Cryptic diversity in supposedly species-poor genera of Enchytraeidae (Annelida: Clitellata)

Cryptic diversity in supposedly species-poor genera of Enchytraeidae (Annelida: Clitellata) Abstract Using a two-step workflow, we test the species boundaries in three genera of enchytraeid worms, Globulidrilus, Hemifridericia and Stercutus (Clitellata: Enchytraeidae), which contains one to three nominal species each. For the species discovery phase, DNA barcode-based clustering analyses in ABGD (Automatic Barcode Gap Discovery) are performed, using mitochondrial cytochrome c oxidase subunit I (COI) data. The clusters from these analyses are then used as input in the species validation phase, where multispecies coalescent-based multilocus species delimitation analyses are performed in BPP (Bayesian Phylogenetics and Phylogeography) using nuclear Histone 3 (H3) and Internal Transcribed Spacer (ITS) data. For all BPP analyses, several species delimitation arrangements are included in the 95% credibility interval, and no complete sets of species are well supported. However, we conclude that our data set comprises at least seven species of Globulidrilus, all attributed to the nominal morpho-species G. riparius, three species of Hemifridericia, whereof two are cryptic lineages within H. parva and one is H. bivesiculata, and at least six species of the previously monotypic Stercutus. The species are not formally described here due to a lack of mature specimens of many of them in combination with low support for some of them in the genetic analyses. However, this is the first step towards a better understanding of the diversity within these groups. BPP, BP&P, Globulidrilus, Hemifridericia, multispecies coalescent, species delimitation, Stercutus INTRODUCTION With the introduction of molecular data, together with the development of species delimitation methods, it has been shown that many nominal species actually are complexes of morphologically very similar, or identical, species, the so-called cryptic species (Bickford et al., 2007). Such species have been found in most animal groups (Pfenninger & Schwenk, 2007), including clitellate annelids (e.g. Erséus & Gustafsson, 2009; Novo et al., 2010; Matamoros, Rota & Erséus, 2012; Donnelly et al., 2013; Liu et al., 2017; Martinsson & Erséus, 2017). In this study, we focus on three assumedly species-poor genera (one to three nominal species in each) of the family Enchytraeidae (Annelida: Clitellata): Hemifridericia Nielsen & Christensen, 1959, Globulidrilus Christensen & Dózsa-Farkas, 2012 and Stercutus Michaelsen, 1888. Today, three species are referred to Hemifridericia, one of which however, H. varanensis Lal, Singh & Prasad, 1981, does not seem to belong to this genus (Dózsa-Farkas & Felföldi, 2015). Hemifridericia parva Nielsen & Christensen, 1959 is widespread in Europe and is also reported from China and USA, whereas the third species, Hemifridericia bivesiculata Christensen & Dózsa-Farkas, 2006, is so far only known from Hungary and Arctic Canada. Hemifridericia is found in a clade together with Buchholzia and Fridericia (Martinsson et al., 2017a). Globulidrilus consists of G. helgei Christensen & Dózsa-Farkas, 2012 (type species) that has so far only been found in South Korea and the Holarctic G. riparius (Bretscher, 1889) that was recently transferred from Marionina (Christensen & Dózsa-Farkas, 2012). Globulidrilus is found in a clade together with Bryodrilus, Henlea, Oconnorella as well as Claparedrilus semifuscoides (as Lumbricillus semifuscus) and Marionina communis; the latter is not found together with the rest of their respective congenerics (Erséus et al., 2010; Klinth, Martinsson & Erséus, 2017; Martinsson et al., 2017a). The third genus in this study, Stercutus, is monotypic for S. niveus Michaelsen, 1888, which is found in both Europe and North America. This genus is found in a clade together with Chamaedrilus and Euenchytreus, two genera that were previously combined into Cognettia (Martinsson et al., 2017a). The species delimitation process can be divided into two steps, species discovery and species validation (Carstens et al., 2013). In the first, specimens are grouped into groups/putative species, usually using a single data source, for example morphology or DNA barcoding, and these species hypotheses are then tested in the latter with additional data and analyses (Carstens et al., 2013). It has been shown that the delimitation success increases with the number of markers and that a single marker is not enough for a solid well-supported delimitation (Dupuis, Roe & Sperling, 2012). The development of the methods used for species delimitation using molecular data has been rapid (see e.g. Sites & Marshall, 2003; Fujita et al., 2012), and in recent years, methods to analyse several loci together in a single analysis for species delimitation has become available (Rannala, 2015). Some of these are based on the multispecies coalescent model (Rannala & Yang, 2003). In this model, genes evolve inside a species phylogeny where the branches are species and the properties of the branches restrict the gene trees. One of these restrictions is that the divergence times between species have to be more recent than the coalescent times for any genes shared between them (Rannala & Yang, 2003). This model can be used for statistical testing of species assignments (Fujita et al., 2012; Rannala, 2015), and it is based on a clearly defined species concept in which a species constitutes a branch of a species tree, which is defined by abrupt speciation and no genetic exchange after the speciation event (Aydin et al., 2014). Under this definition, a species is a separately evolving meta-population lineage, that is, the unified species concept suggested by De Queiroz (2007). Many multilocus species delimitation methods require the user to assign the specimens to putative species that are then tested; the software usually does this by collapsing the species tree and joining sister species, and thereby testing which of the species assignments fits the model the best (Fujita et al., 2012; Rannala, 2015). In theory, it is possible to assign each specimen to its own putative species, but at least for some software products, this may increase the computational time so that the analyses are not practically possible to run (Yang & Rannala, 2014). Additionally, starting with single specimens as input species may lead to low posterior probabilities (PPs) for each of the delimited species, due to the high number of possible species assignments, together with the limited data for each of the putative species (Olave, Sola & Knowles, 2014). In this study, we test the species diversity of Globulidrilus, Hemifridericia and Stercutus, using a combination of DNA-barcode-based clustering analyses for the species discovery phase, followed by multispecies coalescent-based multilocus species delimitation analyses of nuclear markers in the software BPP (also known as BP&P, Bayesian Phylogenetics and Phylogeography; Yang, 2015) for the species validation phase. MATERIAL AND METHODS Specimens, DNA sequencing and assembly DNA data from a total of 101 specimens from Norway, Sweden, Hungary and USA were included in the study: 32 each of S. niveus and G. riparius, 35 of H. parva and 2 of H. bivesiculata (for details, see Table 1). Table 1. Specimens included in the study, with individual specimen numbers, collection data and GenBank accession numbers Specimen no  Museum voucher no  Species  COI cluster  Locality (country, state/province)  Coordinate  GenBank accession number  Latitude  Longitude  COI  H3  ITS  CE694  No voucher  Globulidrilus riparius  A  SE: Uppland  59.3609  18.0802  MF801990  MF802085  MF802180  CE2985  SMNH162823  G. riparius  A  SE: Öland  56.9855  16.8764  MF801976  MF802071  MF802166  CE2987  SMNH162824  G. riparius  A  SE: Öland  56.9855  16.8764  MF801977  MF802072  MF802167  CE3900  SMNH162825  G. riparius  A  SE: Västergötland  57.7761  12.2411  MF801988  MF802083  MF802178  CE11435  SMNH162826  G. riparius  A  SE: Öland  56.5444  16.6095  MF801961  MF802056  MF802151  CE27365  SMNH162827  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801971  MF802065  MF802160  CE27369  SMNH162828  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801973  MF802068  MF802163  CE27370  SMNH162829  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801974  MF802069  MF802164  CE20290  ZMBN110441  G. riparius  A  NO: Østfold  59.0859  11.4359  MF801963  MF802058  MF802153  CE1127  SMNH108427  G. riparius  B  US: Alaska  60.4846  −150.0414  GU902096*  MF802054  MF802149  CE1128  SMNH162830  G. riparius  H  US: Alaska  60.4846  −150.0414  MF801960  MF802055  MF802150  CE23044  ZMBN110952  G. riparius  H  NO: Finnmark  23.3002  69.9497  MF801964  MF802059  MF802154  CE23046  ZMBN120602  G. riparius  C  NO: Finnmark  23.3002  69.9497  MF801965  MF802060  MF802155  CE30036  ZMBN120603  G. riparius  C  NO: Akershus  59.8624  11.2178  MF801979  MF802074  MF802169  CE2923  SMNH162831  G. riparius  C  SE: Öland  56.8621  16.8539  MF801975  MF802070  MF802165  CE11436  SMNH162832  G. riparius  C  SE: Öland  56.5444  16.6095  MF801962  MF802057  MF802152  CE27362  SMNH162833  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801968  MF802063  MF802158  CE27367  SMNH162834  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801969  MF802067  MF802162  CE3595  SMNH162835  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801985  MF802080  MF802175  CE3596  SMNH162836  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801986  MF802081  MF802176  CE3597  SMNH162837  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801987  MF802082  MF802177  CE3956  SMNH162838  G. riparius  D  SE: Västergötland  57.7761  12.2411  MF801989  MF802084  MF802179  CE30115  ZMBN120604  G. riparius  D  NO: Akershus  60.1252  11.4649  MF801982  MF802077  MF802172  CE27360  SMNH162839  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802061  MF802156  CE27361  SMNH162840  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802062  MF802157  CE27363  SMNH162841  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801970  MF802064  MF802159  CE27366  No voucher  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801972  MF802066  MF802161  CE29939  ZMBN120605  G. riparius  G  NO: Østfold  59.3347  11.6375  MF801978  MF802073  MF802168  CE30037  ZMBN120606  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801980  MF802075  MF802170  CE30040  ZMBN120607  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801981  MF802076  MF802171  CE30248  ZMBN120608  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801983  MF802078  MF802173  CE30249  ZMBN120609  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801984  MF802079  MF802174  CE794  No voucher  Hemifridericia parva  A  SE: Västergötland  58.6195  13.4258  GU902081*  KX644882‡  MF802210  CE9509  SMNH162842  H. parva  A  SE: Lappland  66.7996  21.8130  MF801991  MF802115  MF802211  CE9548  SMNH162843  H. parva  A  SE: Lappland  67.8546  20.2173  MF801992  MF802116  MF802212  CE22423  SMNH162844  H. parva  A  SE: Lappland  68.4417  22.4794  MF801997  MF802099  CE22423  CE22424  SMNH162845  H. parva  A  SE: Lappland  68.4417  22.4794  MF801998  MF802100  CE22424  CE22425  SMNH162846  H. parva  A  SE: Lappland  68.4417  22.4794  MF801999  MF802101  CE22425  SM66  SMNH162847  H. parva  A  SE: Uppland  59.7584  17.6461  MF802006  MF802117  MF802213  CE19219  ZMBN110086  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801993  MF802086  MF802196  CE19220  ZMBN110087  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801994  MF802087  MF802197  CE20554  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801995  MF802094  MF802199  CE20563  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801996  MF802097  MF802200  CE23116  ZMBN110966  H. parva  A  NO: Troms  69.7463  21.0604  MF802000  CE23116  MF802204  CE23232  ZMBN110996  H. parva  A  NO: Troms  69.2244  19.5047  MF802001  CE23232  MF802205  CE23235  ZMBN110997  H. parva  A  NO: Troms  69.2244  19.5047  MF802002  CE23235  MF802206  CE23632  ZMBN111071  H. parva  A  NO: Nordland  68.4676  15.8931  MF802003  MF802108  MF802207  CE23633  No voucher  H. parva  A  NO: Nordland  68.4676  15.8931  MF802004  MF802109  MF802208  CE24916  ZMBN111358  H. parva  A  NO: Nordland  67.2352  14.6142  MF802005  MF802114  MF802209  CE19722  ZMBN110253  H. parva  B  NO: Sør-Trøndelag  62.606  11.6599  MF802007  MF802089  MF802182  CE22545  ZMBN110893  H. parva  B  NO: Finnmark  69.1938  23.5756  MF802016  MF802102  MF802190  CE24459  ZMBN111280  H. parva  B  NO: Nordland  67.2784  14.4343  MF802021  MF802112  MF802198  CE24299  ZMBN111246  H. parva  B  NO: Nordland  66.3182  14.1655  MF802020  MF802111  MF802194  CE19496  ZMBN110169  H. parva  C  NO: Møre og Romsdal  62.7558  7.2659  MF802008  MF802088  MF802181  CE19723  ZMBN110256  H. parva  C  NO: Sør-Trøndelag  62.606  11.6599  MF802009  MF802090  MF802183  CE19754  ZMBN110266  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802010  MF802091  MF802184  CE19755  ZMBN110267  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802011  MF802092  MF802185  CE19756  ZMBN110268  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802012  MF802093  MF802186  CE20559  ZMBN110528  H. parva  C  NO: Akershus  59.5645  10.6511  MF802013  MF802095  MF802187  CE20562  ZMBN110529  H. parva  C  NO: Akershus  59.5645  10.6511  MF802014  MF802096  MF802188  CE22224  ZMBN110803  H. parva  C  NO: Hordaland  60.3633  6.743  MF802015  MF802098  MF802189  CE23068  No voucher  H. parva  C  NO: Finnmark  69.9496  23.3003  MF802018  MF802103  MF802191  CE23220  ZMBN110992  H. parva  C  NO: Troms  69.5421  20.5499  MF802017  MF802105  MF802192  CE23653  ZMBN111080  H. parva  C  NO: Nordland  68.5683  14.963  MF802019  MF802110  MF802193  CE24882  ZMBN111348  H. parva  C  NO: Nordland  67.7232  15.8982  MF802022  MF802113  MF802195  511a  –  H. parva  B  HUN      KM591923§  KM591931§  KM591940§  615  –  H. parva  D  HUN      KM591925§  KM591929§  KM591941§  507  –  H. bivesiculata  E  HUN      KM591922§  KM591927§  KM591933§  508b  –  H. bivesiculata  E  HUN      KM591921§  KM591928§  KM591935§  CE841  No voucher  Stercutus niveus  A  SE: Västergötland  57.779  12.286  GU902112*  KF672507†  KF672547†  CE6486  SMNH162848  S. niveus  A  SE: Uppland  59.4967  18.2732  MF802028  MF802121  MF802227  SM54  SMNH162849  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802023  MF802139  MF802221  SM55  SMNH162850  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802024  MF802140  MF802222  SM56  SMNH162851  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802025  MF802141  MF802223  SM60  SMNH162852  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802026  MF802148  MF802224  SM62  No voucher  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802027  MF802142  MF802225  CE11433  SMNH162853  S. niveus  A  SE: Öland  56.5444  16.6095  MF802029  MF802123  MF802228  CE20498  ZMBN110501  S. niveus  A  NO: Akershus  59.5521  10.6912  MF802034  MF802131  MF802226  CE22393  ZMBN110849  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802035  MF802132  MF802214  CE22394  ZMBN110850  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802036  MF802133  MF802215  CE23845  ZMBN111115  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802032  MF802144  MF802218  CE23846  ZMBN111116  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802033  MF802145  MF802219  CE23075  ZMBN110960  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802030  MF802135  MF802216  CE23080  ZMBN110963  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802031  MF802136  MF802217  CE848  SMNH162854  S. niveus  B  SE: Västergötland  57.768  12.255  MF802037  MF802119  MF802230  CE13854  SMNH162855  S. niveus  B  SE: Västergötland  57.768  12.255  MF802038  MF802124  MF802229  CE27637  SMNH162856  S. niveus  B  SE: Dalsland  58.9387  12.4975  MF802039  MF802137  MF802220  CE5803  SMNH162857  S. niveus  C  SE: Västergötland  57.6830  11.9542  MF802045  MF802120  MF802234  CE6647  SMNH162858  S. niveus  C  SE: Södermanland  58.6117  16.7598  MF802041  MF802122  MF802235  CE20421  ZMBN110489  S. niveus  C  NO: Akershus  59.5262  10.69  MF802042  MF802130  MF802233  CE24263  ZMBN111233  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802040  MF802146  MF802231  CE24264  ZMBN111234  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802043  MF802147  MF802232  CE17826  SMNH162859  S. niveus  E  SE: Västergötland  58.069  12.689  MF802046  MF802125  MF802237  CE17827  SMNH162860  S. niveus  E  SE: Västergötland  58.069  12.689  MF802047  MF802126  MF802238  CE17828  SMNH162861  S. niveus  E  SE: Västergötland  58.069  12.689  MF802048  MF802127  MF802239  CE17829  SMNH162862  S. niveus  E  SE: Västergötland  58.069  12.689  MF802049  MF802128  MF802240  CE17830  SMNH162863  S. niveus  E  SE: Västergötland  58.069  12.689  MF802050  MF802118  MF802241  CE17831  SMNH162864  S. niveus  E  SE: Västergötland  58.069  12.689  MF802051  MF802143  MF802242  CE18648  No voucher  S. niveus  D  SE: Skåne  56.0379  13.2524  MF802044  MF802129  MF802236  CE23074  ZMBN110959  S. niveus  F  NO: Finnmark  69.9493  23.299  MF802052  MF802134  MF802243  CE3735  SMNH162865  S. niveus  G  US: Illinois  36.1531  −86.2725  MF802053  MF802138  MF802244  Specimen no  Museum voucher no  Species  COI cluster  Locality (country, state/province)  Coordinate  GenBank accession number  Latitude  Longitude  COI  H3  ITS  CE694  No voucher  Globulidrilus riparius  A  SE: Uppland  59.3609  18.0802  MF801990  MF802085  MF802180  CE2985  SMNH162823  G. riparius  A  SE: Öland  56.9855  16.8764  MF801976  MF802071  MF802166  CE2987  SMNH162824  G. riparius  A  SE: Öland  56.9855  16.8764  MF801977  MF802072  MF802167  CE3900  SMNH162825  G. riparius  A  SE: Västergötland  57.7761  12.2411  MF801988  MF802083  MF802178  CE11435  SMNH162826  G. riparius  A  SE: Öland  56.5444  16.6095  MF801961  MF802056  MF802151  CE27365  SMNH162827  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801971  MF802065  MF802160  CE27369  SMNH162828  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801973  MF802068  MF802163  CE27370  SMNH162829  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801974  MF802069  MF802164  CE20290  ZMBN110441  G. riparius  A  NO: Østfold  59.0859  11.4359  MF801963  MF802058  MF802153  CE1127  SMNH108427  G. riparius  B  US: Alaska  60.4846  −150.0414  GU902096*  MF802054  MF802149  CE1128  SMNH162830  G. riparius  H  US: Alaska  60.4846  −150.0414  MF801960  MF802055  MF802150  CE23044  ZMBN110952  G. riparius  H  NO: Finnmark  23.3002  69.9497  MF801964  MF802059  MF802154  CE23046  ZMBN120602  G. riparius  C  NO: Finnmark  23.3002  69.9497  MF801965  MF802060  MF802155  CE30036  ZMBN120603  G. riparius  C  NO: Akershus  59.8624  11.2178  MF801979  MF802074  MF802169  CE2923  SMNH162831  G. riparius  C  SE: Öland  56.8621  16.8539  MF801975  MF802070  MF802165  CE11436  SMNH162832  G. riparius  C  SE: Öland  56.5444  16.6095  MF801962  MF802057  MF802152  CE27362  SMNH162833  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801968  MF802063  MF802158  CE27367  SMNH162834  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801969  MF802067  MF802162  CE3595  SMNH162835  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801985  MF802080  MF802175  CE3596  SMNH162836  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801986  MF802081  MF802176  CE3597  SMNH162837  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801987  MF802082  MF802177  CE3956  SMNH162838  G. riparius  D  SE: Västergötland  57.7761  12.2411  MF801989  MF802084  MF802179  CE30115  ZMBN120604  G. riparius  D  NO: Akershus  60.1252  11.4649  MF801982  MF802077  MF802172  CE27360  SMNH162839  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802061  MF802156  CE27361  SMNH162840  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802062  MF802157  CE27363  