Research Article |
Corresponding author: David R. Maddison ( david.maddison@science.oregonstate.edu ) Academic editor: James Liebherr
© 2016 David R. Maddison, Roy Anderson.
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:
Maddison DR, Anderson R (2016) Hidden species within the genus Ocys Stephens: the widespread species O. harpaloides (Audinet-Serville) and O. tachysoides (Antoine) (Coleoptera, Carabidae, Bembidiini). Deutsche Entomologische Zeitschrift 63(2): 287-301. https://doi.org/10.3897/dez.63.10748
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Beetles previously considered to be Ocys harpaloides (Audinet-Serville) from northern Africa, Spain, France, the United Kingdom, France, and Belgium belong to two species. These species can be distinguished using DNA sequences of 28S rDNA, 18S rDNA, COI, CAD, and topoisomerase I. A key, diagnoses, and images are provided to enable identification of specimens based upon characteristics of male and female genitalia, as well as microsculpture and other external structures. Through examination of the holotype of Bembidium harpaloides v. tachysoides Antoine, as well as designation of lectotypes of Bembidion harpaloides Audinet-Serville and Ocys melanocephalus Stephens, and designation of a neotype for Tachis rufescens Guérin-Ménéville, the valid names of the two species were determined to be Ocys harpaloides and Ocys tachysoides (Antoine).
DNA taxonomy, cryptic species, Europe, ground beetle, Bembidiini , Ocys
Among European carabid beetles of the tribe Bembidiini Stephens, Ocys harpaloides (Audinet-Serville, 1821) is one of the more distinctive, looking more similar to members of other tribes (for example, the trechine Trechus Clairville, 1806 or the tachyine Porotachys Netolitzky, 1914) than to other bembidiines. As currently circumscribed, Ocys harpaloides is a common and widespread Mediterranean-Atlantic species, ranging from the Azores, Algeria and Morocco north to southern Norway, Britain and Ireland, and east to Italy, the Balkans and Greece (Lindroth 1992).
The only congener with which O. harpaloides is documented to coexist throughout much of this range is Ocys quinquestriatus (Gyllenhal), a species from which it can easily be distinguished by numerous morphological characters (Lindroth 1974; see below). There are more than 20 additional species of Ocys Stephens; almost all of these are rare, and restricted to small geographic areas (e.g.,
In Northern Ireland, two morphologically distinct forms of “Ocys harpaloides”, correlated with habitat, have been reported (
In 2011 we began an examination of the DNA sequences of these forms, and more detailed study of morphological variation. This revealed that two species were indeed present in Northern Ireland, and elsewhere in the range of “Ocys harpaloides”. As this paper was being prepared for publication,
Members of Ocys were examined from the collections listed below. Each collection’s listing begins with the coden used in the text.
BMNH
The
CTVR Luca Toledano Collection, Verona, Italy
DE Dominique Echaroux Collection, Etréchy, France
PB Paolo Bonavita Collection, Rome, Italy
RAC Roy Anderson Collection, Belfast, United Kingdom
Morphological methods. General methods of specimen preparation for morphological work, and terms used, follow
Photographs of male genitalia were taken with a Leica Z6 and JVC KY-F75U camera using Microvision’s Cartograph software for extended depth of field (EDF) processing; the images thus potentially have some artifacts caused by the EDF algorithm.
The following measurements were made:
Length: body length, from apex of the labrum to apex of the longer elytron.
GCR: gonocoxite ratio = gonocoxite 2 length / gonocoxite 1 length (Fig.
Measurements were made either using Microvision’s Cartograph software processing images from a JVC KY-F75U camera on a Leica Z6 lens, or on a Leica M3Z stereomicroscope with a Leitz graticule.
The density of the microsculpture lines on the left elytron was measured by counting the number of microsculpture lines that cross a 0.1 mm longitudinal line placed in the centre of the third elytral interval approximately 1/3 of the distance between the front and back of the elytron.
