Eurydema, Laporte, 1833

1. Differentiation of the species included in the genus Eurydema

Green clade: E. oleracea / sea / nana / herbacea

Together   GoogleMaps , these four species formed a monophyletic group, well supported in both analyses (BP ML = 100, BP NJ = 100). Eurydema herbacea (Herrich-Schaeffer, 1833) samples (all from France) were only weakly separated from the rest of the group (BP ML = 70, BP NJ = 89). None of tested MOTU models separated individuals from these species, and genetic distances between them were low (e.g. between E. oleracea and E. sea 0.007 [0–0.012], the largest distance was found between E. oleracea and E. herbacea , 0.012 [ 0.007 –0.016]).

Pink clade: E. lundbladi / E. ornata : clade 1—specimens from Spain, Canary Islands, Morocco, Madeira and part of those from Corsica (LPRCI1674-21 and LPRCI798-21)

E. lundbladi specimens grouped with all specimens of E. ornata from Spain, Canary Islands, Morocco, Madeira and two Corsican individuals (LPRCI1674-21 and LPRCI798-21) in a well-supported clade (BP ML = 97, BP NJ = 95). However   GoogleMaps , these two groups are only weakly supported: BP ML = 64, BP NJ = 78 for E. lundbladi and BP ML = 56, BP NJ = 78 for clade 1 of E. ornata . The   GoogleMaps genetic distance between E. lundbladi and the E. ornata clade 1 was 0.025 [ 0.011 –0.035], while the maximum intraspecific distances were 0.005 and 0.011, respectively. This interspecific distance was much greater than the observed intraspecific variation and supported the recognition of two distinct and consensual MOTUs corresponding to the two species, as shown in Fig. 5 View FIGURE 5 .

Red clade: E. ornata from Europe and China: clade 2—excluding specimens from Spain, Morocco, Canary Islands, Madeira, and Corsican specimens (LPRCI1674-21 and LPRCI798-21)

All other specimens identified as E. ornata formed a clade weakly supported in both analyses (BP ML = 71, BP NJ = 58), but better supported in Fig. 4 View FIGURE 4 (BP NJ = 87). This clade included all specimens from France, Switzerland, Greece, Crete (labelled as Kriti in the phylogenetic trees), Cyprus and China and the remaining Corsican   GoogleMaps samples. The   GoogleMaps genetic distance between the two E. ornata clades was 0.048 [ 0.038 –0.056], while the maximum intraspecific distance within the “European” E. ornata clade 2 was 0.02.

Blue clade: E. gebleri / dominulus / cyanea

All specimens identified as belonging to these three taxa were recovered in a well-supported clade (BP ML = 91, BP NJ = 96). Eurydema gebleri Kolenati, 1846 formed a distinct and robustly resolved clade (BP ML = 94, BP NJ = 100), clearly separated from specimens as assigned to E. dominulus and E. cyanea (Fieber, 1864) , which themselves clustered into another strongly supported clade (BP ML = 96, BP NJ = 99). The genetic distance between the two clades E. gebleri / E. dominulus from Europe was 0.058 [ 0.051 –0.065]. Specimens identified as Asian E. dominulus formed a loosely-knit clade (BP ML = 57, BP NJ = 68) with a maximum intraspecific distance of 0.027. All MOTU species delimitation analyses (except for bPTP) consistently identified these specimens as a distinctive and consensual MOTU. The genetic distance between the Asian E. dominulus clade and the European clade ( E. dominulus / E. cyanea ) was 0.020 [ 0.015 –0.035]. European specimens identified as E. dominulus or E. cyanea fell within a strongly supported clade (BP ML = 98, BP NJ = 99). The divergence between specimens identified as E. dominulus and those identified as E. cyanea was 0.001 [0–0.005]. Maximum intraspecific variation reached 0.005 in European E. dominulus and 0.003 in E. cyanea . With the exception of the PTP method, all other MOTU species delimitation analyses grouped European E. dominulus and E. cyanea specimens together.

Dark gray clade: E. ventralis / spectabilis / eckerleini

These   GoogleMaps three taxa were grouped in one well-supported clade (BP ML = 97, BP NJ = 100), which was split in two distinct clades, robustly resolved lineages: one comprising specimens identified as E. spectabilis / eckerleini and the other including those identified as E. ventralis (BP ML = 99, BP NJ = 100 and BP ML = 95, BP NJ = 98, respectively). The   GoogleMaps genetic distance between these two clades was approximately 3 to 4 %. The   GoogleMaps specimens identified as E. spectabilis and E. eckerleini , all collected from Crete, clustered tightly together although their identification is still unconfirmed (see Discussion   GoogleMaps ). Maximum   GoogleMaps intraspecific divergence reached 0.008 in E. spectabilis / eckerleini and, 0.012 in E. ventralis . Within   GoogleMaps E. ventralis , individuals were clustered in two supported groups (BP ML = 90, BP NJ = 89 and BP ML = 84, BP NJ = 89). The   GoogleMaps first one included samples from Greece and China, while the second one encompassed specimens from Western Europe   GoogleMaps ( Spain, France, Corsica and Switzerland). The genetic distance between these two clades was 0.019 [ 0.014 –0.026], with a maximum intraspecific variation of 0.009 and 0.012, respectively. A single MOTU was recovered by three out of four species delimitation methods. Bootstrap values (e.g. BP ML = 90 and BP NJ = 84) supporting the separation between the Greek, Chinese and Western European samples were taking into account in the final consensus.

