Corresponding author: Robert B. Angus ( email@example.com )
Academic editor: Dorota Lachowska
© 2017 Robert B. Angus, Ignacio Ribera, Fenglong Jia.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Angus RB, Ribera I, Jia F (2017) Further studies on Boreonectes Angus, 2010, with a molecular phylogeny of the Palaearctic species of the genus. Comparative Cytogenetics 11(2): 189-201. https://doi.org/10.3897/CompCytogen.v11i2.11980
Karyotypes are given for Boreonectes emmerichi (Falkenström, 1936) from its type locality at Kangding, China, and for B. alpestris (Dutton & Angus, 2007) from the St Gotthard and San Bernardino passes in the Swiss Alps. A phylogeny based on sequence data from a combination of mitochondrial and nuclear genes recovered western Palaearctic species of Boreonectes as monophyletic with strong support. Boreonectes emmerichi was placed as sister to the north American forms of B. griseostriatus (De Geer, 1774), although with low support. The diversity of Palaearctic species of the B. griseostriatus species group is discussed.
Dytiscidae, Boreonectes, B. emmerichi, B. alpestris, molecular phylogeny, diversity, karyotypes
The genus Boreonectes Angus, 2010 comprises small diving beetles and most of the species are endemic to the Nearctic Region (
In June–July 2016 one of us (R. B. Angus) had the opportunity to visit the Kangding area and collect B. emmerichi from its type area, for chromosome analysis. This confirms the identity of the Qinghai material as B. emmerichi. Then in August 2016 R. B. Angus was able to visit Switzerland and to collect Boreonectes from the St Gotthard and San Bernardino passes, localities where I. Ribera & A. Cieslak had in 2002 collected material considered to be B. griseostriatus (De Geer, 1774) (
This study aims to clarify the identities of B. emmerichi (Falkenström, 1936) and B. alpestris Dutton & Angus, 2007, from some localities in the Alps using karyotype and molecular data. To establish their phylogenetic positions, we build a molecular phylogeny of the genus Boreonectes including most Palaearctic and a representation of Nearctic species.
The material used for chromosome preparations is listed in Table
Specimens were brought back to the laboratory alive and placed in small aquaria where they were fed with living larvae of Chironomidae (Diptera). Chromosome preparation and photography were as described by
Material giving chromosome preparations.
|B. emmerichi (Falkenström, 1936)||CHINA Sichuan. Kangding County Yalashenshan.30.215°N,101.757°E
Small pools 4052 m a.s.l.
|2 ♂♂, 4 ♀♀|
|B. alpestris (Dutton & Angus, 2007)||SWITZERLAND Ticino. San Bernardino pass. 46.499°N 101.755°E. Pool 2030 m a.s.l.||1♀|
|SWITZERLAND Ticino. St Gotthard pass
46.559°N 8.562°E Pool 2112 m a.s.l.
For DNA extraction and sequencing we used the same methodology as various recent works on the same family Dytiscidae (e.g.
We sequenced four mitochondrial genes in two PCR reactions: 3’ end of cytochrome c oxidase subunit (COI); and a single fragment including the 3’ end of the large ribosomal unit (16S), the whole tRNA–Leu gene and the 5’ end of the NADH dehydrogenase 1. We also amplified fragments of one nuclear gene, histone 3 (H3) (see
Material studied for the molecular phylogeny, with voucher number, locality and collector, and EMBL accession numbers. In bold, sequences newly obtained for this study.