Taxon sampling for DNA studies. We obtained DNA sequence data for 15 Ocys specimens (Table
Locality details for sequenced Ocys exclusive of O. quinquestriatus. Four-digit numbers at the start of each row are D.R. Maddison DNA voucher numbers. Specimen 0569 had been previously sequenced by
Ocys harpaloides | |
2760 | Northern Ireland: Colliery Bay, Ballycastle, Antrim, 55.2116°N 6.1910°W. 5.vii.2011. At grass roots on slope above high water mark. R. Anderson |
2762 | Northern Ireland: Colliery Bay, Ballycastle, Antrim, 55.2116°N 6.1910°W. 5.vii.2011. At grass roots on slope above high water mark. R. Anderson |
2759 | Ireland: Wexford, North Slob. 52.3546°N 6.4082°W. 20.vii.2011. Under driftwood on sandy beach. R. Anderson |
0569 | Belgium: Schorisse, Bos Ter Rijst. Approximately 50.78°N 3.69°E. 17.iv.1997. Temperate broadleaf forest. Konjev Desender |
2937 | Belgium: Schorisse, Bos Ter Rijst. Approximately 50.78°N 3.69°E. 17.iv.1997. Temperate broadleaf forest. Konjev Desender |
2853 | Belgium: Halve Maan, Oostende. 51.237°N 2.932°E. 23.ix.1995. Coastal salt marsh (non tidal). K. Desender. |
4983 | Spain: Sitges. 41.2383°N 01.8244°E. 9.ix.2006. W Maddison. |
Ocys tachysoides | |
2758 | Northern Ireland: Belfast, Belvoir Forest. 54.5544°N, 5.9404°W. 10.iii.2011. Under bark dead alder and willow. R. Anderson |
2761 | Northern Ireland: Belfast, Belvoir Forest. 54.5544°N, 5.9404°W. 10.iii.2011. Under bark dead alder and willow. R. Anderson |
2763 | Northern Ireland: Belfast, Belvoir Forest. 54.5544°N, 5.9404°W. 10.iii.2011. Under bark dead alder and willow. R. Anderson |
2898 | Northern Ireland: Co. Down, Mount Stewart. 54.5491°N 5.6037°W. 12.x.2011. Under bark of sycamore/cedar/pine by sea. R. Anderson |
2899 | Northern Ireland: Co. Down, Murlough NNR. 54.2461°N 5.8330°W. 15.xi.2011. Under bark of sycamore, sandy wood near sea. R. Anderson |
2936 | Belgium: Moerzeke, De Kramp. Approximately 51.06°N 4.15°E. 1.iii.1999. Tidal freshwater marsh along the River Schelde. Konjev Desender |
2938 | Belgium: Kastel, Gespoelde. Approximately 51.05°N 4.16°E. 11.ix.1998 Tidal freshwater marsh along the River Schelde. Konjev Desender |
4606 | Germany: Nordrhein-Westfalen, Landkreis Unna, Selm-Bork, Lippeaue. 51.6533°N 7.4442°E. Under bark of Salix. K. Hannig |
Sampling of Bembidiina species. Four-digit numbers in the “#” column entries for Ocys are D.R. Maddison voucher numbers for sequenced specimens. Data for all non-Ocys specimens, as well as to specimens 1077 and 0569, were obtained from GenBank from previous studies (
DNA sequencing. Genes studied, and abbreviations used in this paper, are: 28S: 28S ribosomal DNA (D1-D3 domains); 18S: 18S ribosomal DNA (near full-length); COI: cytochrome oxidase I; CAD: carbamoyl phosphate synthetase domain of the rudimentary gene; Topo: topoisomerase I. In addition, sequences of the wingless gene and arginine kinase were acquired for specimens Ocys harpaloides 2759 and Ocys tachysoides 2758.
For all except specimen number 4606, DNA was extracted from specimens preserved in 95% ethanol using a Qiagen DNeasy Blood and Tissue Kit. Fragments for the seven genes were amplified using the Polymerase Chain Reaction on an Eppendorf Mastercycler Pro Thermal Cycler, using TaKaRa Ex Taq and the basic protocols recommended by the manufacturers. Primers and details of the cycling reactions used are given in
For specimen 4606, RNA was extracted from an RNA-Later preserved specimen using a Qiagen RNeasy Kit and Trizol (Life Technologies). Isolation of mRNA was done with an NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs). The library was prepared using an NEBNext Ultra RNA (New England BioLabs) kit using 1000 ng of input RNA and NEBNext Multiplex Oligos for Illumina (New England BioLabs). The finished library was quantified using a Qubit Florometer (Life Technologies) and fragment distribution characterized on a 2100 Bioanalyzer (Agilent Technologies). The library was then sequenced on an Illumina 2500 at the Oregon Health Sciences University’s Massively Parallel Sequencing Shared Resource, with reads demultiplexed using CASAVA v1.8. The 100-base, paired-end run produced 36 million reads, which were assembled using CLC Genomics Workbench version 8.5, using the de novo assembly feature with default parameter values. The resulting assembly was converted to a blastable database using NCBI’s makeblastdb tool. Using the Asaphidion yukonense sequences from
Sequences have been deposited in GenBank with accession numbers KX907141 through KX907199 (Table
Alignment and phylogenetic analysis. The appropriate alignment was obvious for COI, CAD, and Topo, as there were no insertion or deletions (indels) evident in the sampled sequences. The two ribosomal genes were aligned by MAFFT version 7.130b (
Models of nucleotide evolution where chosen with the aid of jModelTest version 2.1.7 (
There are two distinct clades in each of the five gene trees (Fig.
