Founder Effects Initiated Rapid Species Radiation in Hawaiian Cave Planthoppers

Founder effects initiated rapid species radiation in Hawaiian cave planthoppers

Andreas Wessela,1, Hannelore Hocha, Manfred Aschea, Thomas von Rintelena, Björn Stelbrinka, Volker Heckb, Fred D. Stonec, and Francis G. Howarthd

aDepartment of Research, Museum für Naturkunde – Leibniz-Institut für Evolutions-und Biodiversitätsforschung an der Humboldt-Universität zu Berlin, D-10115 Berlin, Germany; bGeography, Department of Physics, University of Siegen, D-57068 Siegen, Germany; cMath and Natural Sciences Department, Hawai‘i Community College, Hilo, HI 96720-4091; and dDepartment of Natural Science, Bishop Museum, Honolulu, HI 96817

Edited by Francisco J. Ayala, University of California, Irvine, CA, and approved April 25, 2013 (received for review January 25, 2013) The Hawaiian Islands provide the venue of one of nature’sgrand a unique subterranean environment consisting of interconnected experiments in evolution. Here, we present morphological, behav- systems of air-filled voids of varying sizes up to lava tube caves that ioral, genetic, and geologic data from a young subterranean insect can extend up to several dozen kilometers. These subterranean lineage in lava tube caves on Hawai‘i Island. The Oliarus polyphe- voids host diverse root communities (12–14), with food webs mus species complex has the potential to become a model for largely sustained by living roots of the pioneer plant Metrosideros studying rapid speciation by stochastic events. All species in this polymorpha (Myrtaceae) (SI Appendix,Fig.S1). Roots are ephem- lineage live in extremely similar environments but show strong eral resources, because their abundance decreases with increasing differentiation in behavioral and morphometric characters, which cave age through ecological succession on the surface (SI Appendix, are random with respect to cave age and geographic distribution. Text S2). Our observation that phenotypic variability within populations Species of Oliarus are the only obligatory cave-dwelling pri- decreases with increasing cave age challenges traditional views mary consumers in this ecosystem (15). The Hawaiian Oliarus on founder effects. Furthermore, these cave populations are natu- (Nesoliarus) clade is a monophyletic endemic radiation (16) ral replicates that can be used to test the contradictory hypotheses. comprising about 85 known species. The latter include seven Moreover, Hawaiian cave planthoppers exhibit one of the highest exclusively cave-dwelling species that have been described to EVOLUTION speciation rates among animals and, thus, radically shift our per- date from three islands (17). Three cave taxa are endemic to ception on the evolutionary potential of obligate cavernicoles. Hawai‘i Island, but only O. polyphemus is widely distributed, inhabiting lava tubes on all major volcanoes except Kohala. The density-dependent selection | dynamic adaptive landscape | nonadaptive inhabited caves occur from sea level to about 2,000 m and range speciation | sexual behavior | vibrational communication in age from less than 50 to several thousand years (18) (Fig. 1 and SI Appendix,Fig.S3). O. polyphemus exhibits extreme character he role of extrinsic factors such as environmental changes in reduction (eyes, wings, pigmentation) associated with its trog- Tdriving genetic change in populations is largely undisputed. lobitic habit (SI Appendix,Fig.S2). Genetic drift (i.e., random changes in gene frequency attributable Given the extreme degree of troglomorphy in O. polyphemus, to stochastic allele assortment) also affects all populations, but its dispersal is only possible by subterranean migration, which is con- fi role in yielding signi cant differences between populations in strained by the patchy distribution of resources, both at the intra- interplay with selective forces is controversially discussed. Ge- lava tube (root patches within lava tubes) and interlava tube (and netic drift is most effective in small populations, the best-known lava flow) levels. A certain level of migration is necessary to examples being drastic population bottlenecks or founding indi- maintain populations despite the risk of extinction through eco- viduals (e.g., during the colonization of islands). These observa- logical succession or catastrophic events (see SI Appendix, Text S2 tions led to the development of the much-debated founder-effect for a discussion of modes of subterranean migration). The high concept by Mayr (1). This concept was further developed by in- level of phenotypic and genetic differentiation found between cluding population structure and sexual selection (2, 3) but has – geographically proximate, young caves suggests largely isolated remained contentious (4 6) (for reviews, see refs. 7 and 8). Here, populations and low migration rate through rare and accidental we