Published information: 2n = 54 + X0 (♂), XX (♀) (Dutton and Angus 2007). Mitotic chromosomes, arranged as karyotypes, are shown in Fig. 1a–e. Fig. 1a shows a paratype male, Giemsa stained, Fig. 1b, c shows a male from the Colle del Nivolet, the same nucleus Giemsa stained and C-banded, and Fig. 1d, e shows the same nucleus from a female from the Paso San Bernardino, Giemsa stained and C-banded. The chromosomes in these preparations are in complete agreement with each another. Metaphase I of meiosis from a male from the St Gotthard Pass is shown in Fig. 2a (Giemsa stained) and b (the same nucleus C-banded). The C-banded preparation enables the unpaired X chromosome to be identified. Although preparations from only two of the specimens attempted resulted in complete karyotypes, several other specimens yielded almost complete karyotypes and in no case were more than 55 (♂) or 56 (♀) chromosomes counted. Since B. griseostriatus has a karyotype with 61 or 62 chromosomes (♂, ♀) it is very unlikely that this species was present in either sample.

We extracted and sequenced two specimens of B. alpestris from the same St. Gotthard and San Bernardino populations used to obtain the karyotypes, which had almost identical sequences for the mitochondrial genes (with only 1 mismatch in the COI gene) as two specimens from the same localities collected in 2000 and 2002 respectively (Table 2) and reported as S. griseostriatus in Abellán et al. (2013). These four specimens differ from one sequenced B. alpestris from Colle del Nivolet in five nucleotides in the COI gene (with a length of 826), and one in the 16S gene (with a length of 796 nucleotides). The nuclear gene H3 (with a length of 328 nucleotides) was identical for all sequenced specimens of B. alpestris (Table 1), and identical to the other Palaearctic species of the genus.

Figure 1.

Mitotic chromosomes from mid gut of Boreonectes spp., arranged as karyotypes. a–e B. alpestris, a paratype ♂, Giemsa stained (from Dutton, Angus, (2007)) b, c ♂, Colle del Nivolet b Giemsa stained, c the same nucleus C-banded (from Angus (2010)) d, e San Bernardino, ♀, d Giemsa stained e the same nucleus C-banded f–k B. emmerichi f, g ♂ Yalashenshan f Giemsa stained g the same nucleus C-banded h, i ♀ Yalashenshan h Giemsa stained g the same nucleus C-banded j, k ♂ Mado j Giemsa stained k the same nucleus C-banded (from Angus et al (2015)) l–n B. macedonicus ♂♂ (from Angus (2008)) l Crno ezero Giemsa stained m Karanikoličko ezero Giemsa stained n the same nucleus C-banded o, p B. emmerichi ♂ shown in f, g q, r B. macedonicus Crete q ♂Giemsa stained r ♀C-banded (from Angus (2008). Scale = 5 μm.

Figure 2.

B. alpestris ♂ St Gotthard meiosis metaphase I a Giemsa stained b the same nucleus C-banded. Scale = 5 μm.

Published information: 2n = 52 + X0 (♂), XX (♀) (Angus et al. 2015). Mitotic chromosomes, arranged as karyotypes, are shown in Fig. 1f–k, n, o. The new preparations, from the Kangding area, (Fig. 1f–i) are much better than the Qinghai ones shown by Angus et al. (2015), with the chromosomes less condensed and the C-banding more clearly defined. They confirm the published features of the karyotype and also allow improved comparison of the karyotypes of B. emmerichi and B. macedonicus (Georgiev, 1959). Thus autosome pair 12 was stated to be more evenly metacentric in B. emmerichi, and comparison of the Sichuan male preparation (Fig. 1o, p) with a similarly elongate preparation of B. macedonicus from Karanikoličko ezero (Fig. 1m, n) and with Cretan B. macedonicus (Fig. 1q, r) confirms this. Similarly, the smaller size of autosome pair 26 when compared with pairs 24 and 25 in macedonicus as against the more similar sizes of these three pairs in emmerichi is confirmed. However, the possibly smaller X chromosome in emmerichi, as was suggested by preparations of mitosis but not metaphase II of meiosis, is shown not to be the case. This demonstration that these are chromosomally distinct species is in complete agreement with the degree of difference shown by the COI DNA of the two species (Fig. 5).

Figure 3.

B. emmerichi collecting sites. a, b site 1, Yalashenshan, 4052 m a.s.l, b with Fenglong Jia c site 2, Yalashenshan, 4074 m d site 4, Yajiaheng, 3337 m a.s.l. e site 3, Boij ta, 3495 m a.s.l. with Robert Angus collecting.

Figure 4.

B. emmerichi, habitus. a lectotype b, c males from site 1, Yalashenshan d pale specimen from Maduo e the palest specimen seen, from the western Kun Lun mountains.

Figure 5.

Phylogeny of the Palaearctic species of Boreonectes, obtained with maximum likelihood in RAxML using the combined mitochondrial and nuclear data. Numbers in nodes, bootstrap support. See Table 2 for details of the specimens.

One of the surprises associated with collection of this species in 2016 was that the specimens from the St Gotthard pass appeared, even in the field, as noticeably larger than those from the San Bernardino. At the time it seemed that the St Gotthard specimens might be B. griseostriatus and those from the San Bernardino B. alpestris. In the event this proved not to be the case, but the size differences remain. Thus 10 ♂♂ from the San Bernardino range in length from 4.0–4.4 mm, with a mean length of 4.26 mm, while for 17 ♀♀from the same site the values are 5.2–5.1 mm, mean 4.38. From the St Gotthard the values are: 5 ♂♂, 4.3–4.6 mm, mean 4.51, and 8 ♀♀, 4.4–4.6 mm, mean 4.56 mm.