Phylogenetic trees of the five studied genes. Outgroups not shown. Branch length is shown proportional to relative divergence, as estimated by GARLI. The larger, paler specimens with genitalia as in Fig.
Support for the monophyly of each of the two species in data for each gene. Numbers shown are maximum likelihood bootstrap percentages.
Clade | 28S | 18S | COI | CAD | Topo |
---|---|---|---|---|---|
O. harpaloides | 100 | 100 | 100 | 95 | 95 |
O. tachysoides | 100 | 100 | 84 | 63 | 100 |
In COI, there are 11 sites at which three to six of the O. tachysoides (all specimens except 2899) show a secondary peak in chromatograms; there are three different sites at which 1 to 3 O. harpaloides (2759, 2760, 2853) have a secondary peak. The secondary peaks suggest an alternative version of COI, which is presumably a nuclear copy, or “numt” (
A detailed examination of differences between and within each of these two species also reveals many sites at which they differ (Table
Variation observed in the DNA sequences between and within species. The number of specimens examined per species ranged from 3 to 5, with each species represented by 2 to 3 localities. “nuc”: nucleotides; “aa”: amino acids. The * indicates that one of the fixed differences is an extra base in O. tachysoides relative to O. harpaloides.
fixed differences between species | sites varying within species | % nuc differences between species | |||
---|---|---|---|---|---|
gene | nuc | aa | nuc | aa | |
28S | 22* | - | 1 | - | 2.1-2.2 |
18S | 15 | - | 1 | - | 0.9 |
COI | 50 | 2 | 9 | 0 | 7.2-8.4 |
CAD | 5 | 1 | 6 | 0 | 0.50-1.0 |
Topo | 16 | 3 | 0 | 0 | 2.2 |
Morphological characteristics were in general correlated with DNA sequences. All five males in the Ocys harpaloides clade in Fig.
Morphological features and habitats of the sequenced specimens. “Disc colour” refers to the colour of the elytral disc relative to the apex and lateral margins. Male genitalic type A is that shown in Fig.
Region | Disc colour | Genitalic type | Habitat | |
---|---|---|---|---|
O. harpaloides | Northern Ireland | paler | A | ocean shore |
O. harpaloides | Northern Ireland | paler | A | ocean shore |
O. harpaloides | Republic of Ireland | paler | A | ocean shore |
O. harpaloides | Belgium | slightly paler | FA | broadleaf forest |
O. harpaloides | Belgium | slightly paler | A | coastal salt marsh |
O. harpaloides | Belgium | ± dark | A | broadleaf forest |
O. harpaloides | Spain | paler | A | open woodland |
O. tachysoides | Northern Ireland | dark | C | woodland |
O. tachysoides | Northern Ireland | dark | C | woodland |
O. tachysoides | Northern Ireland | dark | C | woodland |
O. tachysoides | Northern Ireland | dark | C | woodland |
O. tachysoides | Northern Ireland | dark | C | woodland |
O. tachysoides | Belgium | dark | C | freshwater marsh |
O. tachysoides | Belgium | dark | C | freshwater marsh |
O. tachysoides | Germany | dark | C | woodland |
On the island of Ireland, all three O. harpaloides clade males were found on the ocean shore, at two localities, whereas all five O. tachysoides clade males were found in woodland habitats, at three localities (Table
Our data show that specimens in northern Europe referred to “Ocys harpaloides” belong to two species. The DNA data alone strongly suggests that two species live in Northern Ireland and Belgium: the gene trees for two certainly unlinked genes (nuclear 28S and mitochondrial COI) show the same clades for specimens that broadly overlap in geographic range, with specimens of both forms sampled from each region. This pattern, combined with identical patterns for 18S, CAD, and Topo, indicates that the two forms are not currently exchanging genes. This is confirmed by the correlated morphological traits, in particular the distinctive male genitalia.
