Molecular Ecology (2012) 21, 3776–3793 doi: 10.1111/j.1365-294X.2012.05648.x Appearances can be deceptive: different diversification patterns within a group of Mediterranean earthworms (Oligochaeta, Hormogastridae) M A R T A N O V O , * † A N A A L M O D Ó V A R , † R O S A F E R N Á N D E Z , † D O L O R E S T R I G O , † D A R Í O J . D Í A Z - C O S Í N † and G O N Z A L O G I R I B E T * *Department of Organismic and Evolutionary Biology, Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA, †Departamento de Zoologı́a y Antropologı́a Fı́sica, Facultad de Biologı́a, Universidad Complutense de Madrid, C ⁄ José Antonio Nováis, 28040, Madrid, Spain Abstract Many recent studies on invertebrates have shown how morphology not always captures the true diversity of taxa, with cryptic speciation often being discussed in this context. Here, we show how diversification patterns can be very different in two clades of closely related earthworms in the genus Hormogaster stressing the risk of using nonspecific substitution rate values across taxa. On the one hand, the Hormogaster elisae species complex, endemic to the central Iberian Peninsula, shows morphological stasis. On the other hand, a clade of Hormogaster from the NE Iberian Peninsula shows an enormous morphological variability, with 15 described morphospecies. The H. elisae complex, however, evolves faster genetically, and this could be explained by the harsher environmental conditions to which it is confined—as detected in this study, that is, sandier and slightly poorer soils with lower pH values than those of the other species in the family. These extreme conditions could be at the same time limiting morphological evolution and thus be responsible for the observed morphological stasis in this clade. Contrarily, Hormogaster species from the NE Iberian Peninsula, although still inhabiting harsher milieu than other earthworm groups, have had the opportunity to evolve into a greater morphological disparity. An attempt to delimit species within this group following the recently proposed general mixed Yule-coalescent method showed a higher number of entities than expected under the morphospecies concept, most probably due to the low vagility of these animals, which considerably limits gene flow between distant conspecific populations, but also because of the decoupling between morphological and genetic evolution in the H. elisae complex. Keywords: Annelida, environmental analyses, evolutionary rates, general mixed Yulecoalescent species delimitation Received 17 October 2011; revision received 1 April 2012; accepted 4 April 2012 Introduction Recent use of molecular techniques in taxonomy has often led to the detection of unbalanced molecular and morphological evolution, with the study of cryptic speciation becoming an important topic in evolutionary biology (e.g. McGuigan & Sgrò 2009). The discovery of Correspondence: Marta Novo, Fax: +1 617 496 5854; E-mail: [email protected] species difficult to distinguish by morphological traits is not uncommon, especially within invertebrate groups (e.g. Müller 2000; Pfenninger et al. 2003; Stoks et al. 2005; Hogg et al. 2006; Tully et al. 2006; Challis et al. 2007; Finston et al. 2007) and specifically in annelid worms such as leeches (Bely & Weisblat 2006), freshwater oligochaetes (Gustafsson et al. 2009) and earthworms (e.g. Chang et al. 2008; King et al. 2008; James et al. 2010). Mayr (1948) was the first to discuss the difficulties in distinguishing certain species based solely on morphological 2012 Blackwell Publishing Ltd D I V E R S I F I C A T I O N P A T T E R N S I N E A R T H W O R M S 3777 characters and he used this to argue against the morphological species concept (Mayr 1963). This is particularly true in earthworms, whose taxonomy suffers from their structural simplicity (Pop et al. 2003). Earthworms are key organisms for the correct functioning of soil systems. They captured Darwin’s attention who stated that ‘It may be doubted whether there are many other animals which have played so important a part in the history of the world, as have these lowly organized creatures’ (Darwin 1881: 316). Earthworms have also been targeted in applied research for a long time (e.g. Lavelle & Spain 2001; Edwards 2004). More recently, molecular data on earthworms have flourished, including DNA barcoding for understanding taxonomy of these soil organisms (Rougerie et al. 2009), but their evolutionary biology is still poorly known. Several recent studies have shown large genetic diversity, suggesting some interesting cases of cryptic speciation, while others have questioned morphology-based taxonomy (e.g. Briones et al. 2009; Fernández et al. 2011). Discordant patterns of morphological and molecular evolution can be common in the soil environment, where chemical signalling may play a key role in sexual selection (Lee & Frost 2002), perhaps more important than morphology itself. In addition, it has been proposed that extreme subsurface conditions could constrain morphological evolution (e.g. Jones et al. 1992; Caccone & Sbordoni 2001; Wiens et al. 2003). Hormogastrid earthworms, endogeic and endemic to the Mediterranean region (Cobolli-Sbordoni et al. 1992), have shown to be challenging from a genetic perspective. Specifically, a very high genetic diversity was found in the central Iberian Peninsula for Hormogaster elisae (Novo et al. 2009, 2010a), the only morphospecies described in this area, but now thought to constitute a species complex. This contrasts with the much larger species number of the genus in other areas of the Mediterranean. The phylogenetic placement of this group within the family indicates that H. elisae constitutes an independent lineage of the remaining species of Hormogaster, a genus shown to be paraphyletic (Novo et al. 2011). The family Hormogastridae currently comprises four genera and 22 species of large to middle-sized earthworms, most of which inhabit the NE Iberian Peninsula (15 Hormogaster species and some varieties or subspecies), whose phylogeny was studied in Novo et al. (2011). In this work, we markedly increase the sampling for hormogastrids since Novo et al. (2011) with the aim to address an important ecological question—whether the diversity of soil habitats is related to genetic and morphological variability—and its evolutionary consequences. We therefore wanted to test the hypothesis that morphological change in earthworms is limited by their environment (i.e. pH values, poor and sandy 2012 Blackwell Publishing Ltd soils). This constraint of morphological change is exemplified by the H. elisae lineage. In addition, we wanted to compare the substitution rates of the different clades defined in the family Hormogastridae because their morphological diversity and probably the characteristics of the soil they inhabit vary. Finally, we investigated the delimitation of hormogastrid species using a general mixed Yule-coalescent (GMYC) model approach to test whether the limited information provided by morphology could be complemented by this method. Materials and methods Sampling and morphological study We collected 376 mature individuals representing 20 of the 22 described species of Hormogastridae in 46 localities (Table 1, see a similar map in Novo et al. 2011) from the Iberian Peninsula, France (including Corsica) and Italy (Sardinia). Additional data from the study of Novo et al. (2010a) in central Spain were included for cytochrome ca. oxidase subunit I gene (COI) and 16StRNA genes and soil analyses. All individuals were collected by hand, washed in distilled water and preserved in ca. 96% EtOH at )20 C for subsequent molecular work. A portion of the integument (ca. 25 mg) was cleansed under a stereomicroscope to remove soil particles. Subsequently, integument samples were hydrated and preserved at )80 C until DNA extraction. All specimens were dissected and examined morphologically for their taxonomic identification following Qiu & Bouché (1998). DNA extraction, gene amplification and sequencing Total genomic DNA was extracted from the integument tissue sample using the DNeasy Tissue Kit (QIAGEN) eluting twice with 70 lL of buffer. Molecular markers included mitochondrial regions of the COI, 16S rRNA gene and tRNA Leu, Ala, and Ser (16S-tRNA) and two nuclear protein-encoding genes (histone H3 and histone H4). Primer sequences, polymerase chain reactions (PCR) and sequencing reactions are the same as in Novo et al. (2011). Chromatograms were visualized in Sequencher v.4.7 (Gene Codes Corporation, Ann Arbor, MI, USA) to assemble sequences. All amplicons were compared against the GenBank database with the BLAST algorithm (Altschul et al. 1997) for potential contaminants. Genetic diversity Genetic variability estimates and mean genetic differentiation between and within studied populations were Fresno del Torote Lozoyuela Molar H. elisae Sp1 H. elisae Sp1 Navas de Buitrago Paracuellos del Jarama H. elisae Sp1 H. elisae Sp1 H. elisae Sp1 Cubillo de Uceda H. elisae Sp1 Cabrera VIL Villamassargia HE HPA Rı́o Ter Hormogaster pretiosa c.f. hispanica* Hormogaster pretiosa pretiosa Hormogaster elisae Sp1 JAR NAV MOL LOZ FRE UCE CAB ORD Ordal Hormogaster najaformis HCL Code Locality Species Phylogenetic Clade Madrid, Spain Madrid, Spain Madrid, Spain Madrid, Spain Madrid, Spain Guadalajara, Spain CarboniaIglesias, Sardinia, Italy Madrid, Spain Girona, Spain Barcelona, Spain Region, Country GQ409665.1– GQ409670.1 GQ409683.1– GQ409688.1 EF653874.1– EF653880.1 GQ409698.1– GQ409699.1 EF653886.1– EF653890.1, GQ409690.1, GQ409691.1 GQ409692.1– GQ409697.1 HQ621998, JN209576– JN209578 GQ409689.1 HQ621985, JN209571– JN209575 — COI: GenBank Numbers GQ409730.1– GQ409731.1, JN209303– JN209308 GQ409745.1– GQ409749.1, JN209252– JN209256 GQ409732.1– GQ409736.1, JN209294– JN209295 GQ409720.1– GQ409722.1, JN209407– JN209412 GQ409723.1– GQ409724.1 GQ409725.1– GQ409728.1, JN209271– JN209285 HQ621893, JN209439– JN209445 GQ409729.1, JN209221– JN209226 HQ621878, JN209316– JN209321 HQ621892 16S: GenBank Numbers HQ622013, JN209625, JN209628, JN209640 HQ622009, JN209637 HQ622016, JN209650– JN209652, JN209656– JN209658 HQ622019, JN209626– JN209627, JN209629– JN209633, JN209636 HQ622021, JN209653– JN209655 HQ622045, JN209698– JN209700 HQ622007, JN209648– JN209649, JN209659 HQ622039, JN209660– JN209662 HQ622023, JN209695– JN209697 HQ622044 H3: GenBank Numbers HQ622059, JN209724– JN209726 HQ622065, JN209731– JN209732, JN209741– JN209744, JN209746 HQ622067, JN209733– JN209735 HQ622055, JN209723 HQ622062, JN209727– JN209730, JN209748 HQ622085, JN209739– JN209740 HQ622090, JN209771– JN209777 HQ622053, JN209747, JN209752 HQ622069, JN209768– JN209770 — H4: GenBank Numbers N4030¢36.9¢¢ W0331¢59.1¢¢ N4056¢21.0¢¢ W0335¢38.1¢¢ W0333¢53.1¢¢ N4044¢22.9¢¢ N4035¢51.8¢¢ W0324¢42.0¢¢ N4056¢51.9¢¢ W0337¢16.2¢¢ 674 994 753 1036 660 883 1029 N4051¢25.9¢¢ W0337¢18.2¢¢ N4049¢38.7¢¢ W0325¢19.5¢¢ 290 — 391 Altitude (m) N3915¢29.7¢¢ E840¢17.3¢¢ — N4123¢27.3¢¢ E00149¢39.3¢¢ GPS Coordinates Table 1 Hormogastrid species (alphabetically ordered within the clades recovered by Novo et al. 2011), localities of collection and GenBank Accession numbers 3778 M . N O V O E T A L . 