As mentioned in the Introduction, the type material of B. emmerichi has more extensive darker markings, especially on the pronotum, than material from Qinghai, though it is matched by material from Sejilashan in southern Xizang (Angus et al. 2015). It was suggested that this more extensive darkening may be associated with wooded habitats. Latitude and longitude references for the type localities are, for Tatsienlu Tjiji 30°25' (=30.417°) N 102°40'(=102.667°) E (Balke 1992) and for Mukue Tatsienlu 30.05°N 102.03°E (Sykes et al. 2006). Google Earth gives altitudes of 1944 m a.s.l. for Tatsienlu Tjiji and 3935 m a.s.l. for Mukue Tatsienlu, with both localities in wooded areas. The localities from which B. emmerichi were collected in 2016 were: 1: Yalashenshan, 30.215°N 101.757°E, alt. 4052 m a.s.l.; 2: Yalashenshan, 30.205°N 101.755°E, alt. 4074 m a.s.l.; 3: Boij ta car parking area near Xinduqiao on the Kangding–Lhasa road, 30.048°N 101.569°E, alt. 3551 m a.s.l.; and 4: Yajiaheng, 29.927°N 101.995°E, alt. 3337 m a.s.l. Only the Boij ta locality (site 3) was in a wooded zone. In all localities water levels had risen recently due to the ongoing rain (see the silty water in Fig. 3e) but most of the pools had some aquatic vegetation. Only the pool shown in Fig. 3d had few beetles, suggesting that it was only recently flooded. All the chromosome preparations were from specimens collected at site 1. All this material has extensive dark markings. Fig. 4a shows the lectotype of B. emmerichi, while Fig. 4b, c shows two specimens from site 1. The specimen shown in "b" is a very close match for the lectotype and is the specimen from which the chromosomes shown in Fig. 1f, g were obtained. It seems clear that this more extensive darkening of the specimens is a regional phenomenon and not just a response to immediate local conditions.

Material from the northern part of the Tibetan Plateau is generally paler, and Fig. 4d shows a particularly pale specimen from Maduo, original shown as Fig. 1g by Angus et al. (2015). The palest specimen so far seen is from the western end of the Kun Lun mountains, taken by Ying Zhang. I have seen only a photograph of this specimen and this, though not good, does show the pattern (Fig. 4e).

The analysis of the combined mitochondrial and nuclear data recovered a monophyletic Boreonectes with strong support (Fig. 5), although due to the reduced number of outgroups its monophyly cannot be assessed adequately. The Palaearctic species, on the contrary, were not monophyletic, as B. emmerichi was found to be closer to some Nearctic specimens of the B. griseostriatus (De Geer, 1774) complex than to the western Palaearctic species, although with low support (Fig. 5). The Western Palaearctic species were recovered as monophyletic with strong support, and divided into the eastern Mediterranean B. macedonicus (Gerogiev, 1959) and B. riberae (Dutton & Angus, 2007) on one clade and all the western species on the other. Within the latter clade, species seem to be very close to each other, with poor resolution among them and no variation in the nuclear marker (H3, see above), suggesting a very recent expansion and differentiation between them. Contrary to the lack of genetic variation among the Western Palaearctic species of the group, the three studied Nearctic specimens show a high divergence, with ca. 13% of variable positions in the COI gene. There are no available data on the karyotypes of these Nearctic populations, but most probably they represent a complex of undescribed species.

The Palaearctic species show a clearly uneven distribution of their diversity (Fig. 6). Thus, all the material from the Tibetan Plateau is B. emmerichi, with only limited variation in COI haplotypes. This can be compared with the situation in the Iberian Peninsula, occupying an area similar in size to the eastern part of the Tibetan Plateau, from which the B. emmerichi data have been obtained. Here again only one species, B. ibericus (Dutton and Angus 2007) is present, and this species has a wider distribution, also occurring in the French Alpes-Maritimes and Italian Alpi Marittime (Dutton and Angus 2007; Angus 2008), on Corsica (Angus 2010a) and in the Middle Atlas of Morocco (Angus 2010b). The occurrence of B. multilineatus (Falkenström, 1922) in the Pyrenees (Angus 2010b) reflects higher diversity, and this species is also known from inland Sweden and the British Isles (Dutton and Angus 2007) and, from COI data, from the Faroe Islands. The COI of this species appears sufficiently distinct (Fig. 5) to give confidence to the identification, although the study of more material is needed. However, the centre of high diversity is the Alps, where four chromosomally distinct species occur. One of these, B. inexpectatus is known only from a single lake, the Lac de Lauzet Inférieur (Dutton and Angus 2007) but the other three, B. ibericus, B. griseostriatus and B. alpestris, are more widely distributed, though apparently always allopatric. It is possible that the diversity of this group of species in Western Europe is, in part at least, a result of faunal movements associated with Pleistocene climatic fluctuations. B. griseostriatus group species are known as fossils in deposits dating from the Last Glaciation in the English Midlands (e.g. Coope et al. 1961), as well as from Starunia in the Ukrainian Carpathians (Angus 2010a), both areas outside the current known ranges of these species.

Figure 6.

Maps of the distributions of various B. griseostriatus group species. a the Tibetan Plateau showing the localities of studied material of B. emmerichi b the Iberian Peninsula with the distributions of B. ibericus and B. multilineatus c The Alps with the distributions of B. ibericus, inexpectatus, griseostriatus and alpestris.