revisit the founder-effect concepts using the blind plan- Oliarus polyphemus dispersal. This essentially implies a series of many founder events in thopper in the Hawaiian lava tube caves as the establishment of new populations (SI Appendix,Fig.S8and a model system. The Hawaiian cave planthopper system provides Text S2). If the migration rate is low, each new cave population excellent opportunities to test models of stochastic effects in would be descended from a single or few founding events from evolution in a natural setting, because it is simple enough for fl neighboring older established populations. Because even young distinguishing the in uence of many of the major factors involved caves have established populations (SI Appendix,TableS5), the and encompasses natural populations undergoing repeated “ ” age of the founding event probably approximates the age of the events (replicates of natural experiments ) under similar con- lava flow. The minimum speed of dispersal has been estimated ditions where many of the relevant biotic and abiotic factors can at >10 m/y (SI Appendix, Text S2), which is compatible with the be assessed. Grounds and Habits of the Hawaiian Cave Planthoppers Author contributions: A.W., H.H., and F.G.H. designed research; A.W., H.H., M.A., B.S., The Hawaiian archipelago, the most remote group of high islands F.D.S., and F.G.H. performed research; V.H. contributed new reagents/analytic tools; A.W., in the world, hosts a highly diverse endemic fauna. Much of this T.v.R., B.S., and V.H. analyzed data; and A.W. wrote the paper. diversity is the result of radiations following rare colonization The authors declare no conflict of interest. events on the islands (9). The Hawaiian chain was formed by This article is a PNAS Direct Submission. “ ” outpourings of lava from a volcanic hot spot (10), and volcanoes Data deposition: The data reported in this paper have been deposited in the European are still active on its youngest and largest island, Hawai‘i. This Nucleotide Archive (ENA), www.ebi.ac.uk/ena (accession nos. HF674815–HF674838). active volcanism causes rapid landscape dynamics [e.g., 90% of the 1To whom correspondence should be addressed. E-mail: [email protected]. entire surface area of Kllauea Volcano has been replaced within This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the past 1,500 y (11)]. The nearly continuous flow of lava creates 1073/pnas.1301657110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1301657110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 A sp.1 Results fi sp. 2 Phenotypic Differentiation. Signi cant intercave differences in call patterns were observed in all 10 parameters measured for 12 populations (Fig. 1C and SI Appendix, Fig. S5 and Table S7). The significant differentiation found even between caves in close proximity indicates an interruption of gene flow between these caves. In “Pink Pistillaria,” two completely different call patterns were found (SI Appendix, Fig. S6), which, along with morphometric differences, indicate the coexistence of two sympatric species in the oldest (5,000–8,000 y) cave system known to har- bor cave planthoppers (SI Appendix, Table S5). Moreover, in one of the two Pink Pistillaria populations and in the “Kaumana” population, variation of some call parameters did not overlap with these parameters in all other populations studied (SI Ap- Mesonotum width pendix, Fig. S5). The population from “McKenzie Park” even Wing width Wing length showed a unique song structure with a regularly alternating duet, Length of rear tibia Relative wing length again suggesting the existence of a distinct species. Remarkably, McKenzie Park and Kaumana are among the youngest caves [i.e., formed after 1790 (SI Appendix, Fig. S3 and Table S5)]. B Similarly, significant morphometric differentiation between 18 cave populations was found in all 14 parameters measured, albeit to a lesser degree, because no gaps in the ranges of character variation were observed (SI Appendix, Table S7). A discriminant analysis including both song and morphometric parameters variance revealed a 100% assignment for 15 populations in at least one sex for at least one character complex (SI Appendix, Table S8). Historical time (after 1790) The differences between populations do not follow a pattern of 200-750 B.P. clinal variation in either call patterns or morphology (Fig. 1A). In 012345 750-1,500 B.P. addition, no correlation between the degree of morphological cave age [ka] 5,000-10,000 B.P. differentiation and cave age was found. Interestingly, the only C correlation found is a negative one of phenotypic character variability to cave age; this correlation is significant or highly sp.1 sp. 2 significant for 9 of 14 morphological characters (Fig. 1B and SI Appendix, Table S11). None of these morphologic or ethologic differences appears to be adaptations to differences in habitat.