2012 Blackwell Publishing Ltd Phylogenetic Clade Table 1 Continued 2012 Blackwell Publishing Ltd SIG Venturada Siguero H. elisae Sp1 Soto del Real Anchuelo Boadilla del Monte Sevilla la Nueva Pardo H. elisae Sp2 H. elisae Sp3 H. elisae Sp4 H. elisae Sp4 H. elisae Sp5 H. elisae Sp2 VEN Tres Cantos H. elisae Sp1 PAR SEV BOA ANC SOT TRE RED Redueña H. elisae Sp1 Code Locality Species Madrid, Spain Madrid, Spain Madrid, Spain Madrid, Spain Madrid, Spain Segovia, Spain Madrid, Spain Madrid, Spain Madrid, Spain Region, Country EF653898.1– EF653902.1 EF653903.1– EF653905.1 GQ409661.1– GQ409664.1 EF653868.1– EF653873.1 GQ409700.1– GQ409702.1 EF653891.1– EF653897.1 GQ409671.1, GQ409672.1, GQ409674.1 EF653881.1– EF653885.1, GQ409673.1, GQ409675.1– GQ409677.1 GQ409678.1– GQ409682.1 COI: GenBank Numbers GQ409741.1– GQ409744.1, GQ409752.1, JN209338– JN209357 GQ409737.1– GQ409740.1, JN209401– JN209406 GQ409750.1– GQ409751.1, GQ409753.1, JN209420– JN209425 GQ409710.1– GQ409715.1, JN209376– JN209380 GQ409716.1– GQ409719.1, JN209381– JN209386 GQ409754.1, JN209205– JN209207 GQ409704.1– GQ409706.1, JN209215JN209220 GQ409707.1– GQ409708.1, JN209365– JN209370 GQ409709.1, HQ621879, JN209322– JN209325 16S: GenBank Numbers HQ622024, JN209672– JN209673 HQ622031, JN209669– JN209671 HQ622001, JN209663– JN209665 HQ622004, JN209666– JN209668 HQ622034, JN209645– JN209647 HQ622033, JN209642– JN209644 HQ622038, JN209622, JN209639, JN209641 HQ622041, JN209623, JN209635, JN209638 HQ622029, JN209624, JN209634 H3: GenBank Numbers HQ622070, JN209759– JN209761 HQ622077, JN209756– JN209758 HQ622050, JN20975– JN2097553 HQ622047, JN209713–JN209715 HQ622080, JN209720– JN209722 HQ622079, JN209716– JN209719 HQ622087, JN209749– JN209751 HQ622084, JN209737– JN209738, JN209745 HQ622075, JN209736 H4: GenBank Numbers N4031¢11.0¢¢ W0347¢42.7¢¢ N4020¢41.9¢¢ W0400¢48.9¢¢ N4025¢50.2¢¢ W0355¢30.9¢¢ N4028¢50.2¢¢ W0314¢33.5¢¢ N4046¢30.5¢¢ W0346¢42.6¢¢ N4111¢06.1¢¢ W0337¢07.4¢¢ N4048¢07.7¢¢ W0337¢19.6¢¢ N4036¢46.9¢¢ W0340¢41.1¢¢ N4048¢46.7¢¢ W0336¢06.2¢¢ GPS Coordinates 662 644 667 780 985 1073 890 675 797 Altitude (m) D I V E R S I F I C A T I O N P A T T E R N S I N E A R T H W O R M S 3779 HNE HM HIS VPJ ALG GHI Alghero Ghilarza Code Volpajola Locality Sassari, Sardinia, Italy Oristano, Sardinia, Italy Haute-Corse, Corsica, France Region, Country HQ621996, JN209565– JN209570 HQ621971 Graus Banyuls Sur Mer Loporzano Torrecilla en Cameros Hormogaster eserana Hormogaster gallica Hormogaster huescana Hormogaster ireguana TOR LOP BSM GRA La Rioja, Spain PyrénéesOrientales, France Huesca, Spain Huesca, Spain HQ621994, JN209486– JN209492 HQ621980, JN209523– JN209529 HQ621977, JN209530– JN209536 HQ621974 HQ621888, JN209394– JN209400 HQ622037, JN209593– JN209595 HQ622015, JN209614, JN209621 HQ621873, JN209264– JN209270 HQ622028, JN209602– JN209604 HQ621883, JN209331– JN209337 HQ622011, JN209608– JN209610 HQ622006 HQ622010, JN209674– JN209676 HQ622012, JN209677– JN209679 HQ622036, JN209680– JN209682 HQ622014, JN209689– JN209691 HQ622018, JN209692– JN209694 HQ622003, JN209596– JN209598 HQ622005 HQ621869, JN209234– JN209237 HQ621871, JN209245– JN209251 HQ621887, JN209389– JN209393 HQ621872, JN209257– JN209263 HQ621875, JN209287– JN209293 HQ621865, JN209208– JN209214 HQ621866 HQ621870, JN209238– JN209244 HQ621867 HQ622042, JN209683– JN209685 HQ622000 HQ621890, JN209426– JN209431 HQ621863 HQ622061, JN209788– JN209789, JN209829 HQ622083, JN209765– JN209767 HQ622074, JN209793, JN209795– JN209796 HQ622057, JN209785– JN209787 HQ622052 HQ622056, JN209804– JN209806 HQ622058, JN209807– JN209809 HQ622082, JN209810– JN209812 HQ622060, JN209823– JN209825 HQ622064, JN209826, JN209828 HQ622049, JN209820– JN209822 HQ622051 HQ622088, JN209813– JN209815 — 710 N4429¢43.6¢¢ E00327¢41.9¢¢ N4213¢54.7¢¢ W00237¢35.2¢¢ N4228¢08.0¢¢ E00309¢08.2¢¢ N4207¢04.6¢¢ W00014¢54.6¢¢ N4210¢11.8¢¢ E00019¢53.2¢¢ 789 558 105 454 N4151¢ 454 04.6¢¢ E00119¢40.4¢¢ N4148¢04.9¢¢ 1145 E00220¢51.6¢¢ N4033¢49.1¢¢ 957 W00001¢03.5¢¢ 210 54 N4128¢24.4¢¢ E00906¢08.6¢¢ N4418¢46.8¢¢ E00052¢45.0¢¢ 276 247 0 401 Altitude (m) N3919¢23.8¢¢ E00831¢17.7¢¢ N4035¢59.9¢¢ E00817¢56.7¢¢ N4006¢58.9¢¢ E00851¢53.3¢¢ N4231¢18.6¢¢ E00921¢05.7¢¢ COI: GenBank 16S: GenBank H3: GenBank H4: GenBank GPS Numbers Numbers Numbers Numbers Coordinates HQ621976, JN209453– JN209457 Hormogaster Iglesias IGL CarboniaHQ621978, redii redii Iglesias, Sardinia, Italy JN209458– JN209464 Hormogaster Col de la Testa TES Corse-du-Sud, HQ621993, samnitica Corsica, France JN209465– lirapora JN209469 Hemigastrodrilus Cardayre LAC Lot-et-Garonne, France HQ621979, monicae JN209579– JN209585 Hemigastrodrilus Mende MND Lozére, France HQ621982, monicae JN209446– JN209452 Hormogaster Biosca BIO Lleida, Spain HQ621972, arenicola JN209493– JN209499 Hormogaster El Brull BRU Barcelona, Spain HQ621973 catalaunensis Hormogaster Puerto QUE Castellón, Spain HQ621989, castillana Querol JN209507– JN209513 Hormogaster redii insularis Hormogaster redii redii Hormogaster redii redii Phylogenetic Clade Species Table 1 Continued 3780 M . N O V O E T A L . 2012 Blackwell Publishing Ltd 2012 Blackwell Publishing Ltd Table 1 Continued VG Phylogenetic Clade Table 1 Continued QLL PRB MON Vall d’Uixó Quillan Peralba Monrepós Hormogaster pretiosa arrufati* Hormogaster pretiosa nigra* Hormogaster pretiosa var.* Hormogaster pretiosiformis Sant Joan de les Abadesses Vignysa vedovinii SAN Montmajor Hormogaster sylvestris New species (author’s work in progress) Vignysa popi Pignans Saint- Gelydu-Fesc MAJ Cervera del Maestre Hormogaster sp. PIG SGF CER TAL Talarn Hormogaster sp. ALE Alesanco Hormogaster riojana UIX OEL Peña Oroel Hormogaster oroeli Code Locality Species Var, France Hérault, France Girona, Spain Barcelona, Spain Castelló, Spain Lleida, Spain La Rioja, Spain Huesca, Spain Lleida, Spain Aude, France Castelló, Spain Huesca, Spain Region, Country HQ621991, JN209560– JN209564 HQ621986, JN209586– JN209589 HQ621981, JN209552 HQ621990, JN209553– JN209559 HQ621970, JN209477– JN209485 HQ621992, JN209521– JN209522 HQ621975, JN209500– JN209506 HQ621987, JN209537 HQ621983, JN209538– JN209544 HQ621988 HQ621984, JN209545– JN209551 HQ621995, JN209514– JN209520 COI: GenBank Numbers HQ622017, JN209706 HQ622030, JN209707– JN209709 HQ622032, JN209704– JN209705 HQ622025, JN209701– JN209703 HQ621885, JN209371– JN209375 HQ621880, JN209326– JN209329 HQ621999, JN209590– JN209592 HQ622035, JN209611– JN209612 HQ622008, JN209599– JN209601 HQ622026, JN209613 HQ622020, JN209615– JN209617 HQ622027 HQ622022, JN209618– JN209620 HQ622040, JN209605– JN209607 H3: GenBank Numbers HQ621874, JN209286 HQ621884, JN209358– JN209364 HQ621862, JN209196– JN209204 HQ621886, JN209387– JN209388 HQ621868, JN209227– JN209233 HQ621881, JN209330 HQ621876, JN209296– JN209302 HQ621882 HQ621877, JN209309– JN209315 HQ621889, JN209413– JN209419 16S: GenBank Numbers HQ622072, JN209792 HQ622066, JN209791, JN209797, JN209801 HQ622046, JN209762– JN209764 HQ622081, JN209779– JN209780 HQ622054, JN209783– JN209784, JN209827 HQ622063, JN209790 HQ622076, JN209781– JN209782, JN209794 HQ622078, JN209816– JN209817 HQ622071, JN209710– JN209712 HQ622068, JN209798– JN209800 HQ622086, JN209778, JN209802, JN209803 HQ622073 H4: GenBank Numbers N4318¢04.6¢¢ E00612¢35.9¢¢ N4342¢19.0¢¢ E00348¢03.7¢¢ N4201¢43.3¢¢ E00142¢43.7¢¢ N4213¢30.0¢¢ E00214¢57.5¢¢ N4027¢23.1¢¢ E00016¢59.0¢¢ N4211¢05.5¢¢ E00054¢11.7¢¢ N4226¢21.7¢¢ W00250¢18.4¢¢ N4159¢22.7¢¢ E00054¢50.1¢¢ N4223¢26.0¢¢ W00022¢20.9¢¢ N4252¢48.8¢¢ E00210¢12.0¢¢ N3949¢39.4¢¢ W00015¢40.2¢¢ N4231¢20.1¢¢ W000º29́09.1́´ GPS Coordinates 204 152 735 785 214 464 596 860 740 396 168 1090 Altitude (m) D I V E R S I F I C A T I O N P A T T E R N S I N E A R T H W O R M S 3781 HCL, Hormogaster clade used for calibration; HE, Hormogaster elisae species complex; HIS, Hormogaster from the islands (Corsica and Sardinia); HM, Hemigastrodrilus; HNE, Hormogaster from the NE Iberian Peninsula; VG, Vignysa; XN, Xana; COI, cytochrome c oxidase subunit I gene. Sequences in bold are additions for this study. The same species names as in Novo et al. (2011) are used although as indicated in that study, a systematic revision of the family is overdue and some forms named Hormogaster pretiosa (with asterisk) may require a new name (M.N. work in progress). For a specimen donated by Pietro Omodeo, no exact coordinates are available. 644 N4315¢20.9¢¢ W00505¢10.3¢¢ HQ622089, JN209818– JN209819 San Román Xana omodeoi XN XAN Asturias, Spain HQ621997, JN209470– JN209476 HQ621891, JN209432– JN209438 HQ622043, JN209686– JN209688 GPS Coordinates Locality Species Phylogenetic Clade Code Region, Country COI: GenBank Numbers 16S: GenBank Numbers H3: GenBank Numbers H4: GenBank Numbers Altitude (m) 3782 M . N O V O E T A L . calculated using Arlequin v.3.5 (Excoffier & Lischer 2010) and DnaSP v. 5 (Librado & Rozas 2009) using a Kimura 2-parameter correction, as in previous studies. Haplotype networks were constructed for each gene region. The statistical parsimony procedure (Templeton et al. 1992; Crandall et al. 1994) with a 95% connection threshold was used in TCS version 1.2.1 (Clement et al. 2000) for histones. Mitochondrial genes showed large divergence, and thus, these sequences were analysed using SplitsTree4 v.4.10 (Huson & Bryant 2006). Default settings were used, thus constructing a Neighbour-net with uncorrected p-distances. Species boundaries based on GMYC approach and molecular rates Species boundaries were evaluated using a general mixed Yule-coalescent (GMYC) model approach (Pons et al. 2006; Fontaneto et al. 2007) based on a separate analysis of the two mitochondrial genes. A Bayesian analysis was performed in BEAST 1.6.1 for COI and 16StRNA (Drummond & Rambaut 2007) to reconstruct a fully resolved topology with branch length estimates. A relaxed lognormal clock was used with the Coalescence prior. This prior has proven more conservative, thus suggesting more accurate species boundaries, because the GMYC uses a coalescent as the null model for explaining branching patterns (Monaghan et al. 2009). GTR+I+G, the best-fit model as indicated by jModeltest (Posada 2008), was used. The phylogenetic trees were calibrated using the separation between Hormogaster pretiosa pretiosa from Sardinia (VIL) and the continental species clustering in the same clade (named HCL)—Hormogaster pretiosa