Light gray clade: E. fieberi / rotundicollis

Specimens from Spain and France formed a monophyletic, well-supported clade (BP ML = 90, BP NJ = 97), while the Greek specimen (JSTR06848_0101) was clearly different.

Specimens from Spain and Gard ( France) clustered in a highly supported clade (BP ML and NJ = 100). This clade was further subdivided into two and strongly supported subclades, corresponding respectively to the Spanish and Gard specimens (BP ML = 98, BP NJ = 99 for both clades). These two clades were separated by a genetic distance of 0.011 [ 0.009 –0.014]. The maximum intraspecific divergence was 0.003 for Spain and 0.002 for Gard (based on two sequences only). All species delimitation analyses, except ASAP1, consider the Spain and Gard clades as a single MOTU.

French specimens from the Alps identified as E. fieberi or E. rotundicollis were grouped into a strongly supported clade (BP ML = 97, BP NJ = 100), which was recovered as a single MOTU by all species delimitation analyses. Sequences were split in two subclades also well-supported (BP ML = 89, BP NJ = 99 and BP ML = 87, BP NJ = 94) and the intergenetic distance between them was 0.019 [ 0.017 –0.020]. The first subclade was composed of 5 specimens identified as E. fieberi with an intraspecific distance of 0.005. The second comprised 17 specimens identified as E. fieberi or D. rotundicollis with a maximum intraspecific divergence of 0.005.All species delimitation methods retrieved two distinct MOTUs.

Other Eurydema species

We sequenced two additional species, E. nigriceps and E. rugulosa (Dohrn, 1860) . Although only two sequences were available for each, both were clearly differentiated from all other Eurydema species based on maximum bootstrap support (BP ML and NJ = 100) and all MOTU delimitation analyses.

2. Structuration of the genus Eurydema into subgenera

The genus Eurydema is currently divided into three subgenera: Eurydema , Rubrodorsalium Stichel, 1944 and Horvatheurydema Dupuis, 1951 ( Dupuis 1951; Derjanschi & Péricart 2005) ( Table 1 View TABLE 1 ). Most species have been assigned to these subgenera (albeit sometimes unsatisfactorily) but six Asian species remain unassigned ( Rider 2006; Aukema et al. 2013). The currently accepted subgeneric classification of species is as follows:

—Subgenus Eurydema : E. eckerleini , E. gebleri , E. herbacea , E. laticollis Horváth, 1907 , E. lundbladi , E. nana , E. nigriceps , E. oleracea , E. ornata , E. putoni (Jakovlev, 1877) , E. sea , E. syriaca Kirkaldy, 1909 , E. wilkinsi Distant, 1879 .

—Subgenus Rubrodorsalium : E. blanda Horváth, 1903 , E. cyanea , E. dominulus , E. liturifera (Walker, 1867) , E. maracandica Oshanin, 1871 , E. montana Kerzhner, 1972 , E. mrugowskyi Stichel, 1944 , E. spectabilis , E. ventralis .

—Subgenus Horvatheurydema : E. caligata Horváth, 1901 , E. fieberi , E. rotundicollis , E. rugulosa .

In our phylogenetic tree, the placement of species in their respective subgenus was not fully consistent, and the corresponding nodes lacked bootstrap support. Species E oleracea , E. herbacea , E. sea , E. nana , E. ornata and E. lundbladi , all currently assigned to the subgenus Eurydema were recovered as a single clade with moderate support (BP ML = 84, BP NJ = 68). In contrast, E. nigriceps , E. gebleri and E. eckerleini , also assigned to the subgenus Eurydema were not grouped within this clade. E. fieberi and E. rotundicollis (except for one specimen from Greece) formed a clade consistent with their placement in the subgenus Horvatheurydema , with strong bootstrap support (BP ML = 90, BP NJ = 97). Eurydema dominulus and E. cyanea were grouped in a well-supported clade (BP ML and NJ = 96); E. ventralis , E. spectabilis / E. eckerleini also formed a distinct and strongly supported clade (BP ML = 97, BP NJ = 100). The two groups assigned to the subgenus Rubrodorsalium —excluding E. eckerleini , which is currently placed in the subgenus Eurydema —were not grouped together but the bootstraps at the key nodes are not significant.