Genetic Differentiation. An mtDNA-based molecular phylogeny supports the monophyly [i.e., a single initial cave colonization by the ancestor of O. polyphemus (Fig. 2D and SI Appendix, Fig. S7)]. The basal splits involve populations from the western and southernmost caves Pink Pistillaria, “Lanikai,” and “Calabash” and are consistent with the assumption of dispersal from the Fig. 1. Phenotypic variation in the O. polyphemus complex. (A) Morpho- fl metric variation in five characters (population mean, relative scale) plotted older Hualalai Volcano to the southern ows of Mauna Loa. All against geography (compare C). Columns shown for mesonotum width only; other populations in the southeastern part of the island, in- for all other characters, only the resulting 2D projections are shown. (B) cluding those on Kllauea, the youngest volcano, form a mono- Scatterplot of variance in seven morphometric characters (denoted by phyletic group. Although the branching sequence within this symbols) against cave age. (C) Call-pattern variation (schematic represen- clade cannot be resolved, some populations within this group are tation is proportional to mean call length and pulse number) mapped on genetically quite distinct (Fig. 2C and SI Appendix, Table S13). ‘ topography of Hawai i Island. Colors indicate cave age (see the legend in The largest genetic distance (p-distance) between two haplotypes the figure). in the O. polyphemus complex is 4.7%. In contrast to the acoustic and morphological data, genetic distance does correlate with C assumption of underground dispersal across the study area geography (Fig. 2 ) but, again, not with cave age. “ ” within the last 10,000 y. The application of standard, frequently applied substitution Planthoppers worldwide use low-frequency substrate-borne rates for cytochrome c oxidase subunit I (COI) derived from vibration for communication (19–21). The signals are crucial for other insects (2.3% per Ma) (26) and cave arthropods (4.6% per Ma) (27) to the O. polyphemus complex yields divergence time species-specific recognition (22) and have led to the discovery “ ” estimates based on molecular clock analyses for the basal of cryptic acoustic species, which are morphologically indistin- A SI O. polyphemus intracave split ranging from 0.64 to 1.29 Ma (Fig. 2 and guishable (23, 24). In the case of , vibrations are Appendix, Figs. S9 and S10). These dates seem reasonable if transmitted along the roots on which the blind animals feed and are recent estimates for the age of Hawai‘i Island of ca. 1Maare their only means of communication. Differences in male and female accepted prima facie (11) (SI Appendix, Fig. S11). However, it is courtship calls of O. polyphemus from lava tubes on three different not very likely that the O. polyphemus complex is as old as the volcanoes indicate the existence of cryptic species (18, 25). basal rocks of Hawai‘i Island. The extremely complex geologic Here, we use morphological, acoustic, genetic, and geologic history of the island, especially the catastrophic collapses of data to investigate patterns of differentiation in populations of large segments of each volcano in turn, suggests that the current O. polyphemus from 20 caves of varying age (SI Appendix, Text cave Oliarus populations must be much younger than the max- S1, Fig. S3, and Tables S1 and S2). imum age of the island and each volcano. Divergence time

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1301657110 Wessel et al. Downloaded by guest on September 25, 2021 A Discussion Rapid Speciation. Irrespective of the factors leading to the ap- parently high substitution rate in O. polyphemus, some of the fastest speciation processes found to date occur in this clade of subterranean planthoppers. The highest speciation “rate” reported for invertebrates to date is 4.17 species per million years (Sp/ Ma) for the Laupala cricket species of Hawai‘i Island, assuming six species and an island age of 430 ka (30). This is more than an order of magnitude higher than the one usually assumed for arthropods and only surpassed by that of African cichlids (6) or, potentially, Hawaiian drosophilids (31). However, the island age has been B revised to ca. 1 Ma (11). Assuming that the