DOI: 10.5061/dryad.tf7qr
title: Data from: Taxonomist’s nightmare … evolutionist’s delight: an integrative approach resolves species limits in jumping bristletails despite widespread hybridization and parthenogenesis

description: Accurate species delimitation is fundamental to biology. Traditionally,
 species were delimited based on morphological characters, sometimes
 leading to taxonomic uncertainty in morphologically conserved taxa.
 Recently, multiple taxonomically challenging cases have benefited from
 integrative taxonomy – an approach that highlights congruence among
 different disciplines and invokes evolutionary explanations for
 incongruence, acknowledging that different methods can mirror different
 stages of the speciation continuum. Here, we used a cohesive protocol for
 integrative taxonomy to revise species limits in 20 nominal species and
 four morphospecies of an ancestrally wingless insect group, the jumping
 bristletail genus Machilis from the European Eastern Alps. Even though
 morphologically conserved, several small-scale endemic species have been
 described from the Eastern Alps based on variation in hypodermal
 pigmentation patterns – a highly questionable character. As valuable as
 these endemics are for conservation, they have never been verified by
 alternative methods. Using traditional morphometrics, mitochondrial DNA,
 ribosomal DNA, and amplified fragment-length polymorphism markers, we
 identify six nominal species as taxonomic junior synonyms (Machilis
 alpicola Janetschek, 1953 syn. n. under M. vagans Wygodzinsky, 1941; M.
 ladensis Janetschek, 1950 syn. n., M. robusta Wygodzinsky, 1941 syn. n.,
 and M. vicina Wygodzinsky, 1941 syn. n. under M. inermis Wygodzinsky,
 1941; M. aleamaculata Wygodzinsky, 1941 syn n. under M. montana
 Wygodzinsky 1941; M. pulchra Janetschek 1950 syn. n. under M. helleri
 Verhoeff 1910) and describe two new species (Machilis cryptoglacialis sp.
 n. and Machilis albida sp. n.), one uncovered from morphological crypsis
 and one never sampled before. Building on numerous cases of incongruence
 among data sources, we further shed light on complex evolutionary
 histories including hybrid speciation, historical and recent
 hybridization, and ongoing speciation. We hypothesize that an inherent
 affinity to hybridization, combined with parallel switches to
 parthenogenesis and repeated postglacial colonization events may have
 boosted endemicity in Eastern Alpine Machilis. We thus emphasize the
 importance of integrative taxonomy for rigorous species delimitation and
 its implication for evolutionary research and conservation in
 taxonomically challenging taxa.
description: Supplementary_material1_TM_datasetTraditional morphometrics datasetSupplementary_material2_CO1_alignmentCytochrome Oxidase 1 haplotype alignmentSupplementary_material3_ITS2_alignmentInternal transcribed spacer 2 final alignmentSupplementary_material4_AFLP_matrixFinal AFLP matrix of four combined primer combinationsList of specimensList of specimens used in this study, including voucher IDs, sampling locality, GPS coordinates of sampling sites, GenBank accessions, and information whether an individual was included (1) in the traditional morphometrics (TM) and / or amplified fragment-length polymorphism (AFLP) dataset. The fifteen specimens of M. cryptoglacialis sp. n. that were listed in Dejaco et al. (2012) under M. glacialis are highlighted with bold letters.TabS1_revised2.docxTM character definitionsDefinition of characters used in traditional morphometrics analysisTabS2_revised.docxOptimized AFLP scoring parametersOptimized parameters used for the final scoring of the amplified fragment-length polymorphism profiles. The four dyes (FAM, HEX, NED and PET) represent four different primer combinations.TabS3_revised.docxResults of NewHybrids - engiadina groupResults of the hybrid test conducted in New Hybrids based on 466 amplified fragment-length polymorphism markers for the M. engiadina species group. Individuals were defined as parental species (z1 or z0) or putative hybrids (unknowns; highlighted red). Columns 4 to 9 represent six admixture classes and values are the posterior probabilities of belonging to one of these classes for each individual. P1 = parental species 1; P2 = parental species 2; F1 = first-generation hybrid; F2 = second-generation hybrid; BX1 = backcross to parental species 1; BX2 = backcross to parental species 2. (a) Machilis rubrofusca and sexual M. ticinensis as parental species and just M. engiadina as unknowns. (b) Machilis rubrofusca and sexual M. ticinensis as parental species and all other members of the M. engiadina species group as unknowns.TabS4_revised.docxResults of NewHybrids - helleri groupResults of the pairwise hybrid test conducted in NewHybrids based on 466 amplified fragment-length polymorphism markers for the M. helleri species group. Individuals were defined as parental species (z1 or z0) or putative hybrids (unknowns; highlighted red). Columns 4 to 9 represent six admixture classes and values are the posterior probabilities of belonging to one of these classes for each individual. P1 = parental species 1; P2 = parental species 2; F1 = first-generation hybrid; F2 = second-generation hybrid; BX1 = backcross to parental species 1; BX2 = backcross to parental species 2. (a) Machilis hrabei vs. M. helleri; note that individual #25 is likely a F2 hybrid even though no mito-nuclear discordance was found (see Discussion). (b) Machilis hrabei vs. M. lehnhoferi. (c) Machilis lehnhoferi vs. M. helleri.TabS5_revised.docxOptimal combinations of TM charactersOptimal combination of traditional morphometrics characters (identified by an exhaustive search) giving the highest classification rate in each species group.TabS6_revised.docxReanalysis of TM characters including type specimensReanalysis of traditional morphometric characters for discriminating among final species hypotheses and classifying type specimens (when available). Classification results based on discriminant functions calculated from an optimal combination of characters (see Table S6 for details), separately for four species groups. We defined these groups according to ovipositor length (i.e., number of articles on gonapophysis VIII). This corresponded to the first steps in the Machilis identification key of Palissa (1964). Type specimens were not included in the training set. Classification success is given as the actual number (absolute) and the percentage (%) of individuals classified in original and leave-one-out cross-validated (LOOCV) discriminant analyses. Correct and wrong classifications are highlighted in green and red, respectively. dis = M. distincta, eng = M. engiadina, rub = M. rubrofusca, tic = M. ticinensis, tir = M. tirolensis, vag = M. vagans, spE = M. sp. E, spF = M. sp. F.TabS7_revised2.docxNJ trees of individual AFLP primer combinationsNJ trees for each of the four primer combinations (each represented by the four dyes FAM, HEX, NED, and PET) used to produce amplified fragment-length polymorphism profiles. Sample names include the following information: ’plate number’_’well number’_’specimen ID’_’species code’.FigS1_revised.pdfReanalysis of traditional morphometric characters.Principal component analysis (PCA) scatterplots based on optimal character combinations for each of the four species groups. Whenever available, type specimens were included and are represented by stars.FigS2_revised.pdfDelimitation results of bPTP algorithmSpecies delimitation results of the Bayesian Poisson tree processes (bPTP) algorithm. Values on branches are posterior delimitation probabilities.FigS3_revised2.pdfEstimating the number of K from STRUCTURE results.Graphical representation of Evanno’s ∆K (circles) and the mean marginal likelihood across K (diamonds) from each 10 replicated runs from K=5 to K=27.FigS4_revised.pdfGeographical distributions of M. helleri species group.Geographical distributions of M. helleri species group. AT = Austria, CZ = Czech Republic, DE = Germany, IT = Italy.FigS5_revised.pdf