SMNH162841  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801970  MF802064  MF802159  CE27366  No voucher  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801972  MF802066  MF802161  CE29939  ZMBN120605  G. riparius  G  NO: Østfold  59.3347  11.6375  MF801978  MF802073  MF802168  CE30037  ZMBN120606  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801980  MF802075  MF802170  CE30040  ZMBN120607  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801981  MF802076  MF802171  CE30248  ZMBN120608  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801983  MF802078  MF802173  CE30249  ZMBN120609  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801984  MF802079  MF802174  CE794  No voucher  Hemifridericia parva  A  SE: Västergötland  58.6195  13.4258  GU902081*  KX644882‡  MF802210  CE9509  SMNH162842  H. parva  A  SE: Lappland  66.7996  21.8130  MF801991  MF802115  MF802211  CE9548  SMNH162843  H. parva  A  SE: Lappland  67.8546  20.2173  MF801992  MF802116  MF802212  CE22423  SMNH162844  H. parva  A  SE: Lappland  68.4417  22.4794  MF801997  MF802099  CE22423  CE22424  SMNH162845  H. parva  A  SE: Lappland  68.4417  22.4794  MF801998  MF802100  CE22424  CE22425  SMNH162846  H. parva  A  SE: Lappland  68.4417  22.4794  MF801999  MF802101  CE22425  SM66  SMNH162847  H. parva  A  SE: Uppland  59.7584  17.6461  MF802006  MF802117  MF802213  CE19219  ZMBN110086  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801993  MF802086  MF802196  CE19220  ZMBN110087  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801994  MF802087  MF802197  CE20554  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801995  MF802094  MF802199  CE20563  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801996  MF802097  MF802200  CE23116  ZMBN110966  H. parva  A  NO: Troms  69.7463  21.0604  MF802000  CE23116  MF802204  CE23232  ZMBN110996  H. parva  A  NO: Troms  69.2244  19.5047  MF802001  CE23232  MF802205  CE23235  ZMBN110997  H. parva  A  NO: Troms  69.2244  19.5047  MF802002  CE23235  MF802206  CE23632  ZMBN111071  H. parva  A  NO: Nordland  68.4676  15.8931  MF802003  MF802108  MF802207  CE23633  No voucher  H. parva  A  NO: Nordland  68.4676  15.8931  MF802004  MF802109  MF802208  CE24916  ZMBN111358  H. parva  A  NO: Nordland  67.2352  14.6142  MF802005  MF802114  MF802209  CE19722  ZMBN110253  H. parva  B  NO: Sør-Trøndelag  62.606  11.6599  MF802007  MF802089  MF802182  CE22545  ZMBN110893  H. parva  B  NO: Finnmark  69.1938  23.5756  MF802016  MF802102  MF802190  CE24459  ZMBN111280  H. parva  B  NO: Nordland  67.2784  14.4343  MF802021  MF802112  MF802198  CE24299  ZMBN111246  H. parva  B  NO: Nordland  66.3182  14.1655  MF802020  MF802111  MF802194  CE19496  ZMBN110169  H. parva  C  NO: Møre og Romsdal  62.7558  7.2659  MF802008  MF802088  MF802181  CE19723  ZMBN110256  H. parva  C  NO: Sør-Trøndelag  62.606  11.6599  MF802009  MF802090  MF802183  CE19754  ZMBN110266  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802010  MF802091  MF802184  CE19755  ZMBN110267  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802011  MF802092  MF802185  CE19756  ZMBN110268  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802012  MF802093  MF802186  CE20559  ZMBN110528  H. parva  C  NO: Akershus  59.5645  10.6511  MF802013  MF802095  MF802187  CE20562  ZMBN110529  H. parva  C  NO: Akershus  59.5645  10.6511  MF802014  MF802096  MF802188  CE22224  ZMBN110803  H. parva  C  NO: Hordaland  60.3633  6.743  MF802015  MF802098  MF802189  CE23068  No voucher  H. parva  C  NO: Finnmark  69.9496  23.3003  MF802018  MF802103  MF802191  CE23220  ZMBN110992  H. parva  C  NO: Troms  69.5421  20.5499  MF802017  MF802105  MF802192  CE23653  ZMBN111080  H. parva  C  NO: Nordland  68.5683  14.963  MF802019  MF802110  MF802193  CE24882  ZMBN111348  H. parva  C  NO: Nordland  67.7232  15.8982  MF802022  MF802113  MF802195  511a  –  H. parva  B  HUN      KM591923§  KM591931§  KM591940§  615  –  H. parva  D  HUN      KM591925§  KM591929§  KM591941§  507  –  H. bivesiculata  E  HUN      KM591922§  KM591927§  KM591933§  508b  –  H. bivesiculata  E  HUN      KM591921§  KM591928§  KM591935§  CE841  No voucher  Stercutus niveus  A  SE: Västergötland  57.779  12.286  GU902112*  KF672507†  KF672547†  CE6486  SMNH162848  S. niveus  A  SE: Uppland  59.4967  18.2732  MF802028  MF802121  MF802227  SM54  SMNH162849  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802023  MF802139  MF802221  SM55  SMNH162850  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802024  MF802140  MF802222  SM56  SMNH162851  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802025  MF802141  MF802223  SM60  SMNH162852  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802026  MF802148  MF802224  SM62  No voucher  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802027  MF802142  MF802225  CE11433  SMNH162853  S. niveus  A  SE: Öland  56.5444  16.6095  MF802029  MF802123  MF802228  CE20498  ZMBN110501  S. niveus  A  NO: Akershus  59.5521  10.6912  MF802034  MF802131  MF802226  CE22393  ZMBN110849  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802035  MF802132  MF802214  CE22394  ZMBN110850  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802036  MF802133  MF802215  CE23845  ZMBN111115  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802032  MF802144  MF802218  CE23846  ZMBN111116  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802033  MF802145  MF802219  CE23075  ZMBN110960  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802030  MF802135  MF802216  CE23080  ZMBN110963  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802031  MF802136  MF802217  CE848  SMNH162854  S. niveus  B  SE: Västergötland  57.768  12.255  MF802037  MF802119  MF802230  CE13854  SMNH162855  S. niveus  B  SE: Västergötland  57.768  12.255  MF802038  MF802124  MF802229  CE27637  SMNH162856  S. niveus  B  SE: Dalsland  58.9387  12.4975  MF802039  MF802137  MF802220  CE5803  SMNH162857  S. niveus  C  SE: Västergötland  57.6830  11.9542  MF802045  MF802120  MF802234  CE6647  SMNH162858  S. niveus  C  SE: Södermanland  58.6117  16.7598  MF802041  MF802122  MF802235  CE20421  ZMBN110489  S. niveus  C  NO: Akershus  59.5262  10.69  MF802042  MF802130  MF802233  CE24263  ZMBN111233  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802040  MF802146  MF802231  CE24264  ZMBN111234  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802043  MF802147  MF802232  CE17826  SMNH162859  S. niveus  E  SE: Västergötland  58.069  12.689  MF802046  MF802125  MF802237  CE17827  SMNH162860  S. niveus  E  SE: Västergötland  58.069  12.689  MF802047  MF802126  MF802238  CE17828  SMNH162861  S. niveus  E  SE: Västergötland  58.069  12.689  MF802048  MF802127  MF802239  CE17829  SMNH162862  S. niveus  E  SE: Västergötland  58.069  12.689  MF802049  MF802128  MF802240  CE17830  SMNH162863  S. niveus  E  SE: Västergötland  58.069  12.689  MF802050  MF802118  MF802241  CE17831  SMNH162864  S. niveus  E  SE: Västergötland  58.069  12.689  MF802051  MF802143  MF802242  CE18648  No voucher  S. niveus  D  SE: Skåne  56.0379  13.2524  MF802044  MF802129  MF802236  CE23074  ZMBN110959  S. niveus  F  NO: Finnmark  69.9493  23.299  MF802052  MF802134  MF802243  CE3735  SMNH162865  S. niveus  G  US: Illinois  36.1531  −86.2725  MF802053  MF802138  MF802244  Accession numbers in bold are newly generated in this study. *From Erséus et al. (2010). †From Martinsson & Erséus (2014). ‡From Martinsson et al. (2017a). §From Dózsa-Farkas & Felföldi (2015). View Large Table 1. Specimens included in the study, with individual specimen numbers, collection data and GenBank accession numbers Specimen no  Museum voucher no  Species  COI cluster  Locality (country, state/province)  Coordinate  GenBank accession number  Latitude  Longitude  COI  H3  ITS  CE694  No voucher  Globulidrilus riparius  A  SE: Uppland  59.3609  18.0802  MF801990  MF802085  MF802180  CE2985  SMNH162823  G. riparius  A  SE: Öland  56.9855  16.8764  MF801976  MF802071  MF802166  CE2987  SMNH162824  G. riparius  A  SE: Öland  56.9855  16.8764  MF801977  MF802072  MF802167  CE3900  SMNH162825  G. riparius  A  SE: Västergötland  57.7761  12.2411  MF801988  MF802083  MF802178  CE11435  SMNH162826  G. riparius  A  SE: Öland  56.5444  16.6095  MF801961  MF802056  MF802151  CE27365  SMNH162827  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801971  MF802065  MF802160  CE27369  SMNH162828  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801973  MF802068  MF802163  CE27370  SMNH162829  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801974  MF802069  MF802164  CE20290  ZMBN110441  G. riparius  A  NO: Østfold  59.0859  11.4359  MF801963  MF802058  MF802153  CE1127  SMNH108427  G. riparius  B  US: Alaska  60.4846  −150.0414  GU902096*  MF802054  MF802149  CE1128  SMNH162830  G. riparius  H  US: Alaska  60.4846  −150.0414  MF801960  MF802055  MF802150  CE23044  ZMBN110952  G. riparius  H  NO: Finnmark  23.3002  69.9497  MF801964  MF802059  MF802154  CE23046  ZMBN120602  G. riparius  C  NO: Finnmark  23.3002  69.9497  MF801965  MF802060  MF802155  CE30036  ZMBN120603  G. riparius  C  NO: Akershus  59.8624  11.2178  MF801979  MF802074  MF802169  CE2923  SMNH162831  G. riparius  C  SE: Öland  56.8621  16.8539  MF801975  MF802070  MF802165  CE11436  SMNH162832  G. riparius  C  SE: Öland  56.5444  16.6095  MF801962  MF802057  MF802152  CE27362  SMNH162833  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801968  MF802063  MF802158  CE27367  SMNH162834  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801969  MF802067  MF802162  CE3595  SMNH162835  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801985  MF802080  MF802175  CE3596  SMNH162836  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801986  MF802081  MF802176  CE3597  SMNH162837  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801987  MF802082  MF802177  CE3956  SMNH162838  G. riparius  D  SE: Västergötland  57.7761  12.2411  MF801989  MF802084  MF802179  CE30115  ZMBN120604  G. riparius  D  NO: Akershus  60.1252  11.4649  MF801982  MF802077  MF802172  CE27360  SMNH162839  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802061  MF802156  CE27361  SMNH162840  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802062  MF802157  CE27363  SMNH162841  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801970  MF802064  MF802159  CE27366  No voucher  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801972  MF802066  MF802161  CE29939  ZMBN120605  G. riparius  G  NO: Østfold  59.3347  11.6375  MF801978  MF802073  MF802168  CE30037  ZMBN120606  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801980  MF802075  MF802170  CE30040  ZMBN120607  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801981  MF802076  MF802171  CE30248  ZMBN120608  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801983  MF802078  MF802173  CE30249  ZMBN120609  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801984  MF802079  MF802174  CE794  No voucher  Hemifridericia parva  A  SE: Västergötland  58.6195  13.4258  GU902081*  KX644882‡  MF802210  CE9509  SMNH162842  H. parva  A  SE: Lappland  66.7996  21.8130  MF801991  MF802115  MF802211  CE9548  SMNH162843  H. parva  A  SE: Lappland  67.8546  20.2173  MF801992  MF802116  MF802212  CE22423  SMNH162844  H. parva  A  SE: Lappland  68.4417  22.4794  MF801997  MF802099  CE22423  CE22424  SMNH162845  H. parva  A  SE: Lappland  68.4417  22.4794  MF801998  MF802100  CE22424  CE22425  SMNH162846  H. parva  A  SE: Lappland  68.4417  22.4794  MF801999  MF802101  CE22425  SM66  SMNH162847  H. parva  A  SE: Uppland  59.7584  17.6461  MF802006  MF802117  MF802213  CE19219  ZMBN110086  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801993  MF802086  MF802196  CE19220  ZMBN110087  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801994  MF802087  MF802197  CE20554  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801995  MF802094  MF802199  CE20563  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801996  MF802097  MF802200  CE23116  ZMBN110966  H. parva  A  NO: Troms  69.7463  21.0604  MF802000  CE23116  MF802204  CE23232  ZMBN110996  H. parva  A  NO: Troms  69.2244  19.5047  MF802001  CE23232  MF802205  CE23235  ZMBN110997  H. parva  A  NO: Troms  69.2244  19.5047  MF802002  CE23235  MF802206  CE23632  ZMBN111071  H. parva  A  NO: Nordland  68.4676  15.8931  MF802003  MF802108  MF802207  CE23633  No voucher  H. parva  A  NO: Nordland  68.4676  15.8931  MF802004  MF802109  MF802208  CE24916  ZMBN111358  H. parva  A  NO: Nordland  67.2352  14.6142  MF802005  MF802114  MF802209  CE19722  ZMBN110253  H. parva  B  NO: Sør-Trøndelag  62.606  11.6599  MF802007  MF802089  MF802182  CE22545  ZMBN110893  H. parva  B  NO: Finnmark  69.1938  23.5756  MF802016  MF802102  MF802190  CE24459  ZMBN111280  H. parva  B  NO: Nordland  67.2784  14.4343  MF802021  MF802112  MF802198  CE24299  ZMBN111246  H. parva  B  NO: Nordland  66.3182  14.1655  MF802020  MF802111  MF802194  CE19496  ZMBN110169  H. parva  C  NO: Møre og Romsdal  62.7558  7.2659  MF802008  MF802088  MF802181  CE19723  ZMBN110256  H. parva  C  NO: Sør-Trøndelag  62.606  11.6599  MF802009  MF802090  MF802183  CE19754  ZMBN110266  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802010  MF802091  MF802184  CE19755  ZMBN110267  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802011  MF802092  MF802185  CE19756  ZMBN110268  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802012  MF802093  MF802186  CE20559  ZMBN110528  H. parva  C  NO: Akershus  59.5645  10.6511  MF802013  MF802095  MF802187  CE20562  ZMBN110529  H. parva  C  NO: Akershus  59.5645  10.6511  MF802014  MF802096  MF802188  CE22224  ZMBN110803  H. parva  C  NO: Hordaland  60.3633  6.743  MF802015  MF802098  MF802189  CE23068  No voucher  H. parva  C  NO: Finnmark  69.9496  23.3003  MF802018  MF802103  MF802191  CE23220  ZMBN110992  H. parva  C  NO: Troms  69.5421  20.5499  MF802017  MF802105  MF802192  CE23653  ZMBN111080  H. parva  C  NO: Nordland  68.5683  14.963  MF802019  MF802110  MF802193  CE24882  ZMBN111348  H. parva  C  NO: Nordland  67.7232  15.8982  MF802022  MF802113  MF802195  511a  –  H. parva  B  HUN      KM591923§  KM591931§  KM591940§  615  –  H. parva  D  HUN      KM591925§  KM591929§  KM591941§  507  –  H. bivesiculata  E  HUN      KM591922§  KM591927§  KM591933§  508b  –  H. bivesiculata  E  HUN      KM591921§  KM591928§  KM591935§  CE841  No voucher  Stercutus niveus  A  SE: Västergötland  57.779  12.286  GU902112*  KF672507†  KF672547†  CE6486  SMNH162848  S. niveus  A  SE: Uppland  59.4967  18.2732  MF802028  MF802121  MF802227  SM54  SMNH162849  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802023  MF802139  MF802221  SM55  SMNH162850  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802024  MF802140  MF802222  SM56  SMNH162851  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802025  MF802141  MF802223  SM60  SMNH162852  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802026  MF802148  MF802224  SM62  No voucher  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802027  MF802142  MF802225  CE11433  SMNH162853  S. niveus  A  SE: Öland  56.5444  16.6095  MF802029  MF802123  MF802228  CE20498  ZMBN110501  S. niveus  A  NO: Akershus  59.5521  10.6912  MF802034  MF802131  MF802226  CE22393  ZMBN110849  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802035  MF802132  MF802214  CE22394  ZMBN110850  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802036  MF802133  MF802215  CE23845  ZMBN111115  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802032  MF802144  MF802218  CE23846  ZMBN111116  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802033  MF802145  MF802219  CE23075  ZMBN110960  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802030  MF802135  MF802216  CE23080  ZMBN110963  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802031  MF802136  MF802217  CE848  SMNH162854  S. niveus  B  SE: Västergötland  57.768  12.255  MF802037  MF802119  MF802230  CE13854  SMNH162855  S. niveus  B  SE: Västergötland  57.768  12.255  MF802038  MF802124  MF802229  CE27637  SMNH162856  S. niveus  B  SE: Dalsland  58.9387  12.4975  MF802039  MF802137  MF802220  CE5803  SMNH162857  S. niveus  C  SE: Västergötland  57.6830  11.9542  MF802045  MF802120  MF802234  CE6647  SMNH162858  S. niveus  C  SE: Södermanland  58.6117  16.7598  MF802041  MF802122  MF802235  CE20421  ZMBN110489  S. niveus  C  NO: Akershus  59.5262  10.69  MF802042  MF802130  MF802233  CE24263  ZMBN111233  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802040  MF802146  MF802231  CE24264  ZMBN111234  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802043  MF802147  MF802232  CE17826  SMNH162859  S. niveus  E  SE: Västergötland  58.069  12.689  MF802046  MF802125  MF802237  CE17827  SMNH162860  S. niveus  E  SE: Västergötland  58.069  12.689  MF802047  MF802126  MF802238  CE17828  SMNH162861  S. niveus  E  SE: Västergötland  58.069  12.689  MF802048  MF802127  MF802239  CE17829  SMNH162862  S. niveus  E  SE: Västergötland  58.069  12.689  MF802049  MF802128  MF802240  CE17830  SMNH162863  S. niveus  E  SE: Västergötland  58.069  12.689  MF802050  MF802118  MF802241  CE17831  SMNH162864  S. niveus  E  SE: Västergötland  58.069  12.689  MF802051  MF802143  MF802242  CE18648  No voucher  S. niveus  D  SE: Skåne  56.0379  13.2524  MF802044  MF802129  MF802236  CE23074  ZMBN110959  S. niveus  F  NO: Finnmark  69.9493  23.299  MF802052  MF802134  MF802243  CE3735  SMNH162865  S. niveus  G  US: Illinois  36.1531  −86.2725  MF802053  MF802138  MF802244  Specimen no  Museum voucher no  Species  COI cluster  Locality (country, state/province)  Coordinate  GenBank accession number  Latitude  Longitude  COI  H3  ITS  CE694  No voucher  Globulidrilus riparius  A  SE: Uppland  59.3609  18.0802  MF801990  MF802085  MF802180  CE2985  SMNH162823  G. riparius  A  SE: Öland  56.9855  16.8764  MF801976  MF802071  MF802166  CE2987  SMNH162824  G. riparius  A  SE: Öland  56.9855  16.8764  MF801977  MF802072  MF802167  CE3900  SMNH162825  G. riparius  A  SE: Västergötland  57.7761  12.2411  MF801988  MF802083  MF802178  CE11435  SMNH162826  G. riparius  A  SE: Öland  56.5444  16.6095  MF801961  MF802056  MF802151  CE27365  SMNH162827  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801971  MF802065  MF802160  CE27369  SMNH162828  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801973  MF802068  MF802163  CE27370  SMNH162829  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801974  MF802069  MF802164  CE20290  ZMBN110441  G. riparius  A  NO: Østfold  59.0859  11.4359  MF801963  MF802058  MF802153  CE1127  SMNH108427  G. riparius  B  US: Alaska  60.4846  −150.0414  GU902096*  MF802054  MF802149  CE1128  SMNH162830  G. riparius  H  US: Alaska  60.4846  −150.0414  MF801960  MF802055  MF802150  CE23044  ZMBN110952  G. riparius  H  NO: Finnmark  23.3002  69.9497  MF801964  MF802059  MF802154  CE23046  ZMBN120602  G. riparius  C  NO: Finnmark  23.3002  69.9497  MF801965  MF802060  MF802155  CE30036  ZMBN120603  G. riparius  C  NO: Akershus  59.8624  11.2178  MF801979  MF802074  MF802169  CE2923  SMNH162831  G. riparius  C  SE: Öland  56.8621  16.8539  MF801975  MF802070  MF802165  CE11436  SMNH162832  G. riparius  C  SE: Öland  56.5444  16.6095  MF801962  MF802057  MF802152  CE27362  SMNH162833  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801968  MF802063  MF802158  CE27367  SMNH162834  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801969  MF802067  MF802162  CE3595  SMNH162835  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801985  MF802080  MF802175  CE3596  SMNH162836  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801986  MF802081  MF802176  CE3597  SMNH162837  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801987  MF802082  MF802177  CE3956  SMNH162838  G. riparius  D  SE: Västergötland  57.7761  12.2411  MF801989  MF802084  MF802179  CE30115  ZMBN120604  G. riparius  D  NO: Akershus  60.1252  11.4649  MF801982  MF802077  MF802172  CE27360  SMNH162839  