|1||B. alpestris (Dutton & Angus, 2007)||
||Italy||Piemonte. Gran Paradiso Nat. Park. Colle del Nivolet, roadside lake at ca 2500 m. 3.9.2010||R.B. Angus||LT796525||LT796551||LT796537|
||Switzerland||Col de San Bernardino, ponds||R.B. Angus||LT796523||-||LT796535|
||Switzerland||St. Gottardo pass, summit||R.B. Angus||LT796524||-||LT796536|
|4||B. alpestris||MNCN-AI298||Switzerland||Col de San Bernardino, ponds 13.10.2002||I. Ribera &
|5||B. alpestris||NHM-IR309||Switzerland||St. Gottardo pass, summit, 27.7.00||I. Ribera &
|6||B. emmerichi (Falkenström, 1936)||
||China||Sichuan, Kangding County, Yalashenshan. Small pools 4052m 27.6.2016||R.B. Angus, F-L Jia, Z-Q. Li & K. Chen||LT796531||-||LT796541|
||China||CHINA Sichuan.Kangding County.Yalashenshan, Small pools 4074 m a.s.l. 27.6.2016||R.B. Angus, F-L Jia, Z-Q. Li & K. Chen||LT796528||-||LT796538|
||China||Sichuan, Kangding County Boij ta Car parking zone, Grassy and weedy pools. 3531 m a.s.l. 28.6.2016||R.B. Angus, F-L Jia, Z-Q. Li & K. Chen||LT796529||-||LT796539|
||China||Sichuan, Kangding County Yajiaheng, Shallow stony & peaty pools 3337 m a.s.l. 30.6.2016||R.B. Angus, F-L Jia, Z-Q. Li & K. Chen||LT796530||-||LT796540|
||China||N. Qinghai Hu, Gangca, 1 km SE of Gangca Dasi, stream, 3464m 5.6.2013||R.B. Angus, F.L. Jia & Y. Zhang.||LT796526||LT796555||-|
||China||Qinghai, Golo, Maduo, Roadside pools on river flats ca 20 km SE Maduo. 8.6.2013||R.B. Angus, F.L. Jia & Y. Zhang.||LT796527||-||-|
||Tibet||S Tibet, S Namtso lake 4750m, banks, 21.7.2010||J. Schmidt||LT796532||LT796552||LT796542|
|13||B. funereus (Crotch, 1873)||MNCN-AI1208||California (US)||California, 9.2000||Y. Alarie||HF931173||HF931393||LT796543|
|14||B. griseostriatus (De Geer, 1774)||MNCN-AI952||Sweden||prov. Angermanland, Hörnefors parish, Norrbyskäv island, rock pool, 23.6.2006||A.N. Nilsson||LT796533||LT796553||LT796544|
|15||B. griseostriatus (De Geer, 1774) cplx||MNCN-AI1160||California (US)||Napa Co., Knoxville Recreation Area, 2000||T. Berendonk||HF931168||HF931387||LT796547|
|16||B. griseostriatus cplx||MNCN-AI1150||California (US)||Sacramento Co., Clay Station Rd., 20.6.1999||W.D. Shepard
& C.B. Barr
|17||B. griseostriatus cplx||
||California (US)||Marin Co., Seasonal Pools in Dillans Beach Dunes, Spring 2011.||D. Post||HF931317||HF931541||LT796546|
|18||B. griseostriatus strandi (Brinck, 1943)||MNCN-AI1082||Norway||Bugöynes, 29.7.2006||S. Ligaard
& B. Andrén
|19||B. ibericus (Dutton & Angus, 2007)||NHM-IR22||Portugal||Sa. Da Estrela, Torre, lagoon 25.7.1998||I. Ribera||EF670064||EF670030||EF670157|
|20||B. macedonicus (Georgiev, 1959)||MNCN-AI1120||Macedonia||Macedonia, Sar (Shar) Planina, Karanikolicko ezero Black Nick’s Lake, 6.9.2006||R.B. Angus||LT796534||LT796554||LT796549|
|21||B. multilineatus (Falkenström, 1922)||MNCN-AI115||Faroes (Isl.)||20.9.2004||J. Hansen||HF931165||HF931384||-|
|22||B. riberae (Dutton & Angus, 2007)||MNCN-AI829||Turkey||Düzce, between Kartalkaya and Çaydurt, pools in mountain pass, 1700m 23.4.2006||I. Ribera||HF931232||HF931461||LT796550|
|23||B. striatellus (Le Conte, 1852)||NHM-IR295||California (US)||Mono co., Long Valley 19.6.2000||I. Ribera
& A. Cieslak
|24||Stictotarsus falli Nilsson, 2001||NHM-IR334||New Mexico (US)||New Mexico, 9.2000||Y. Alarie||EF670063||EF670029||EF670155|
|25||Stictotarsus roffii (Clark, 1862)||NHM-IR335||Texas (US)||Texas, 9.2000||Y. Alarie||AJ850607||AJ850355||EF670158|
We aligned the sequences using the MAFFT online v.6 and the Q–INS–i algorithm (
Published information: 2n = 54 + X0 (♂), XX (♀) (
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
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
Published information: 2n = 52 + X0 (♂), XX (♀) (
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.
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.
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 (
Material from the northern part of the Tibetan Plateau is generally paler, and Fig.
The analysis of the combined mitochondrial and nuclear data recovered a monophyletic Boreonectes with strong support (Fig.
The Palaearctic species show a clearly uneven distribution of their diversity (Fig.
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.
We thank the Natural History Museum London, Royal Holloway University of London and the Biology School of Sun Yat-sen University, Guangzhou for research facilities, Dandan Zhang for her help for booking hotel and flight tickets for the Kangding collection, Zhiqiang Li and Mr. Kai Chen for their help when we were working on field, A. Izquierdo, R. Alonso and A. Cardoso (