variation in the out groups fell within the morphological space  of Oreoderus in all four characters. The similarity among species in these four characters is also reflected in the Minimal Spanning Tree (MST) (Suppl. material 1: Fig. A.1). Based on the PCA results, the morphological diversity in these four characters suggest they are not equally diagnostic in Oreoderus ( Table 2). The highest morphological variation and clearest differentiation among species is found in the parameres. The second most diagnostic character is the protibia and the third is the pronotum. The elytron provides the least morphological resolution among Oreoderus species.

Validity of the new species
To extend the results of the comparative morphological analysis, a canonical variate analysis (CVA) of Chinese Oreoderus including the new species was conducted to quantitatively assess the differences among species, with a particular focus on differentiating the new species. The canonical variates scores of pronotum variables showed the 90% equal frequency ellipse, although there is some overlap between O. maculipennis and O. oblongus (Fig. 8A). The morphological differences based on Mahalanobis distances among the seven species are all highly significant in all pairwise comparisons (p<0.05). Similar results were found for the other morphological variables, as Mahalanobis distances based on the morphology of elytra, protibia and aedeagus were all highly significant in all pairwise comparisons (p<0.05) (Suppl. material 1: Table A.1-4; Fig. 8B-D).
Most obtained p-values from permutation tests (10000 permutation rounds) for Procrustes distances based on the morphology of four characters (pronotum, elytra, protibia and aedeagus, respectively) among the seven species were smaller than 0.05 (Suppl. material 1: Table A.1-4).

Discussion
In this study, the taxonomy values of four characters (pronotum, elytra, protibia and aedeagus) were evaluated in Oreoderus. According the CVA analyses above, the studied specimens were clustered into seven groups for all four morphological characters. Comparisons among species were significantly different when quantified by Mahalanobis distances. This meant the morphological boundary of species based on the sample specimens from the Oreoderus species were distinctly separated from each other. In other words, the Oreoderus species could be statistically separated and determined based on Mahalanobis distances of the four characters. However, the average shape of these four characters was not always significantly different when measured by Procrustes distance, and may not be a useful metric for taxonomic evaluations of Oreoderus. Our results demonstrate that geometric morphometric analysis of external and internal characters can enhance species diagnosis in cryptic species.
The taxonomical value of the four characters examined was not equivalent. The aedeagus of Oreoderus is very distinctive in Valgini, with an overall longer and more robust shape. Also, the aedeagus contains the most morphological variation in Oreoderus, and provides the best character for taxonomic determination in this genus.
The GM analysis suggests that the pronotum and protibia can also be informative in the taxonomy of Oreoderus. The shape of pronotum in Oreoderus is nearly trapezoidal, longer than it is wide. The apical part is usually narrow and the lateral margin is sinuate. The main shape variations are the outline of pronotum, length and relative position of the carinae on the pronotum according to the result of PCA. The protibia of Oreoderus is usually short and flat, dentate with only one spur. The main variation of the protibia is on the external teeth according to the result of PCA.
The numbers of the external teeth of protibia are already used in the taxonomy of Valgini (Paulian 1961, Ricchiardi 1994, Krajčík 2011. For example, the presence of two to three teeth on the outer margin of the protibia is diagnostic of Oreoderus, whereas five or more teeth are found in other genera within Valgini. For other members of the Cetoniinae, the male and female usually differ in the number of protibial teeth and the last teeth are always absent or very small in the females. Compared with the common use of the numbers of teeth, the shape of the protibia is rarely used in the taxonomy of Oreoderus and other Valgini. Our results suggest that the shape of the protibia is diagnostic among the species of Oreoderus.
The elytra of Oreoderus are flat and covered with scales. The elytra contained the least morphological variation among four characters in this study. Additionally, the out groups were not separated from Oreoderus. The taxonomic value of the elytral shape is not highly supported.
Traditionally, discrete characters are commonly used in taxonomy. However it is often difficult to find enough discrete characters to resolve confusing taxonomic problem, such as the morphological convergence of Oreoderus. In such a case, geometric morphometric (GM) can been used (Villemant et al. 2007, Hájek and Fikáček 2010, Xu et al. 2013, Zúñiga-Reinoso and Benitez 2015. Our study is the first to apply this approach to analyze shape variation in Valgini and demonstrate that this tool can be used to resolve this sort of problem. Based on our results, we suggest that future studies will benefit from by incorporating geometric morphometric techniques, and could, for example, examine unknown species of Oreoderus in combination with our data to investigate the possible status of an unknown specimen. Additionally, other characters, such as continuously variable characters, could be examined in addition to those we studied, in order to help resolve morphological differences in other species.