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802061  MF802156  CE27361  SMNH162840  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802062  MF802157  CE27363  SMNH162841  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801970  MF802064  MF802159  CE27366  No voucher  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801972  MF802066  MF802161  CE29939  ZMBN120605  G. riparius  G  NO: Østfold  59.3347  11.6375  MF801978  MF802073  MF802168  CE30037  ZMBN120606  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801980  MF802075  MF802170  CE30040  ZMBN120607  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801981  MF802076  MF802171  CE30248  ZMBN120608  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801983  MF802078  MF802173  CE30249  ZMBN120609  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801984  MF802079  MF802174  CE794  No voucher  Hemifridericia parva  A  SE: Västergötland  58.6195  13.4258  GU902081*  KX644882‡  MF802210  CE9509  SMNH162842  H. parva  A  SE: Lappland  66.7996  21.8130  MF801991  MF802115  MF802211  CE9548  SMNH162843  H. parva  A  SE: Lappland  67.8546  20.2173  MF801992  MF802116  MF802212  CE22423  SMNH162844  H. parva  A  SE: Lappland  68.4417  22.4794  MF801997  MF802099  CE22423  CE22424  SMNH162845  H. parva  A  SE: Lappland  68.4417  22.4794  MF801998  MF802100  CE22424  CE22425  SMNH162846  H. parva  A  SE: Lappland  68.4417  22.4794  MF801999  MF802101  CE22425  SM66  SMNH162847  H. parva  A  SE: Uppland  59.7584  17.6461  MF802006  MF802117  MF802213  CE19219  ZMBN110086  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801993  MF802086  MF802196  CE19220  ZMBN110087  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801994  MF802087  MF802197  CE20554  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801995  MF802094  MF802199  CE20563  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801996  MF802097  MF802200  CE23116  ZMBN110966  H. parva  A  NO: Troms  69.7463  21.0604  MF802000  CE23116  MF802204  CE23232  ZMBN110996  H. parva  A  NO: Troms  69.2244  19.5047  MF802001  CE23232  MF802205  CE23235  ZMBN110997  H. parva  A  NO: Troms  69.2244  19.5047  MF802002  CE23235  MF802206  CE23632  ZMBN111071  H. parva  A  NO: Nordland  68.4676  15.8931  MF802003  MF802108  MF802207  CE23633  No voucher  H. parva  A  NO: Nordland  68.4676  15.8931  MF802004  MF802109  MF802208  CE24916  ZMBN111358  H. parva  A  NO: Nordland  67.2352  14.6142  MF802005  MF802114  MF802209  CE19722  ZMBN110253  H. parva  B  NO: Sør-Trøndelag  62.606  11.6599  MF802007  MF802089  MF802182  CE22545  ZMBN110893  H. parva  B  NO: Finnmark  69.1938  23.5756  MF802016  MF802102  MF802190  CE24459  ZMBN111280  H. parva  B  NO: Nordland  67.2784  14.4343  MF802021  MF802112  MF802198  CE24299  ZMBN111246  H. parva  B  NO: Nordland  66.3182  14.1655  MF802020  MF802111  MF802194  CE19496  ZMBN110169  H. parva  C  NO: Møre og Romsdal  62.7558  7.2659  MF802008  MF802088  MF802181  CE19723  ZMBN110256  H. parva  C  NO: Sør-Trøndelag  62.606  11.6599  MF802009  MF802090  MF802183  CE19754  ZMBN110266  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802010  MF802091  MF802184  CE19755  ZMBN110267  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802011  MF802092  MF802185  CE19756  ZMBN110268  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802012  MF802093  MF802186  CE20559  ZMBN110528  H. parva  C  NO: Akershus  59.5645  10.6511  MF802013  MF802095  MF802187  CE20562  ZMBN110529  H. parva  C  NO: Akershus  59.5645  10.6511  MF802014  MF802096  MF802188  CE22224  ZMBN110803  H. parva  C  NO: Hordaland  60.3633  6.743  MF802015  MF802098  MF802189  CE23068  No voucher  H. parva  C  NO: Finnmark  69.9496  23.3003  MF802018  MF802103  MF802191  CE23220  ZMBN110992  H. parva  C  NO: Troms  69.5421  20.5499  MF802017  MF802105  MF802192  CE23653  ZMBN111080  H. parva  C  NO: Nordland  68.5683  14.963  MF802019  MF802110  MF802193  CE24882  ZMBN111348  H. parva  C  NO: Nordland  67.7232  15.8982  MF802022  MF802113  MF802195  511a  –  H. parva  B  HUN      KM591923§  KM591931§  KM591940§  615  –  H. parva  D  HUN      KM591925§  KM591929§  KM591941§  507  –  H. bivesiculata  E  HUN      KM591922§  KM591927§  KM591933§  508b  –  H. bivesiculata  E  HUN      KM591921§  KM591928§  KM591935§  CE841  No voucher  Stercutus niveus  A  SE: Västergötland  57.779  12.286  GU902112*  KF672507†  KF672547†  CE6486  SMNH162848  S. niveus  A  SE: Uppland  59.4967  18.2732  MF802028  MF802121  MF802227  SM54  SMNH162849  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802023  MF802139  MF802221  SM55  SMNH162850  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802024  MF802140  MF802222  SM56  SMNH162851  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802025  MF802141  MF802223  SM60  SMNH162852  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802026  MF802148  MF802224  SM62  No voucher  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802027  MF802142  MF802225  CE11433  SMNH162853  S. niveus  A  SE: Öland  56.5444  16.6095  MF802029  MF802123  MF802228  CE20498  ZMBN110501  S. niveus  A  NO: Akershus  59.5521  10.6912  MF802034  MF802131  MF802226  CE22393  ZMBN110849  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802035  MF802132  MF802214  CE22394  ZMBN110850  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802036  MF802133  MF802215  CE23845  ZMBN111115  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802032  MF802144  MF802218  CE23846  ZMBN111116  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802033  MF802145  MF802219  CE23075  ZMBN110960  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802030  MF802135  MF802216  CE23080  ZMBN110963  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802031  MF802136  MF802217  CE848  SMNH162854  S. niveus  B  SE: Västergötland  57.768  12.255  MF802037  MF802119  MF802230  CE13854  SMNH162855  S. niveus  B  SE: Västergötland  57.768  12.255  MF802038  MF802124  MF802229  CE27637  SMNH162856  S. niveus  B  SE: Dalsland  58.9387  12.4975  MF802039  MF802137  MF802220  CE5803  SMNH162857  S. niveus  C  SE: Västergötland  57.6830  11.9542  MF802045  MF802120  MF802234  CE6647  SMNH162858  S. niveus  C  SE: Södermanland  58.6117  16.7598  MF802041  MF802122  MF802235  CE20421  ZMBN110489  S. niveus  C  NO: Akershus  59.5262  10.69  MF802042  MF802130  MF802233  CE24263  ZMBN111233  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802040  MF802146  MF802231  CE24264  ZMBN111234  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802043  MF802147  MF802232  CE17826  SMNH162859  S. niveus  E  SE: Västergötland  58.069  12.689  MF802046  MF802125  MF802237  CE17827  SMNH162860  S. niveus  E  SE: Västergötland  58.069  12.689  MF802047  MF802126  MF802238  CE17828  SMNH162861  S. niveus  E  SE: Västergötland  58.069  12.689  MF802048  MF802127  MF802239  CE17829  SMNH162862  S. niveus  E  SE: Västergötland  58.069  12.689  MF802049  MF802128  MF802240  CE17830  SMNH162863  S. niveus  E  SE: Västergötland  58.069  12.689  MF802050  MF802118  MF802241  CE17831  SMNH162864  S. niveus  E  SE: Västergötland  58.069  12.689  MF802051  MF802143  MF802242  CE18648  No voucher  S. niveus  D  SE: Skåne  56.0379  13.2524  MF802044  MF802129  MF802236  CE23074  ZMBN110959  S. niveus  F  NO: Finnmark  69.9493  23.299  MF802052  MF802134  MF802243  CE3735  SMNH162865  S. niveus  G  US: Illinois  36.1531  −86.2725  MF802053  MF802138  MF802244  Accession numbers in bold are newly generated in this study. *From Erséus et al. (2010). †From Martinsson & Erséus (2014). ‡From Martinsson et al. (2017a). §From Dózsa-Farkas & Felföldi (2015). View Large A vast majority of the sequences were newly generated (Table 1). DNA was extracted from the posterior ends of ethanol-preserved worms, and the anterior parts were mounted in Canada balsam to be used as physical vouchers. DNA was extracted using either Qiagen’s DNeasy Blood & Tissue Kit or Epicentre QuickExtract DNA Extraction Solution 1.0, following the manufacturer’s instructions. Three markers, the mitochondrial cytochrome c oxidase subunit I (COI) gene, the complete nuclear ribosomal Internal Transcribed Spacer (ITS) region and the nuclear gene Histone 3 (H3), were amplified using primers and PCR programmes listed in Supporting Information, Table S1. Sequencing was carried out by Macrogen Inc. (Seoul, Korea) and Eurofins MWG Operon (Ebersberg, Germany). As specified in Table 1, a few sequences are from previously published works (Erséus et al., 2010; Martinsson & Erséus, 2014; Dózsa-Farkas & Felföldi, 2015; Martinsson et al., 2017a) and were all downloaded from GenBank. Sequences were assembled in Geneious Pro v. 7.1. (Biomatters Ltd.; http://www.geneious.com) and aligned separately for each genus using MAFFT v7.017 (Katoh et al., 2002) as implemented in Geneious Pro v. 7.1., using the auto-algorithm and default settings. Information about the alignments (length and number of variable positions) can be found in Table 2. All sequences produced in this study are deposited in GenBank, and the vouchers are deposited in either the Swedish Museum of Natural History (SMNH), Stockholm, Sweden, or the University Museum of Bergen (ZMBN), Bergen, Norway (accession numbers in Table 1). Table 2. Details of alignments   Alignment length  Number of variable positions  Globulidrilus   COI  658  221   H3  328  15   ITS  949  56  Hemifridericia   COI  658  151   H3  328  8   ITS  877  39  Stercutus   COI  625  179   H3  320  11   ITS  949  36    Alignment length  Number of variable positions  Globulidrilus   COI  658  221   H3  328  15   ITS  949  56  Hemifridericia   COI  658  151   H3  328  8   ITS  877  39  Stercutus   COI  625  179   H3  320  11   ITS  949  36  The variation in length in COI and H3 between genera is due to trimming of the ends of the alignments to minimize the amount of missing data; this is also true for ITS, but this marker also varies in length between taxa. View Large Table 2. Details of alignments   Alignment length  Number of variable positions  Globulidrilus   COI  658  221   H3  328  15   ITS  949  56  Hemifridericia   COI  658  151   H3  328  8   ITS  877  39  Stercutus   COI  625  179   H3  320  11   ITS  949  36    Alignment length  Number of variable positions  Globulidrilus   COI  658  221   H3  328  15   ITS  949  56  Hemifridericia   COI  658  151   H3  328  8   ITS  877  39  Stercutus   COI  625  179   H3  320  11   ITS  949  36  The variation in length in COI and H3 between genera is due to trimming of the ends of the alignments to minimize the amount of missing data; this is also true for ITS, but this marker also varies in length between taxa. View Large Species discovery The three COI alignments were used to divide the specimens into clusters representing hypothetical species. The COI alignments were analysed in the web version of ABGD (Automatic Barcode Gap Discovery) (Puillandre et al., 2012) (available at http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html), using simple distances, Pmin = 0.001, Pmax = 0.10 and X (relative gap width) = 0.5. The highest numbers of clusters from the initial partition were selected as input for the species validation step, as BBP (see Species validation) only can lump groups into more inclusive species, not split them into smaller groups; therefore, we rather risk starting with too many species than with few input species. However, we compare the result with the clusters given using a maximum intraspecific p-distance of 7%, this value is chosen, based on the genetic distances in the data sets, to represent a more reasonable threshold between intraspecific and interspecific genetic distances. However, as for most threshold values, it is somewhat arbitrarily chosen. Uncorrected intra-cluster and inter-cluster p-distances were calculated in MEGA v6.06 (Tamura et al., 2013), missing data and gap was excluded using pairwise deletions, for between-cluster comparisons the minimum distances were used and for within-cluster comparison the maximum distances were used. The clusters were visualized on COI gene trees estimated with maximum likelihood in PhyML (Anisimova & Gascuel, 2006; Guindon et al., 2010) as implemented at the South of France Bioinformatics platform (http://www.atgc-montpellier.fr/). The automatic model selection using Smart Model Selection with Bayesian information criterion as the selection criterion was chosen; Subtree Pruning and Regrafting was used for tree improvement. Branch support was calculated with the chi-square-based approximate likelihood ratio test (Anisimova & Gascuel, 2006) in PhyML. The same settings were used for all three COI analyses. The trees (Fig. 1) were drawn in FigTree 1.4.2 (Rambaut, 2014) and further edited in Adobe Illustrator. The variation in H3 and ITS was visualized by haplotype networks created in PopART v1 (Leigh & Bryant, 2015) using statistical parsimony (Templeton, Crandall & Sing, 1992; Clement et al., 2002); sites with missing data or gaps were masked and not included in the networks (Fig. 2). Figure 1. View largeDownload slide COI gene trees, showing clusters from ABGD analyses. All trees are rooted using midpoint rooting. A, Globulidrilus, all clusters represent G. riparius s.l. B, Hemifridericia, cluster E corresponds with H. bivesiculata, the other clusters represent H. parva s.l. C, Stercutus, all clusters represent S. niveus s.l. Numbers at branches denote branch support by the approximate likelihood ratio test. Scale bars show estimated numbers of nucleotide substitutions per site. Figure 1. View largeDownload slide COI gene trees, showing clusters from ABGD analyses. All trees are rooted using midpoint rooting. A, Globulidrilus, all clusters represent G. riparius s.l. B, Hemifridericia, cluster E corresponds with H. bivesiculata, the other clusters represent H. parva s.l. C, Stercutus, all clusters represent S. niveus s.l. Numbers at branches denote branch support by the approximate likelihood ratio test. Scale bars show estimated numbers of nucleotide substitutions per site. Figure 2. View largeDownload slide Statistical parsimony haplotype networks. A, B, Globulidrilus. C, D, Hemifridericia. E, F, Stercutus. A, C, D, H3 networks. B, D, F, ITS networks. The size of the circles is relative to the number of sequences sharing that haplotype, the colours correspond to COI clusters and the hatch marks correspond to substitutions. Figure 2. View largeDownload slide Statistical parsimony haplotype networks. A, B, Globulidrilus. C, D, Hemifridericia. E, F, Stercutus. A, C, D, H3 networks. B, D, F, ITS networks. The size of the circles is relative to the number of sequences sharing that haplotype, the colours correspond to COI clusters and the hatch marks correspond to substitutions. Species validation The two nuclear markers (H3 and ITS) were used for the validation step and were analysed using the BPP v.3.3 program (Yang, 2015). Including the COI data in the validation phase would make the result more robust. However, doing so would allow the COI data to take over, as the COI data set matches the groups found in the discovery phase perfectly. There is a risk that the reasoning would be circular when the same data are used to find and validate species; therefore, we perform the BPP analyses with the nuclear data only. Joint Bayesian species delimitations and species tree estimations were conducted, a method using the multispecies coalescent model to compare different arrangements of species delimitation and species phylogeny in a Bayesian framework, accounting for incomplete lineage sorting due to ancestral polymorphism and gene tree-species tree conflicts (Yang & Rannala, 2010; Rannala & Yang, 2013; Yang & Rannala, 2014). Three analyses were run for each genus, varying the population size (θs) and divergence time (τ0) priors. The priors in the different analyses, respectively, were the same for all genera: In analysis A, the population size parameters (θs) were assigned the gamma prior G(2, 400), with mean 2/400 = 0.005. The divergence time at the root of the species tree (τ0) was assigned the gamma prior G(2, 200), while the other divergence time parameters were assigned the Dirichlet prior (Yang & Rannala, 2010: equation 2). In analysis B, the population size parameters (θs) were assigned the gamma prior G(2, 1000), with mean 2/1000 = 0.002. The divergence time at the root of the species tree (τ0) was assigned the gamma prior G(2, 200), while the other divergence time parameters were assigned the Dirichlet prior (Yang & Rannala, 2010: equation 2). In analysis C, the population size parameters (θs) were assigned the gamma prior G(2, 2000), with mean 2/2000 = 0.001. The divergence time at the root of the species tree (τ0) was assigned the gamma prior G(2, 200), while the other divergence time parameters were assigned the Dirichlet prior (Yang & Rannala, 2010: equation 2). Each analysis was run three times to confirm consistency between runs. For the species arrangements, the 95% credibility intervals, that is the number of arrangements with the highest PP that together makes up 95% of the PP, were calculated. With regard to the individual species, we considered species delimited with a PP > 0.90 in all analyses to be well supported. For clusters with a PP < 0.90, we used a conservative approach and instead accepted the best-supported more inclusive species. The PP values for the species were mapped on the species tree with the highest PP in the majority of analyses. Genetic uncorrected p-distances were calculated for H3 and ITS the same way as for COI (see Species discovery), but sorted on the delimited species instead of the initial clusters. RESULTS Species discovery The ABGD analyses of COI divided the Globulidrilus data set into eight clusters, the Hemifridericia data set into five clusters and the Stercutus data set into nine clusters (Fig. 1A–C) using the maximum number of clusters. Using the 7% threshold, the Globulidrilus data set is divided into two groups, combining clusters A, D and F into one group and clusters B, C, E, G and H into another group; the Hemifridericia data set was divided into three groups, combining groups A and D into one group and groups B and C into another group, and group E still being a separate group. The Stercutus data set was divided into seven groups, clusters A and H were combined into one group and C and I into another group, remaining clusters were unchanged. The uncorrected p-distances presented here are the maximum intra-cluster distances and the minimum intra-cluster distances for each comparison; the values are summarized in Supporting Information, Tables S2–S4. For the Globulidrilus clusters (Supporting Information, Table S2), the largest intra-cluster p-distance was 6.1% (within cluster C), the smallest inter-cluster p-distance was 8.5% (between clusters D and F) and the largest inter-cluster p-distance was 18.3% (between clusters D and E). For the Hemifridericia data set (Supporting Information, Table S3), the largest intra-cluster p-distance was 3.0% (within cluster B), the smallest inter-cluster p-distance was 3.0% (between clusters B and C) and the largest inter-cluster p-distance was 15.9% (between clusters B and E). For the Stercutus data set (Supporting Information, Table S4), the largest intra-cluster p-distance was 1.0% (within clusters C), the smallest inter-cluster p-distance was 3.2% (between clusters A and H) and the largest inter-cluster p-distance was 15.7% (between clusters B and G). The haplotype networks (Fig. 2) showed that the specimens of most COI clusters have unique haplotypes in both their nuclear genes (H3 and ITS), not shared with other clusters, and that the H3/ITS haplotypes from the same COI-based cluster are close to each other. In a few cases, however, the H3/ITS haplotypes are shared by two or three clusters. Moreover, one specimen (CE24459) of H. parva cluster B is not even found together with the other cluster B specimens, neither in H3 nor in ITS; instead, it shares its nuclear