O. polyphemus clade comprises seven species (see SI Appendix, Text S5 for a discussion of species numbers), a slightly higher rate of speciation as for Laupala is obtained under any estimate of island age. The assumption of clade age corresponding to island age is the most conservative estimate possible. If the transition to postshield volcanics of the Hualalai (11, 32) is used as a somewhat more realistic estimate of the maximum age of the clade, a roughly 10-fold higher rate emerges. Assuming, in addition, that dispersal and cave colonization originated from the Hualalai, and using the three species C of the Mauna Loa/Kllauea clade and a maximum age of 10 ka, the resulting rate estimate is again 10 times higher. This estimate, which exceeds the highest yet-recorded speciation rate in any taxon—75.6 Sp/Ma in Lake Victoria cichlids (33)—seems not overly extreme given the speciﬁc conditions of the Hawaiian

cave system. EVOLUTION O. polyphemus D Founder Effects and Adaptively Neutral Change. The species complex has a very fast substitution rate and possibly the highest diversification rate in animals. The responsible mecha- nisms are still largely unknown, but our data suggest that stochastic events, along with changing selective forces, may play an important role in this system. The lack of any correlation of phenotypic evolution with distance and habitat differences precludes a major role of geography and adaptation in diversification. In addition, the predominant role of random individual dispersal (i.e., where single or very few individuals found new populations) results in parallel and frequent sequential genetic bottlenecks. These naturally occurring “replicates” can be used to test the contradictory Fig. 2. Genetic variation in the O. polyphemus complex. (A) Bayesian In- hypotheses of the different founder-effect concepts. ference (BEAST) phylogram; the three scales represent the three different The observed genetic changes associated with founder events rates used. Red triangles indicate the origin of Hawai‘i Island ca. 1 Ma for led to the original founder-effect concept, which refers to the each rate. The two unlabeled bottom lineages have been used as the out- changes brought about by genetic drift on the reduced and arbi- group (see Material and Methods for details). The red dashed bar indicates trary sample of a population’s total genetic variation carried by the the 95% confidence interval for the age of the O. polyphemus complex. (B) few individuals that establish a new population. In its classical Distribution of cavernicolous Oliarus species on Hawai‘i Island. Unlabeled formulation by Mayr (1), these changes principally involve a de- fl ows are inhabited by O. polyphemus s.str. (C) Haplotype network. Circle crease in genetic variation and the increased fixation of rare and size is relative to number of sequences per haplotype. (D) Bayesian Inference (MrBayes) phylogram. Three-letter codes in C and D are cave abbreviations common alleles. Subsequently, selection in the altered genetic (SI Appendix, Table S6). environment causes the breakup and reorganization of coadapted gene complexes, leading to a hypothesized “genetic revolution.” Mayr’s founder-effect concept has been criticized for requiring estimates gained from a much faster COI rate of 17.2% per Ma unrealistic conditions to work (4), such as an exceedingly small (28) would, for example, be compatible with assuming that the number of founding individuals surviving for several generations. maximum age of the species complex is constrained by the Moreover, it is barely possible to distinguish the impact of pop- transition from shield to post shield volcanics of Hualalai ca. ulation bottlenecks in small founder populations from the effects 130–100 ka ago (11) (Fig. 2A and SI Appendix, Fig. S12). The of isolation, environmental differences, and genetic drift in mod- volcanic dynamics of the postshield phase might even constrain erately sized populations. the maximum age further to 25 ka (see SI Appendix, Text S3 and In recognition of these problems, Carson (2, 5, 34) developed Text S4 for additional information). If the dispersal of O. poly- the “founder-flush” concept that proposes a mechanism for the phemus