By comparing our sequences with those presented by
The following key allows one to identify Ocys specimens from much of Europe, outside of those regions containing localized endemics (that is, outside of higher elevations in mountains in Spain, northern Africa, or Italy (
1 | Pronotum with obtuse and rounded hind angles, with the hind margin protruding posteriorly in the middle relative to the hind angles; colour dark brown with bluish or greenish reflections | Ocys quinquestriatus (Gyllenhal) |
– | Pronotum with sharp hind angles, approximately a right angle, with the hind margin more or less straight; colour without notable metallic reflections | 2 |
2 | Elytra rufous or brown, in most specimens paler centrally with darker apical and lateral margins. Microsculpture of elytra consisting of more closely spaced, transverse lines with less of a tendency to form meshes (Fig. |
Ocys harpaloides (Audinet-Serville) |
– | Elytra dark brown to black in colour, in some specimens paler along the suture, but entire discal region not paler than margins. Microsculpture of elytra consisting of less closely spaced, transverse lines with more of a tendency to form meshes (Fig. |
Ocys tachysoides (Antoine) |
Microsculpture at center of disc of left elytron, third and fourth intervals. A, B, O. harpaloides, both from Colliery Bay, Ballycastle, Northern Ireland (A: voucher V100670, B: voucher V100672). C, D. O. tachysoides (C: Murlough NNR, Co. Down, Northern Ireland, voucher DNA2899, D: Mount Stewart, Co. Down, Northern Ireland, voucher DNA2898). Scale bar 0.1 mm.
Spermatheca. A, B, O. harpaloides (A: Bos Ter Rijst, Schorisse, Belgium, voucher DNA0569, B: Beuda, Girona, Spain, voucher V100984) C, D. O. tachysoides (B: Murlough NNR, Co. Down, Northern Ireland, voucher V100983, D: Mount Stewart, Co. Down, Northern Ireland, voucher V100982). A bubble of air has been digitally removed from within the spermatheca shown in C. Scale bar 0.1 mm.
Bembidion
harpaloides
Audinet-Serville, 1821:78. Lectotype male, here designated, in the Dufour collection of the
Tachis
rufescens
Guérin-Ménéville, 1823:123. Type specimens lost (see below). Type locality as specified in the original description: an island on the Seine River (presumably Île Seguin or Île Saint-Germain), Meudon, France. Neotype male, here designated, in the
Ocys melanocephalus Stephens, 1828:10. Lectotype male, here designated, in BMNH, examined, with six labels: “BRITISH ISLES J. Stephens Coll. BM 1853- 46”, “O melanocephalum” [handwritten], “♂”, “harpaloides det Netolitzky” [handwritten], “NHMUK010363535” [with matrix barcode], “LECTOTYPE Ocys melanocephalus Stephens designated 2016 D.R. Maddison and R. Anderson” [partly handwritten, with red lines around the border]. Type locality: British Isles.
Bembidium dubium Wollaston, 1857:23. Holotype female, in BMNH, examined. Type locality: Madeira. Junior primary homonym of Bembidium dubium Heer, 1838. Synonymy tentative, based primarily on the colour of the type.
Interpretation of Audinet-Serville’s name rests in part on discovery of his specimens, as his description is not sufficient to distinguish between the two species. Audinet-Serville notes that his specimen or specimens of Bembidion harpaloides are from “M. de Brébisson”, presumably the botanist Louis Alphonse de Brébisson. Audinet-Serville’s Coleoptera specimens were acquired by Léon Jean Marie Dufour (Thierry Deuve, pers. comm.), whose collection was incorporated into the Muséum National d’Histoire Naturelle in Paris. In March 2016, David H. Kavanaugh searched for several hours for the type series of Bembidion harpaloides Audinet-Serville in Dufour’s collection at the
The collection of Félix Édouard Guérin-Méneville was incorporated into the Chaudoir collection at the
In the BMNH, under “melanocephalum”, are seven specimens (four males, three females) in the J. Stephens collection, all with the label “British Isles J. Stephens Coll. BM. 1853 – 46”. We consider this to be the type series of Ocys melanocephalus Stephens. Three of the males in this series were dissected, and genitalia examined; they all match genitalic type A (i.e., as in Fig.
