The identities of two species in the Pterostichus macrogenys species group of subterranean carabid beetles (Coleoptera, Carabidae) revealed by external morphometric analysis and comparative genital morphology

The identities of two species in the species group of subterranean carabid beetles (Coleoptera, Carabidae) revealed by external analysis and comparative Abstract The Pterostichus macrogenys species group is an endemic subterranean Japanese carabid lineage that shows marked regional differentiation, but unresolved taxonomic issues remain, even at the species level. Based on morphological examinations of the genital structures of newly collected specimens and an external morphometric analysis of all the species concerned, P. falcispinus Sasakawa, 2005 syn. nov. is synonymized with P. asahinus Habu & Baba, 1960, and P. awashimaensis sp. nov. is described from Awashima Island, a small island off the coast of Honshu. Based on external morphometrics, P. awashimaensis was determined to be most similar to P . yahikosanus Sasakawa, 2009, but its external and genital features differ distinctly from the latter species and it is thought to be more ancestral.


Introduction
The macrogenys species group of Pterostichus subgenus Nialoe Tanaka, 1958 (s. lat., i.e., Nialoe sensu Sasakawa 2021) is a Japanese endemic subterranean lineage in the beetle family Carabidae. This group shows marked differentiation in mountainous areas of Honshu, and ca. 30 species are currently recognized (Sasakawa et al. 2020). Members of the species group are flightless due to their atrophied hind wings, and are characterized by a large head with long mandibles and flattened body, which is thought to be associated with a subterranean lifestyle. Sasakawa et al. (2020) were the last to revise the group and revealed that coexisting different-sized species and disjunct distributions are more common than previously recognized (Fig. 1). However, some issues remained unresolved.
This study sought to resolve two taxonomic issues in the macrogenys species group: the relationship between P. asahinus Habu & Baba, 1960 and P. falcispinus Sasakawa, 2005, and the identity of the population on Awashima Island, a small island off the coast of Honshu. The fact that the holotype of P. asahinus and the available specimen from the Awashima population are both females, which provide less taxonomic information than males, has hampered resolution of these taxonomic issues. To overcome this, we performed external morphometric analysis, a useful taxonomic method that can also be applied to females (e.g., Sasakawa and Kubota 2007;Ober, and Connolly 2015;Ortuño et al. 2021), in addition to the usual genital morphology examinations.