indeed involved a series of thousands or tens of thou- successful shift of the balanced genotype in founder populations. sands of founder events, this would lead to the amplified Under Carson’s model, a strong increase in variability occurs accumulation of mutations in a neutral marker and might pro- during the process of rapid population growth during which the vide a plausible explanation for the high genetic distances ob- founding population expands to occupy the new habitat (the served. This interpretation is corroborated by comparative founder flush) after a founder event and the associated bottle- studies on Hawaiian Drosophila lineages experiencing founder neck. This increase in variation is the result of relaxed selection events (29). resulting from reduced intraspecific competition for resources

Wessel et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 and, especially, of relaxed sexual selection on both sexes resulting A from low availability of mating partners (see also ref. 35). With further population growth and accompanying increase in population density, strong selection is resumed, and the population

collapses. The envisioned population collapse may not be a de- Fitness crease in the census population size but, rather, a collapse of the effective population size after strong sexual selection has resumed Y and random mating is replaced once more by assortative mating X (see SI Appendix, Fig. S13, time b → d). The breakup of some B Source population Founder population coadapted gene complexes in the founder-flush phase and random processes can cause the state of some characters in the now- fi stabilized population to differ signi cantly from those of the stage 1 parental population (5). Carson’s founder-flush concept was subsequently complemented with Templeton’s genetic-transilience theory, which also invokes an increase of selectable genetic vari- Fitness Fitness ation after a founder event (3, 8). Carson’s and Templeton’scon- cepts are compatible and even synergistic with one another, but YY they are both incompatible with Mayr’s genetic revolution theory XX (5, 8). Repeated attempts to test Carson’s and Templeton’s concepts in the laboratory yielded ambiguous results (36–38), although stage 2 a statistical reanalysis of the individual experimental results found strong support for the predictions made by this founder fi fi concept (8). However, these ndings are dif cult to interpret Fitness Fitness with respect to the role of founder effects in evolution because YY this can only be tested using a natural population with an un- XX disturbed mating system. O. polyphemus offers just these conditions, and the system is simple enough to distinguish between the influence of most factors involved. The populations of O. polyphemus essentially constitute naturally occurring replicates stage 3 of an evolutionary experiment at different time stages, permit- ting a space-for-time approach to test the predictions of the

founder-effect models of Mayr (1) and Carson and Templeton Fitness Fitness (2, 5, 34), which differ in a crucial aspect. Mayr predicts that the YY initial founder event is followed by an immediate further de- XX crease in genetic variability from which the population will only recover slowly. In contrast, Carson proposed that a founder Fig. 3. Peak move in a fitness landscape. Schematic representation of the fl Wrightian peak shift problem (A) and the envisioned Carsonian peak move event is immediately followed by the founder ush, leading to an fi fi instant rise in variability. The variance of morphological char- in a tness landscape (B) (see Discussion for further explanations). The tness landscape is a diagrammatic representation of the field of gene combina- acters, which is used here as a proxy of genetic variability, is tions in two dimensions (axes x and y represent allele frequencies), graded highest in the youngest caves and decreases with increasing age with respect to adaptive value under a particular set of conditions (60). of the caves and thus presumably the age of the populations of the O. polyphemus complex (Fig. 1B). This pattern would be expected under the founder-flush model but does not fit the perspective or is defined by