haplotypes with specimens from clusters A and D (Fig. 2C, D). Species validation The 95% credibility intervals from the BPP analyses of Globulidrilus contain three species delimitation arrangements in analysis A and two arrangements in analyses B and C (Table 3). Either a seven-species or an eight-species arrangement was preferred, the best seven-species arrangement had a mean PP for the three runs of each analysis of 0.58, 0.50 or 0.39 in analyses A, B and C, respectively, whereas the eight-species arrangement had a mean PP of 0.35, 0.47 or 0.60 in analyses A, B and C, respectively (Table 3). The difference between the seven-species and eight-species arrangements pertains to whether clusters B and H are united into one species or treated as two separate species. All clusters (putative species) except B and H were delimited with a PP > 0.90 in all analyses (Table 4). Table 3. List of species delimitations and their mean posterior probability   Species delimitations  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  7 (A, BH, C, D, E, F, G)  0.58  0.50  0.38  8 (A, B, C, D, E, F, G, H)  0.35  0.47  0.60  6 (A, BH, C, DF, E, G)  0.04  0.02  0.01  7 (A, B, C, DF, E, G, H)  0.02  0.01  0.01  Hemifridericia  3 (AD, BC, E)  0.64  0.56  0.57  4 (AD, B, C, E)  0.30  0.32  0.34  4 (A, BC, D, E)  0.03  0.03  0.03  5 (A, B, C, D, E)  0.02  0.03  0.02  4 (A, BD, C, E)  0.01  0.02  0.02  3 (A, BCD, E)  0.01  0.04  0.01  Stercutus  7 (AH B CE D F G I)  0.55  0.52  0.51  6 (AH BD CE F G I)  0.12  0.07  0.05  8 (A B CE D F G H I)  0.11  0.15  0.17  8 (AH B C D E F G I)  0.10  0.16  0.18  7 (AH BD C E F G I)  0.02  0.02  0.02  7 (A BD CE F G H I)  0.02  0.02  0.02  9 (A B C D E F G H I)  0.02  0.04  0.06  6 (AH B CE DF G I)  0.01  0.00  0.00  6 (AH B CE DG F I)  0.01  0.00  0.00  6 (AH B CE D FG I)  0.01  0.00  0.00    Species delimitations  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  7 (A, BH, C, D, E, F, G)  0.58  0.50  0.38  8 (A, B, C, D, E, F, G, H)  0.35  0.47  0.60  6 (A, BH, C, DF, E, G)  0.04  0.02  0.01  7 (A, B, C, DF, E, G, H)  0.02  0.01  0.01  Hemifridericia  3 (AD, BC, E)  0.64  0.56  0.57  4 (AD, B, C, E)  0.30  0.32  0.34  4 (A, BC, D, E)  0.03  0.03  0.03  5 (A, B, C, D, E)  0.02  0.03  0.02  4 (A, BD, C, E)  0.01  0.02  0.02  3 (A, BCD, E)  0.01  0.04  0.01  Stercutus  7 (AH B CE D F G I)  0.55  0.52  0.51  6 (AH BD CE F G I)  0.12  0.07  0.05  8 (A B CE D F G H I)  0.11  0.15  0.17  8 (AH B C D E F G I)  0.10  0.16  0.18  7 (AH BD C E F G I)  0.02  0.02  0.02  7 (A BD CE F G H I)  0.02  0.02  0.02  9 (A B C D E F G H I)  0.02  0.04  0.06  6 (AH B CE DF G I)  0.01  0.00  0.00  6 (AH B CE DG F I)  0.01  0.00  0.00  6 (AH B CE D FG I)  0.01  0.00  0.00  For each analysis and genus, the PP values in bold make up the 95% credibility interval. View Large Table 3. List of species delimitations and their mean posterior probability   Species delimitations  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  7 (A, BH, C, D, E, F, G)  0.58  0.50  0.38  8 (A, B, C, D, E, F, G, H)  0.35  0.47  0.60  6 (A, BH, C, DF, E, G)  0.04  0.02  0.01  7 (A, B, C, DF, E, G, H)  0.02  0.01  0.01  Hemifridericia  3 (AD, BC, E)  0.64  0.56  0.57  4 (AD, B, C, E)  0.30  0.32  0.34  4 (A, BC, D, E)  0.03  0.03  0.03  5 (A, B, C, D, E)  0.02  0.03  0.02  4 (A, BD, C, E)  0.01  0.02  0.02  3 (A, BCD, E)  0.01  0.04  0.01  Stercutus  7 (AH B CE D F G I)  0.55  0.52  0.51  6 (AH BD CE F G I)  0.12  0.07  0.05  8 (A B CE D F G H I)  0.11  0.15  0.17  8 (AH B C D E F G I)  0.10  0.16  0.18  7 (AH BD C E F G I)  0.02  0.02  0.02  7 (A BD CE F G H I)  0.02  0.02  0.02  9 (A B C D E F G H I)  0.02  0.04  0.06  6 (AH B CE DF G I)  0.01  0.00  0.00  6 (AH B CE DG F I)  0.01  0.00  0.00  6 (AH B CE D FG I)  0.01  0.00  0.00    Species delimitations  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  7 (A, BH, C, D, E, F, G)  0.58  0.50  0.38  8 (A, B, C, D, E, F, G, H)  0.35  0.47  0.60  6 (A, BH, C, DF, E, G)  0.04  0.02  0.01  7 (A, B, C, DF, E, G, H)  0.02  0.01  0.01  Hemifridericia  3 (AD, BC, E)  0.64  0.56  0.57  4 (AD, B, C, E)  0.30  0.32  0.34  4 (A, BC, D, E)  0.03  0.03  0.03  5 (A, B, C, D, E)  0.02  0.03  0.02  4 (A, BD, C, E)  0.01  0.02  0.02  3 (A, BCD, E)  0.01  0.04  0.01  Stercutus  7 (AH B CE D F G I)  0.55  0.52  0.51  6 (AH BD CE F G I)  0.12  0.07  0.05  8 (A B CE D F G H I)  0.11  0.15  0.17  8 (AH B C D E F G I)  0.10  0.16  0.18  7 (AH BD C E F G I)  0.02  0.02  0.02  7 (A BD CE F G H I)  0.02  0.02  0.02  9 (A B C D E F G H I)  0.02  0.04  0.06  6 (AH B CE DF G I)  0.01  0.00  0.00  6 (AH B CE DG F I)  0.01  0.00  0.00  6 (AH B CE D FG I)  0.01  0.00  0.00  For each analysis and genus, the PP values in bold make up the 95% credibility interval. View Large Table 4. List of delimited species and their mean posterior probabilities   Species  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  G  1.00  1.00  1.00  C  1.00  1.00  1.00  E  1.00  1.00  1.00  A  0.99  1.00  1.00  F  0.94  0.97  0.98  D  0.94  0.97  0.98  BH  0.62  0.51  0.39  H  0.38  0.49  0.61  B  0.38  0.49  0.61  DF  0.06  0.03  0.02  Hemifridericia  E  1.00  1.00  1.00  AD  0.93  0.88  0.91  BC  0.67  0.59  0.61  C  0.32  0.37  0.38  B  0.32  0.35  0.36  A  0.07  0.12  0.09  D  0.05  0.06  0.06  BD  0.01  0.02  0.02  BCD  0.01  0.04  0.01  CD  0.00  0.00  0.00  Stercutus  I  0.99  1.00  1.00  G  0.97  0.99  1.00  F  0.96  0.99  1.00  CE  0.85  0.77  0.74  AH  0.84  0.79  0.75  B  0.82  0.88  0.92  D  0.80  0.87  0.91  A  0.16  0.21  0.25  H  0.16  0.21  0.25  BD  0.17  0.12  0.08  E  0.15  0.23  0.26  C  0.15  0.23  0.26  DF  0.02  0.01  0.00  FG  0.01  0.00  0.00  DG  0.01  0.00  0.00    Species  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  G  1.00  1.00  1.00  C  1.00  1.00  1.00  E  1.00  1.00  1.00  A  0.99  1.00  1.00  F  0.94  0.97  0.98  D  0.94  0.97  0.98  BH  0.62  0.51  0.39  H  0.38  0.49  0.61  B  0.38  0.49  0.61  DF  0.06  0.03  0.02  Hemifridericia  E  1.00  1.00  1.00  AD  0.93  0.88  0.91  BC  0.67  0.59  0.61  C  0.32  0.37  0.38  B  0.32  0.35  0.36  A  0.07  0.12  0.09  D  0.05  0.06  0.06  BD  0.01  0.02  0.02  BCD  0.01  0.04  0.01  CD  0.00  0.00  0.00  Stercutus  I  0.99  1.00  1.00  G  0.97  0.99  1.00  F  0.96  0.99  1.00  CE  0.85  0.77  0.74  AH  0.84  0.79  0.75  B  0.82  0.88  0.92  D  0.80  0.87  0.91  A  0.16  0.21  0.25  H  0.16  0.21  0.25  BD  0.17  0.12  0.08  E  0.15  0.23  0.26  C  0.15  0.23  0.26  DF  0.02  0.01  0.00  FG  0.01  0.00  0.00  DG  0.01  0.00  0.00  PP > 0.90 are marked in bold. Accepted species are also marked in bold; note, for example, that although the two Stercutus clusters B and D have PP values between 0.80 and 0.92 in all analyses, the more conservative alternative of merging them into a single species (BD) is preferred. View Large Table 4. List of delimited species and their mean posterior probabilities   Species  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  G  1.00  1.00  1.00  C  1.00  1.00  1.00  E  1.00  1.00  1.00  A  0.99  1.00  1.00  F  0.94  0.97  0.98  D  0.94  0.97  0.98  BH  0.62  0.51  0.39  H  0.38  0.49  0.61  B  0.38  0.49  0.61  DF  0.06  0.03  0.02  Hemifridericia  E  1.00  1.00  1.00  AD  0.93  0.88  0.91  BC  0.67  0.59  0.61  C  0.32  0.37  0.38  B  0.32  0.35  0.36  A  0.07  0.12  0.09  D  0.05  0.06  0.06  BD  0.01  0.02  0.02  BCD  0.01  0.04  0.01  CD  0.00  0.00  0.00  Stercutus  I  0.99  1.00  1.00  G  0.97  0.99  1.00  F  0.96  0.99  1.00  CE  0.85  0.77  0.74  AH  0.84  0.79  0.75  B  0.82  0.88  0.92  D  0.80  0.87  0.91  A  0.16  0.21  0.25  H  0.16  0.21  0.25  BD  0.17  0.12  0.08  E  0.15  0.23  0.26  C  0.15  0.23  0.26  DF  0.02  0.01  0.00  FG  0.01  0.00  0.00  DG  0.01  0.00  0.00    Species  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  G  1.00  1.00  1.00  C  1.00  1.00  1.00  E  1.00  1.00  1.00  A  0.99  1.00  1.00  F  0.94  0.97  0.98  D  0.94  0.97  0.98  BH  0.62  0.51  0.39  H  0.38  0.49  0.61  B  0.38  0.49  0.61  DF  0.06  0.03  0.02  Hemifridericia  E  1.00  1.00  1.00  AD  0.93  0.88  0.91  BC  0.67  0.59  0.61  C  0.32  0.37  0.38  B  0.32  0.35  0.36  A  0.07  0.12  0.09  D  0.05  0.06  0.06  BD  0.01  0.02  0.02  BCD  0.01  0.04  0.01  CD  0.00  0.00  0.00  Stercutus  I  0.99  1.00  1.00  G  0.97  0.99  1.00  F  0.96  0.99  1.00  CE  0.85  0.77  0.74  AH  0.84  0.79  0.75  B  0.82  0.88  0.92  D  0.80  0.87  0.91  A  0.16  0.21  0.25  H  0.16  0.21  0.25  BD  0.17  0.12  0.08  E  0.15  0.23  0.26  C  0.15  0.23  0.26  DF  0.02  0.01  0.00  FG  0.01  0.00  0.00  DG  0.01  0.00  0.00  PP > 0.90 are marked in bold. Accepted species are also marked in bold; note, for example, that although the two Stercutus clusters B and D have PP values between 0.80 and 0.92 in all analyses, the more conservative alternative of merging them into a single species (BD) is preferred. View Large The 95% credibility intervals from the BPP analyses of Hemifridericia contain three different species delimitation arrangements in analysis A and four arrangements in analyses B and C (Table 3). In all analyses, a three-species arrangement, uniting clusters A and D as well as B and C, was preferred with a mean PP of 0.64, 0.56 and 0.57 in analyses A, B and C, respectively (Table 3). Cluster E, that is H. bivesiculata, was delimited with a PP of 1 in all analyses, species A + D was delimited with a PP > 0.90 in analyses A and C and with a PP of 0.88 in analyses B and species B + C had a PP of 0.67, 0.59 and 0.61 in analyses A, B and C, respectively (Fig. 3B; Table 4). Figure 3. View largeDownload slide Species trees from BPP analyses, the trees shown are the species trees and species delimitations with the highest PP in a majority of analyses. A, Globulidrilus. B, Hemifridericia. C, Stercutus. Numbers at branches are mean PP for species in analyses A, B and C, respectively. Figure 3. View largeDownload slide Species trees from BPP analyses, the trees shown are the species trees and species delimitations with the highest PP in a majority of analyses. A, Globulidrilus. B, Hemifridericia. C, Stercutus. Numbers at branches are mean PP for species in analyses A, B and C, respectively. The 95% credibility intervals from the BPP analyses of Stercutus contain nine different species delimitation arrangements in analysis A, six arrangements in analyses B and five arrangements in analysis C (Table 3). In all analyses, a seven-species arrangement, uniting clusters A and H as well as clusters C and E, was preferred with a mean PP of 0.55, 0.52 or 0.54 in analyses A, B and C, respectively (Table 3). Clusters F, G and I were delimited with a PP > 0.95 in all analyses, and clusters B and D were delimited with a PP > 0.90 in analysis C; species C + E had a PP of 0.85, 0.77 and 0.74 in analyses A, B and C, respectively, and species A + H had a PP of 0.84, 0.79 and 0.75 in analyses A, B and C, respectively (Fig. 3C; Table 4). For Globulidrilus, the genetic distances within the delimited species in H3 are maximally 0.3% (in BH) and in ITS are 0.6% (in C); the minimum distances between species vary in H3 between 0.0 (between A, D and F) and 3.5% (between A and BH, C and F) and in ITS between 0.1 (between C and G) and 2.8% (between C and F) (Supporting Information, Table S5). For Hemifridericia, the genetic distances within the delimited species in H3 are maximally 0.3% (in BC) and in ITS are 0.8% (in BC); the minimum distances between species vary in H3 between 0.0 (between AD and BC) and 2.1% (between E and the other species) and in ITS between 0.0 (between AD and BC) and 2.4% (between AD and E) (Supporting Information, Table S6). If specimen CE24459, which is found together with different groups in the COI and the nuclear data sets, would be excluded, there would be a difference between species AD and BC in both data sets (0.3% in H3 and 0.8% in ITS). For Stercutus, the maximum genetic distances within the delimited species in H3 are 0.3% (in BD) and in ITS are 0.7% (in BD); the minimum distances between species vary in H3 between 0.3 (between BD and AH, CE and F, and between CD and I) and 2.6% (between G and I) and in ITS between 0.3 (between AH and CE) and 1.5% (between BD and G) (Supporting Information, Table S7). To summarize, the results of the BPP analyses suggest that our data set includes at least seven species of Globulidrilus, for the time being all attributed to the nominal species G. riparius; three species of Hemifridericia, whereof two are referred to as H. parva and the third as H. bivesiculata; and at least six species of Stercutus. DISCUSSION We have found that the four nominal taxa that we included in the analyses actually represent at least 16 species, with varying support. It is reasonable to question whether all these recognized units are species or if they represent something else. The multispecies coalescent approach has been criticized for delimiting population structure and not real species (Sukumaran & Knowles, 2017). However, the latter authors do not specify what they mean by ‘species’, and as so many species concepts have been put forward in the past (see e.g. De Queiroz, 2007), it is hard to understand what they mean with their claims. One of the strengths of the multispecies coalescent model is that it is based on a well-defined concept of species being lineages that no longer exchange genes following divergence from a common ancestor (Toprak et al., 2016). It is possible that some of these lineages, in the future, will merge and hybridize to such extent that they will despeciate (as described by Turner, 2002). It is also possible that addition of more data, either in the form of more specimens or additional markers, will lead to another result than the one we have found in this study. Species assignments are hypotheses and as such may change over time, but, for the time being, the results of this study are the best hypotheses that we have for the species boundaries in our three genera. The high number of species arrangements within the 95% credibility intervals, especially for Stercutus (see Table 2), as well as the low support for some of the delimited species (Table 3) are probably due to a lack of phylogenetic signal in our nuclear data, and it is possible that including more variable loci would give better support for some species. However, in other studies, BPP has successfully been used with an amount of data similar to what we have herein (e.g. Hambäck et al., 2013; Parmakelis et al., 2013; Fossen et al., 2016; Martinsson, Rhodén & Erséus, 2017b). When using a higher, fixed threshold (7%) in the species discovery part the clusters changes, for Hemifridericia, the resulting clusters were the same as the delimited species, whereas for the other genera, it gave a different result, for Globulidrilus, a 7% threshold only gave two clusters, and interestingly, these two groups can be seen in the nuclear data as well, where there is a larger distance between groups ADF and BCEH than within the groups, nevertheless the BPP analyses gave strong support for at least seven species. For Stercutus, a 7% threshold gave mainly the same result as using the highest number of cluster, with the exception that it united clusters A and H as well as clusters C and I, and AD was found to be one species in the BPP analyses, but there is no support for combining C and I, instead C and E are found in the same species. This highlights the problem of using strict thresholds for single loci in species delimitation, even if it still can be a useful rule of thumb. Of the nominal taxa in focus in this study, G. riparius has already been suggested to be a species complex based on morphological observations (Christensen & Dózsa-Farkas, 2012), and for S. niveus, morphological variation has been observed as well (Rota, 1995). In H. parva, there is no notion of morphological variation, at least not in Europe. However, a description of this alleged species from China (Wang, Xie & Liang, 1999) differs from the description of material from Europe (e.g. Nielsen & Christensen, 1959) in detail about the septal glands; it could very well represent a different species. The odd specimen of H. parva (CE24459), which was found together with specimens of different clusters in the mitochondrial (cluster B) and nuclear data (in clusters A and D) sets, could be a case of hybridization and mitochondrial introgression between the two H. parva species. Hybridization has been reported between cryptic earthworm species (Dupont et al., 2016; Martinsson & Erséus, 2017). At the locality where the specimen corresponding to CE24459 was found, there was also a specimen of cluster A (C.E., unpubl. data). In the material used in our present work, we had co-occurrence of more than one cluster at the same location in all species (see Table 1). In Globulidrilus, clusters A, C and F, clusters A and D, clusters B and H and clusters C and G are found together. In Hemifridericia, A and C are found together, and in Stercutus, F and H are found together. Despite this co-occurrence, only one case of mismatch between mitochondrial and nuclear markers is observed, indicating that reproductive barriers between the delimited species exist. It is worth noting that our two lineages of H. parva are found in Hungary as well as in Scandinavia and they are probably well spread in Europe. To summarize, we found high genetic diversity in the studied enchytraeid groups, compared to the previously recognized four species. We suggest that these three genera comprise at least 16 species. The delimited species are not formally revised, described and named here, mainly due to the combination of low support for some of them and lack of mature specimens of many of them, which makes morphological comparisons and good-quality descriptions difficult. However, it is likely that some of these species are truly cryptic and that it will be impossible to identify them based on morphology. Nevertheless, this study is a first step towards understanding the species diversity of these groups, and hopefully, more material suitable for morphological studies will be collected in the future. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Primers, sequences and PCR programs used for amplification of the mitochondrial COI and nuclear ITS and H3. Table S2. Uncorrected pairwise COI distances for Globulidrilus riparius, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. Table S3. Uncorrected pairwise COI distances for Hemifridericia, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. Clusters A–D represent H. parva and cluster E, H. bivesiculata. All distances are expressed as percents. Table S4. Uncorrected pairwise COI distances for Stercutus niveus, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. Table S5. Uncorrected pairwise H3 and ITS distances for Globulidrilus riparius, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. The H3 distances are at the lower left side, and the ITS distances are at the upper right side. Table S6. Uncorrected pairwise H3 and ITS distances for Hemifridericia, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. The H3 distances are at the lower left side, and the ITS distances are at the upper right side. Clusters A–D represent H. parva and cluster E, H. bivesiculata. All distances are expressed as percents. Table S7. Uncorrected pairwise H3 and ITS distances for Stercutus niveus, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. The H3 distances are at the lower left side, and the ITS distances are at the upper right side. ACKNOWLEDGEMENTS We are grateful to Leyla Arsan, Stephen Atkinson, Mårten Klinth, Tryggve Persson, Mark Wetzel and Magdalena Zarowiecki for providing specimens and to Anna Ansebo, Marcus Carlberg, Daniel Gustafsson, Per Hjelmstedt, Jeff Hunt, Mårten Klinth, Emelie Lindquist, Maria Lindström and Urban Olsson for laboratory assistance. We thank Kerryn Elliott and two anonymous reviewers for valuable comments, which helped to improve the manuscript. 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Cryptic diversity in supposedly species-poor genera of Enchytraeidae (Annelida: Clitellata)