cf. hispanica collected near the shore of the river Ter in Girona (HPA) and Hormogaster najaformis (ORD)—assuming a divergence time for these lineages at least dating back to the separation of the Corso-Sardinian microplate from continental Europe. As a calibration point, we use the most current estimates for the separation of the occidental Mediterranean microplates (33 Ma; Schettino & Turco 2006). Bayesian analyses were run from for 50 million generations, sampling from every 5000th generation, always resulting in 10 000 trees. After checking for stationarity with Tracer v. 1.5 (Rambaut & Drummond 2007), 20% of the trees were discarded. A maximum clade credibility tree was then built with the remaining trees and analysed in the R package SPLITS (http:// r-forge.r-project.org/projects/splits), following the GMYC approach for species delimitation with single and multiple threshold optimizations. Molecular rates (in terms of substitution rates) were calculated for the four genes combined to compare the 2012 Blackwell Publishing Ltd D I V E R S I F I C A T I O N P A T T E R N S I N E A R T H W O R M S 3783 evolution of the different clades. A similar approach as for the GMYC was followed by constructing a fully resolved topology with branch length estimates in BEAST 1.6.1. In this case, two priors were compared (Coalescence with constant size and Speciation-Yule) with the aim to evaluate the credibility of the rates yielded by the different analysis. We also calculated relative timings by calibrating the tree with a 1 in the root for Hormogastridae. Differences between the substitution rates (recovered by BEAST 1.6.1 for each node) of distinct clades within Hormogastridae (namely HCL, HE, HIS, HM, HNE, VG, XN, see Table 1) were analysed by means of a one-way ANOVA in STATISTICA v. 6.1 (StatSoft, Inc. (2001), http://www.statsoft.com). ables and sampling localities to reduce these variables to Factors. This analysis was executed via a correlation matrix after standardization (43 localities · 8 soil variables), which permits a visualization of the variations among localities. Localities belonging to different phylogenetic clades (previously recovered by Novo et al. 2011; i.e. HCL, HE, HIS, HM, HNE, VG, XN, see Table 1) were represented with different colours in the PCA graph. Afterwards, the mean values of factor scores and separate soil variables for these phylogenetic clades were compared by an ANOVA (STATISTICA v. 6.1, StatSoft, Inc. 2001, http://www.statsoft.com). This is aimed to help ascertain whether the type of soil has an influence on the composition of the earthworms inhabiting each locality. Geographical and environmental assessment Results A pattern of isolation-by-distance was tested for the big clades HE and HNE by means of a Mantel (1967) test, correlating the matrix of genetic distance between localities (FST with Kimura 2-parameter correction) based on the most variable gene, COI, and geographical distance (here, straight line between study sites). The test was performed in Arlequin v. 3.5 (Excoffier & Lischer 2010), and the significance of matrices correlation was evaluated comparing the Mantel test statistic Z, for which random distributions were obtained with 10 000 permutations. Some soil factors were measured in the sampled locations to explore the role of the environment in the genetic diversification of this group of Mediterranean earthworms. These were selected because they are among the most important factors affecting earthworm distribution (Edwards & Bohlen 1996; Hernández et al. 2003). Soil texture and pH were determined as described by Guitián & Carballas (1976). Total nitrogen content was determined by the Kjeldahl method as indicated in Page et al. (1982) and expressed as a percentage. Organic oxidable carbon analysis was based on Anne’s (1945) method, adapted for a microplate reader (Microplate Bio-Rad, 590 nm), employed using glucose as a standard and expressed as a percentage. A Mantel test with 10 000 permutations was implemented in Arlequin v.3.5 (Excoffier & Lischer 2010) to correlate the genetic distances (FST with a Kimura 2parameter correction), based on all the genes separately, with the differences between localities for soil factors (in absolute values). This was performed to indentify the soil factors that could be related to the earthworm genetic diversity (i.e. do the most genetically distinct populations live in the most unique soils? what soil characteristics could be involved in the evolution of the group?) As the Mantel test including the genetic sequence information was not conclusive, a principal component analysis (PCA) was performed only with the soil vari 2012 Blackwell Publishing Ltd Characteristics of the used genes and genetic variability Genetic variability values for each gene region are shown in Table 2 for the whole data set of Hormogastridae. Detailed diversity descriptions by locality are shown in Table S1 (Supporting information). Also the mean genetic divergence between localities (Kimura 2-parameter corrected) can be found in Table S2 (mitochondrial data) and Table S3 (nuclear data) (Supporting information). Mitochondrial regions showed to be more variable than the nuclear genes, with a mean divergence in the range of 13.6–16.4%. Histones were the least variable markers, showing a mean divergence of ca. 3%. Indel events were only detected in the 16S-tRNA fragment. Haplotypic networks are shown in Fig. 1. A neighbour-net constructed with SplitsTree and based on mitochondrial data shows how the HE clade presents much deeper subdivisions than the HNE clade, despite the former including a single morphospecies and the latter many. Regarding the histone networks, both histones H3 and H4 presented unconnected networks for each of the main clades. The histone H4 for those networks with more than two terminals is presented as an example (Fig. 1), showing that the patterns of diversification for HE and HNE are similar for histones. Species boundaries based on GMYC approach and molecular rates General mixed Yule-coalescent analyses exhibited a higher subdivision for Hormogastridae than expected under a morphospecies concept. Results from both mitochondrial genes are similar, with 56 entities identified. The number of entities was artificially high in the HE clade, separating individuals collected in the same locale as different entities (24 entities in 16 populations). 3784 M . N O V O E T A L . Table 2 Genetic diversity values of the gene fragments used for the study N NHAP H p Positions Substitutions Indels S Ts Tv Dp (N) Dp (%) COI 16S-tRNA H3 H4 375 170 0.99 (0.0001) 0.164 (0.078) 648 514 0 266 366 148 106.35 (45.77) 16.41 (7.06) 374 162 0.99 (0.001) 0.136 (0.065) 808 572 78 380 358 214 110.20 (47.41) 13.64 (5.87) 166 (332) 65 0.98 (0.002) 0.035 (0.018) 328 95 0 68 59 36 11.47 (5.21) 3.50 (1.59) 164 (328) 37 0.92 (0.01) 0.028 (0.015) 183 47 0 32 31 16 5.12 (2.49) 2.80 (1.36) N, Number of individuals analysed (in the case of the histones, two alleles per individual were analysed, because of the presence of ambiguities); NHAP, number of identified haplotypes; H, haplotypic diversity; p, nucleotidic diversity; S, number of polymorphic sites; Ts, number of transitions; Tv, number of transversions, Dp, mean number of pairwise differences (N, total number, %, percentage); COI, cytochrome c oxidase subunit I gene. Standard deviation of the estimates is shown in parenteses. The HM clade also exhibited unexpected subdivisions (four entities in two populations). Regarding the remaining clades, nearly all localities were identified as a separate entity. The multiple threshold approach resulted in an even higher number of entities (126 with 16S-tRNA and 118 with COI). Molecular rates were suitable for ANOVA analyses after the Log10 transformation. Only differences between the rates of clades HE and HNE were statistically significant, as the remaining clades had few values and great variability. Absolute molecular rates (after constructing the trees with the calibration in HCL at 33 Ma) were significantly higher (ANOVA: F1,36 = 6.6988, P = 0.01) in the HE clade (Mean rate = 0.002941, SD = 0.000735) than in the HNE clade (Mean rate = 0.002363, SD = 0.000779) for the Coalescent prior. No significant differences were found for the Speciation-Yule prior (ANOVA: F1,35 = 1.3407, P = 0.254), although mean values for HE (Mean rate = 0.00803, SD = 0.007611) were higher than in HNE (Mean rate = 0.005506, SD = 0.005808). Similar results were obtained when comparing the relative molecular rate values (obtained without calibration, setting a value of 1 in the root of Hormogastridae) showing significant differences with the Coalescent prior (ANOVA Coalescence F1,35 = 13.499, P = 0.0008) but not with the Speciation-Yule prior (F1,33 = 2.9994, P = 0.092). Note that the absolute molecular rate values are difficult to compare between different priors as they are very different. Geographical and environmental assessment A pattern of isolation-by-distance was found for the HE clade, as shown by the positive correlation of the Man- tel test (r = 0.372, P = 0.003). No such pattern was detected for the HNE clade (r = 0.077, P = 0.334). Soil characteristics for each locality are shown in Table 3. The results of the Mantel test between matrices of genetic distance and absolute differences in soil properties are shown in Table 4. All factors, except for the percentage of fine sand, are significantly correlated with each genetic fragment analysed. Principal component analysis revealed three factors (Table 5) that explained the 77.52% of the total variance in the soil characteristics between localities. The first factor (36.35% of the total variance) was highly and positively correlated to the level of nutrients (carbon and nitrogen) and clay, and negatively correlated to the percentage of sand. pH and fine silt were positively related to the second factor (25.02% of the total variance). The graphical representation of the localities in the first two axes obtained is presented in Fig. 2, where localities are coloured according to the phylogenetic clade recovered in previous analyses by Novo et al. (2011). Localities included in the clade HE exhibit a tendency towards the more acidic soils, with higher content of coarse sand, and slightly poorer, whereas clade HNE is found in soils with finer texture and higher pH. Xana inhabits soils with higher content of clay than the ones of other hormogastrids and soil characteristics in Corsica and Sardinia (clade HIS) resulted very heterogeneous. These differences between the main clades (HE and HNE) are confirmed by the ANOVA. Scores of Factor 1 showed significant differences (F6,36 = 4.55, P = 0.002) only among XN and HE ⁄ HNE