population structure, respectively. prediction of Mayr. Furthermore, it corroborates the counter- There is no “objectively” recognizable landscape that is indepen- intuitive prediction of Carson’s and Templeton’s theories that dent from the population nor does a landscape for a species or an founder events can actually increase the additive phenotypic individual exist. Thus, the adaptive landscape can change even in fi variance when dealing with epistatic systems, as con rmed by a stable environment if the population structure changes (bottle- diverse experimental systems (e.g., ref. 39; for review, see ref. 8). neck, founder flush), so the landscape is in continuous flux through “ However, as succinctly pointed out by Mayr, the real problem extrinsic and intrinsic (population) factors. of speciation is not how to produce difference but rather to es- A founder event would accordingly involve the establishment cape from the cohesion of the gene complex” (ref. 40, p. 518). B ’ of a new landscape (Fig. 3 , stage 1). The relaxation of sexual According to Carson s model (5), coadapted gene complexes can selection during the flush phase allows the temporary existence of be broken up in the flush phase, leading to new combinations. a fitness plateau instead of a fitness peak for the characters in- This model can be translated into Sewall Wright’s heuristic an- volved in sexual selection; in other words, the plateau is the local alytical tool of fitness landscapes (41–43) to integrate concepts of fitness maximization and adaptive change. At the core of this relaxation in attractors establishing a section in the multidimen- sional fitness space, which can be freely occupied by the pop- approach is the interpretation of speciation as a shift of a pop- fl B ulation from one adaptive peak to the next. Assuming a static ulation during the ush phase (Fig. 3 , stage 2). When sexual landscape surface this would pose the classical problem for the selection sets in again, accompanied by a breakdown of effective founder-flush models, how and why the founders leave an population size (i.e., effective breeding size), the plateau collap- → adaptive peak and cross the adaptive valley (Fig. 3A). Some reviews ses into a peak (stage 2 3), which will, with some probability, even unjustifiably subsumed the concepts of Mayr, Carson, and occupy a different place from the one in the source population Templeton under the term “peak shift models” (see, for example, (Fig. 3B, stage 3). This can be regarded as a “peak move” rather ref. 6), ignoring differing conceptual approaches to the problem than as a peak shift. The movements of the population on the that led us to a new thinking about fitness landscapes. plateau are almost entirely random, and this generates an un- In sexually reproducing species under sexual selection, the directed peak movement in a series of founder events. This would structure of the landscape can only be estimated from a population explain why there is no observable cline or trend in the changes

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1301657110 Wessel et al. Downloaded by guest on September 25, 2021 of the O. polyphemus populations (Fig. 1). Similarities between three pulses of the call, the parameters 18–24 were included in the analysis as populations can also be attributable to random peak movements parameters 25–31 to assess intraindividual variability (SI Appendix, Table S4 (i.e., may have originated in parallel). and ref. 25). Our observation that phenotypic variability decreases with in- fl Statistical Analyses. Statistical analyses were performed with SPSS Version creasing cave age is consistent with the founder- ush model (2, 5) fi but challenges traditional views on founder effects (1). The in- 17.0 for Windows. A suf cient set of measured parameters for statistical fl “ ” analyses was available for 18 (morphometry) and 12 (acoustics) populations, terpretation of the founder- ush concept as a peak-move model respectively (SI Appendix, Tables S1 and S2). Discriminant analyses were in the framework of the adaptive landscape (Fig. 3) allows an conducted using all measured morphological and acoustic parameters for 18 “ ” sensu understanding of adaptively neutral [i.e., nonadaptive (2)] populations (SI Appendix, Tables S8–S10). Confidence for correlation be- evolutionary change. The O. polyphemus species complex now tween character variability and population age was tested using Kendall’s offers an opportunity to develop a compelling quantitative con- τ (47) and correlation strength using Spearman’s ρ (48, 49). The statistical cept further toward numerical simulations using data from natural treatment of the DNA sequence data are described below. populations to model the interplay of stochastic effects and density-dependent selection in evolution. Molecular Genetics and Sequence Analyses. Specimens from 18 caves were used for the genetic analyses (SI Appendix, Table S1). DNA was purified from whole Methods nymphs (fourth- and fifth-instar larvae) with a Qiagen DNeasy Tissue kits. A fi Cave Exploration, Sampling, Mapping, and Age Determination. Since the dis- 658-bp COI fragment was ampli ed and sequenced (primers LCO1490 and μ covery of the cave ecosystems in July 1971 (12)—in the course of the In- HCO2198) (50). PCR was performed in 25- L volumes containing double-dis- × μ – ternational Biological Program, Hawaii Subprogramme—the caves were tilled H2O, 1 Taq buffer, 1.5 mM MgCl2, 200 M each dNTP, 1 2.5 U of Taq systematically explored through the Hawaii Biological Survey (14). To protect polymerase, and ca. 100 nM DNA, with an initial denaturation step of 3 min the sensitive ecosystems and cultural sites, the cave entrances have not been at 94 °C; cycling conditions of 35 cycles of 1 min each at 94 °C, 40–45 °C, and officially mapped, and the respective data are treated as confidential. Two 72 °C; and a final elongation step of 5 min. PCR products were purified with caves are open to the public as designated tourist caves and can be found on QiaQuick PCR purification kits and cycle-sequenced with Big Dye Terminator maps: Thurston Lava Tube and Kaumana Cave. O. polyphemus occurs in Chemistry Version 1.1 (Applied Biosystems). Sequences were assembled and both. Overall, populations of O. polyphemus are known from more than 30 corrected using CodonCode Aligner Version 3.7.1. Sequences have been de- caves, of which 21 were included in this study (SI Appendix, Tables S1 and posited in the ENA (see SI Appendix, Table S12 for accession numbers). Sub- S5). The position of the cave entrances was determined with a global posi- stitution model was estimated with jModeltest Version 0.1.1 (24 models; tioning system (GPS) device (Garmin GPS 12XL; 12 channels). The Digital Akaike information criteria: GTR+G) (51). Haplotypes were identified using Terrain Model (DTM) (SI Appendix, Fig. S4) was calculated from data pro- DAMBE (software package for extensive Data Analysis in Molecular Biology EVOLUTION vided by the US Geological Survey (USGS): http://hawaii.wr.usgs.gov/oahu/ and Evolution) Version 5.1.1 (52), uncorrected genetic p-distances were data.html. All data were calculated in the North American Datum of 1983 calculated using MEGA (Molecular Evolutionary Genetics Analysis) Version (NAD83), universal transverse Mercator grid zone 4 coordinate system. The 4.1 (53). compressed Shape file providing an equidistance of 100 feet was processed using LISA, to calculate DTM from contour lines (see www.lisa-geosoftware. Phylogenetic Analyses. Phylogenetic analyses were performed using maxi- de/prod_1e.htm for more information). The fundamental 2D and 3D terrain mum parsimony (MP) as implemented in PAUP* Version 4.0b010 for Win- fl models were overlaid with the lava ows mapped by USGS. In all cases, it dows [heuristic search with 10 random addition cycles, tree bisection and fl was possible to assign cave positions unambiguously to a dated lava ow reconnection (TBR)] (54); maximum likelihood (ML) using TREEFINDER (see Figs. 1 and 2 and SI Appendix, Fig. S3). The course of the ca. 60-km-long Version June 2008 (search depth: 2; bootstrap replicates: 1,000) (55); and Kazumura master tube was mapped using data from (44), and the course Bayesian inference (BI) using MrBayes Version 3.1.2 (ngen: 1,000,000; ’ of Carson s Cave could be charted for about 3.7 km using measurements samplefreq: 20; burnin: 35,001) (56). Trees were rooted using two epigean by A.W. species of Oliarus from Hawai‘i Island. A minimum spanning COI network was generated using TCS Version 1.21 (57) with a connection limit (parsi- Morphology (Morphometry). For the morphometric analyses, individuals from mony criterion) of 96%. 