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© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society
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Abstract

Abstract Using a two-step workflow, we test the species boundaries in three genera of enchytraeid worms, Globulidrilus, Hemifridericia and Stercutus (Clitellata: Enchytraeidae), which contains one to three nominal species each. For the species discovery phase, DNA barcode-based clustering analyses in ABGD (Automatic Barcode Gap Discovery) are performed, using mitochondrial cytochrome c oxidase subunit I (COI) data. The clusters from these analyses are then used as input in the species validation phase, where multispecies coalescent-based multilocus species delimitation analyses are performed in BPP (Bayesian Phylogenetics and Phylogeography) using nuclear Histone 3 (H3) and Internal Transcribed Spacer (ITS) data. For all BPP analyses, several species delimitation arrangements are included in the 95% credibility interval, and no complete sets of species are well supported. However, we conclude that our data set comprises at least seven species of Globulidrilus, all attributed to the nominal morpho-species G. riparius, three species of Hemifridericia, whereof two are cryptic lineages within H. parva and one is H. bivesiculata, and at least six species of the previously monotypic Stercutus. The species are not formally described here due to a lack of mature specimens of many of them in combination with low support for some of them in the genetic analyses. However, this is the first step towards a better understanding of the diversity within these groups. BPP, BP&P, Globulidrilus, Hemifridericia, multispecies coalescent, species delimitation, Stercutus INTRODUCTION With the introduction of molecular data, together with the development of species delimitation methods, it has been shown that many nominal species actually are complexes of morphologically very similar, or identical, species, the so-called cryptic species (Bickford et al., 2007). Such species have been found in most animal groups (Pfenninger & Schwenk, 2007), including clitellate annelids (e.g. Erséus & Gustafsson, 2009; Novo et al., 2010; Matamoros, Rota & Erséus, 2012; Donnelly et al., 2013; Liu et al., 2017; Martinsson & Erséus, 2017). In this study, we focus on three assumedly species-poor genera (one to three nominal species in each) of the family Enchytraeidae (Annelida: Clitellata): Hemifridericia Nielsen & Christensen, 1959, Globulidrilus Christensen & Dózsa-Farkas, 2012 and Stercutus Michaelsen, 1888. Today, three species are referred to Hemifridericia, one of which however, H. varanensis Lal, Singh & Prasad, 1981, does not seem to belong to this genus (Dózsa-Farkas & Felföldi, 2015). Hemifridericia parva Nielsen & Christensen, 1959 is widespread in Europe and is also reported from China and USA, whereas the third species, Hemifridericia bivesiculata Christensen & Dózsa-Farkas, 2006, is so far only known from Hungary and Arctic Canada. Hemifridericia is found in a clade together with Buchholzia and Fridericia (Martinsson et al., 2017a). Globulidrilus consists of G. helgei Christensen & Dózsa-Farkas, 2012 (type species) that has so far only been found in South Korea and the Holarctic G. riparius (Bretscher, 1889) that was recently transferred from Marionina (Christensen & Dózsa-Farkas, 2012). Globulidrilus is found in a clade together with Bryodrilus, Henlea, Oconnorella as well as Claparedrilus semifuscoides (as Lumbricillus semifuscus) and Marionina communis; the latter is not found together with the rest of their respective congenerics (Erséus et al., 2010; Klinth, Martinsson & Erséus, 2017; Martinsson et al., 2017a). The third genus in this study, Stercutus, is monotypic for S. niveus Michaelsen, 1888, which is found in both Europe and North America. This genus is found in a clade together with Chamaedrilus and Euenchytreus, two genera that were previously combined into Cognettia (Martinsson et al., 2017a). The species delimitation process can be divided into two steps, species discovery and species validation (Carstens et al., 2013). In the first, specimens are grouped into groups/putative species, usually using a single data source, for example morphology or DNA barcoding, and these species hypotheses are then tested in the latter with additional data and analyses (Carstens et al., 2013). It has been shown that the delimitation success increases with the number of markers and that a single marker is not enough for a solid well-supported delimitation (Dupuis, Roe & Sperling, 2012). The development of the methods used for species delimitation using molecular data has been rapid (see e.g. Sites & Marshall, 2003; Fujita et al., 2012), and in recent years, methods to analyse several loci together in a single analysis for species delimitation has become available (Rannala, 2015). Some of these are based on the multispecies coalescent model (Rannala & Yang, 2003). In this model, genes evolve inside a species phylogeny where the branches are species and the properties of the branches restrict the gene trees. One of these restrictions is that the divergence times between species have to be more recent than the coalescent times for any genes shared between them (Rannala & Yang, 2003). This model can be used for statistical testing of species assignments (Fujita et al., 2012; Rannala, 2015), and it is based on a clearly defined species concept in which a species constitutes a branch of a species tree, which is defined by abrupt speciation and no genetic exchange after the speciation event (Aydin et al., 2014). Under this definition, a species is a separately evolving meta-population lineage, that is, the unified species concept suggested by De Queiroz (2007). Many multilocus species delimitation methods require the user to assign the specimens to putative species that are then tested; the software usually does this by collapsing the species tree and joining sister species, and thereby testing which of the species assignments fits the model the best (Fujita et al., 2012; Rannala, 2015). In theory, it is possible to assign each specimen to its own putative species, but at least for some software products, this may increase the computational time so that the analyses are not practically possible to run (Yang & Rannala, 2014). Additionally, starting with single specimens as input species may lead to low posterior probabilities (PPs) for each of the delimited species, due to the high number of possible species assignments, together with the limited data for each of the putative species (Olave, Sola & Knowles, 2014). In this study, we test the species diversity of Globulidrilus, Hemifridericia and Stercutus, using a combination of DNA-barcode-based clustering analyses for the species discovery phase, followed by multispecies coalescent-based multilocus species delimitation analyses of nuclear markers in the software BPP (also known as BP&P, Bayesian Phylogenetics and Phylogeography; Yang, 2015) for the species validation phase. MATERIAL AND METHODS Specimens, DNA sequencing and assembly DNA data from a total of 101 specimens from Norway, Sweden, Hungary and USA were included in the study: 32 each of S. niveus and G. riparius, 35 of H. parva and 2 of H. bivesiculata (for details, see Table 1). Table 1. Specimens included in the study, with individual specimen numbers, collection data and GenBank accession numbers Specimen no  Museum voucher no  Species  COI cluster  Locality (country, state/province)  Coordinate  GenBank accession number  Latitude  Longitude  COI  H3  ITS  CE694  No voucher  Globulidrilus riparius  A  SE: Uppland  59.3609  18.0802  MF801990  MF802085  MF802180  CE2985  SMNH162823  G. riparius  A  SE: Öland  56.9855  16.8764  MF801976  MF802071  MF802166  CE2987  SMNH162824  G. riparius  A  SE: Öland  56.9855  16.8764  MF801977  MF802072  MF802167  CE3900  SMNH162825  G. riparius  A  SE: Västergötland  57.7761  12.2411  MF801988  MF802083  MF802178  CE11435  SMNH162826  G. riparius  A  SE: Öland  56.5444  16.6095  MF801961  MF802056  MF802151  CE27365  SMNH162827  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801971  MF802065  MF802160  CE27369  SMNH162828  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801973  MF802068  MF802163  CE27370  SMNH162829  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801974  MF802069  MF802164  CE20290  ZMBN110441  G. riparius  A  NO: Østfold  59.0859  11.4359  MF801963  MF802058  MF802153  CE1127  SMNH108427  G. riparius  B  US: Alaska  60.4846  −150.0414  GU902096*  MF802054  MF802149  CE1128  SMNH162830  G. riparius  H  US: Alaska  60.4846  −150.0414  MF801960  MF802055  MF802150  CE23044  ZMBN110952  G. riparius  H  NO: Finnmark  23.3002  69.9497  MF801964  MF802059  MF802154  CE23046  ZMBN120602  G. riparius  C  NO: Finnmark  23.3002  69.9497  MF801965  MF802060  MF802155  CE30036  ZMBN120603  G. riparius  C  NO: Akershus  59.8624  11.2178  MF801979  MF802074  MF802169  CE2923  SMNH162831  G. riparius  C  SE: Öland  56.8621  16.8539  MF801975  MF802070  MF802165  CE11436  SMNH162832  G. riparius  C  SE: Öland  56.5444  16.6095  MF801962  MF802057  MF802152  CE27362  SMNH162833  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801968  MF802063  MF802158  CE27367  SMNH162834  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801969  MF802067  MF802162  CE3595  SMNH162835  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801985  MF802080  MF802175  CE3596  SMNH162836  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801986  MF802081  MF802176  CE3597  SMNH162837  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801987  MF802082  MF802177  CE3956  SMNH162838  G. riparius  D  SE: Västergötland  57.7761  12.2411  MF801989  MF802084  MF802179  CE30115  ZMBN120604  G. riparius  D  NO: Akershus  60.1252  11.4649  MF801982  MF802077  MF802172  CE27360  SMNH162839  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802061  MF802156  CE27361  SMNH162840  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802062  MF802157  CE27363  SMNH162841  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801970  MF802064  MF802159  CE27366  No voucher  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801972  MF802066  MF802161  CE29939  ZMBN120605  G. riparius  G  NO: Østfold  59.3347  11.6375  MF801978  MF802073  MF802168  CE30037  ZMBN120606  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801980  MF802075  MF802170  CE30040  ZMBN120607  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801981  MF802076  MF802171  CE30248  ZMBN120608  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801983  MF802078  MF802173  CE30249  ZMBN120609  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801984  MF802079  MF802174  CE794  No voucher  Hemifridericia parva  A  SE: Västergötland  58.6195  13.4258  GU902081*  KX644882‡  MF802210  CE9509  SMNH162842  H. parva  A  SE: Lappland  66.7996  21.8130  MF801991  MF802115  MF802211  CE9548  SMNH162843  H. parva  A  SE: Lappland  67.8546  20.2173  MF801992  MF802116  MF802212  CE22423  SMNH162844  H. parva  A  SE: Lappland  68.4417  22.4794  MF801997  MF802099  CE22423  CE22424  SMNH162845  H. parva  A  SE: Lappland  68.4417  22.4794  MF801998  MF802100  CE22424  CE22425  SMNH162846  H. parva  A  SE: Lappland  68.4417  22.4794  MF801999  MF802101  CE22425  SM66  SMNH162847  H. parva  A  SE: Uppland  59.7584  17.6461  MF802006  MF802117  MF802213  CE19219  ZMBN110086  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801993  MF802086  MF802196  CE19220  ZMBN110087  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801994  MF802087  MF802197  CE20554  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801995  MF802094  MF802199  CE20563  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801996  MF802097  MF802200  CE23116  ZMBN110966  H. parva  A  NO: Troms  69.7463  21.0604  MF802000  CE23116  MF802204  CE23232  ZMBN110996  H. parva  A  NO: Troms  69.2244  19.5047  MF802001  CE23232  MF802205  CE23235  ZMBN110997  H. parva  A  NO: Troms  69.2244  19.5047  MF802002  CE23235  MF802206  CE23632  ZMBN111071  H. parva  A  NO: Nordland  68.4676  15.8931  MF802003  MF802108  MF802207  CE23633  No voucher  H. parva  A  NO: Nordland  68.4676  15.8931  MF802004  MF802109  MF802208  CE24916  ZMBN111358  H. parva  A  NO: Nordland  67.2352  14.6142  MF802005  MF802114  MF802209  CE19722  ZMBN110253  H. parva  B  NO: Sør-Trøndelag  62.606  11.6599  MF802007  MF802089  MF802182  CE22545  ZMBN110893  H. parva  B  NO: Finnmark  69.1938  23.5756  MF802016  MF802102  MF802190  CE24459  ZMBN111280  H. parva  B  NO: Nordland  67.2784  14.4343  MF802021  MF802112  MF802198  CE24299  ZMBN111246  H. parva  B  NO: Nordland  66.3182  14.1655  MF802020  MF802111  MF802194  CE19496  ZMBN110169  H. parva  C  NO: Møre og Romsdal  62.7558  7.2659  MF802008  MF802088  MF802181  CE19723  ZMBN110256  H. parva  C  NO: Sør-Trøndelag  62.606  11.6599  MF802009  MF802090  MF802183  CE19754  ZMBN110266  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802010  MF802091  MF802184  CE19755  ZMBN110267  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802011  MF802092  MF802185  CE19756  ZMBN110268  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802012  MF802093  MF802186  CE20559  ZMBN110528  H. parva  C  NO: Akershus  59.5645  10.6511  MF802013  MF802095  MF802187  CE20562  ZMBN110529  H. parva  C  NO: Akershus  59.5645  10.6511  MF802014  MF802096  MF802188  CE22224  ZMBN110803  H. parva  C  NO: Hordaland  60.3633  6.743  MF802015  MF802098  MF802189  CE23068  No voucher  H. parva  C  NO: Finnmark  69.9496  23.3003  MF802018  MF802103  MF802191  CE23220  ZMBN110992  H. parva  C  NO: Troms  69.5421  20.5499  MF802017  MF802105  MF802192  CE23653  ZMBN111080  H. parva  C  NO: Nordland  68.5683  14.963  MF802019  MF802110  MF802193  CE24882  ZMBN111348  H. parva  C  NO: Nordland  67.7232  15.8982  MF802022  MF802113  MF802195  511a  –  H. parva  B  HUN      KM591923§  KM591931§  KM591940§  615  –  H. parva  D  HUN      KM591925§  KM591929§  KM591941§  507  –  H. bivesiculata  E  HUN      KM591922§  KM591927§  KM591933§  508b  –  H. bivesiculata  E  HUN      KM591921§  KM591928§  KM591935§  CE841  No voucher  Stercutus niveus  A  SE: Västergötland  57.779  12.286  GU902112*  KF672507†  KF672547†  CE6486  SMNH162848  S. niveus  A  SE: Uppland  59.4967  18.2732  MF802028  MF802121  MF802227  SM54  SMNH162849  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802023  MF802139  MF802221  SM55  SMNH162850  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802024  MF802140  MF802222  SM56  SMNH162851  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802025  MF802141  MF802223  SM60  SMNH162852  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802026  MF802148  MF802224  SM62  No voucher  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802027  MF802142  MF802225  CE11433  SMNH162853  S. niveus  A  SE: Öland  56.5444  16.6095  MF802029  MF802123  MF802228  CE20498  ZMBN110501  S. niveus  A  NO: Akershus  59.5521  10.6912  MF802034  MF802131  MF802226  CE22393  ZMBN110849  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802035  MF802132  MF802214  CE22394  ZMBN110850  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802036  MF802133  MF802215  CE23845  ZMBN111115  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802032  MF802144  MF802218  CE23846  ZMBN111116  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802033  MF802145  MF802219  CE23075  ZMBN110960  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802030  MF802135  MF802216  CE23080  ZMBN110963  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802031  MF802136  MF802217  CE848  SMNH162854  S. niveus  B  SE: Västergötland  57.768  12.255  MF802037  MF802119  MF802230  CE13854  SMNH162855  S. niveus  B  SE: Västergötland  57.768  12.255  MF802038  MF802124  MF802229  CE27637  SMNH162856  S. niveus  B  SE: Dalsland  58.9387  12.4975  MF802039  MF802137  MF802220  CE5803  SMNH162857  S. niveus  C  SE: Västergötland  57.6830  11.9542  MF802045  MF802120  MF802234  CE6647  SMNH162858  S. niveus  C  SE: Södermanland  58.6117  16.7598  MF802041  MF802122  MF802235  CE20421  ZMBN110489  S. niveus  C  NO: Akershus  59.5262  10.69  MF802042  MF802130  MF802233  CE24263  ZMBN111233  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802040  MF802146  MF802231  CE24264  ZMBN111234  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802043  MF802147  MF802232  CE17826  SMNH162859  S. niveus  E  SE: Västergötland  58.069  12.689  MF802046  MF802125  MF802237  CE17827  SMNH162860  S. niveus  E  SE: Västergötland  58.069  12.689  MF802047  MF802126  MF802238  CE17828  SMNH162861  S. niveus  E  SE: Västergötland  58.069  12.689  MF802048  MF802127  MF802239  CE17829  SMNH162862  S. niveus  E  SE: Västergötland  58.069  12.689  MF802049  MF802128  MF802240  CE17830  SMNH162863  S. niveus  E  SE: Västergötland  58.069  12.689  MF802050  MF802118  MF802241  CE17831  SMNH162864  S. niveus  E  SE: Västergötland  58.069  12.689  MF802051  MF802143  MF802242  CE18648  No voucher  S. niveus  D  SE: Skåne  56.0379  13.2524  MF802044  MF802129  MF802236  CE23074  ZMBN110959  S. niveus  F  NO: Finnmark  69.9493  23.299  MF802052  MF802134  MF802243  CE3735  SMNH162865  S. niveus  G  US: Illinois  36.1531  −86.2725  MF802053  MF802138  MF802244  Specimen no  Museum voucher no  Species  COI cluster  Locality (country, state/province)  Coordinate  GenBank accession number  Latitude  Longitude  COI  H3  ITS  CE694  No voucher  Globulidrilus riparius  A  SE: Uppland  59.3609  18.0802  MF801990  MF802085  MF802180  CE2985  SMNH162823  G. riparius  A  SE: Öland  56.9855  16.8764  MF801976  MF802071  MF802166  CE2987  SMNH162824  G. riparius  A  SE: Öland  56.9855  16.8764  MF801977  MF802072  MF802167  CE3900  SMNH162825  G. riparius  A  SE: Västergötland  57.7761  12.2411  MF801988  MF802083  MF802178  CE11435  SMNH162826  G. riparius  A  SE: Öland  56.5444  16.6095  MF801961  MF802056  MF802151  CE27365  SMNH162827  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801971  MF802065  MF802160  CE27369  SMNH162828  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801973  MF802068  MF802163  CE27370  SMNH162829  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801974  MF802069  MF802164  CE20290  ZMBN110441  G. riparius  A  NO: Østfold  59.0859  11.4359  MF801963  MF802058  MF802153  CE1127  SMNH108427  G. riparius  B  US: Alaska  60.4846  −150.0414  GU902096*  MF802054  MF802149  CE1128  SMNH162830  G. riparius  H  US: Alaska  60.4846  −150.0414  MF801960  MF802055  MF802150  CE23044  ZMBN110952  G. riparius  H  NO: Finnmark  23.3002  69.9497  MF801964  MF802059  MF802154  CE23046  ZMBN120602  G. riparius  C  NO: Finnmark  23.3002  69.9497  MF801965  MF802060  MF802155  CE30036  ZMBN120603  G. riparius  C  NO: Akershus  59.8624  11.2178  MF801979  MF802074  MF802169  CE2923  SMNH162831  G. riparius  C  SE: Öland  56.8621  16.8539  MF801975  MF802070  MF802165  CE11436  SMNH162832  G. riparius  C  SE: Öland  56.5444  16.6095  MF801962  MF802057  MF802152  CE27362  SMNH162833  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801968  MF802063  MF802158  CE27367  SMNH162834  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801969  MF802067  MF802162  CE3595  SMNH162835  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801985  MF802080  MF802175  CE3596  SMNH162836  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801986  MF802081  MF802176  CE3597  SMNH162837  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801987  MF802082  MF802177  CE3956  SMNH162838  G. riparius  D  SE: Västergötland  57.7761  12.2411  MF801989  MF802084  MF802179  CE30115  ZMBN120604  G. riparius  D  NO: Akershus  60.1252  11.4649  MF801982  MF802077  MF802172  CE27360  SMNH162839  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802061  MF802156  CE27361  SMNH162840  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802062  MF802157  CE27363  