Materials and methods
Specimens newly examined here were collected using subterranean baited traps, which were also used in our previous studies (Sasakawa and Itô 2017). This study applied discriminant analysis using external morphometrics of female specimens, morphological examinations of the endophallus of male genitalia of a male specimen newly collected near the type locality of P. asahinus, and morphological examinations of genital membranous parts of the female specimen from Awashima Island.
The discriminant analysis was used to investigate morphometrically whether the identity of the P. asahinus holotype and our newly defined P. asahinus is supported and to which species the Awashima specimen is most similar. The following specimens were examined together with the Awashima specimen and P. asahinus holotype: P. adatarasanus 3♀, P. asahinus (redefined here to include P. falcispinus) 17♀, P. chokaisanus 2♀, P. eboshiyamanus 3♀, P. iwakiensis 7♀, P. monolineatus 1♀, P. ohsawacavus 1♀, P. takadateyamanus 25♀, and P. yahikosanus 6♀. These samples include all species to which the Awashima specimen and P. asahinus holotype may belong and for which female specimens are available. Scaled photos obtained previously (Sasakawa et al. 2020), from the specimens newly examined here, and of the P. asahinus holotype in the type-specimen database of the National Agriculture and Food Research Organization (Division of Informatics and Inventory, Insect Systematics Unit, Institute for Agro-Environmental Sciences, National Agriculture and Food Research Organization 2011) that were size-calibrated using information on the body length in the original description (15.2 mm; Habu and Baba 1960) were analyzed. Using these photos, four parts of the pronotum were measured with ImageJ v.1.50i (Rasband 2016) to the nearest 0.01 mm: pronotum length along the median line (PL), pronotal anterior margin width (PAW), pronotum width at the widest part (PW), and pronotal posterior margin width (PPW). Next, linear discriminant analysis was performed for a dataset excluding the Awashima  (2021)  specimen and P. asahinus holotype, with "species" as the response variable and the four measurements as explanatory variables. Then, the species identities of the Awashima specimen and P. asahinus holotype were determined using the obtained function. To capture the results visually, scatterplots based on the first two canonical variates were created. All statistical analyses were performed in R v.3.4.3 (R Development Core Team 2017). Information on specimens and the raw data are in Appendix 1.
The male endophallus was everted and fully inflated by injecting toothpaste from the base of the aedeagus (Berlov 1992). For the female genitalia, muscles around the genitalia were dissolved using 5% potassium hydroxide, and the organs were cleaned and observed in pure water. The terminology of the male genitalia followed Sasakawa et al. (2020) and Sasakawa and Itô (2015). Both male and female genitalia were compared with photos of the organs of related species, which were taken during previous studies (Sasakawa andItô 2015, 2017;Sasakawa et al. 2020). Notes. Sasakawa et al. (2020) suggested that P. asahinus and P. falcispinus might be conspecific. However, this hypothesis remained untested because males from the type locality of P. asahinus have not been examined. The male specimen examined here was obtained from a valley southwest of Mt. Dôrokujinpô, which can virtually be regarded as the type locality (Fig. 1). The structures of the endophallus and right paramere of this specimen are identical to those of the eastern type of P. falcispinus in Sasakawa et al. (2020). Its body length is intermediate between that of the eastern and western types of P. falcispinus as follows: body length from mandible apices to elytral end (BLm) 16.32 mm, that from anterior margin of labrum (BLl) 14.72 mm, and that from clypeal apex (BLc) 14.26 mm. Based on these results, we regard P. asahinus and P. falcispinus as conspecific. The results of the discriminant analysis support this conclusion. The P. asahinus holotype was classified in this newly defined P. asahinus in the discriminant function analysis, and was within the area of P. asahinus on the scatterplot of the first two canonical variates (Fig. 3).  Pterostichus (Paralianoe) macrogenys macrogenys: Habu and Baba (1972): 19 (part). Pterostichus macrogenys: Tanaka (1985): 114 (part?); Shimizu (2001): 23.
Notes. In the discriminant function analysis, the Awashima specimen was classified as P. yahikosanus. On the scatterplots of the first two canonical variates, the specimen was located outside of, but close to, the area of P. yahikosanus (Fig. 3). These results suggest that the Awashima specimen is most similar in external morphology to, but different from, P. yahikosanus. Differences between the Awashima specimen and P. yahikosanus were also observed in other morphological features: the body length of the Awashima specimen (see below) is smaller than that of P. yahikosanus (n=6 from Mt. Yahikosan; BLm 15.32-14.35, mean 14.94 mm; BLl 14.22-13.46, mean 13.73 mm; BLc 13.64-12.89, mean 13.18 mm) (Fig. 4A, B); in the pronotum, anterior angles are barely produced, hind angles are obtuse, and posterior margin is arcuate posteriorly behind the laterobasal impressions in the Awashima specimen (Fig. 4C), whereas in P. yahikosanus, anterior angles are notably produced, hind angles are right-angled to acute, and the posterior margin behind the laterobasal impressions is almost straight (Fig. 4D); the body of the Awashima specimen is darker than that of P. yahikosanus (Fig. 4A-D); and pigmentation on the innermost part of vagina is smaller in the Awashima specimen, less than half the size of median and seminal apophyses (Fig. 4E), while it is larger than these apophyses in P. yahikosanus (Fig. 4F). In the macrogenys species group, pronotum shape and the pigmentation on the innermost part of vagina differ distinctly among species and are used as important species-level diagnostic characters (e.g., Sasakawa 2005;Sugimura 2005;Morita et al. 2013). Because these characters differ distinctly between the Awashima specimen and P. yahikosanus, we describe the Awashima specimen as a new species. Based on information on the morphological features of the pronotum and female genitalia in related species (Sasakawa et al. 2020 and references therein), notably produced pronotal anterior angles and developed vaginal pigmentation are limited to some species and considered to be derived character states in the macrogenys species group. Therefore, the Awashima species is considered more ancestral than P. yahikosanus with respect to the pronotal and genital features.
Description. BLm 13.79 mm; BLl 12.66 mm; BLc 12.13 mm. Head, pronotum, and elytra dark brown; appendages reddish brown. Dorsal surface almost smooth except for laterobasal impressions of the pronotum, the anterior half of which bear several transverse wrinkles and the posterior half of which are weakly punctate.
Head large, widest at tempora, which are distinctly swollen; width at the widest point larger than pronotal posterior margin width; length from clypeal apex to neck base longer than pronotum length along the median line. Left mandible larger than the right and curved at the apical 1/4; length between mandible apex and posterolateral end on dorsal side slightly shorter than 2.5 times the anterior width of the clypeus. Eyes weakly convex, with the anterior-posterior length longer than 1/2 length of antennal segment 1. Antennal segment 2 with two setae.
Pronotum cordate, notably flat, widest at apical 1/5. Lateral margins arcuate on apical 2/3, slightly sinuate on basal 1/3; two marginal setae on each lateral side, anterior setae near widest pronotal point, and posterior setae near hind angles. Anterior margin emarginated, with curvature approximately the same as that of apical 2/3 of lateral margins; anterior angles widely rounded and barely produced. Hind angles obtuse. Posterior margin only slightly emarginated at median area, weakly but distinctly arcuate posteriorly behind the laterobasal impressions. Median line impressed in the middle, not reaching either the anterior or posterior margins; laterobasal impressions single, shallow.
Elytra sides almost parallel, less convex; shoulder distinct, but not denticulate; apices rounded; scutellar stria present, connected to stria 1 on the right side but not on the left; 1 setigerous puncture on stria 1 at the level of the posterior end of scutellum; two setigerous punctures on interval 3, anterior one slightly before the middle and posterior one on apical 1/5, both adjoining stria 2. Hind wings completely atrophied. First fore tarsomere no adhesive hairs on ventral side.
Female genital structures identical to those of other consubgeners; apophyses of seminal canal and median oviduct fully sclerotized; pigmentation on the innermost part of the vagina present, but less than half the size of apophyses of seminal canal and median oviduct; other parts of vagina lack conspicuous pigmentation.