In the Wollaston collection of the BMNH, in Insecta Maderensia, Drawer 2, is a single teneral female, without a determination label. This specimen is presumed to be the holotype of Bembidium dubium Wollaston, 1854. Its colour suggests that it belongs to this species. However, the species membership of the type is of no nomenclatural importance, as the name is a junior primary homonym of Bembidium dubium Heer, 1838, itself a junior synonym of Bemidion assimile (Gyllenhal, 1810).
An additional name (Carabus tempestivus Panzer, 1799) is listed as a synonym of O. harpaloides by some authors (e.g. Stephens, 1828). However, this name is a synonym of Trechus quadristriatus (Schrank, 1781), as documented by
Body length 4.2–6.1 mm (
Hind margin of pronotum. A, B, O. harpaloides (A: North Slob, Wexford, Ireland, voucher DNA2759, B: Halve Maan, Oostende, Belgium, voucher DNA2853). C, D. O. tachysoides (B: Belvoir Forest, Belfast, Northern Ireland, voucher DNA2763, D: Murlough NNR, Co. Down, Northern Ireland, voucher DNA2899). Scale bar 0.1 mm.
Left shoulder. A, B, O. harpaloides (A: North Slob, Wexford, Ireland, voucher DNA2759, B: Bos Ter Rijst, Schorisse, Belgium, voucher DNA2937) C, D. O. tachysoides (B: River Schelde, De Kramp, Moerzeke, Belgium, voucher DNA2936, D: Murlough NNR, Co. Down, Northern Ireland, voucher DNA2899). Scale bar 0.1 mm.
(Fig.
Distribution map of Ocys harpaloides and O. tachysoides. Only records of which we are confident are included. In addition to specimens we have examined, and those from
In addition to type specimens, and those listed in Table
Bembidium
harpaloides
v.
tachysoides
Antoine, 1933:79. Holotype female, in the
The type of Bembidium harpaloides v. tachysoides Antoine belongs to this species. We have examined external features of the holotype, including elytral microsculpture (which has a density of 21 lines per 0.1 mm), as well as shape of the spermatheca. Both second gonocoxites are broken in the holotype, and thus we were not able to measure their lengths.
Body length 4.0–5.8 mm (
(Fig.
In addition to the type specimen, and those listed in Table
We are extremely thankful to the individuals who worked so hard on our behalf as we were attempting to locate original Audinet-Serville and Guérin-Ménéville specimens at the Muséum National d’Histoire Naturelle, Paris, and to designate primary types for the associated names. Most importantly, Thierry Deuve patiently dealt with our many queries, examined numerous specimens, and visited Dominique Echaroux. His thoughtful responses, as we puzzled through the best approach to resolving the nomenclatural issues, were invaluable. Azadeh Taghavian’s help in this regard can also not go unmentioned. In addition, David Kavanaugh spent many valuable hours while in Paris personally looking for Ocys types, and we could have not completed the project without his help; we are also thankful for his advice as we considered the best path, and for hand-carrying the Audinet-Serville material back from Paris. Several others aided these efforts as well, and we wish to thank them: Antoine Mantilleri (for his advice about Audinet-Serville material), and Paul Johnson (for providing copies of pages from Horn & Kahle). Finally, we wish to thank Mr. Dominique Echaroux, from Etréchy (Essonne, France), for providing specimens of Ocys harpaloides from his collection, and for allowing us to designate one of them as a neotype of Tachis rufescens.
We are also very thankful for Beulah Garner’s stewardship of the specimens at the Natural History Museum in London, and for sending a portion of the type series of Ocys melanocephalus Stephens so that we might designate a lectotype.
For various helpful discussions we thank Marcos Toribio, Luca Toledano, Paolo Bonavita, and Paolo Neri. For their thoughtful reviews of the manuscript, we thank Michael Raupach, Pier Mauro Giachino, and Joachim Schmidt. We also thank Luca Toledano and Paolo Bonavita for examining the specimens in their collections and providing additional localities for Fig.
We thank all those who provided material preserved for DNA studies, including Joachim Schmidt, Frederik Hendrickx, and Wayne Maddison, and in particular the late Konjev Desender. We also thank John S. Sproul for extracting the RNA of specimen DNA4606, James M. Pflug for preparing the sequencing library, and Illumina Inc. for helping resolve some sequencing issues.
This work was funded in part by the Harold E. and Leona M. Rice Endowment Fund at Oregon State University.