according to post hoc comparisons (Tukey’s test). But when excluding XN and HCL (Phylogenetic clades represented only by one locality) the differences among 2012 Blackwell Publishing Ltd D I V E R S I F I C A T I O N P A T T E R N S I N E A R T H W O R M S 3785 Hormogaster CL Xana H. najaformis_ORD H. pretiosa pretiosa_VIL X. omodeoi_XAN H. elisae_SOT H. elisae_SIG H. elisae_FRE H. sylvestris_MAJ 0.01 H. sp nov._SAN H. elisae_CAB H. elisae_NAV H. elisae_LOZ H. arenicola_BIO Hormogaster NE H. elisae_TRE H. elisae_MOL H. eserana_GRA H. elisae_TRE H. pretiosa var_PRB H. elisae_RED H. oroeli_OEL H. elisae_RED_VEN H. elisae_JAR_VEN H. pretiosiformis_MON H. huescana_LOP H. pretiosa arrufati_UIX H. castillanaQUE Hormogaster sp_CER Hormogaster sp_TAL H. elisae_UCE H. ireguana_TOR H. riojana_ALE H. elisae_ANC H pretiosa nigra_QLL H. catalaunensis_BRU H. gallica_BSM V. popi_SGF Vignysa H. elisae_SEV H. saminitica lirapora_TES H. elisae_BOA V. vedovinii_PIG H. elisae_PAR H. redii redii_IGL H. redii redii_GHI H. redii redii_ALG He monicae_MND Hormogaster IS H. elisae species complex H. redii insularis_VPJ He. monicae_LAC H. redii redii_GHI H. redii redii_IGL Hemigastrodrilus H. saminitica lirapora_TES N ae_VE H. elis H. elisae_CAB H. redii insularis_VPJ H. castillana_QUE Hormogaster sp_TAL H. pretiosa arrufati_UIX H. sylvestris_MAJ H. elisae_TRE H. elisae_JAR H. elisae_RED H. elisae_LOZ H. ireguana_TOR H. elisae_TRE H. sp nov._SAN H. eserana_GRA H. elisae_NAV Hormogaster sp_CER H. elisae_TRE H. elisae_MOL H. castillana_QUE H. pretiosiformis_MON H. pretiosa var_PRB H. elisae_UCE H. elisae_MOL H. elisae_CAB H. riojana_ALE H. elisae_FRE H. huescana_LOP H. pretiosiformis_MON H. elisae_SIG H. elisae_VEN H. elisae_SOT H. oroeli_OEL H. arenicola_BIO H. arenicola_BIO H. elisae_ANC H. catalaunensis_BRU H. elisae_SEV H. elisae_BOA H pretiosa nigra_QLL H. gallica_BSM H. elisae_PAR Fig. 1 Illustration of the genetic diversity within the different clades of Hormogastridae. In the upper part of the Figure, a network recovered by SplitsTree4 and based on the mitochondrial data is shown. Branch length is proportional to the genetic distance. Different clades recovered by Novo et al. (2011) are indicated. TCS network for some of the clades is shown, based on histone H4 data. The size of the circles reflects haplotype frequency. Circles with no names are intermediate inferred haplotypes. Each branch represents one mutational step, branch lenght being meaningless. 2012 Blackwell Publishing Ltd 3786 M . N O V O E T A L . Table 3 Soil properties of the sampled locations of hormogastrids Clade Code %Coarse sand %Fine Sand %Total Sand %Coarse Silt %Fine Silt %Total Silt %Clay Texture classification %C %N vC ⁄ N pH HCL HE ORD CAB UCE FRE LOZ MOL NAV JAR RED TRE VEN SIG SOT ANC BOA SEV PAR VPJ GHI IGL TES LAC MND BIO BRU QUE GRA BSM LOP TOR OEL UIX QLL PRB MON ALE TAL CER MAJ SAN SGF PIG XAN 19.24 65.46 22.94 45.34 50.37 52.47 51.76 36.01 38.15 56.29 40.69 44.99 45.11 11.69 59.67 66.19 65.51 30.61 11.32 30.54 69.76 23.67 38.47 7.53 47.50 34.18 25.03 25.21 33.99 19.78 12.90 33.24 11.04 15.37 10.21 9.24 11.28 20.41 11.71 13.57 16.68 20.21 9.01 9.73 9.28 16.53 18.24 20.21 19.91 18.22 11.48 21.36 13.16 16.85 10.9 25.74 17.48 11.93 8.22 8.65 17.82 10.15 14.40 12.69 8.26 23.95 14.10 12.61 26.25 20.86 8.33 12.37 25.04 28.49 16.93 11.09 15.83 15.17 25.12 16.33 14.22 6.50 9.62 23.62 11.09 5.67 28.97 74.75 39.47 63.58 70.58 72.38 69.97 47.49 59.51 69.45 57.54 55.89 70.84 29.16 71.6 74.41 74.16 48.43 21.47 44.94 82.45 31.92 62.42 21.62 60.11 60.43 45.89 33.55 46.36 44.82 41.38 50.17 22.13 31.20 25.38 34.36 27.61 34.64 18.22 23.18 40.30 31.31 14.68 9.35 4.3 26.2 12.41 10.23 2.92 10.1 12.36 8.63 6.08 7.95 10.74 6.5 14.58 2.32 2.93 3.16 7.57 12.49 19.20 2.23 5.45 5.95 4.36 5.25 4.65 13.08 7.11 4.50 15.87 12.32 4.57 3.46 6.85 5.61 55.38 8.81 9.24 6.88 6.27 13.19 7.78 1.84 32.72 11.62 20.14 12.28 10.76 9.61 10.5 13.69 19.83 10.97 18.07 18.13 13.97 36.49 7.61 6.58 7.49 34.68 39.85 5.34 7.92 43.64 27.61 69.01 17.72 32.83 32.31 36.49 30.10 17.87 21.80 21.41 72.08 59.30 66.64 1.86 61.27 40.34 69.02 32.37 28.82 33.55 22.00 42.07 15.92 46.34 24.69 21 12.53 20.6 26.06 28.46 17.05 26.03 28.88 20.46 51.08 9.93 9.52 10.65 42.25 52.34 24.54 10.16 49.09 33.55 73.37 22.97 37.47 45.39 43.60 34.60 33.74 34.12 25.98 75.54 66.15 72.25 57.24 70.08 49.58 75.90 38.64 42.00 41.33 23.84 28.97 9.34 14.19 11.74 8.43 15.1 9.42 26.45 12.03 13.5 16.44 15.23 8.69 19.76 18.47 16.08 15.19 9.31 26.19 30.52 7.40 18.99 4.03 5.01 16.92 2.09 8.72 22.85 19.05 21.44 24.50 23.85 2.33 2.66 2.37 8.40 2.31 15.78 5.88 38.18 17.70 27.36 61.48 Clay loam Sandy loam Loam Sandy loam Sandy loam Sandy loam Sandy loam Sandy clay loam Sandy loam Sandy loam Sandy loam Sandy loam Sandy loam Clay Sandy loam Sandy loam Sandy loam Loam Silt loam Clay loam Loamy Sand Loam Sandy loam Silt loam Sandy loam Sandy loam Loam Loam Loam Loam Loam Sandy clay loam Silt loam Silt loam Silt loam Silt loam Silt loam Loam Silt loam Clay loam Loam Loam Clay 3.72 2.15 2.3 0.86 2 0.96 1.88 1.82 1.77 1.86 2.08 2.14 1.87 1.78 1.18 1.28 1.5 2.59 5.81 1.83 1.21 4.44 2.15 3.86 4.76 3.79 2.20 3.88 1.59 2.32 2.76 3.19 1.81 3.44 1.78 1.63 2.23 2.70 2.98 4.48 2.34 4.26 3.34 0.76 0.514 0.476 0.179 0.424 0.16 0.452 0.343 0.399 0.41 0.543 0.606 0.326 0.391 0.237 0.199 0.263 0.29 1.37 0.51 0.37 0.93 0.52 1.20 1.32 0.84 0.68 0.94 0.37 0.53 0.53 0.39 0.52 0.71 0.35 0.30 1.41 0.54 0.83 1.32 0.67 0.85 1.25 4.91 4.18 4.83 4.79 4.72 5.92 4.16 5.31 4.45 4.55 3.83 3.54 5.74 4.55 4.98 6.46 5.69 8.93 4.24 3.56 3.31 4.79 4.14 3.21 3.61 4.49 3.23 4.13 4.36 4.37 5.16 8.18 3.52 4.87 5.15 5.33 1.58 5.01 3.60 3.39 3.48 5.02 2.68 7.38 5.68 5.57 7.45 5.49 6.4 5.35 7 7.62 6.24 7.41 5.05 5.43 7.9 6.42 5.93 6.19 5.73 5.6 7.26 5.78 7.44 7.6 7.19 4.76 7.47 7.49 5.51 7.79 7.45 7.3 7.56 7.62 7.42 7.59 7.33 7.57 7.5 7.39 7.09 7.55 7.4 5.26 HIS HM HNE VG XN C, Carbon; N, Nitrogen. ALG, HPA and VIL values are not included because specimens from those localities were not collected by the authors and soil samples were not available for study. For complete names see Table 1. HE and HNE were significant (F4,36 = 3.03, P = 0.03). Scores of Factor 2 showed significant differences (F6,36 = 6.11, P = 0.0002) among HE and HNE according to post hoc comparisons. All the individually analysed soil variables showed significant differences among clades (P < 0.05) excepting fine sand and coarse silt content. Post hoc comparisons showed that coarse sand content was significantly higher in localities included in the HE clade than in the HNE clade, whereas fine silt content, Carbon, Nitrogen and pH were lower in the HE than in the HNE clade (P < 0.05 in all the cases). Locality XN showed a significantly higher content of clay when compared to the rest (P < 0.01, except for HCL). 2012 Blackwell Publishing Ltd D I V E R S I F I C A T I O N P A T T E R N S I N E A R T H W O R M S 3787 Table 4 Results from the Mantel test of the correlation between FST based on different genes among localities, and the differences (in absolute values) of their soil factors COI Coarse sand Fine sand Total sand Coarse silt Fine silt Total silt Clay C N pH 16S-tRNA H3 H4 P r P r P r P r 0.023 0.630 0.076 0.003 0.014 0.011 0.052 0.118 0.040 0.034 0.125 )0.023 0.086 0.158 0.155 0.158 0.127 0.108 0.143 0.119 0.120 0.350 0.038 0.009 0.031 0.080 0.011 0.023 0.034 0.022 0.070 0.030 0.087 0.130 0.121 0.094 0.133 0.130 0.123 0.112 0.004 0.199 0.001 0.313 0.000 0.001 0.004 0.000 0.004 0.117 0.146 0.050 0.157 0.056 0.194 0.173 0.150 0.227 0.175 0.066 0.001 0.060 0.003 0.119 0.010 0.046 0.311 0.009 0.097 0.003 0.173 0.080 0.135 0.085 0.135 0.090 0.039 0.136 0.074 0.147 C, carbon; N, nitrogen; COI, cytochrome c oxidase subunit I gene. Significant P values (<0.05) and the associated correlation coefficients (r) are shown in bold and italics. Table 5 Correlation coefficients between soil variables and principal component analysis factors Variables Factor 1 Factor 2 Factor 3 %Coarse sand %Fine sand %Coarse silt %Fine silt %Clay %C %N pH Explained variance (%) )0.580 )0.423 0.032 0.277 0.688 0.843 0.837 )0.068 36.348 )0.666 0.196 )0.031 0.881 )0.477 0.200 0.256 0.721 25.024 0.373 )0.643 )0.886 0.303 )0.108 0.155 0.201 )0.321 16.147 C, carbon; N, nitrogen. Values in bold and italics are significant (P < 0.05). Discussion Characteristics of the used genes and genetic variability The genetic variability found in these endogeic Mediterranean earthworms has proven to be high, when compared with other studied annelids at similar taxonomic levels. However, the evolutionary pattern does not seem constant along the different lineages in the family, as we have detected important differences between clades regarding genetic and morphological evolution. On one hand, the Hormogaster elisae complex (HE), whose distribution is limited to the central Iberian Peninsula, includes only one morphospecies showing a constancy in all morphological diagnostic characters, but exhibiting great genetic variability and a deep genetic subdivision. Other examples of large genetic divergence 2012 Blackwell Publishing Ltd across short geographical distances (in the order of a few kilometres or even in the same site) have been reported for earthworms (James et al. 2010; Dupont et al. 2011; Férnandez 2011), in which cryptic speciation could be common. On the other hand, the clade including Hormogaster from the NE Iberian Peninsula (HNE), with a similar sampling effort, includes 15 described species (in this work, we include 13 described species and some varieties, as well as a possible new species, but H. multilamella and H. lleidana were not found). Qiu & Bouché (1998) described the morphological diversity of this clade that occupies a slightly larger area in the northeastern part of the Iberian Peninsula. The genetic variability of HNE is not as great as in the HE clade and it does not present such deep subdivisions in mitochondrial data (see Splitstree in Fig. 1), despite showing much larger morphological diversity and including highly autapomorphic taxa, currently considered monotypic genera. Regarding the nuclear data, both clades show a core of sequence diversity that seems to be the origin of the remaining sequence types (see TCS networks for H4 in Fig. 1). All this evidence suggests that these two clades are undergoing different evolutionary processes and these could be attributed to the particular characteristics of the soil that they inhabit (see below), or to some rarely documented evolutionary processes. For the remaining clades, no sound conclusions can be drawn due to the lack of sufficient information available. It would be particularly interesting to explore the hormogastrid fauna from Corsica and Sardinia to shed light on the potential cryptic speciation processes occurring there, as indicated by the great genetic divergences between sampled localities of Hormogaster redii and also 3788 M . N O V O E T A L . 