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.
Supplementary_material1_TM_dataset
Traditional morphometrics dataset
Supplementary_material2_CO1_alignment
Cytochrome Oxidase 1 haplotype alignment
Supplementary_material3_ITS2_alignment
Internal transcribed spacer 2 final alignment
Supplementary_material4_AFLP_matrix
Final AFLP matrix of four combined primer combinations
List of specimens
List 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.docx
TM character definitions
Definition of characters used in traditional morphometrics analysis
TabS2_revised.docx
Optimized AFLP scoring parameters
Optimized 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.docx
Results of NewHybrids - engiadina group
Results 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.docx
Results of NewHybrids - helleri group
Results 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.docx
Optimal combinations of TM characters
Optimal combination of traditional morphometrics characters (identified by an exhaustive search) giving the highest classification rate in each species group.
TabS6_revised.docx
Reanalysis of TM characters including type specimens
Reanalysis 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.docx
NJ trees of individual AFLP primer combinations
NJ 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.pdf
Reanalysis 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.pdf
Delimitation results of bPTP algorithm
Species delimitation results of the Bayesian Poisson tree processes (bPTP) algorithm. Values on branches are posterior delimitation probabilities.
FigS3_revised2.pdf
Estimating 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.pdf
Geographical 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