22 populations were studied (SI Appendix, Table S1). Nine parameters (SI Appendix, Table S3) were measured using a measuring ocular with an Molecular Clock Analysis. Relaxed lognormal molecular clock analyses were Olympus SZH 10 at 50-fold magnification (accuracy ± 10 μm). In the analyses, performed using BEAST (Bayesian Evolutionary Analysis Sampling Trees) these measurements were complemented by five indices computed from Version 1.6.2 (HKY+I; Yule/birth–death process; ngen: 10,000,000; log: 200; seven of the measured parameters (see also ref. 25). burnin: 35,001) (58); clock rates: 2.3%/Ma [mitochondrial insect rate (26)]; 4.6%/Ma [cave amphipods; COI and cytochrome c oxidase subunit II (COII) Acoustic Recording and Analyses. For the sound recordings, adults and fifth- (27)]; 17.2%/Ma [Coleoptera; COI (28)], and in-group calibration [1.0 ± instar nymphs from 15 caves (SI Appendix, Table S2) were taken to the 0.0001 Ma (11), resulting in a mean rate of 3.19%/Ma]. Bayes factor analysis laboratory and kept under controlled conditions closely resembling those of was conducted in Tracer Version 1.5 (1,000 bootstrap replicates) (59) to test their natural habitat (complete darkness; constant temperature, about 18 °C). support for a Yules vs. birth–death process. Bayes factor analyses resulted in Adults were kept individually on roots of Metrosideros or fresh sprouts low positive values and, thus, slightly favored a birth–death process (Bayes of soybeans, as a substitute; the nymphs were separated following final factors: 2.3%/Ma, 0.542; 4.6%/Ma, 0.343; 17.2%/Ma, 0.514; calibration, molting (for details, see refs. 45 and 18). 0.444; however, node ages were almost identical). For recording of vibrational signals, a male and female (if available) from the same population were placed together onto the substrate. The natural substrate (living Metrosideros roots) was substituted by fresh Metrosideros ACKNOWLEDGMENTS. We thank all of the individuals who have supported the study on O. polyphemus in Hawai‘i; the staff of Hawai‘i Volcanoes Na- leaves. Light exposition of the recording area was considerably reduced; tional Park, US Geological Survey, and Department of Land and Natural however, enough light was retained to observe the animals and record their Resources for the permits under which these studies were conducted; and behavior. The vibrational signals were received with a magnetodynamic all private landowners who generously granted access to cave entrances on induction converter system [“MD-system” sensu (46)] and amplified ∼1,000 their properties, regardless of the risks involved. We also thank numerous times in the process. The signals were recorded with a Sony TCD-D8 digital colleagues from different disciplines, especially the late naturalists Hampton audio tape (DAT) recorder (on TDK DA-RXG DAT tapes; sampling rate, L. Carson, Ernst Mayr, and Günter Tembrock for inspiring discussions, and Kari 48 kHz). Roesch Goodman for helpful comments on the manuscript. We thank two For time-pattern measurements, signals were digitized using Mac Lab/4s anonymous reviewers for their constructive comments, which helped us to improve the manuscript. This study was supported by the Graduate School (ADInstruments) running on a Power Macintosh 7600/132 (Apple) with a “Evolutionary Transformations and Extinction Events” [Deutsche Forschungs- sampling rate of 44 kHz. Measurements were taken using Chart Version gemeinschaft (DFG) Research Training Group 503 scholarship to A.W.], DFG ± 3.5.4/s with an accuracy of 0.15 ms. Ten time-pattern parameters of single Grants HO 1004/3-1 and HO 1004/7-1 (to H.H.), a grant from the Hawaii calls (composed of more or less homogenous pulse trains; SI Appendix, Bishop Research Institute (to. F.G.H.), and National Science Foundation Grants Fig. S6) were taken (SI Appendix, Table S4). After omission of the first and last GB 23075, GB 75-23106, DEB 79-04760, and BSR 85-15183 (to F.G.H.).

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