2.5 MON 2.0 QLL TAL BIO PRB MAJ 1.5 QUE Factor 2 (25.02%) 1.0 MND 0.5 GRA ANC CER LOP RED VPJ 0.0 –0.5 –1.0 FRE VEN ALE MOL SOT TRE TES BOA LOZ PAR –1.5 –2.0 –1.5 NAV CAB LAC SGF TOR OEL PIG ORD SAN UIX GHI BSM UCE JAR IGL BRU SIG SEV XAN –1.0 –0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Factor 1 (36.35%) 1.0 0.8 pH %Fine Silt 0.6 0.4 Factor 2 0.2 % Nitrogen %Fine Sand % Carbon 0.0 %Coarse Silt –0.2 –0.4 % Clay –0.6 %Coarse Sand –0.8 –1.0 –1.0 –0.8 –0.6 –0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 Factor 1 Fig. 2 Plot of the factor scores for soil analyses on the first two principal components for hormogastrid localities. Graphical illustration of the localities, indicating the variance explained by each axis (above) and correlation circle representation of the soil variables (below). Localities were coloured according to the clade in which they were included in previous phylogenetic analyses. Localities included in the clade HE are represented in pink, HCL in yellow, HIS in red, HM in orange, HNE in green, VG in blue and XN in brown. See codes for the localities and complete name of species and clades in Table 1. Barycentre coordinates of each clade are represented by a star of the corresponding colour. Note that the clades that include only one locality (HCL and XN) are only represented by this star. by the GMYC results that identify each locality as a putative entity. Species boundaries Recently, the use of coalescent-based models of species delineation has flourished for studying difficult groups based on morphology alone (Nekola et al. 2009), for assigning immature specimens to their adult counterparts (Gattolliat & Monaghan 2010), to match spider genders where males and females are often described based on independent sets of character (M.A. Arnedo & G. Hormiga, unpublished data), or to perform rapid biodiversity estimates for taxonomic groups in areas where little taxonomic information is available (Pons et al. 2006; Kaya et al. 2009; Monaghan et al. 2009). The GMYC method has proven to work well for many of these examples, including arthropods and molluscs (e.g. Monaghan et al. 2009; Nekola et al. 2009; Gattolliat & Monaghan 2010), but almost no published account exists for annelids or oligochaetes. Only a very recent publication has explored the method in anecic earthworms 2012 Blackwell Publishing Ltd D I V E R S I F I C A T I O N P A T T E R N S I N E A R T H W O R M S 3789 showing surprising results when compared to traditional taxonomy (Fernández et al. in press), and we found interesting to further test this method with these animals. Papadopoulou et al. (2008) compared two groups of beetles occupying different habitats and with different dispersal habilities showing that in stable habitats (expected to have lower dispersal rates) there tends to be a higher subdivision of clades when applying the GMYC model, and lineage branching occurred more deeply in the tree. Also, the lineages from the more stable habitats had higher levels of nucleotide and haplotype diversity and greater geographical structure, perhaps indicating that the populations had had more time to diversify. This is indeed the case of hormogastrid earthworms. The soil is a very stable milieu, the dispersal rates of earthworms are thought to be very low (Ligthart & Peek 1997; Hale et al. 2005), and these animals tend to stay put in the same place for long periods of time, following the fate of their habitat and geographical area (Omodeo & Rota 2008). Specifically, in hormogastrid earthworms, this pattern is magnified because their active dispersal capabilities and passive dispersal opportunities are even lower (Novo et al. 2010b). Still, this is not an explanation for why the individuals from the same populations in clade HE are considered different entities by the GMYC model, but just an indication that they indeed show deep genetic divergences. Following Wiens & Penkrot’s (2002) phylogenetic concept, these subdivisions make little sense. The genetic variability in this complex of species exceeds the predictable values and seems to be undergoing different evolutionary processes, as shown in some soil arthropods (Boyer et al. 2007). The HE clade already shows extreme subdivision when constructing statistical parsimony networks (with TCS, Novo et al. 2010a), also separating individuals from the same populations. Other authors stated that independent haplotype networks generally agree with named species or species subgroups (Pons et al. 2006; Hart & Sunday 2007), but again the HE clade may not follow this rule. Lohse (2009) argued that incomplete sampling of demes involved in the coalescent process could artificially overestimate species numbers by the GMYC method. Papadopoulou et al. (2009) responded that indeed, if populations (demes) with intermediate haplotype composition are left unsampled, these results are an overestimate of species (oversplitting). Nevertheless, they argue that the problems raised by Lohse (2009) result from the fact that the offspring is produced in the vicinity of the parents, which in turn produces greater similarity of genotypes at a site compared with other sites. This is surely the case of hormogastrid earthworms (Novo et al. 2010b) but it only could explain the subdi 2012 Blackwell Publishing Ltd vision pattern found as one population corresponding to one entity. Species delimitation within earthworms is still an open question because their morphology not always captures the true diversity owing to their structural simplicity and specific adaptations to the soil. Here, we show that coalescent methods overestimate the number of species, probably due to the marked genetic structure and scarce dispersion capacity. Further research in this important topic is therefore needed. Molecular rates The first conclusion regarding molecular rates is that a unique value cannot be generalized for this particular family. As shown in Fig. 2, these values not only vary along the tree, but they also differ depending on the prior used for the analyses. Thus, general statements such as the one proposed by Omodeo (2000), who indicated (based on continental drift) the need of 180 MY for an earthworm genus to differenciate, cannot be accepted and the rates proposed by other authors (e.g. Chang & Chen 2005; Chang et al. 2008; Buckley et al. 2011) working on megascolecids, should be taken with a grain of salt and limited to the species studied and to the concrete scenario of each particular case. They can definitely not be extrapolated to hormogastrids in general or to any particular clade. Our data suggest that the HE lineage has undergone quicker diversification than HNE as it presents higher rates, as shown by the ANOVA results. Conclusions about the remaining clades should await further collecting effort. These results confirm cryptic speciation within the HE lineage in the central Iberian Peninsula, where a mismatch between morphological and molecular evolution has been reported (Novo et al. 2009, 2010a). Here, we propose that the characteristics of the soils where the members of clade HE live limits their morphological change, and thus, even molecular evolution is fast morphological changes do not have time to fixate. The harsher soil conditions (i.e. dryer and with less organic matter than other soil typically inhabited by earthworms) they inhabit could be implied also in this rate acceleration. Geographical and environmental assessment Hormogastrid evolutionary history is largely shaped by the geographical history of the landmasses they inhabit, probably because of their low vagility. Although confined to their particular areas, soil conditions could also be shaping the genetic processes of these earthworms, as shown by the correlation between the genetic divergences and the differences in soil characteristics 3790 M . N O V O E T A L . among collection sites. However, no single soil property is responsible for these differences, and instead, it is the combined effect of these properties what shows a correlation. Novo et al. (2010a) found that some soil texture characteristics have a larger influence in the differenciation between the H. elisae lineages. The higher diversity of soils included in this work shows relevance of other factors, such as carbon or nitrogen content, when taking into account the whole family Hormogastridae, as opposed to a single lineage. The PCA permits a better understanding of the ecological scenario by combining the soil variables in factors. In general, hormogastrids are confined to poor soils, with low nitrogen and organic matter content, being relegated to the soils where other earthworms cannot survive, sometimes by means of exclusive competition (Ramajo 2010). The percentages in organic matter found in the studied localities were low in comparison with those observed by other authors studying earthworms in other areas of the Iberian Peninsula (Mariño et al. 1985). The localities included in the HE clade exhibit a tendency towards more acidic soils, with higher content of coarse sand, and slightly poorer, when compared to the localities included in the HNE clade, whose members inhabit soils with finer texture and higher pH. The characteristics of the soil may be the reason why morphological adaptations are generally maintained in earthworms, causing thus morphological stasis. This is certainly the case of hormogastrids, as they are adapted to harsh soil conditions, and magnified in the case of H. elisae (HE), enduring very harsh soil conditions among oligochaetes. This, together with the extreme climatic characteristics (cold winters and hot and dry summers) in the central Iberian Peninsula makes the habitat of HE unsuited for most species of earthworms (Hernández et al. 2007). Other authors already proposed that extreme subsurface conditions could constrain morphological evolution and be responsible for convergence over large periods of time (e.g. for amphipods, beetles and salamanders: Jones et al. 1992; Caccone & Sbordoni 2001; Wiens et al. 2003). The morphological adaptations already evolved to inhabit these harsh habitats are probably optimal and any change could be nonadaptive. The specialized environment can impose stabilizing selection, thus minimizing or eliminating the morphological change that can occur during speciation (Bickford et al. 2007). Therefore, the high genetic divergences found within HE could be related to biochemical changes not detectable morphologically. We propose here, as observed for clade HE that the harsh soil conditions these