SMNH162841  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801970  MF802064  MF802159  CE27366  No voucher  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801972  MF802066  MF802161  CE29939  ZMBN120605  G. riparius  G  NO: Østfold  59.3347  11.6375  MF801978  MF802073  MF802168  CE30037  ZMBN120606  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801980  MF802075  MF802170  CE30040  ZMBN120607  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801981  MF802076  MF802171  CE30248  ZMBN120608  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801983  MF802078  MF802173  CE30249  ZMBN120609  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801984  MF802079  MF802174  CE794  No voucher  Hemifridericia parva  A  SE: Västergötland  58.6195  13.4258  GU902081*  KX644882‡  MF802210  CE9509  SMNH162842  H. parva  A  SE: Lappland  66.7996  21.8130  MF801991  MF802115  MF802211  CE9548  SMNH162843  H. parva  A  SE: Lappland  67.8546  20.2173  MF801992  MF802116  MF802212  CE22423  SMNH162844  H. parva  A  SE: Lappland  68.4417  22.4794  MF801997  MF802099  CE22423  CE22424  SMNH162845  H. parva  A  SE: Lappland  68.4417  22.4794  MF801998  MF802100  CE22424  CE22425  SMNH162846  H. parva  A  SE: Lappland  68.4417  22.4794  MF801999  MF802101  CE22425  SM66  SMNH162847  H. parva  A  SE: Uppland  59.7584  17.6461  MF802006  MF802117  MF802213  CE19219  ZMBN110086  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801993  MF802086  MF802196  CE19220  ZMBN110087  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801994  MF802087  MF802197  CE20554  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801995  MF802094  MF802199  CE20563  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801996  MF802097  MF802200  CE23116  ZMBN110966  H. parva  A  NO: Troms  69.7463  21.0604  MF802000  CE23116  MF802204  CE23232  ZMBN110996  H. parva  A  NO: Troms  69.2244  19.5047  MF802001  CE23232  MF802205  CE23235  ZMBN110997  H. parva  A  NO: Troms  69.2244  19.5047  MF802002  CE23235  MF802206  CE23632  ZMBN111071  H. parva  A  NO: Nordland  68.4676  15.8931  MF802003  MF802108  MF802207  CE23633  No voucher  H. parva  A  NO: Nordland  68.4676  15.8931  MF802004  MF802109  MF802208  CE24916  ZMBN111358  H. parva  A  NO: Nordland  67.2352  14.6142  MF802005  MF802114  MF802209  CE19722  ZMBN110253  H. parva  B  NO: Sør-Trøndelag  62.606  11.6599  MF802007  MF802089  MF802182  CE22545  ZMBN110893  H. parva  B  NO: Finnmark  69.1938  23.5756  MF802016  MF802102  MF802190  CE24459  ZMBN111280  H. parva  B  NO: Nordland  67.2784  14.4343  MF802021  MF802112  MF802198  CE24299  ZMBN111246  H. parva  B  NO: Nordland  66.3182  14.1655  MF802020  MF802111  MF802194  CE19496  ZMBN110169  H. parva  C  NO: Møre og Romsdal  62.7558  7.2659  MF802008  MF802088  MF802181  CE19723  ZMBN110256  H. parva  C  NO: Sør-Trøndelag  62.606  11.6599  MF802009  MF802090  MF802183  CE19754  ZMBN110266  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802010  MF802091  MF802184  CE19755  ZMBN110267  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802011  MF802092  MF802185  CE19756  ZMBN110268  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802012  MF802093  MF802186  CE20559  ZMBN110528  H. parva  C  NO: Akershus  59.5645  10.6511  MF802013  MF802095  MF802187  CE20562  ZMBN110529  H. parva  C  NO: Akershus  59.5645  10.6511  MF802014  MF802096  MF802188  CE22224  ZMBN110803  H. parva  C  NO: Hordaland  60.3633  6.743  MF802015  MF802098  MF802189  CE23068  No voucher  H. parva  C  NO: Finnmark  69.9496  23.3003  MF802018  MF802103  MF802191  CE23220  ZMBN110992  H. parva  C  NO: Troms  69.5421  20.5499  MF802017  MF802105  MF802192  CE23653  ZMBN111080  H. parva  C  NO: Nordland  68.5683  14.963  MF802019  MF802110  MF802193  CE24882  ZMBN111348  H. parva  C  NO: Nordland  67.7232  15.8982  MF802022  MF802113  MF802195  511a  –  H. parva  B  HUN      KM591923§  KM591931§  KM591940§  615  –  H. parva  D  HUN      KM591925§  KM591929§  KM591941§  507  –  H. bivesiculata  E  HUN      KM591922§  KM591927§  KM591933§  508b  –  H. bivesiculata  E  HUN      KM591921§  KM591928§  KM591935§  CE841  No voucher  Stercutus niveus  A  SE: Västergötland  57.779  12.286  GU902112*  KF672507†  KF672547†  CE6486  SMNH162848  S. niveus  A  SE: Uppland  59.4967  18.2732  MF802028  MF802121  MF802227  SM54  SMNH162849  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802023  MF802139  MF802221  SM55  SMNH162850  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802024  MF802140  MF802222  SM56  SMNH162851  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802025  MF802141  MF802223  SM60  SMNH162852  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802026  MF802148  MF802224  SM62  No voucher  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802027  MF802142  MF802225  CE11433  SMNH162853  S. niveus  A  SE: Öland  56.5444  16.6095  MF802029  MF802123  MF802228  CE20498  ZMBN110501  S. niveus  A  NO: Akershus  59.5521  10.6912  MF802034  MF802131  MF802226  CE22393  ZMBN110849  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802035  MF802132  MF802214  CE22394  ZMBN110850  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802036  MF802133  MF802215  CE23845  ZMBN111115  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802032  MF802144  MF802218  CE23846  ZMBN111116  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802033  MF802145  MF802219  CE23075  ZMBN110960  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802030  MF802135  MF802216  CE23080  ZMBN110963  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802031  MF802136  MF802217  CE848  SMNH162854  S. niveus  B  SE: Västergötland  57.768  12.255  MF802037  MF802119  MF802230  CE13854  SMNH162855  S. niveus  B  SE: Västergötland  57.768  12.255  MF802038  MF802124  MF802229  CE27637  SMNH162856  S. niveus  B  SE: Dalsland  58.9387  12.4975  MF802039  MF802137  MF802220  CE5803  SMNH162857  S. niveus  C  SE: Västergötland  57.6830  11.9542  MF802045  MF802120  MF802234  CE6647  SMNH162858  S. niveus  C  SE: Södermanland  58.6117  16.7598  MF802041  MF802122  MF802235  CE20421  ZMBN110489  S. niveus  C  NO: Akershus  59.5262  10.69  MF802042  MF802130  MF802233  CE24263  ZMBN111233  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802040  MF802146  MF802231  CE24264  ZMBN111234  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802043  MF802147  MF802232  CE17826  SMNH162859  S. niveus  E  SE: Västergötland  58.069  12.689  MF802046  MF802125  MF802237  CE17827  SMNH162860  S. niveus  E  SE: Västergötland  58.069  12.689  MF802047  MF802126  MF802238  CE17828  SMNH162861  S. niveus  E  SE: Västergötland  58.069  12.689  MF802048  MF802127  MF802239  CE17829  SMNH162862  S. niveus  E  SE: Västergötland  58.069  12.689  MF802049  MF802128  MF802240  CE17830  SMNH162863  S. niveus  E  SE: Västergötland  58.069  12.689  MF802050  MF802118  MF802241  CE17831  SMNH162864  S. niveus  E  SE: Västergötland  58.069  12.689  MF802051  MF802143  MF802242  CE18648  No voucher  S. niveus  D  SE: Skåne  56.0379  13.2524  MF802044  MF802129  MF802236  CE23074  ZMBN110959  S. niveus  F  NO: Finnmark  69.9493  23.299  MF802052  MF802134  MF802243  CE3735  SMNH162865  S. niveus  G  US: Illinois  36.1531  −86.2725  MF802053  MF802138  MF802244  Accession numbers in bold are newly generated in this study. *From Erséus et al. (2010). †From Martinsson & Erséus (2014). ‡From Martinsson et al. (2017a). §From Dózsa-Farkas & Felföldi (2015). View Large Table 1. Specimens included in the study, with individual specimen numbers, collection data and GenBank accession numbers Specimen no  Museum voucher no  Species  COI cluster  Locality (country, state/province)  Coordinate  GenBank accession number  Latitude  Longitude  COI  H3  ITS  CE694  No voucher  Globulidrilus riparius  A  SE: Uppland  59.3609  18.0802  MF801990  MF802085  MF802180  CE2985  SMNH162823  G. riparius  A  SE: Öland  56.9855  16.8764  MF801976  MF802071  MF802166  CE2987  SMNH162824  G. riparius  A  SE: Öland  56.9855  16.8764  MF801977  MF802072  MF802167  CE3900  SMNH162825  G. riparius  A  SE: Västergötland  57.7761  12.2411  MF801988  MF802083  MF802178  CE11435  SMNH162826  G. riparius  A  SE: Öland  56.5444  16.6095  MF801961  MF802056  MF802151  CE27365  SMNH162827  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801971  MF802065  MF802160  CE27369  SMNH162828  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801973  MF802068  MF802163  CE27370  SMNH162829  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801974  MF802069  MF802164  CE20290  ZMBN110441  G. riparius  A  NO: Østfold  59.0859  11.4359  MF801963  MF802058  MF802153  CE1127  SMNH108427  G. riparius  B  US: Alaska  60.4846  −150.0414  GU902096*  MF802054  MF802149  CE1128  SMNH162830  G. riparius  H  US: Alaska  60.4846  −150.0414  MF801960  MF802055  MF802150  CE23044  ZMBN110952  G. riparius  H  NO: Finnmark  23.3002  69.9497  MF801964  MF802059  MF802154  CE23046  ZMBN120602  G. riparius  C  NO: Finnmark  23.3002  69.9497  MF801965  MF802060  MF802155  CE30036  ZMBN120603  G. riparius  C  NO: Akershus  59.8624  11.2178  MF801979  MF802074  MF802169  CE2923  SMNH162831  G. riparius  C  SE: Öland  56.8621  16.8539  MF801975  MF802070  MF802165  CE11436  SMNH162832  G. riparius  C  SE: Öland  56.5444  16.6095  MF801962  MF802057  MF802152  CE27362  SMNH162833  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801968  MF802063  MF802158  CE27367  SMNH162834  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801969  MF802067  MF802162  CE3595  SMNH162835  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801985  MF802080  MF802175  CE3596  SMNH162836  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801986  MF802081  MF802176  CE3597  SMNH162837  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801987  MF802082  MF802177  CE3956  SMNH162838  G. riparius  D  SE: Västergötland  57.7761  12.2411  MF801989  MF802084  MF802179  CE30115  ZMBN120604  G. riparius  D  NO: Akershus  60.1252  11.4649  MF801982  MF802077  MF802172  CE27360  SMNH162839  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802061  MF802156  CE27361  SMNH162840  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802062  MF802157  CE27363  SMNH162841  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801970  MF802064  MF802159  CE27366  No voucher  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801972  MF802066  MF802161  CE29939  ZMBN120605  G. riparius  G  NO: Østfold  59.3347  11.6375  MF801978  MF802073  MF802168  CE30037  ZMBN120606  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801980  MF802075  MF802170  CE30040  ZMBN120607  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801981  MF802076  MF802171  CE30248  ZMBN120608  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801983  MF802078  MF802173  CE30249  ZMBN120609  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801984  MF802079  MF802174  CE794  No voucher  Hemifridericia parva  A  SE: Västergötland  58.6195  13.4258  GU902081*  KX644882‡  MF802210  CE9509  SMNH162842  H. parva  A  SE: Lappland  66.7996  21.8130  MF801991  MF802115  MF802211  CE9548  SMNH162843  H. parva  A  SE: Lappland  67.8546  20.2173  MF801992  MF802116  MF802212  CE22423  SMNH162844  H. parva  A  SE: Lappland  68.4417  22.4794  MF801997  MF802099  CE22423  CE22424  SMNH162845  H. parva  A  SE: Lappland  68.4417  22.4794  MF801998  MF802100  CE22424  CE22425  SMNH162846  H. parva  A  SE: Lappland  68.4417  22.4794  MF801999  MF802101  CE22425  SM66  SMNH162847  H. parva  A  SE: Uppland  59.7584  17.6461  MF802006  MF802117  MF802213  CE19219  ZMBN110086  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801993  MF802086  MF802196  CE19220  ZMBN110087  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801994  MF802087  MF802197  CE20554  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801995  MF802094  MF802199  CE20563  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801996  MF802097  MF802200  CE23116  ZMBN110966  H. parva  A  NO: Troms  69.7463  21.0604  MF802000  CE23116  MF802204  CE23232  ZMBN110996  H. parva  A  NO: Troms  69.2244  19.5047  MF802001  CE23232  MF802205  CE23235  ZMBN110997  H. parva  A  NO: Troms  69.2244  19.5047  MF802002  CE23235  MF802206  CE23632  ZMBN111071  H. parva  A  NO: Nordland  68.4676  15.8931  MF802003  MF802108  MF802207  CE23633  No voucher  H. parva  A  NO: Nordland  68.4676  15.8931  MF802004  MF802109  MF802208  CE24916  ZMBN111358  H. parva  A  NO: Nordland  67.2352  14.6142  MF802005  MF802114  MF802209  CE19722  ZMBN110253  H. parva  B  NO: Sør-Trøndelag  62.606  11.6599  MF802007  MF802089  MF802182  CE22545  ZMBN110893  H. parva  B  NO: Finnmark  69.1938  23.5756  MF802016  MF802102  MF802190  CE24459  ZMBN111280  H. parva  B  NO: Nordland  67.2784  14.4343  MF802021  MF802112  MF802198  CE24299  ZMBN111246  H. parva  B  NO: Nordland  66.3182  14.1655  MF802020  MF802111  MF802194  CE19496  ZMBN110169  H. parva  C  NO: Møre og Romsdal  62.7558  7.2659  MF802008  MF802088  MF802181  CE19723  ZMBN110256  H. parva  C  NO: Sør-Trøndelag  62.606  11.6599  MF802009  MF802090  MF802183  CE19754  ZMBN110266  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802010  MF802091  MF802184  CE19755  ZMBN110267  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802011  MF802092  MF802185  CE19756  ZMBN110268  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802012  MF802093  MF802186  CE20559  ZMBN110528  H. parva  C  NO: Akershus  59.5645  10.6511  MF802013  MF802095  MF802187  CE20562  ZMBN110529  H. parva  C  NO: Akershus  59.5645  10.6511  MF802014  MF802096  MF802188  CE22224  ZMBN110803  H. parva  C  NO: Hordaland  60.3633  6.743  MF802015  MF802098  MF802189  CE23068  No voucher  H. parva  C  NO: Finnmark  69.9496  23.3003  MF802018  MF802103  MF802191  CE23220  ZMBN110992  H. parva  C  NO: Troms  69.5421  20.5499  MF802017  MF802105  MF802192  CE23653  ZMBN111080  H. parva  C  NO: Nordland  68.5683  14.963  MF802019  MF802110  MF802193  CE24882  ZMBN111348  H. parva  C  NO: Nordland  67.7232  15.8982  MF802022  MF802113  MF802195  511a  –  H. parva  B  HUN      KM591923§  KM591931§  KM591940§  615  –  H. parva  D  HUN      KM591925§  KM591929§  KM591941§  507  –  H. bivesiculata  E  HUN      KM591922§  KM591927§  KM591933§  508b  –  H. bivesiculata  E  HUN      KM591921§  KM591928§  KM591935§  CE841  No voucher  Stercutus niveus  A  SE: Västergötland  57.779  12.286  GU902112*  KF672507†  KF672547†  CE6486  SMNH162848  S. niveus  A  SE: Uppland  59.4967  18.2732  MF802028  MF802121  MF802227  SM54  SMNH162849  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802023  MF802139  MF802221  SM55  SMNH162850  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802024  MF802140  MF802222  SM56  SMNH162851  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802025  MF802141  MF802223  SM60  SMNH162852  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802026  MF802148  MF802224  SM62  No voucher  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802027  MF802142  MF802225  CE11433  SMNH162853  S. niveus  A  SE: Öland  56.5444  16.6095  MF802029  MF802123  MF802228  CE20498  ZMBN110501  S. niveus  A  NO: Akershus  59.5521  10.6912  MF802034  MF802131  MF802226  CE22393  ZMBN110849  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802035  MF802132  MF802214  CE22394  ZMBN110850  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802036  MF802133  MF802215  CE23845  ZMBN111115  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802032  MF802144  MF802218  CE23846  ZMBN111116  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802033  MF802145  MF802219  CE23075  ZMBN110960  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802030  MF802135  MF802216  CE23080  ZMBN110963  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802031  MF802136  MF802217  CE848  SMNH162854  S. niveus  B  SE: Västergötland  57.768  12.255  MF802037  MF802119  MF802230  CE13854  SMNH162855  S. niveus  B  SE: Västergötland  57.768  12.255  MF802038  MF802124  MF802229  CE27637  SMNH162856  S. niveus  B  SE: Dalsland  58.9387  12.4975  MF802039  MF802137  MF802220  CE5803  SMNH162857  S. niveus  C  SE: Västergötland  57.6830  11.9542  MF802045  MF802120  MF802234  CE6647  SMNH162858  S. niveus  C  SE: Södermanland  58.6117  16.7598  MF802041  MF802122  MF802235  CE20421  ZMBN110489  S. niveus  C  NO: Akershus  59.5262  10.69  MF802042  MF802130  MF802233  CE24263  ZMBN111233  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802040  MF802146  MF802231  CE24264  ZMBN111234  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802043  MF802147  MF802232  CE17826  SMNH162859  S. niveus  E  SE: Västergötland  58.069  12.689  MF802046  MF802125  MF802237  CE17827  SMNH162860  S. niveus  E  SE: Västergötland  58.069  12.689  MF802047  MF802126  MF802238  CE17828  SMNH162861  S. niveus  E  SE: Västergötland  58.069  12.689  MF802048  MF802127  MF802239  CE17829  SMNH162862  S. niveus  E  SE: Västergötland  58.069  12.689  MF802049  MF802128  MF802240  CE17830  SMNH162863  S. niveus  E  SE: Västergötland  58.069  12.689  MF802050  MF802118  MF802241  CE17831  SMNH162864  S. niveus  E  SE: Västergötland  58.069  12.689  MF802051  MF802143  MF802242  CE18648  No voucher  S. niveus  D  SE: Skåne  56.0379  13.2524  MF802044  MF802129  MF802236  CE23074  ZMBN110959  S. niveus  F  NO: Finnmark  69.9493  23.299  MF802052  MF802134  MF802243  CE3735  SMNH162865  S. niveus  G  US: Illinois  36.1531  −86.2725  MF802053  MF802138  MF802244  Specimen no  Museum voucher no  Species  COI cluster  Locality (country, state/province)  Coordinate  GenBank accession number  Latitude  Longitude  COI  H3  ITS  CE694  No voucher  Globulidrilus riparius  A  SE: Uppland  59.3609  18.0802  MF801990  MF802085  MF802180  CE2985  SMNH162823  G. riparius  A  SE: Öland  56.9855  16.8764  MF801976  MF802071  MF802166  CE2987  SMNH162824  G. riparius  A  SE: Öland  56.9855  16.8764  MF801977  MF802072  MF802167  CE3900  SMNH162825  G. riparius  A  SE: Västergötland  57.7761  12.2411  MF801988  MF802083  MF802178  CE11435  SMNH162826  G. riparius  A  SE: Öland  56.5444  16.6095  MF801961  MF802056  MF802151  CE27365  SMNH162827  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801971  MF802065  MF802160  CE27369  SMNH162828  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801973  MF802068  MF802163  CE27370  SMNH162829  G. riparius  A  SE: Skåne  55.5321  14.2701  MF801974  MF802069  MF802164  CE20290  ZMBN110441  G. riparius  A  NO: Østfold  59.0859  11.4359  MF801963  MF802058  MF802153  CE1127  SMNH108427  G. riparius  B  US: Alaska  60.4846  −150.0414  GU902096*  MF802054  MF802149  CE1128  SMNH162830  G. riparius  H  US: Alaska  60.4846  −150.0414  MF801960  MF802055  MF802150  CE23044  ZMBN110952  G. riparius  H  NO: Finnmark  23.3002  69.9497  MF801964  MF802059  MF802154  CE23046  ZMBN120602  G. riparius  C  NO: Finnmark  23.3002  69.9497  MF801965  MF802060  MF802155  CE30036  ZMBN120603  G. riparius  C  NO: Akershus  59.8624  11.2178  MF801979  MF802074  MF802169  CE2923  SMNH162831  G. riparius  C  SE: Öland  56.8621  16.8539  MF801975  MF802070  MF802165  CE11436  SMNH162832  G. riparius  C  SE: Öland  56.5444  16.6095  MF801962  MF802057  MF802152  CE27362  SMNH162833  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801968  MF802063  MF802158  CE27367  SMNH162834  G. riparius  C  SE: Skåne  55.5321  14.2701  MF801969  MF802067  MF802162  CE3595  SMNH162835  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801985  MF802080  MF802175  CE3596  SMNH162836  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801986  MF802081  MF802176  CE3597  SMNH162837  G. riparius  E  US: Tennessee  36.1540  −86.2723  MF801987  MF802082  MF802177  CE3956  SMNH162838  G. riparius  D  SE: Västergötland  57.7761  12.2411  MF801989  MF802084  MF802179  CE30115  ZMBN120604  G. riparius  D  NO: Akershus  60.1252  11.4649  MF801982  MF802077  MF802172  CE27360  SMNH162839  