earthworms have to endure contribute to accelerate genetic evolution (i.e. higher molecular rates). Principal component analysis detected that Xana inhabits soils that differ from those of other hormogastrids, with a much higher clay content. Thus, the hypothesis of Novo et al. (2011) suggesting the reduction from three to two gizzards as an adaptation to the environment remains plausible. In that study, Xana and Vignysa (the only hormogastrids presenting two gizzards, while the others have three) were not related phylogenetically in most analyses (but they clustered in the analyses based on nuclear genes). The soil collected in Corsica and Sardinia (where the clade HIS inhabits) is heterogeneous, as shown by the PCA, where the samples from both islands appear mixed without a clear tendency. This heterogeneity could be explained by the geological origin of the islands (Bacchetta et al. 2007; Omodeo & Rota 2008) and could also be the cause of the unexpected genetic diversity found within this clade. More populations of H. redii should be collected to study the real scope of this diversity. A pattern of isolation-by-distance has been found in the HE clade but not for the HNE clade. Nevertheless, the relationships recovered within HNE show clusters related to geographical location. As mentioned earlier, hormogastrids show strong biogeographical patterns of vicariance and a diffusive-like colonization is expected via active dispersal (very slow) or via nonanthropogenic passive dispersal (e.g. by birds, mammals, wind or waterways) (Cameron et al. 2008). The fact that the isolation-by-distance pattern is significant in the case of HE but not for HNE demonstrates that in the former the great genetic diversity concentrated in a small geographical area has a deep genetic basis and that the animals show low vagility, as postulated for the members of this family. The geographical distribution of HE could also explain its high diversity, as it is located in the westernmost limit of the Hormogastridae distribution (Cobolli-Sbordoni et al. 1992), exploiting and colonizing marginal habitats. Among sampled populations of HE, the ones in the mountainous area (Guadarrama Mountains, see Novo et al. 2009, 2010a for a detailed map) exhibit higher diversity and were easier to locate, which could be a sign of greater population densities and probably a more favourable area for hormogastrid development. Meanwhile, the conditions in the southern area may be less optimal, as reflected in the difficulties locating populations and their lower diversity values. These populations seem to represent the vanguard of this evolutionary lineage, which is the reason why the speciation and diversification is increasing in this southern area (including the localities PAR, SEV, BOA in the Tajo fluvial valley, see Novo et al. 2009, 2010a for a detailed map and more information). These hypotheses are consistent with the pattern found 2012 Blackwell Publishing Ltd D I V E R S I F I C A T I O N P A T T E R N S I N E A R T H W O R M S 3791 by Novo et al. (2010a) who showed expansion processes in the southern populations and stability in mountainous populations. Conclusions Morphological changes in such a restrictive milieu as the soil are unlikely once the animals are adapted, and thus, morphology-based studies could confound the evolutionary patterns in certain groups of invertebrates, such as earthworms, showing sometimes high levels of homoplasy because of the convergence in morphological solutions to specific conditions. In this study, we found how within hormogastrid earthworms, morphological and molecular evolution can be decoupled in some clades. The high genetic diversification and morphological constancy in the HE clade from the central Iberian Peninsula contrasts with the higher morphological diversity in the HNE clade of NE Iberia, and these differences could be attributed to several factors. Higher molecular rates in HE could be the cause of this decoupling between the rapid molecular evolution and the morphological stasis found in this clade, and this could be accentuated by the extreme conditions it is subjected to, which at the same time could be forcing the higher evolutionary rates leading to changes only at the biochemical level. The GMYC analyses yielded a higher number of entities than expected under a morphospecies concept within Hormogastridae, generally showing each population as one different entity (i.e. species) probably due to the low vagility of these earthworms. The method yielded particularly high numbers of entities for HE, probably because of its high genetic diversity. Further studies for different invertebrates inhabiting similarly uniform and isolated environments would be necessary to shed light on these interesting evolutionary processes. Acknowledgements We are indebted to Carlos Bayón, Pablo Cabo, Inés Garcı́a, Noa Novo and Juan José Molina, for assisting with fieldwork, Maigualida Ricoy and Pietro Omodeo for sharing additional samples, Irene Parra, Carlos Granado, Miquel Arnedo and Joan Pons for offering their suggestions in data analyses and Daniel Ayllón and Sheila Leal for technical support. The members from Giribet laboratory offered help for laboratory work and data analyses, especially Sebastián Vélez, Sónia Andrade and Prashant Sharma. The Editors and anonymous reviewers provided insightful comments that contributed to improve an earlier version of this work. M.N. was supported by a Grant from Fundación Caja Madrid. This research was funded by projects CGL2007-60715 ⁄ BOS and CGL2010 ⁄ 16032 from the Spanish Government. 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A.A. is interested in molecular and evolutionary ecology of freshwater fishes and phylogeography of invertebrate animals. D.T. is interested in soil ecology and the role of earthworms in restoration. G.G. is interested in the origins and maintenance of animal diversity, using invertebrates as model organisms. Data accessibility DNA sequences: GenBank Accession nos: JN209196–JN209829. See Table 1 for specific Accession nos on different loci and samples. Supporting information Additional supporting information may be found in the online version of this article. Table S1 Intrapopulation diversity values for hormogastrids. Table S2 Mean values of genetic divergence (K2P in percentage of changes) between pairs of populations within hormogastrids, based on mitochondrial data (COI below the diagonal; 16S-tRNAs above the diagonal). Table S3 Mean values of genetic divergence (K2P in percentage of changes) between pairs of populations within hormogastrids, based on nuclear data (H4 below the diagonal; H3 above the diagonal). Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. COI CLADE HCL HE HIS HM HNE VG XN Code ORD HPA VIL CAB UCE FRE LOZ MOL NAV JAR RED TRE VEN SIG SOT ANC BOA SEV PAR VPJ ALG GHI IGL TES LAC MND BIO BRU QUE GRA BSM LOP TOR OEL UIX QLL PRB MON ALE TAL CER MAJ SAN SGF PIG XAN 16S H3 H4 N NHAP K2P N NHAP K2P N NALL K2P N NALL K2P 6 4 12 12 2 22 12 12 12 26 12 12 11 12 9 12 12 12 7 1 6 8 6 8 8 8 1 8 8 1 8 8 8 8 1 2 8 10 3 8 2 8 6 5 8 2 1 1 6 2 7 7 6 6 9 5 3 7 3 6 4 3 5 5 1 5 5 4 6 5 3 1 2 7 1 3 2 2 5 1 2 4 1 1 7 2 3 3 3 3 0.10 0 0 0.99 3.67 1.60 1.03 0.22 1.45 3.90 4.77 2.08 0.92 0.32 0.27 0.10 0.17 0.25 0.67 0 0.76 0.32 0.79 3.68 7.06 0.10 0 1.18 0.47 0 0.10 0.33 0.88 0.86 0 0.62 0.47 0 0 1.14 0.47 0.10 0.14 0.12 0.37 7 1 8 12 12 2 22 12 12 11 26 12 12 11 12 9 12 12 7 7 1 5 8 6 8 8 8 1 8 8 1 8 8 8 8 1 2 8 10 3 8 2 8 6 5 8 5 1 6 3 7 2 6 5 5 7 6 5 4 9 7 1 4 2 2 4 1 4 4 4 5 4 3 1 2 7 1 2 2 1 3 1 2 5 3 1 4 2 2 2 3 3 0.18 0 0.29 0.06 0.44 1.03 0.60 0.24 0.04 0.99 1.14 1.17 0.64 0.30 0.53 0 0.12 0.05 6.03 0.28 0.00 0.18 0.31 0.21 1.92 2.14 0.11 0.00 0.41 0.21 0.00 0.61 0.00 0.00 0.28 0.00 0.13 0.83 0.03 0.00 0.28 0.25 0.05 0.00 0.10 0.17 4 1 4 4 4 2 7 9 4 4 3 4 4 4 4 4 4 4 3 4 1 4 4 4 4 4 4 1 4 4 1 3 4 4 4 1 2 4 4 3 4 2 4 3 4 4 2 1 1 3 2 2 3 6 1 7 3 3 4 3 3 2 1 1 4 1 1 1 2 1 1 3 2 1 1 1 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 1 0.13 0 0 0.25 0.26 0.20 0.42 0.35 0 0.60 0.51 0.31 0.51 0.47 0.29 0.16 0 0 0.43 0 0 0 0.16 0 0 0.53 0.16 0 0 0 0 0.33 0 0 0 0 0 0.08 0 0 0 0 0 0 0 0 4 8 3 3 2 6 8 4 4 2 4 4 5 4 4 4 4 4 4 1 2 3 1 1 2 3 1 1 1 3 2 2 2 2 1 1 1 2 1 1 1 1 2 3 1 2 1 1 1 1 1 3 1 1 2 1 1 1 1 1 1 2 1 0 0.27 0.85 0 0 0.09 0.36 0 0 0 0.55 0.14 0.11 0.31 0.14 0 0 0 0.29 0 0 0 0 1.18 0.61 0 0.29 0 0 0 0 0 0.27 0 0 0.29 0 0 0 0 0 0 0.14 0 4 4 4 4 3 4 1 4 4 1 4 4 4 4 1 2 4 4 4 4 2 4 3 4 3 Table S1. Intrapopulation diversity values for hormogastrids. N: Number of individuals analyzed (two alleles per individual were analyzed for histones due to the presence of ambiguities); NHAP: Number of identified haplotypes; NALL: Number of identified alleles; K2P: Mean number of pairwise differences in percentage corrected with Kimura 2-parameter model. See Table 1 for complete names of localities and species. Mean values of genetic divergence (K2P in percentage of changes) between pairs of populations within hormogastrids, based on mitochondrial data (COI below the diagonal; 16S-tRNAs above the diagonal). See complete names of localities and species in Table 1. CLADE HCL HE CODE ORD HPA VIL ORD x 5.73 9.78 HCL HPA x 8.94 VIL 19.89 x CAB UCE FRE LOZ MOL NAV JAR HIS RED TRE VEN SIG SOT ANC BOA SEV PAR VPJ HM ALG GHI IGL TES HNE LAC MND BIO BRU QUE GRA BSM LOP TOR OEL UIX VG QLL PRB MON ALE TAL CER MAJ SAN SGF PIG XN XAN 18.51 18.44 19.33 18.98 19.01 18.30 19.00 18.66 18.49 18.51 19.75 19.68 18.50 18.28 18.86 17.89 14.87 16.36 15.37 15.79 15.26 17.51 14.91 14.24 14.18 12.94 13.77 13.07 13.48 15.58 13.76 14.13 15.15 14.16 13.57 14.70 14.49 13.61 14.97 15.71 16.50 15.91 13.79 19.05 19.46 19.49 19.53 18.71 19.01 18.50 19.03 18.93 18.57 18.97 18.90 18.34 18.32 19.06 17.46 14.11 15.08 14.11 14.35 13.90 16.20 14.69 13.73 14.47 13.88 14.36 13.68 14.07 16.02 14.19 14.25 14.59 14.59 14.08 15.21 14.77 14.52 15.02 15.89 14.91 14.09 12.01 17.66 18.43 18.08 18.12 17.61 18.27 17.42 17.57 17.31 17.32 16.79 16.91 17.20 17.33 18.68 15.46 14.44 14.64 14.12 15.64 14.54 16.49 13.26 12.93 14.19 13.14 13.47 13.70 12.94 13.95 13.31 13.08 13.71 13.70 12.54 14.12 14.12 13.76 13.86 14.94 14.34 14.14 10.75 CAB UCE FRE LOZ MOL NAV JAR HE RED TRE VEN SIG SOT ANC BOA SEV PAR 22.25 24.97 21.44 20.80 23.75 21.76 21.36 21.26 20.73 22.04 20.42 21.16 21.10 22.17 22.31 21.46 22.94 25.78 20.57 21.53 24.62 21.15 23.33 22.99 22.45 23.01 23.23 22.26 23.66 22.34 23.08 21.87 x 17.95 15.33 5.00 12.38 4.00 10.81 9.64 10.79 10.49 17.70 18.45 16.29 18.99 20.07 20.27 7.92 x 16.41 17.67 16.39 17.15 14.35 12.75 14.15 13.52 19.19 19.64 18.75 22.06 21.04 19.94 6.20 7.13 x 14.95 12.65 14.18 12.15 11.57 12.04 11.94 15.51 15.37 16.37 20.15 17.95 19.86 1.36 7.44 6.37 x 13.11 4.73 11.53 10.83 10.86 11.34 17.28 17.59 17.22 18.88 18.46 19.57 5.08 7.19 6.02 5.08 x 11.19 6.08 5.84 3.45 5.69 15.19 15.88 16.93 19.99 20.19 18.97 1.65 7.30 5.91 1.76 4.88 x 11.46 10.15 10.09 10.60 17.82 17.92 16.96 19.30 19.36 19.31 5.16 6.89 5.48 4.84 1.96 4.97 x 3.50 5.69 1.45 14.48 15.48 15.23 18.96 17.83 19.89 4.81 6.21 5.27 4.46 1.43 4.50 0.90 x 4.37 2.05 12.85 14.05 14.91 17.52 16.58 17.72 4.63 6.58 5.16 4.74 1.15 4.51 1.21 0.87 x 4.55 12.71 13.31 14.89 16.72 17.28 16.05 4.85 6.62 5.53 4.71 1.64 4.91 0.46 0.62 0.98 x 14.54 15.19 15.34 18.46 17.59 18.88 8.11 7.51 9.07 8.11 7.70 8.03 7.58 7.07 7.52 7.48 x 2.03 15.22 19.71 18.30 17.97 8.06 7.37 8.86 8.02 7.69 7.94 7.46 7.11 7.36 7.36 0.44 x 15.73 18.95 18.57 18.73 10.47 10.35 9.31 11.14 9.71 11.35 9.43 9.36 9.64 9.47 10.33 10.32 x 17.81 17.06 18.31 13.37 12.52 12.93 13.36 12.22 13.48 12.24 12.11 12.18 11.98 13.64 13.79 12.50 x 6.55 10.99 12.84 11.57 12.37 12.82 12.03 12.82 12.04 12.17 12.36 11.93 12.94 12.93 12.09 2.86 x 11.41 12.06 11.56 12.22 12.49 11.28 11.96 11.03 10.79 11.29 11.11 11.08 11.20 11.53 8.16 7.81 x 19.50 18.43 18.10 19.28 18.06 19.13 18.69 18.65 18.63 18.76 17.99 17.56 18.65 17.01 17.40 18.28 18.57 18.06 17.75 17.74 17.67 18.21 17.35 17.39 17.56 17.44 16.99 17.27 17.89 17.58 18.77 17.49 18.36 19.36 18.41 17.87 17.99 17.83 18.23 18.08 18.37 18.60 16.69 16.73 18.74 18.47 19.69 17.63 18.27 17.27 16.91 17.53 17.35 17.75 17.36 17.08 17.33 17.40 16.34 16.12 18.12 16.85 18.04 17.45 18.09 17.63 16.79 17.32 17.69 17.09 17.23 17.25 17.31 17.61 16.78 16.72 18.39 17.49 18.56 16.98 17.09 17.97 16.87 16.72 17.82 17.00 17.02 17.00 17.26 17.27 16.92 17.11 18.87 18.77 19.39 17.00 16.29 16.41 15.16 15.96 16.12 16.28 16.11 15.82 15.87 16.25 16.16 16.41 15.86 17.16 17.69 15.95 18.37 18.37 17.15 18.62 18.27 18.66 17.65 17.98 17.45 17.73 17.72 17.46 16.86 17.24 18.11 16.89 18.37 19.48 18.24 18.73 18.04 18.65 18.09 18.24 17.99 17.96 17.75 17.56 18.49 16.56 17.26 16.74 17.62 18.04 16.91 17.84 17.65 17.72 17.57 17.32 17.15 17.45 17.76 17.79 16.23 17.27 18.29 16.23 19.13 18.32 17.68 19.08 18.99 19.15 18.69 18.70 18.66 18.90 18.58 18.68 16.96 18.28 18.89 17.51 18.19 18.64 18.37 18.54 18.19 18.29 18.20 18.07 17.95 18.08 17.31 17.38 17.35 16.54 17.24 15.94 17.14 17.47 16.84 17.38 17.13 17.25 17.17 16.85 16.65 16.96 17.56 17.58 16.51 17.27 18.69 16.31 19.51 20.30 18.90 19.52 19.02 19.61 18.90 18.83 18.84 19.04 18.62 18.95 18.31 17.97 19.49 18.09 18.28 18.05 17.75 18.68 18.31 18.55 18.08 18.23 17.81 18.19 18.17 18.35 16.92 17.86 18.57 17.14 18.46 18.74 17.12 18.45 18.47 18.55 18.24 18.14 18.03 18.11 18.35 18.45 17.05 18.17 18.91 17.07 19.17 18.85 19.53 19.39 18.61 19.13 18.84 18.77 18.85 18.89 18.01 18.32 17.51 16.41 17.10 16.25 19.05 18.65 17.69 19.12 19.08 18.99 19.08 18.71 18.69 18.97 18.58 18.68 17.81 18.93 19.65 17.71 17.98 17.78 17.16 18.36 17.60 18.09 17.80 17.71 17.33 17.70 17.64 17.65 16.78 17.60 18.41 16.63 17.80 19.74 18.70 17.78 18.29 18.04 18.52 18.40 18.41 18.69 19.05 19.09 17.56 18.36 19.72 17.62 17.91 18.01 17.69 17.94 17.45 18.02 17.54 17.44 17.17 17.42 17.96 18.15 17.04 17.49 18.51 16.82 17.11 17.98 16.92 17.30 17.60 17.37 17.21 16.92 16.79 17.10 17.79 17.95 16.81 17.63 18.65 16.53 19.49 19.10 19.03 19.85 19.85 19.90 19.81 19.57 19.62 20.08 18.65 19.04 17.15 17.73 19.58 16.29 19.66 18.94 19.35 19.65 20.17 19.90 20.00 19.73 19.72 20.42 18.97 19.23 17.63 18.02 19.17 16.64 16.62 17.36 18.58 16.88 16.87 17.05 16.71 17.03 16.78 16.89 16.63 16.80 16.94 16.40 17.44 15.36 17.53 18.40 17.47 17.74 18.01 17.63 17.84 18.15 18.09 18.14 16.52 16.72 18.11 17.57 18.47 16.72 16.71 15.96 16.07 16.33 16.05 16.81 16.04 15.99 15.60 15.99 16.39 16.48 15.78 16.66 17.44 16.57 VPJ ALG HIS GHI IGL TES 22.10 20.94 21.96 23.07 23.23 24.72 22.06 22.82 20.38 20.68 20.54 20.01 18.87 22.06 18.65 24.21 21.04 20.70 22.34 21.04 20.18 18.37 18.71 17.84 17.32 22.06 18.90 18.23 21.28 19.17 22.09 18.88 19.13 21.93 19.93 21.93 19.55 18.26 20.87 19.25 21.47 16.92 17.41 22.06 19.38 20.93 15.23 15.44 19.49 17.84 21.56 15.93 17.14 19.17 17.62 21.96 16.32 16.29 21.06 18.57 25.28 18.24 18.06 20.64 17.44 26.00 18.92 17.78 21.04 17.44 21.76 21.50 20.94 21.38 19.82 22.79 21.57 21.68 21.31 18.78 23.21 20.78 20.72 20.54 17.94 23.18 20.87 18.43 18.15 17.41 x 19.87 19.09 23.02 23.09 10.25 x 12.64 16.56 19.74 9.70 5.73 x 16.07 18.50 11.26 8.13 8.47 x 18.79 9.53 10.14 9.60 10.35 x 17.28 14.71 14.84 15.89 15.18 14.54 14.13 13.73 14.71 13.04 13.59 14.37 14.91 14.37 13.33 13.00 14.63 12.95 14.67 13.78 13.93 14.52 13.85 14.95 12.78 13.86 15.07 14.20 15.51 12.88 14.05 14.31 12.78 14.58 13.29 14.10 15.17 13.91 15.58 13.08 16.83 16.32 15.11 17.54 14.30 13.96 15.67 14.14 16.61 13.84 13.75 15.01 14.07 15.61 13.28 13.28 14.94 14.02 15.60 13.29 14.03 15.58 14.47 16.47 13.14 14.03 15.17 14.01 16.13 13.23 17.36 16.20 15.78 16.50 14.33 14.55 15.01 14.77 15.62 13.97 14.45 14.64 14.12 15.05 13.37 15.39 14.66 14.96 16.45 12.98 16.42 15.03 15.29 16.04 12.28 13.84 14.47 14.04 16.83 13.06 13.94 12.92 12.54 15.66 12.20 13.50 14.18 14.57 15.82 12.68 HM LAC 21.49 MND 18.39 21.22 19.93 21.30 19.02 18.88 21.06 18.61 20.03 18.65 17.85 19.50 22.03 21.47 19.44 19.74 19.60 17.64 19.37 22.24 18.93 21.00 20.52 x 7.17 18.86 16.53 19.30 15.90 17.06 16.75 16.71 16.10 15.01 13.59 16.11 16.43 16.69 17.26 15.85 14.41 14.71 19.28 17.01 15.41 17.33 17.52 15.10 x 15.01 14.72 15.05 15.32 14.55 13.66 15.20 15.04 14.52 16.17 15.77 14.41 15.66 14.88 14.54 16.14 16.18 16.00 16.78 15.29 12.73 12.86 13.17 13.16 13.07 12.56 13.83 12.90 12.73 13.96 13.95 12.64 13.83 12.81 13.09 12.34 14.22 15.64 15.97 13.00 BIO BRU QUE GRA BSM LOP TOR OEL HNE UIX QLL PRB MON ALE TAL CER MAJ SAN 20.89 24.35 19.53 20.99 21.88 19.20 21.35 21.26 19.34 21.52 20.42 20.79 21.59 19.30 19.58 24.14 20.58 x 22.42 16.68 16.18 20.39 18.38 19.96 18.04 18.03 21.58 15.96 17.80 19.04 18.81 17.96 22.10 18.26 22.38 24.66 21.15 23.43 22.72 21.42 23.88 23.91 22.69 24.09 21.09 22.30 23.63 22.39 22.09 24.72 21.43 VG SGF 24.00 PIG 23.84 XN XAN 23.22 24.58 20.94 20.81 19.54 20.86 19.04 21.40 20.93 21.43 23.89 20.31 20.52 20.07 19.76 20.50 20.72 20.21 24.41 23.86 22.03 21.81 21.95 20.80 22.59 22.06 23.45 21.91 21.62 21.42 23.21 21.66 24.06 22.72 21.67 23.87 21.58 20.83 19.00 20.63 17.25 19.93 21.38 20.60 20.31 18.04 22.23 21.41 19.61 21.11 21.81 19.53 23.95 20.42 20.92 20.19 20.30 18.71 21.57 21.98 22.04 21.74 19.87 20.12 22.04 21.21 20.83 20.40 18.94 25.63 20.71 21.39 20.92 19.48 19.14 20.70 22.12 21.85 21.42 20.00 21.59 20.87 20.18 21.36 21.00 21.66 23.64 19.92 19.82 19.10 20.94 17.63 20.62 21.16 20.74 22.42 18.51 20.40 21.07 20.20 19.55 19.94 19.84 24.07 20.93 20.31 19.16 20.18 18.23 19.56 20.83 20.08 21.30 19.32 20.64 20.23 18.77 20.88 21.53 20.55 23.63 19.42 18.97 18.82 17.85 17.82 18.44 19.57 19.04 19.93 18.61 18.43 19.60 18.68 20.01 20.26 18.61 21.63 18.94 19.44 18.29 17.55 17.00 18.34 19.59 19.75 20.06 18.12 18.74 18.36 19.24 19.40 19.33 18.74 23.81 19.88 19.95 19.22 19.09 17.99 19.80 20.34 19.68 20.56 18.75 19.84 20.06 18.83 20.50 21.05 19.66 23.15 23.64 20.94 21.18 20.76 20.86 21.78 22.26 22.33 23.73 19.95 21.34 20.94 22.00 22.58 22.01 20.90 24.37 23.93 21.53 21.21 20.70 21.33 21.39 22.93 22.10 23.06 20.32 22.23 21.34 22.21 22.68 23.02 20.11 23.20 22.86 19.89 19.38 21.73 18.15 21.26 18.67 19.07 22.49 18.04 18.15 19.73 20.20 21.09 20.70 19.93 26.71 23.94 20.29 21.77 23.23 21.26 20.86 21.26 22.58 24.96 22.57 20.78 18.64 22.74 21.98 22.02 22.21 25.24 21.65 19.90 22.48 22.47 20.35 20.69 22.39 21.10 23.04 21.74 20.77 18.82 22.54 22.48 19.82 21.14 20.16 21.88 18.80 19.72 20.53 19.11 19.76 18.79 21.81 25.04 18.58 18.19 17.45 21.10 20.64 20.27 18.33 24.04 21.33 21.31 22.57 24.33 20.81 23.12 23.17 22.32 23.11 21.68 22.72 26.01 22.24 21.74 21.40 23.29 21.43 22.63 19.45 20.39 22.06 18.24 18.60 20.50 20.14 19.48 19.30 18.70 19.34 19.74 20.80 22.02 22.99 20.65 20.06 17.63 18.54 19.87 17.28 17.54 17.93 17.22 20.64 18.52 17.27 17.72 19.07 19.20 22.35 18.57 20.63 21.00 20.62 20.85 18.96 20.41 21.14 19.81 22.42 21.03 20.34 18.58 18.95 21.47 21.93 20.20 20.83 19.88 20.59 16.55 16.45 19.01 18.28 17.92 17.32 17.28 20.48 15.58 17.07 17.32 17.61 17.65 20.59 19.98 20.22 22.38 18.80 18.78 20.91 19.14 19.12 19.93 17.83 22.10 18.52 18.45 22.44 20.39 19.37 21.06 19.60 18.16 16.72 16.97 17.56 16.95 16.41 17.89 16.86 16.65 18.66 17.87 16.70 17.38 17.21 16.96 19.13 17.50 9.55 x 18.70 19.48 11.49 16.79 21.71 16.76 17.81 13.87 17.87 17.78 20.40 20.60 19.14 20.55 22.11 5.34 9.01 x 13.49 19.44 8.13 16.17 13.01 6.72 18.00 12.64 11.48 16.12 7.79 5.19 16.14 17.02 5.38 9.39 3.03 x 17.17 11.35 17.19 12.16 13.79 17.77 6.05 12.35 16.21 13.96 12.07 19.25 17.20 9.38 2.85 8.27 8.94 x 17.32 19.70 20.16 19.86 15.07 17.89 18.92 19.99 21.06 19.16 20.50 18.49 5.41 8.30 1.87 2.95 7.91 x 16.58 11.08 9.61 16.31 9.53 9.96 17.15 10.12 8.45 16.34 16.79 8.96 11.46 7.42 7.84 10.85 7.15 x 15.74 16.18 20.11 16.40 17.19 10.15 15.55 15.80 21.19 18.02 5.86 8.89 3.36 2.81 8.88 2.36 8.20 x 13.10 15.97 11.05 6.29 15.50 12.17 12.51 17.65 18.04 4.99 8.89 2.08 2.67 8.48 2.01 7.66 3.64 x 16.65 11.63 12.28 16.15 7.70 7.50 17.69 18.41 9.02 5.97 8.81 8.96 5.28 8.36 12.34 8.72 8.95 x 17.34 17.84 21.32 18.41 18.54 22.85 21.19 5.16 9.39 3.20 1.49 8.94 2.77 7.83 2.80 2.90 9.08 x 10.40 15.74 12.04 10.70 16.99 15.64 5.10 7.62 2.37 2.32 7.55 1.30 6.89 1.16 2.47 8.10 2.06 x 15.48 11.57 11.22 15.98 18.23 8.98 11.62 7.29 8.57 10.86 7.00 4.05 7.91 8.33 11.46 8.41 6.68 x 15.25 15.26 19.70 20.46 5.49 9.45 2.27 3.63 8.71 2.41 8.31 4.20 2.70 8.85 3.59 3.20 8.03 x 6.61 18.35 18.89 5.66 9.13 1.12 3.58 8.40 1.83 7.21 3.50 2.75 8.83 3.43 2.50 6.69 2.25 x 17.66 16.76 11.59 12.05 11.04 10.28 11.57 9.92 12.77 10.07 10.67 12.19 10.60 10.09 11.82 10.92 11.30 x 12.67 12.10 12.12 11.31 10.75 11.19 9.80 13.12 10.35 11.10 12.25 11.17 10.15 12.10 11.50 11.31 4.07 x 14.89 13.85 14.94 15.30 14.13 14.75 17.07 14.68 14.93 14.80 15.71 14.36 16.63 15.29 15.10 15.14 14.99 14.49 13.04 14.38 13.66 13.93 13.99 16.66 14.10 13.92 15.22 13.86 13.39 15.50 15.03 14.66 12.76 14.18 13.81 14.10 12.35 12.66 13.77 12.40 13.96 12.71 13.05 13.90 12.93 12.07 14.69 12.73 12.35 14.18 13.33 22.19 21.47 21.66 20.17 20.64 21.64 21.70 20.09 18.52 20.67 19.71 21.57 22.24 23.01 23.05 20.67 22.29 22.51 21.37 19.56 21.70 19.21 19.73 18.38 23.42 21.45 21.99 22.51 21.53 22.81 20.23 19.88 21.74 21.19 22.66 20.96 19.21 21.67 22.25 21.62 20.72 x 12.37 15.03 23.76 23.79 21.33 21.83 23.09 20.71 21.71 20.69 20.92 18.57 20.95 22.78 22.77 24.66 23.46 24.80 21.34 25.07 23.67 21.97 25.44 21.80 19.57 20.64 21.07 23.36 19.41 21.15 23.19 21.39 23.99 23.06 20.02 23.53 19.57 23.45 23.36 19.62 21.86 24.66 22.35 24.21 x 15.10 20.25 21.25 22.37 20.85 20.99 21.02 20.42 19.86 18.19 18.31 19.39 21.89 20.85 20.77 21.26 20.92 19.13 21.46 17.52 18.85 19.34 17.85 22.06 17.50 20.78 23.35 19.22 18.93 21.96 18.83 19.89 