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802061  MF802156  CE27361  SMNH162840  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801966  MF802062  MF802157  CE27363  SMNH162841  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801970  MF802064  MF802159  CE27366  No voucher  G. riparius  F  SE: Skåne  55.5321  14.2701  MF801972  MF802066  MF802161  CE29939  ZMBN120605  G. riparius  G  NO: Østfold  59.3347  11.6375  MF801978  MF802073  MF802168  CE30037  ZMBN120606  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801980  MF802075  MF802170  CE30040  ZMBN120607  G. riparius  G  NO: Akershus  59.8624  11.2178  MF801981  MF802076  MF802171  CE30248  ZMBN120608  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801983  MF802078  MF802173  CE30249  ZMBN120609  G. riparius  G  NO: Hedmark  60.1930  12.0288  MF801984  MF802079  MF802174  CE794  No voucher  Hemifridericia parva  A  SE: Västergötland  58.6195  13.4258  GU902081*  KX644882‡  MF802210  CE9509  SMNH162842  H. parva  A  SE: Lappland  66.7996  21.8130  MF801991  MF802115  MF802211  CE9548  SMNH162843  H. parva  A  SE: Lappland  67.8546  20.2173  MF801992  MF802116  MF802212  CE22423  SMNH162844  H. parva  A  SE: Lappland  68.4417  22.4794  MF801997  MF802099  CE22423  CE22424  SMNH162845  H. parva  A  SE: Lappland  68.4417  22.4794  MF801998  MF802100  CE22424  CE22425  SMNH162846  H. parva  A  SE: Lappland  68.4417  22.4794  MF801999  MF802101  CE22425  SM66  SMNH162847  H. parva  A  SE: Uppland  59.7584  17.6461  MF802006  MF802117  MF802213  CE19219  ZMBN110086  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801993  MF802086  MF802196  CE19220  ZMBN110087  H. parva  A  NO: Sogn og Fjordane  60.8308  7.1196  MF801994  MF802087  MF802197  CE20554  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801995  MF802094  MF802199  CE20563  No voucher  H. parva  A  NO: Akershus  59.5645  10.6511  MF801996  MF802097  MF802200  CE23116  ZMBN110966  H. parva  A  NO: Troms  69.7463  21.0604  MF802000  CE23116  MF802204  CE23232  ZMBN110996  H. parva  A  NO: Troms  69.2244  19.5047  MF802001  CE23232  MF802205  CE23235  ZMBN110997  H. parva  A  NO: Troms  69.2244  19.5047  MF802002  CE23235  MF802206  CE23632  ZMBN111071  H. parva  A  NO: Nordland  68.4676  15.8931  MF802003  MF802108  MF802207  CE23633  No voucher  H. parva  A  NO: Nordland  68.4676  15.8931  MF802004  MF802109  MF802208  CE24916  ZMBN111358  H. parva  A  NO: Nordland  67.2352  14.6142  MF802005  MF802114  MF802209  CE19722  ZMBN110253  H. parva  B  NO: Sør-Trøndelag  62.606  11.6599  MF802007  MF802089  MF802182  CE22545  ZMBN110893  H. parva  B  NO: Finnmark  69.1938  23.5756  MF802016  MF802102  MF802190  CE24459  ZMBN111280  H. parva  B  NO: Nordland  67.2784  14.4343  MF802021  MF802112  MF802198  CE24299  ZMBN111246  H. parva  B  NO: Nordland  66.3182  14.1655  MF802020  MF802111  MF802194  CE19496  ZMBN110169  H. parva  C  NO: Møre og Romsdal  62.7558  7.2659  MF802008  MF802088  MF802181  CE19723  ZMBN110256  H. parva  C  NO: Sør-Trøndelag  62.606  11.6599  MF802009  MF802090  MF802183  CE19754  ZMBN110266  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802010  MF802091  MF802184  CE19755  ZMBN110267  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802011  MF802092  MF802185  CE19756  ZMBN110268  H. parva  C  NO: Sør-Trøndelag  62.5763  11.3745  MF802012  MF802093  MF802186  CE20559  ZMBN110528  H. parva  C  NO: Akershus  59.5645  10.6511  MF802013  MF802095  MF802187  CE20562  ZMBN110529  H. parva  C  NO: Akershus  59.5645  10.6511  MF802014  MF802096  MF802188  CE22224  ZMBN110803  H. parva  C  NO: Hordaland  60.3633  6.743  MF802015  MF802098  MF802189  CE23068  No voucher  H. parva  C  NO: Finnmark  69.9496  23.3003  MF802018  MF802103  MF802191  CE23220  ZMBN110992  H. parva  C  NO: Troms  69.5421  20.5499  MF802017  MF802105  MF802192  CE23653  ZMBN111080  H. parva  C  NO: Nordland  68.5683  14.963  MF802019  MF802110  MF802193  CE24882  ZMBN111348  H. parva  C  NO: Nordland  67.7232  15.8982  MF802022  MF802113  MF802195  511a  –  H. parva  B  HUN      KM591923§  KM591931§  KM591940§  615  –  H. parva  D  HUN      KM591925§  KM591929§  KM591941§  507  –  H. bivesiculata  E  HUN      KM591922§  KM591927§  KM591933§  508b  –  H. bivesiculata  E  HUN      KM591921§  KM591928§  KM591935§  CE841  No voucher  Stercutus niveus  A  SE: Västergötland  57.779  12.286  GU902112*  KF672507†  KF672547†  CE6486  SMNH162848  S. niveus  A  SE: Uppland  59.4967  18.2732  MF802028  MF802121  MF802227  SM54  SMNH162849  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802023  MF802139  MF802221  SM55  SMNH162850  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802024  MF802140  MF802222  SM56  SMNH162851  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802025  MF802141  MF802223  SM60  SMNH162852  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802026  MF802148  MF802224  SM62  No voucher  S. niveus  A  SE: Uppland  59.7584  17.6461  MF802027  MF802142  MF802225  CE11433  SMNH162853  S. niveus  A  SE: Öland  56.5444  16.6095  MF802029  MF802123  MF802228  CE20498  ZMBN110501  S. niveus  A  NO: Akershus  59.5521  10.6912  MF802034  MF802131  MF802226  CE22393  ZMBN110849  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802035  MF802132  MF802214  CE22394  ZMBN110850  S. niveus  A  NO: Oppland  61.1488  8.7387  MF802036  MF802133  MF802215  CE23845  ZMBN111115  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802032  MF802144  MF802218  CE23846  ZMBN111116  S. niveus  A  NO: Akershus  60.3139  11.1799  MF802033  MF802145  MF802219  CE23075  ZMBN110960  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802030  MF802135  MF802216  CE23080  ZMBN110963  S. niveus  H  NO: Finnmark  69.9493  23.299  MF802031  MF802136  MF802217  CE848  SMNH162854  S. niveus  B  SE: Västergötland  57.768  12.255  MF802037  MF802119  MF802230  CE13854  SMNH162855  S. niveus  B  SE: Västergötland  57.768  12.255  MF802038  MF802124  MF802229  CE27637  SMNH162856  S. niveus  B  SE: Dalsland  58.9387  12.4975  MF802039  MF802137  MF802220  CE5803  SMNH162857  S. niveus  C  SE: Västergötland  57.6830  11.9542  MF802045  MF802120  MF802234  CE6647  SMNH162858  S. niveus  C  SE: Södermanland  58.6117  16.7598  MF802041  MF802122  MF802235  CE20421  ZMBN110489  S. niveus  C  NO: Akershus  59.5262  10.69  MF802042  MF802130  MF802233  CE24263  ZMBN111233  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802040  MF802146  MF802231  CE24264  ZMBN111234  S. niveus  I  NO: Nordland  66.3181  14.1658  MF802043  MF802147  MF802232  CE17826  SMNH162859  S. niveus  E  SE: Västergötland  58.069  12.689  MF802046  MF802125  MF802237  CE17827  SMNH162860  S. niveus  E  SE: Västergötland  58.069  12.689  MF802047  MF802126  MF802238  CE17828  SMNH162861  S. niveus  E  SE: Västergötland  58.069  12.689  MF802048  MF802127  MF802239  CE17829  SMNH162862  S. niveus  E  SE: Västergötland  58.069  12.689  MF802049  MF802128  MF802240  CE17830  SMNH162863  S. niveus  E  SE: Västergötland  58.069  12.689  MF802050  MF802118  MF802241  CE17831  SMNH162864  S. niveus  E  SE: Västergötland  58.069  12.689  MF802051  MF802143  MF802242  CE18648  No voucher  S. niveus  D  SE: Skåne  56.0379  13.2524  MF802044  MF802129  MF802236  CE23074  ZMBN110959  S. niveus  F  NO: Finnmark  69.9493  23.299  MF802052  MF802134  MF802243  CE3735  SMNH162865  S. niveus  G  US: Illinois  36.1531  −86.2725  MF802053  MF802138  MF802244  Accession numbers in bold are newly generated in this study. *From Erséus et al. (2010). †From Martinsson & Erséus (2014). ‡From Martinsson et al. (2017a). §From Dózsa-Farkas & Felföldi (2015). View Large A vast majority of the sequences were newly generated (Table 1). DNA was extracted from the posterior ends of ethanol-preserved worms, and the anterior parts were mounted in Canada balsam to be used as physical vouchers. DNA was extracted using either Qiagen’s DNeasy Blood & Tissue Kit or Epicentre QuickExtract DNA Extraction Solution 1.0, following the manufacturer’s instructions. Three markers, the mitochondrial cytochrome c oxidase subunit I (COI) gene, the complete nuclear ribosomal Internal Transcribed Spacer (ITS) region and the nuclear gene Histone 3 (H3), were amplified using primers and PCR programmes listed in Supporting Information, Table S1. Sequencing was carried out by Macrogen Inc. (Seoul, Korea) and Eurofins MWG Operon (Ebersberg, Germany). As specified in Table 1, a few sequences are from previously published works (Erséus et al., 2010; Martinsson & Erséus, 2014; Dózsa-Farkas & Felföldi, 2015; Martinsson et al., 2017a) and were all downloaded from GenBank. Sequences were assembled in Geneious Pro v. 7.1. (Biomatters Ltd.; http://www.geneious.com) and aligned separately for each genus using MAFFT v7.017 (Katoh et al., 2002) as implemented in Geneious Pro v. 7.1., using the auto-algorithm and default settings. Information about the alignments (length and number of variable positions) can be found in Table 2. All sequences produced in this study are deposited in GenBank, and the vouchers are deposited in either the Swedish Museum of Natural History (SMNH), Stockholm, Sweden, or the University Museum of Bergen (ZMBN), Bergen, Norway (accession numbers in Table 1). Table 2. Details of alignments   Alignment length  Number of variable positions  Globulidrilus   COI  658  221   H3  328  15   ITS  949  56  Hemifridericia   COI  658  151   H3  328  8   ITS  877  39  Stercutus   COI  625  179   H3  320  11   ITS  949  36    Alignment length  Number of variable positions  Globulidrilus   COI  658  221   H3  328  15   ITS  949  56  Hemifridericia   COI  658  151   H3  328  8   ITS  877  39  Stercutus   COI  625  179   H3  320  11   ITS  949  36  The variation in length in COI and H3 between genera is due to trimming of the ends of the alignments to minimize the amount of missing data; this is also true for ITS, but this marker also varies in length between taxa. View Large Table 2. Details of alignments   Alignment length  Number of variable positions  Globulidrilus   COI  658  221   H3  328  15   ITS  949  56  Hemifridericia   COI  658  151   H3  328  8   ITS  877  39  Stercutus   COI  625  179   H3  320  11   ITS  949  36    Alignment length  Number of variable positions  Globulidrilus   COI  658  221   H3  328  15   ITS  949  56  Hemifridericia   COI  658  151   H3  328  8   ITS  877  39  Stercutus   COI  625  179   H3  320  11   ITS  949  36  The variation in length in COI and H3 between genera is due to trimming of the ends of the alignments to minimize the amount of missing data; this is also true for ITS, but this marker also varies in length between taxa. View Large Species discovery The three COI alignments were used to divide the specimens into clusters representing hypothetical species. The COI alignments were analysed in the web version of ABGD (Automatic Barcode Gap Discovery) (Puillandre et al., 2012) (available at http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html), using simple distances, Pmin = 0.001, Pmax = 0.10 and X (relative gap width) = 0.5. The highest numbers of clusters from the initial partition were selected as input for the species validation step, as BBP (see Species validation) only can lump groups into more inclusive species, not split them into smaller groups; therefore, we rather risk starting with too many species than with few input species. However, we compare the result with the clusters given using a maximum intraspecific p-distance of 7%, this value is chosen, based on the genetic distances in the data sets, to represent a more reasonable threshold between intraspecific and interspecific genetic distances. However, as for most threshold values, it is somewhat arbitrarily chosen. Uncorrected intra-cluster and inter-cluster p-distances were calculated in MEGA v6.06 (Tamura et al., 2013), missing data and gap was excluded using pairwise deletions, for between-cluster comparisons the minimum distances were used and for within-cluster comparison the maximum distances were used. The clusters were visualized on COI gene trees estimated with maximum likelihood in PhyML (Anisimova & Gascuel, 2006; Guindon et al., 2010) as implemented at the South of France Bioinformatics platform (http://www.atgc-montpellier.fr/). The automatic model selection using Smart Model Selection with Bayesian information criterion as the selection criterion was chosen; Subtree Pruning and Regrafting was used for tree improvement. Branch support was calculated with the chi-square-based approximate likelihood ratio test (Anisimova & Gascuel, 2006) in PhyML. The same settings were used for all three COI analyses. The trees (Fig. 1) were drawn in FigTree 1.4.2 (Rambaut, 2014) and further edited in Adobe Illustrator. The variation in H3 and ITS was visualized by haplotype networks created in PopART v1 (Leigh & Bryant, 2015) using statistical parsimony (Templeton, Crandall & Sing, 1992; Clement et al., 2002); sites with missing data or gaps were masked and not included in the networks (Fig. 2). Figure 1. View largeDownload slide COI gene trees, showing clusters from ABGD analyses. All trees are rooted using midpoint rooting. A, Globulidrilus, all clusters represent G. riparius s.l. B, Hemifridericia, cluster E corresponds with H. bivesiculata, the other clusters represent H. parva s.l. C, Stercutus, all clusters represent S. niveus s.l. Numbers at branches denote branch support by the approximate likelihood ratio test. Scale bars show estimated numbers of nucleotide substitutions per site. Figure 1. View largeDownload slide COI gene trees, showing clusters from ABGD analyses. All trees are rooted using midpoint rooting. A, Globulidrilus, all clusters represent G. riparius s.l. B, Hemifridericia, cluster E corresponds with H. bivesiculata, the other clusters represent H. parva s.l. C, Stercutus, all clusters represent S. niveus s.l. Numbers at branches denote branch support by the approximate likelihood ratio test. Scale bars show estimated numbers of nucleotide substitutions per site. Figure 2. View largeDownload slide Statistical parsimony haplotype networks. A, B, Globulidrilus. C, D, Hemifridericia. E, F, Stercutus. A, C, D, H3 networks. B, D, F, ITS networks. The size of the circles is relative to the number of sequences sharing that haplotype, the colours correspond to COI clusters and the hatch marks correspond to substitutions. Figure 2. View largeDownload slide Statistical parsimony haplotype networks. A, B, Globulidrilus. C, D, Hemifridericia. E, F, Stercutus. A, C, D, H3 networks. B, D, F, ITS networks. The size of the circles is relative to the number of sequences sharing that haplotype, the colours correspond to COI clusters and the hatch marks correspond to substitutions. Species validation The two nuclear markers (H3 and ITS) were used for the validation step and were analysed using the BPP v.3.3 program (Yang, 2015). Including the COI data in the validation phase would make the result more robust. However, doing so would allow the COI data to take over, as the COI data set matches the groups found in the discovery phase perfectly. There is a risk that the reasoning would be circular when the same data are used to find and validate species; therefore, we perform the BPP analyses with the nuclear data only. Joint Bayesian species delimitations and species tree estimations were conducted, a method using the multispecies coalescent model to compare different arrangements of species delimitation and species phylogeny in a Bayesian framework, accounting for incomplete lineage sorting due to ancestral polymorphism and gene tree-species tree conflicts (Yang & Rannala, 2010; Rannala & Yang, 2013; Yang & Rannala, 2014). Three analyses were run for each genus, varying the population size (θs) and divergence time (τ0) priors. The priors in the different analyses, respectively, were the same for all genera: In analysis A, the population size parameters (θs) were assigned the gamma prior G(2, 400), with mean 2/400 = 0.005. The divergence time at the root of the species tree (τ0) was assigned the gamma prior G(2, 200), while the other divergence time parameters were assigned the Dirichlet prior (Yang & Rannala, 2010: equation 2). In analysis B, the population size parameters (θs) were assigned the gamma prior G(2, 1000), with mean 2/1000 = 0.002. The divergence time at the root of the species tree (τ0) was assigned the gamma prior G(2, 200), while the other divergence time parameters were assigned the Dirichlet prior (Yang & Rannala, 2010: equation 2). In analysis C, the population size parameters (θs) were assigned the gamma prior G(2, 2000), with mean 2/2000 = 0.001. The divergence time at the root of the species tree (τ0) was assigned the gamma prior G(2, 200), while the other divergence time parameters were assigned the Dirichlet prior (Yang & Rannala, 2010: equation 2). Each analysis was run three times to confirm consistency between runs. For the species arrangements, the 95% credibility intervals, that is the number of arrangements with the highest PP that together makes up 95% of the PP, were calculated. With regard to the individual species, we considered species delimited with a PP > 0.90 in all analyses to be well supported. For clusters with a PP < 0.90, we used a conservative approach and instead accepted the best-supported more inclusive species. The PP values for the species were mapped on the species tree with the highest PP in the majority of analyses. Genetic uncorrected p-distances were calculated for H3 and ITS the same way as for COI (see Species discovery), but sorted on the delimited species instead of the initial clusters. RESULTS Species discovery The ABGD analyses of COI divided the Globulidrilus data set into eight clusters, the Hemifridericia data set into five clusters and the Stercutus data set into nine clusters (Fig. 1A–C) using the maximum number of clusters. Using the 7% threshold, the Globulidrilus data set is divided into two groups, combining clusters A, D and F into one group and clusters B, C, E, G and H into another group; the Hemifridericia data set was divided into three groups, combining groups A and D into one group and groups B and C into another group, and group E still being a separate group. The Stercutus data set was divided into seven groups, clusters A and H were combined into one group and C and I into another group, remaining clusters were unchanged. The uncorrected p-distances presented here are the maximum intra-cluster distances and the minimum intra-cluster distances for each comparison; the values are summarized in Supporting Information, Tables S2–S4. For the Globulidrilus clusters (Supporting Information, Table S2), the largest intra-cluster p-distance was 6.1% (within cluster C), the smallest inter-cluster p-distance was 8.5% (between clusters D and F) and the largest inter-cluster p-distance was 18.3% (between clusters D and E). For the Hemifridericia data set (Supporting Information, Table S3), the largest intra-cluster p-distance was 3.0% (within cluster B), the smallest inter-cluster p-distance was 3.0% (between clusters B and C) and the largest inter-cluster p-distance was 15.9% (between clusters B and E). For the Stercutus data set (Supporting Information, Table S4), the largest intra-cluster p-distance was 1.0% (within clusters C), the smallest inter-cluster p-distance was 3.2% (between clusters A and H) and the largest inter-cluster p-distance was 15.7% (between clusters B and G). The haplotype networks (Fig. 2) showed that the specimens of most COI clusters have unique haplotypes in both their nuclear genes (H3 and ITS), not shared with other clusters, and that the H3/ITS haplotypes from the same COI-based cluster are close to each other. In a few cases, however, the H3/ITS haplotypes are shared by two or three clusters. Moreover, one specimen (CE24459) of H. parva cluster B is not even found together with the other cluster B specimens, neither in H3 nor in ITS; instead, it shares its nuclear haplotypes with specimens from clusters