21.05 20.30 22.58 18.18 18.70 20.74 20.13 19.15 22.49 22.19 21.12 21.85 x Mean values of genetic divergence (K2P in percentage of changes) between pairs of populations within hormogastrids, based on nuclear data (H4 below the diagonal; H3 above the diagonal). See complete names of localities and species in Table 1. CLADE HCL HE HIS HM HNE VG XN CODE ORD HPA VIL CAB UCE FRE LOZ MOL NAV JAR RED TRE VEN SIG SOT ANC BOA SEV PAR VPJ ALG GHI IGL TES LAC MND BIO BRU QUE GRA BSM LOP TOR OEL UIX QLL PRB MON ALE TAL CER MAJ SAN SGF PIG XAN ORD x HCL HPA 0.01 0.62 3.97 4.78 4.64 3.95 4.92 4.09 4.49 4.77 4.68 4.61 4.22 3.67 4.54 4.42 3.77 4.36 4.41 4.74 4.42 5.11 4.42 4.75 4.32 3.81 4.74 3.45 3.13 4.10 3.07 3.77 2.81 3.77 4.43 3.45 2.81 3.77 3.13 3.45 3.45 3.77 3.13 3.45 4.42 x 4.47 4.75 4.66 4.60 4.21 3.65 3.82 4.10 4.01 4.03 3.56 3.01 4.52 4.40 3.75 4.35 4.40 3.87 3.76 3.11 3.70 3.75 4.73 4.40 5.09 4.40 4.73 4.30 4.08 3.76 4.77 3.76 4.08 3.66 3.79 4.73 3.15 4.08 3.44 3.11 2.80 2.48 4.08 3.06 3.75 2.80 3.76 3.44 2.42 3.11 2.17 3.12 4.41 3.44 2.80 3.75 3.11 3.44 3.44 3.76 3.12 3.76 2.80 2.17 3.11 2.48 2.80 2.80 3.12 2.48 3.44 4.40 2.80 3.76 0.42 0.99 0.60 0.62 0.33 0.19 1.26 2.99 2.67 2.62 4.60 4.93 5.25 5.95 4.60 4.96 4.54 3.38 4.64 3.03 2.71 3.99 2.86 2.71 3.03 3.35 4.32 3.03 3.03 2.71 2.71 3.03 3.35 3.03 3.99 4.32 4.64 1.63 2.21 1.81 1.83 1.53 1.38 2.31 3.79 3.47 3.42 5.42 5.75 6.08 6.79 5.42 5.49 5.06 4.15 5.42 3.79 3.46 4.77 3.62 3.46 3.79 4.11 5.10 3.79 3.79 3.46 3.46 3.79 3.87 3.54 4.76 5.09 5.08 0.17 0.36 0.36 0.10 0.78 0.63 1.87 3.66 3.34 3.18 5.28 5.61 5.94 6.65 5.29 4.95 4.52 4.01 5.28 3.65 3.33 4.63 3.49 3.33 3.65 3.98 4.96 3.65 3.66 3.33 3.33 3.65 3.98 3.65 4.63 4.96 4.78 0.63 1.14 0.75 0.82 0.60 0.50 1.43 2.97 2.65 2.60 4.58 4.91 5.23 5.94 4.58 5.14 4.71 3.56 4.82 3.20 2.88 4.17 3.04 2.88 3.20 3.52 4.50 3.20 3.20 2.88 2.88 3.20 3.52 3.20 4.17 4.49 4.81 0.13 0.21 0.18 0.10 0.94 0.89 2.13 3.93 3.60 3.45 5.56 5.90 6.22 6.94 5.57 5.22 4.79 4.28 5.56 3.92 3.60 4.91 3.76 3.60 3.92 4.25 5.24 3.92 3.93 3.60 3.60 3.92 4.25 3.92 4.90 5.23 4.93 VIL 1.18 0.61 3.95 4.76 4.63 3.93 4.90 4.07 x 3.31 4.11 3.98 3.29 4.25 3.43 CAB 3.31 4.55 x 1.17 0.58 0.13 0.83 0.07 UCE 3.93 5.17 0.22 x 1.78 1.34 2.05 1.26 FRE 3.93 5.17 0.22 0.00 x LOZ 3.83 5.08 0.22 0.00 0.00 x 0.90 0.22 0.82 1.04 0.07 MOL 4.04 5.29 0.32 0.10 0.10 0.10 x 1.07 NAV 3.93 5.17 0.22 0.00 0.00 0.00 0.10 x 0.66 1.24 0.85 0.86 0.57 0.43 1.35 3.11 2.80 2.74 4.72 5.05 5.38 6.08 4.73 5.05 4.62 3.47 4.72 3.11 2.80 4.08 2.95 2.80 3.11 3.43 4.40 3.11 3.11 2.80 2.80 3.11 3.43 3.11 4.07 4.40 4.72 JAR 3.93 5.17 0.22 0.00 0.00 0.00 0.10 0.00 x 1.71 3.50 3.18 3.05 4.96 5.29 5.62 6.33 4.97 4.95 4.53 3.86 5.13 3.50 3.18 4.48 3.33 3.17 3.50 3.82 4.81 3.50 3.50 3.17 3.18 3.50 3.82 3.50 4.47 4.80 4.79 HE RED TRE 3.93 5.17 0.22 0.00 0.00 0.00 0.10 0.00 0.00 x 2.30 4.10 3.78 3.62 5.41 5.74 6.07 6.78 5.41 5.07 4.64 4.13 5.41 3.77 3.45 4.76 3.61 3.77 3.77 4.10 5.09 3.77 3.77 3.77 3.45 3.77 4.10 4.10 4.75 5.08 4.85 4.09 5.35 0.36 0.14 0.14 0.14 -0.01 0.14 0.14 0.14 x 1.90 3.69 3.37 3.24 5.32 5.65 5.98 6.69 5.32 5.14 4.72 4.05 5.32 3.69 3.36 4.67 3.52 3.36 3.69 4.01 5.00 3.69 3.69 3.36 3.36 3.69 4.01 3.69 4.66 4.99 4.98 VEN 3.36 4.60 0.63 0.41 0.41 0.33 0.51 0.41 0.41 0.41 0.55 x 1.91 3.71 3.38 3.23 5.33 5.67 6.00 6.54 5.34 5.00 4.57 4.06 5.33 3.70 3.38 4.68 3.54 3.38 3.70 4.03 5.02 3.70 3.70 3.38 3.38 3.70 4.03 3.70 4.68 5.01 4.91 SIG 3.93 5.19 1.33 1.10 1.10 1.01 1.20 1.10 1.10 1.10 1.25 0.55 x 1.46 3.41 2.92 2.71 4.86 5.19 5.52 6.22 4.87 4.90 4.47 3.60 4.86 3.24 2.92 4.21 3.08 2.92 3.24 3.56 4.54 3.24 3.24 2.92 2.92 3.24 3.56 3.24 3.64 3.96 4.85 SOT 3.48 4.73 0.89 0.67 0.67 0.58 0.77 0.67 0.67 0.67 0.81 0.12 0.12 x 1.47 2.85 2.53 2.48 4.30 4.62 4.95 5.65 4.30 4.62 4.19 3.44 4.49 3.09 2.77 3.85 2.92 2.93 3.09 3.41 4.18 3.09 3.09 2.93 2.77 3.09 3.21 3.05 3.65 3.97 4.29 ANC 4.38 5.65 2.46 2.22 2.22 2.13 2.33 2.22 2.22 2.22 2.37 1.66 1.10 1.23 x 5.17 5.50 6.20 4.85 4.84 5.07 3.71 4.32 3.35 3.03 3.67 2.76 3.03 3.35 3.67 4.00 3.35 3.27 3.03 3.03 3.35 3.67 3.35 3.87 4.19 5.17 BOA 3.35 4.59 2.27 2.78 2.78 2.69 2.89 2.78 2.78 2.78 2.94 2.22 1.66 1.78 2.79 x 0.92 1.59 5.71 5.71 6.03 6.42 5.71 6.71 6.27 5.42 6.36 5.38 5.05 5.71 5.21 4.72 5.38 5.05 6.04 5.38 5.38 4.72 5.05 5.38 5.38 5.05 5.71 6.05 6.03 SEV PAR 3.35 3.93 4.59 2.27 2.78 2.78 2.69 2.89 2.78 5.18 2.84 3.35 3.35 3.26 3.47 3.35 2.78 2.78 2.94 2.22 1.66 1.78 3.35 3.35 3.51 2.79 2.22 2.35 2.79 0.00 x 0.66 5.05 3.36 0.55 0.55 x 5.00 5.05 5.37 5.75 5.05 6.03 5.60 5.00 5.33 6.03 5.00 5.88 5.45 4.76 5.70 4.71 5.65 4.72 4.39 4.67 4.34 5.05 4.56 4.07 4.72 4.39 5.00 4.51 4.02 4.67 4.34 5.38 4.72 4.72 4.07 4.39 4.72 4.72 4.39 5.05 5.33 4.67 4.67 4.02 4.34 4.67 4.67 4.34 5.00 5.38 5.37 5.34 5.33 VPJ 2.43 3.64 3.53 4.14 4.14 4.05 4.25 4.14 4.14 4.14 4.30 3.57 4.15 3.70 5.17 3.56 3.56 4.14 x 1.23 1.54 2.20 0.92 5.38 5.28 4.96 5.04 4.72 4.40 4.40 4.34 5.05 4.72 5.05 4.72 4.72 4.68 5.05 4.40 4.72 4.72 5.05 4.73 5.05 5.05 x 0.31 1.58 0.92 5.71 5.61 4.96 5.37 5.05 4.72 4.72 4.67 5.05 5.05 5.38 5.05 5.05 5.01 5.05 4.72 5.05 4.40 4.73 5.05 5.39 4.73 0.17 0.01 0.01 0.52 0.47 0.13 0.11 1.04 0.97 0.05 0.65 0.66 0.72 0.67 0.39 4.20 3.23 3.18 4.84 ALG HIS GHI 1.66 2.86 2.74 3.35 3.35 3.26 3.46 3.35 3.35 3.35 3.51 2.79 3.36 2.91 4.37 2.78 2.78 3.35 0.75 x 1.89 1.23 5.38 5.28 4.64 5.05 4.73 4.40 4.40 4.34 4.72 4.73 5.05 4.73 4.73 4.69 4.72 4.40 4.73 4.08 4.41 4.73 5.06 4.40 IGL TES 1.66 1.66 2.86 2.74 3.35 3.35 3.26 3.46 3.35 2.86 2.74 3.35 3.35 3.26 3.46 3.35 3.35 3.35 3.51 2.79 3.36 2.91 3.35 3.35 3.51 2.79 3.36 2.91 4.37 2.78 2.78 3.35 0.75 4.37 2.78 2.78 3.35 0.75 0.00 x 1.89 6.41 6.31 0.00 0.00 x 5.38 5.28 5.99 6.40 4.97 5.05 6.08 5.74 4.73 4.40 5.74 5.69 6.07 6.08 6.41 4.40 4.34 5.05 4.73 5.05 6.08 6.08 6.04 6.07 5.74 6.08 6.08 6.41 6.08 4.73 4.73 4.69 5.05 4.40 4.73 4.73 5.05 4.73 6.42 5.75 5.06 5.06 HM LAC 5.11 MND 5.09 6.41 6.17 6.90 6.90 6.86 7.03 6.90 6.31 6.16 6.89 6.89 6.80 6.98 6.89 6.90 6.90 7.09 6.86 7.56 7.08 6.89 6.89 7.08 6.30 6.91 6.44 8.00 6.31 6.31 6.93 5.34 7.99 5.69 5.69 6.29 3.56 5.13 5.13 5.13 x 0.66 3.35 3.35 3.35 3.92 x 4.64 4.72 4.13 4.95 4.40 4.07 3.89 3.57 4.07 3.80 4.72 4.40 4.73 4.30 3.73 4.21 3.89 4.22 4.40 4.07 4.32 4.72 4.07 4.40 3.75 4.07 3.75 4.14 3.57 3.89 4.21 3.57 3.89 3.33 3.65 3.98 4.08 5.38 4.30 4.95 BIO 1.84 3.06 2.84 3.55 3.55 3.46 3.65 3.55 3.55 3.55 3.72 2.99 3.57 3.12 4.58 2.98 2.98 3.56 2.05 1.28 1.28 1.28 5.37 3.92 x 0.95 0.65 3.04 0.79 0.65 0.95 1.27 2.72 0.95 0.96 0.65 0.65 0.95 2.52 2.84 3.79 4.12 3.47 BRU 2.79 4.03 3.70 4.52 4.52 4.42 4.64 4.52 4.52 4.52 4.69 3.95 3.37 3.50 4.38 2.79 2.79 3.36 3.00 2.22 2.22 2.22 5.15 5.12 1.29 x 3.44 3.12 0.61 2.85 3.76 3.44 3.76 1.54 3.44 3.36 3.76 3.12 3.44 3.12 3.44 4.07 3.75 5.05 QUE 1.72 2.93 2.81 3.42 3.42 3.33 3.53 3.42 3.42 3.42 3.58 2.85 2.85 2.69 3.86 2.28 2.28 2.85 1.93 1.16 1.16 1.16 5.21 4.58 1.35 1.72 x 2.81 0.45 0.92 0.61 0.31 2.49 0.61 0.61 0.92 0.31 0.00 2.17 2.49 3.44 3.76 4.07 GRA 1.66 2.86 2.74 3.35 3.35 3.26 3.46 3.35 3.35 3.35 3.51 2.79 2.79 2.63 3.79 2.22 2.22 2.78 1.86 1.10 1.10 1.10 5.13 4.51 1.28 1.66 0.06 x 2.49 0.14 0.61 0.31 0.61 2.17 0.31 0.31 0.61 0.00 0.31 1.85 2.17 3.11 3.44 3.75 BSM 3.36 4.62 4.29 5.11 5.11 5.02 5.23 5.11 5.11 5.11 5.28 4.54 3.95 4.08 4.97 3.36 3.36 3.94 3.00 2.80 2.80 2.80 4.55 4.52 1.86 0.55 2.29 2.22 x 0.92 2.81 2.73 3.12 2.49 2.81 2.49 2.81 3.44 3.11 4.73 LOP 1.66 2.86 2.74 3.35 3.35 3.26 3.46 3.35 3.35 3.35 3.51 2.79 2.79 2.63 3.79 2.22 2.22 2.78 1.86 1.10 1.10 1.10 5.13 4.51 1.28 1.66 0.06 0.00 2.22 x 1.90 0.45 0.19 0.76 0.14 0.45 2.01 2.32 2.63 2.95 3.91 TOR 1.10 2.30 3.31 3.93 3.93 3.83 4.04 3.93 3.93 3.93 4.09 3.36 3.36 3.20 4.36 2.78 2.78 3.35 2.43 1.66 1.66 1.66 5.71 5.09 1.84 2.22 0.61 0.55 2.79 0.55 x 2.80 0.92 0.92 0.00 0.61 0.92 2.48 2.17 3.75 4.08 4.07 OEL 2.22 3.43 3.31 3.93 3.93 3.83 4.04 3.93 3.93 3.93 4.09 3.36 3.36 3.20 4.36 2.78 2.78 3.35 2.43 1.66 1.66 1.66 5.71 5.09 1.84 2.22 0.61 0.55 2.79 0.55 1.10 x 2.49 0.61 0.00 0.92 0.31 0.61 2.17 2.49 2.80 3.11 4.07 HNE UIX 1.66 2.87 2.65 3.29 3.29 3.19 3.40 3.29 3.29 3.29 3.45 2.72 2.72 2.56 3.72 2.15 2.15 2.72 1.87 1.10 1.10 1.10 5.14 4.44 1.22 1.59 0.06 0.00 2.16 0.00 0.55 0.55 x 2.81 0.92 0.92 1.23 0.61 0.31 2.49 2.81 3.76 4.08 4.40 QLL 3.35 4.60 4.28 5.10 5.10 5.00 5.22 5.10 5.10 5.10 5.27 4.52 3.94 4.07 4.95 3.35 3.35 3.93 3.57 2.79 2.79 2.79 5.73 5.70 1.85 0.55 2.29 2.22 1.10 2.22 2.78 1.66 2.15 x 4.08 5.05 PRB 1.66 2.86 2.74 3.35 3.35 3.26 3.46 3.35 3.35 3.35 3.51 2.79 2.79 2.63 3.79 2.22 2.22 2.78 1.86 1.10 1.10 1.10 5.13 4.51 1.28 1.66 0.06 0.00 2.22 0.00 0.55 0.55 0.00 2.22 x MON 1.72 2.93 2.81 3.42 3.42 3.33 3.53 3.42 3.42 3.42 3.58 2.85 2.85 2.69 3.86 2.28 2.28 2.85 1.93 1.16 1.16 1.16 5.20 4.58 1.35 1.72 0.12 0.06 2.29 0.06 0.61 0.20 0.06 1.86 0.06 x ALE 1.10 2.30 3.31 3.93 3.93 3.83 4.04 3.93 3.93 3.93 4.09 3.36 3.36 3.20 4.36 2.78 2.78 3.35 2.43 1.66 1.66 1.66 5.71 5.09 1.84 2.22 0.61 0.55 2.79 0.55 0.00 1.10 0.55 2.78 0.55 0.61 x TAL 1.66 2.86 2.74 3.35 3.35 3.26 3.46 3.35 3.35 3.35 3.51 2.79 2.79 2.63 3.79 2.22 2.22 2.78 1.86 1.10 1.10 1.10 5.13 4.51 1.28 1.66 0.06 0.00 2.22 0.00 0.55 0.55 0.00 2.22 0.00 0.06 0.55 x CER 1.66 2.86 2.74 3.35 3.35 3.26 3.46 3.35 3.35 3.35 3.51 2.79 2.79 2.63 3.79 2.22 2.22 2.78 1.86 1.10 1.10 1.10 5.13 4.51 1.28 1.66 0.06 0.00 2.22 0.00 0.55 0.55 0.00 2.22 0.00 0.06 0.55 0.00 x 2.17 2.49 3.44 3.76 4.07 MAJ 1.66 SAN 1.66 2.86 2.74 3.35 3.35 3.26 3.46 3.35 2.86 2.74 3.35 3.35 3.26 3.46 3.35 3.35 3.35 3.51 2.79 2.79 2.63 3.35 3.35 3.51 2.79 2.79 2.63 3.79 2.22 2.22 2.78 1.86 3.79 2.22 2.22 2.78 1.86 1.10 1.10 1.10 5.13 4.51 1.10 1.10 1.10 5.13 4.51 1.28 1.66 1.28 1.66 0.06 0.00 0.06 0.00 2.22 0.00 0.55 0.55 0.00 2.22 0.00 0.55 0.55 0.00 2.22 0.00 0.06 0.55 0.00 0.00 x 0.31 3.44 2.22 0.00 0.06 0.55 0.00 0.00 0.00 x 3.76 3.76 4.07 4.08 4.40 1.10 4.44 2.30 2.17 2.78 2.78 2.69 2.89 2.78 5.71 4.89 5.61 5.61 5.52 5.70 5.61 2.78 2.78 2.94 2.22 2.79 2.35 5.61 5.61 5.79 5.03 5.63 5.17 3.79 2.22 2.22 2.79 1.30 6.67 5.03 5.03 5.62 4.66 0.55 0.55 0.55 4.55 3.93 3.86 3.86 3.86 6.92 5.33 0.73 1.67 2.68 3.87 0.61 0.55 3.94 3.86 2.24 0.55 1.10 1.10 0.55 4.46 3.86 4.44 3.86 3.73 2.22 0.55 0.61 1.10 0.55 0.55 0.55 0.55 x 3.86 3.86 3.72 4.44 3.86 3.86 3.86 3.86 3.29 1.54 4.40 x 4.42 1.10 2.30 2.17 2.78 2.78 2.69 2.89 2.78 2.78 2.78 2.94 2.22 2.79 2.35 3.79 2.22 2.22 2.79 1.30 0.55 0.55 0.55 4.55 3.93 0.73 1.67 0.61 0.55 2.24 0.55 1.10 1.10 0.55 2.22 0.55 0.61 1.10 0.55 0.55 0.55 0.55 0.00 3.29 x VG SGF PIG XN XAN 3.67 0.31 2.22 3.12 2.81 3.13 0.76 0.25 0.76 0.92 1.23 0.92 2.49 2.41 2.80 2.17 2.49 2.81 3.13 3.76 0.61 0.92 0.31 0.61 2.17 2.49 3.44 0.92 0.31 0.61 2.17 2.49 2.72 0.61 0.92 2.48 2.17 3.75 0.31 1.85 2.17 3.11 3.76 4.07 3.04 4.08 4.08 4.07 3.44 3.75
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