A and D (Fig. 2C, D). Species validation The 95% credibility intervals from the BPP analyses of Globulidrilus contain three species delimitation arrangements in analysis A and two arrangements in analyses B and C (Table 3). Either a seven-species or an eight-species arrangement was preferred, the best seven-species arrangement had a mean PP for the three runs of each analysis of 0.58, 0.50 or 0.39 in analyses A, B and C, respectively, whereas the eight-species arrangement had a mean PP of 0.35, 0.47 or 0.60 in analyses A, B and C, respectively (Table 3). The difference between the seven-species and eight-species arrangements pertains to whether clusters B and H are united into one species or treated as two separate species. All clusters (putative species) except B and H were delimited with a PP > 0.90 in all analyses (Table 4). Table 3. List of species delimitations and their mean posterior probability   Species delimitations  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  7 (A, BH, C, D, E, F, G)  0.58  0.50  0.38  8 (A, B, C, D, E, F, G, H)  0.35  0.47  0.60  6 (A, BH, C, DF, E, G)  0.04  0.02  0.01  7 (A, B, C, DF, E, G, H)  0.02  0.01  0.01  Hemifridericia  3 (AD, BC, E)  0.64  0.56  0.57  4 (AD, B, C, E)  0.30  0.32  0.34  4 (A, BC, D, E)  0.03  0.03  0.03  5 (A, B, C, D, E)  0.02  0.03  0.02  4 (A, BD, C, E)  0.01  0.02  0.02  3 (A, BCD, E)  0.01  0.04  0.01  Stercutus  7 (AH B CE D F G I)  0.55  0.52  0.51  6 (AH BD CE F G I)  0.12  0.07  0.05  8 (A B CE D F G H I)  0.11  0.15  0.17  8 (AH B C D E F G I)  0.10  0.16  0.18  7 (AH BD C E F G I)  0.02  0.02  0.02  7 (A BD CE F G H I)  0.02  0.02  0.02  9 (A B C D E F G H I)  0.02  0.04  0.06  6 (AH B CE DF G I)  0.01  0.00  0.00  6 (AH B CE DG F I)  0.01  0.00  0.00  6 (AH B CE D FG I)  0.01  0.00  0.00    Species delimitations  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  7 (A, BH, C, D, E, F, G)  0.58  0.50  0.38  8 (A, B, C, D, E, F, G, H)  0.35  0.47  0.60  6 (A, BH, C, DF, E, G)  0.04  0.02  0.01  7 (A, B, C, DF, E, G, H)  0.02  0.01  0.01  Hemifridericia  3 (AD, BC, E)  0.64  0.56  0.57  4 (AD, B, C, E)  0.30  0.32  0.34  4 (A, BC, D, E)  0.03  0.03  0.03  5 (A, B, C, D, E)  0.02  0.03  0.02  4 (A, BD, C, E)  0.01  0.02  0.02  3 (A, BCD, E)  0.01  0.04  0.01  Stercutus  7 (AH B CE D F G I)  0.55  0.52  0.51  6 (AH BD CE F G I)  0.12  0.07  0.05  8 (A B CE D F G H I)  0.11  0.15  0.17  8 (AH B C D E F G I)  0.10  0.16  0.18  7 (AH BD C E F G I)  0.02  0.02  0.02  7 (A BD CE F G H I)  0.02  0.02  0.02  9 (A B C D E F G H I)  0.02  0.04  0.06  6 (AH B CE DF G I)  0.01  0.00  0.00  6 (AH B CE DG F I)  0.01  0.00  0.00  6 (AH B CE D FG I)  0.01  0.00  0.00  For each analysis and genus, the PP values in bold make up the 95% credibility interval. View Large Table 3. List of species delimitations and their mean posterior probability   Species delimitations  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  7 (A, BH, C, D, E, F, G)  0.58  0.50  0.38  8 (A, B, C, D, E, F, G, H)  0.35  0.47  0.60  6 (A, BH, C, DF, E, G)  0.04  0.02  0.01  7 (A, B, C, DF, E, G, H)  0.02  0.01  0.01  Hemifridericia  3 (AD, BC, E)  0.64  0.56  0.57  4 (AD, B, C, E)  0.30  0.32  0.34  4 (A, BC, D, E)  0.03  0.03  0.03  5 (A, B, C, D, E)  0.02  0.03  0.02  4 (A, BD, C, E)  0.01  0.02  0.02  3 (A, BCD, E)  0.01  0.04  0.01  Stercutus  7 (AH B CE D F G I)  0.55  0.52  0.51  6 (AH BD CE F G I)  0.12  0.07  0.05  8 (A B CE D F G H I)  0.11  0.15  0.17  8 (AH B C D E F G I)  0.10  0.16  0.18  7 (AH BD C E F G I)  0.02  0.02  0.02  7 (A BD CE F G H I)  0.02  0.02  0.02  9 (A B C D E F G H I)  0.02  0.04  0.06  6 (AH B CE DF G I)  0.01  0.00  0.00  6 (AH B CE DG F I)  0.01  0.00  0.00  6 (AH B CE D FG I)  0.01  0.00  0.00    Species delimitations  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  7 (A, BH, C, D, E, F, G)  0.58  0.50  0.38  8 (A, B, C, D, E, F, G, H)  0.35  0.47  0.60  6 (A, BH, C, DF, E, G)  0.04  0.02  0.01  7 (A, B, C, DF, E, G, H)  0.02  0.01  0.01  Hemifridericia  3 (AD, BC, E)  0.64  0.56  0.57  4 (AD, B, C, E)  0.30  0.32  0.34  4 (A, BC, D, E)  0.03  0.03  0.03  5 (A, B, C, D, E)  0.02  0.03  0.02  4 (A, BD, C, E)  0.01  0.02  0.02  3 (A, BCD, E)  0.01  0.04  0.01  Stercutus  7 (AH B CE D F G I)  0.55  0.52  0.51  6 (AH BD CE F G I)  0.12  0.07  0.05  8 (A B CE D F G H I)  0.11  0.15  0.17  8 (AH B C D E F G I)  0.10  0.16  0.18  7 (AH BD C E F G I)  0.02  0.02  0.02  7 (A BD CE F G H I)  0.02  0.02  0.02  9 (A B C D E F G H I)  0.02  0.04  0.06  6 (AH B CE DF G I)  0.01  0.00  0.00  6 (AH B CE DG F I)  0.01  0.00  0.00  6 (AH B CE D FG I)  0.01  0.00  0.00  For each analysis and genus, the PP values in bold make up the 95% credibility interval. View Large Table 4. List of delimited species and their mean posterior probabilities   Species  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  G  1.00  1.00  1.00  C  1.00  1.00  1.00  E  1.00  1.00  1.00  A  0.99  1.00  1.00  F  0.94  0.97  0.98  D  0.94  0.97  0.98  BH  0.62  0.51  0.39  H  0.38  0.49  0.61  B  0.38  0.49  0.61  DF  0.06  0.03  0.02  Hemifridericia  E  1.00  1.00  1.00  AD  0.93  0.88  0.91  BC  0.67  0.59  0.61  C  0.32  0.37  0.38  B  0.32  0.35  0.36  A  0.07  0.12  0.09  D  0.05  0.06  0.06  BD  0.01  0.02  0.02  BCD  0.01  0.04  0.01  CD  0.00  0.00  0.00  Stercutus  I  0.99  1.00  1.00  G  0.97  0.99  1.00  F  0.96  0.99  1.00  CE  0.85  0.77  0.74  AH  0.84  0.79  0.75  B  0.82  0.88  0.92  D  0.80  0.87  0.91  A  0.16  0.21  0.25  H  0.16  0.21  0.25  BD  0.17  0.12  0.08  E  0.15  0.23  0.26  C  0.15  0.23  0.26  DF  0.02  0.01  0.00  FG  0.01  0.00  0.00  DG  0.01  0.00  0.00    Species  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  G  1.00  1.00  1.00  C  1.00  1.00  1.00  E  1.00  1.00  1.00  A  0.99  1.00  1.00  F  0.94  0.97  0.98  D  0.94  0.97  0.98  BH  0.62  0.51  0.39  H  0.38  0.49  0.61  B  0.38  0.49  0.61  DF  0.06  0.03  0.02  Hemifridericia  E  1.00  1.00  1.00  AD  0.93  0.88  0.91  BC  0.67  0.59  0.61  C  0.32  0.37  0.38  B  0.32  0.35  0.36  A  0.07  0.12  0.09  D  0.05  0.06  0.06  BD  0.01  0.02  0.02  BCD  0.01  0.04  0.01  CD  0.00  0.00  0.00  Stercutus  I  0.99  1.00  1.00  G  0.97  0.99  1.00  F  0.96  0.99  1.00  CE  0.85  0.77  0.74  AH  0.84  0.79  0.75  B  0.82  0.88  0.92  D  0.80  0.87  0.91  A  0.16  0.21  0.25  H  0.16  0.21  0.25  BD  0.17  0.12  0.08  E  0.15  0.23  0.26  C  0.15  0.23  0.26  DF  0.02  0.01  0.00  FG  0.01  0.00  0.00  DG  0.01  0.00  0.00  PP > 0.90 are marked in bold. Accepted species are also marked in bold; note, for example, that although the two Stercutus clusters B and D have PP values between 0.80 and 0.92 in all analyses, the more conservative alternative of merging them into a single species (BD) is preferred. View Large Table 4. List of delimited species and their mean posterior probabilities   Species  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  G  1.00  1.00  1.00  C  1.00  1.00  1.00  E  1.00  1.00  1.00  A  0.99  1.00  1.00  F  0.94  0.97  0.98  D  0.94  0.97  0.98  BH  0.62  0.51  0.39  H  0.38  0.49  0.61  B  0.38  0.49  0.61  DF  0.06  0.03  0.02  Hemifridericia  E  1.00  1.00  1.00  AD  0.93  0.88  0.91  BC  0.67  0.59  0.61  C  0.32  0.37  0.38  B  0.32  0.35  0.36  A  0.07  0.12  0.09  D  0.05  0.06  0.06  BD  0.01  0.02  0.02  BCD  0.01  0.04  0.01  CD  0.00  0.00  0.00  Stercutus  I  0.99  1.00  1.00  G  0.97  0.99  1.00  F  0.96  0.99  1.00  CE  0.85  0.77  0.74  AH  0.84  0.79  0.75  B  0.82  0.88  0.92  D  0.80  0.87  0.91  A  0.16  0.21  0.25  H  0.16  0.21  0.25  BD  0.17  0.12  0.08  E  0.15  0.23  0.26  C  0.15  0.23  0.26  DF  0.02  0.01  0.00  FG  0.01  0.00  0.00  DG  0.01  0.00  0.00    Species  PP analysis A  PP analysis B  PP analysis C  Globulidrilus  G  1.00  1.00  1.00  C  1.00  1.00  1.00  E  1.00  1.00  1.00  A  0.99  1.00  1.00  F  0.94  0.97  0.98  D  0.94  0.97  0.98  BH  0.62  0.51  0.39  H  0.38  0.49  0.61  B  0.38  0.49  0.61  DF  0.06  0.03  0.02  Hemifridericia  E  1.00  1.00  1.00  AD  0.93  0.88  0.91  BC  0.67  0.59  0.61  C  0.32  0.37  0.38  B  0.32  0.35  0.36  A  0.07  0.12  0.09  D  0.05  0.06  0.06  BD  0.01  0.02  0.02  BCD  0.01  0.04  0.01  CD  0.00  0.00  0.00  Stercutus  I  0.99  1.00  1.00  G  0.97  0.99  1.00  F  0.96  0.99  1.00  CE  0.85  0.77  0.74  AH  0.84  0.79  0.75  B  0.82  0.88  0.92  D  0.80  0.87  0.91  A  0.16  0.21  0.25  H  0.16  0.21  0.25  BD  0.17  0.12  0.08  E  0.15  0.23  0.26  C  0.15  0.23  0.26  DF  0.02  0.01  0.00  FG  0.01  0.00  0.00  DG  0.01  0.00  0.00  PP > 0.90 are marked in bold. Accepted species are also marked in bold; note, for example, that although the two Stercutus clusters B and D have PP values between 0.80 and 0.92 in all analyses, the more conservative alternative of merging them into a single species (BD) is preferred. View Large The 95% credibility intervals from the BPP analyses of Hemifridericia contain three different species delimitation arrangements in analysis A and four arrangements in analyses B and C (Table 3). In all analyses, a three-species arrangement, uniting clusters A and D as well as B and C, was preferred with a mean PP of 0.64, 0.56 and 0.57 in analyses A, B and C, respectively (Table 3). Cluster E, that is H. bivesiculata, was delimited with a PP of 1 in all analyses, species A + D was delimited with a PP > 0.90 in analyses A and C and with a PP of 0.88 in analyses B and species B + C had a PP of 0.67, 0.59 and 0.61 in analyses A, B and C, respectively (Fig. 3B; Table 4). Figure 3. View largeDownload slide Species trees from BPP analyses, the trees shown are the species trees and species delimitations with the highest PP in a majority of analyses. A, Globulidrilus. B, Hemifridericia. C, Stercutus. Numbers at branches are mean PP for species in analyses A, B and C, respectively. Figure 3. View largeDownload slide Species trees from BPP analyses, the trees shown are the species trees and species delimitations with the highest PP in a majority of analyses. A, Globulidrilus. B, Hemifridericia. C, Stercutus. Numbers at branches are mean PP for species in analyses A, B and C, respectively. The 95% credibility intervals from the BPP analyses of Stercutus contain nine different species delimitation arrangements in analysis A, six arrangements in analyses B and five arrangements in analysis C (Table 3). In all analyses, a seven-species arrangement, uniting clusters A and H as well as clusters C and E, was preferred with a mean PP of 0.55, 0.52 or 0.54 in analyses A, B and C, respectively (Table 3). Clusters F, G and I were delimited with a PP > 0.95 in all analyses, and clusters B and D were delimited with a PP > 0.90 in analysis C; species C + E had a PP of 0.85, 0.77 and 0.74 in analyses A, B and C, respectively, and species A + H had a PP of 0.84, 0.79 and 0.75 in analyses A, B and C, respectively (Fig. 3C; Table 4). For Globulidrilus, the genetic distances within the delimited species in H3 are maximally 0.3% (in BH) and in ITS are 0.6% (in C); the minimum distances between species vary in H3 between 0.0 (between A, D and F) and 3.5% (between A and BH, C and F) and in ITS between 0.1 (between C and G) and 2.8% (between C and F) (Supporting Information, Table S5). For Hemifridericia, the genetic distances within the delimited species in H3 are maximally 0.3% (in BC) and in ITS are 0.8% (in BC); the minimum distances between species vary in H3 between 0.0 (between AD and BC) and 2.1% (between E and the other species) and in ITS between 0.0 (between AD and BC) and 2.4% (between AD and E) (Supporting Information, Table S6). If specimen CE24459, which is found together with different groups in the COI and the nuclear data sets, would be excluded, there would be a difference between species AD and BC in both data sets (0.3% in H3 and 0.8% in ITS). For Stercutus, the maximum genetic distances within the delimited species in H3 are 0.3% (in BD) and in ITS are 0.7% (in BD); the minimum distances between species vary in H3 between 0.3 (between BD and AH, CE and F, and between CD and I) and 2.6% (between G and I) and in ITS between 0.3 (between AH and CE) and 1.5% (between BD and G) (Supporting Information, Table S7). To summarize, the results of the BPP analyses suggest that our data set includes at least seven species of Globulidrilus, for the time being all attributed to the nominal species G. riparius; three species of Hemifridericia, whereof two are referred to as H. parva and the third as H. bivesiculata; and at least six species of Stercutus. DISCUSSION We have found that the four nominal taxa that we included in the analyses actually represent at least 16 species, with varying support. It is reasonable to question whether all these recognized units are species or if they represent something else. The multispecies coalescent approach has been criticized for delimiting population structure and not real species (Sukumaran & Knowles, 2017). However, the latter authors do not specify what they mean by ‘species’, and as so many species concepts have been put forward in the past (see e.g. De Queiroz, 2007), it is hard to understand what they mean with their claims. One of the strengths of the multispecies coalescent model is that it is based on a well-defined concept of species being lineages that no longer exchange genes following divergence from a common ancestor (Toprak et al., 2016). It is possible that some of these lineages, in the future, will merge and hybridize to such extent that they will despeciate (as described by Turner, 2002). It is also possible that addition of more data, either in the form of more specimens or additional markers, will lead to another result than the one we have found in this study. Species assignments are hypotheses and as such may change over time, but, for the time being, the results of this study are the best hypotheses that we have for the species boundaries in our three genera. The high number of species arrangements within the 95% credibility intervals, especially for Stercutus (see Table 2), as well as the low support for some of the delimited species (Table 3) are probably due to a lack of phylogenetic signal in our nuclear data, and it is possible that including more variable loci would give better support for some species. However, in other studies, BPP has successfully been used with an amount of data similar to what we have herein (e.g. Hambäck et al., 2013; Parmakelis et al., 2013; Fossen et al., 2016; Martinsson, Rhodén & Erséus, 2017b). When using a higher, fixed threshold (7%) in the species discovery part the clusters changes, for Hemifridericia, the resulting clusters were the same as the delimited species, whereas for the other genera, it gave a different result, for Globulidrilus, a 7% threshold only gave two clusters, and interestingly, these two groups can be seen in the nuclear data as well, where there is a larger distance between groups ADF and BCEH than within the groups, nevertheless the BPP analyses gave strong support for at least seven species. For Stercutus, a 7% threshold gave mainly the same result as using the highest number of cluster, with the exception that it united clusters A and H as well as clusters C and I, and AD was found to be one species in the BPP analyses, but there is no support for combining C and I, instead C and E are found in the same species. This highlights the problem of using strict thresholds for single loci in species delimitation, even if it still can be a useful rule of thumb. Of the nominal taxa in focus in this study, G. riparius has already been suggested to be a species complex based on morphological observations (Christensen & Dózsa-Farkas, 2012), and for S. niveus, morphological variation has been observed as well (Rota, 1995). In H. parva, there is no notion of morphological variation, at least not in Europe. However, a description of this alleged species from China (Wang, Xie & Liang, 1999) differs from the description of material from Europe (e.g. Nielsen & Christensen, 1959) in detail about the septal glands; it could very well represent a different species. The odd specimen of H. parva (CE24459), which was found together with specimens of different clusters in the mitochondrial (cluster B) and nuclear data (in clusters A and D) sets, could be a case of hybridization and mitochondrial introgression between the two H. parva species. Hybridization has been reported between cryptic earthworm species (Dupont et al., 2016; Martinsson & Erséus, 2017). At the locality where the specimen corresponding to CE24459 was found, there was also a specimen of cluster A (C.E., unpubl. data). In the material used in our present work, we had co-occurrence of more than one cluster at the same location in all species (see Table 1). In Globulidrilus, clusters A, C and F, clusters A and D, clusters B and H and clusters C and G are found together. In Hemifridericia, A and C are found together, and in Stercutus, F and H are found together. Despite this co-occurrence, only one case of mismatch between mitochondrial and nuclear markers is observed, indicating that reproductive barriers between the delimited species exist. It is worth noting that our two lineages of H. parva are found in Hungary as well as in Scandinavia and they are probably well spread in Europe. To summarize, we found high genetic diversity in the studied enchytraeid groups, compared to the previously recognized four species. We suggest that these three genera comprise at least 16 species. The delimited species are not formally revised, described and named here, mainly due to the combination of low support for some of them and lack of mature specimens of many of them, which makes morphological comparisons and good-quality descriptions difficult. However, it is likely that some of these species are truly cryptic and that it will be impossible to identify them based on morphology. Nevertheless, this study is a first step towards understanding the species diversity of these groups, and hopefully, more material suitable for morphological studies will be collected in the future. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Primers, sequences and PCR programs used for amplification of the mitochondrial COI and nuclear ITS and H3. Table S2. Uncorrected pairwise COI distances for Globulidrilus riparius, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. Table S3. Uncorrected pairwise COI distances for Hemifridericia, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. Clusters A–D represent H. parva and cluster E, H. bivesiculata. All distances are expressed as percents. Table S4. Uncorrected pairwise COI distances for Stercutus niveus, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. Table S5. Uncorrected pairwise H3 and ITS distances for Globulidrilus riparius, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. The H3 distances are at the lower left side, and the ITS distances are at the upper right side. Table S6. Uncorrected pairwise H3 and ITS distances for Hemifridericia, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. The H3 distances are at the lower left side, and the ITS distances are at the upper right side. Clusters A–D represent H. parva and cluster E, H. bivesiculata. All distances are expressed as percents. Table S7. Uncorrected pairwise H3 and ITS distances for Stercutus niveus, the values shown are the minimum values for the inter-cluster comparisons and the maximum for the intra-cluster comparisons. All distances are expressed as percents. The H3 distances are at the lower left side, and the ITS distances are at the upper right side. ACKNOWLEDGEMENTS We are grateful to Leyla Arsan, Stephen Atkinson, Mårten Klinth, Tryggve Persson, Mark Wetzel and Magdalena Zarowiecki for providing specimens and to Anna Ansebo, Marcus Carlberg, Daniel Gustafsson, Per Hjelmstedt, Jeff Hunt, Mårten Klinth, Emelie Lindquist, Maria Lindström and Urban Olsson for laboratory assistance. We thank Kerryn Elliott and two anonymous reviewers for valuable comments, which helped to improve the manuscript. 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