Molecular Phylogenetics and Evolution 91 (2015) 56–67
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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Multilocus sequence data reveal dozens of putative cryptic species in a radiation of endemic Californian mygalomorph spiders (Araneae, Mygalomorphae, Nemesiidae) q ⇑ Dean H. Leavitt a, , James Starrett a, Michael F. Westphal b, Marshal Hedin a a Department of Biology, San Diego State University, San Diego, CA 92182, United States b Bureau of Land Management, Hollister Field Office, Hollister, CA 95023, United States article info abstract
Article history: We use mitochondrial and multi-locus nuclear DNA sequence data to infer both species boundaries and Received 6 March 2015 species relationships within California nemesiid spiders. Higher-level phylogenetic data show that the Revised 11 May 2015 California radiation is monophyletic and distantly related to European members of the genus Accepted 19 May 2015 Brachythele. As such, we consider all California nemesiid taxa to belong to the genus Calisoga Available online 27 May 2015 Chamberlin, 1937. Rather than find support for one or two taxa as previously hypothesized, genetic data reveal Calisoga to be a species-rich radiation of spiders, including perhaps dozens of species. This conclu- Keywords: sion is supported by multiple mitochondrial barcoding analyses, and also independent analyses of DNA barcoding nuclear data that reveal general genealogical congruence. We discovered three instances of sympatry, Cryptic species Genealogical concordance and genetic data indicate reproductive isolation when in sympatry. An examination of female reproduc- Species tree tive morphology does not reveal species-specific characters, and observed male morphological differ- Sympatry ences for a subset of putative species are subtle. Our coalescent species tree analysis of putative species lays the groundwork for future research on the taxonomy and biogeographic history of this remarkable endemic radiation. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction recovered support for some previous morphology-based hypothe- ses, but have also demonstrated the limitations of morphological Mygalomorph spiders, the radiation that includes tarantulas data and revealed some surprising relationships (Hedin and and trapdoors spiders, are ancient and diverse. Because of general Bond, 2006; Bond et al., 2012; Starrett et al., 2013; Opatova dispersal limitation and high levels of regional endemism, several et al., 2013; Opatova and Arnedo, 2014a). research groups from different continents have developed mygalo- In addition to improving our understanding of deeper phyloge- morphs as models for studies of species delimitation, cryptic spe- netic relationships, DNA sequence data have played a crucial role ciation, and historical biogeography (e.g., Bond and Stockman, in uncovering and recognizing species-level diversity in mygalo- 2008; Satler et al., 2013; Hedin et al., 2013; Hendrixson et al., morphs. Recent genetic studies have revealed ancient but morpho- 2013; Castalanelli et al., 2014; Hamilton et al., 2014; Opatova logically cryptic species diversity in various mygalomorph groups and Arnedo, 2014a). Still, many features of mygalomorph biology (Starrett and Hedin, 2007; Stockman and Bond, 2007; Hedin and make them challenging to study. These spiders are largely subter- Carlson, 2011; Graham et al., 2015). Thus far, most genetic studies ranean and spend much of their lives out of sight; also, much of have relied principally on one or two fragments of mitochondrial their diversity is obfuscated by conserved morphology across both DNA. Theory has long emphasized the shortcomings of relying on species and higher taxonomic groups. Raven (1985) used morphol- a single marker (Maddison, 1997), and empirical data increasingly ogy to revise taxonomy and contributed greatly to our understand- demonstrate that inferences based on mtDNA alone can be mis- ing of higher-level mygalomorph phylogenetic relationships. leading (e.g., McGuire et al., 2007). For example, the mitochondrial Recent phylogenetic studies using DNA sequence data have genome is particularly prone to asymmetric introgression across species boundaries (Schwenk et al., 2008), leading to both misun- derstanding species boundaries and gene tree/species tree incon- q This paper has been recommended for acceptance by Marcos Perez-Losada. gruence if mtDNA is the sole locus employed (e.g., Harrington ⇑ Corresponding author. and Near, 2012). The need for accompanying nuclear markers is E-mail address: [email protected] (D.H. Leavitt). http://dx.doi.org/10.1016/j.ympev.2015.05.016 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67 57 generally well appreciated, and while nuclear DNA data have the known distribution both to the north and the southwest. We become standard in many taxonomic groups, thus far only a few attempted to collect 3–5 individuals per locality, although some recent studies of mygalomorphs have included multiple nuclear localities yielded fewer individuals. In total, this study includes loci (e.g., Satler et al., 2013; Hedin et al., 2013; Opatova and genetic data for 197 California nemesiid specimens from 106 dis- Arnedo, 2014b). tinct localities (Fig. 1). Whole spiders were vouchered and depos- The mygalomorph family Nemesiidae has a global distribution, ited in the San Diego State University Terrestrial Arthropods with particularly high generic and species diversity in Australia Collection, and typically one leg was cryopreserved for subsequent and South America (Raven, 1985; Goloboff, 1995; World Spider genetic work. DNA was extracted from ethanol-preserved tissue Catalog, 2015). While recent phylogenetic data reveal uncertainty using the Qiagen DNeasy kit. in the composition of the family (Bond et al., 2012), North American nemesiid diversity is known to be depauperate relative 2.2. Molecular protocols to other continental regions. Mexico is home to Mexentypesa chia- pas (Raven, 1987) and at least one undescribed species (Hedin and For the higher-level phylogenetic analysis of Nemesiidae, we Bond, 2006). Other North American representatives include five downloaded published data from Genbank for 28S and elongation nominal species from California: Brachythele longitarsis Simon factor 1 c (EF-1c), nuclear loci that are widely used in phylogenetic 1891, B. anomala Schenkel 1950, Calisoga theveneti Simon 1891, analyses of mygalomorphs (Hedin and Bond, 2006; Ayoub et al., Calisoga sacra Chamberlin 1937 and Calisoga centronetha 2007; Bond et al., 2012; Opatova et al., 2013). We also included Chamberlin and Ivie, 1939 (World Spider Catalog, 2015). additional data from transcriptomes and supplementary PCR reac- In his unpublished dissertation, Bentzien (1976) found tions (see Supplementary Material, which also includes 28S and California Brachythele to be morphologically distinct from EF-1c primers). All PCR products were purified with Millipore European members of that genus and assigned all California spe- Multiscreen PCR filter plates and Sanger sequenced in both direc- cies to the genus Calisoga. These spiders are robust, aggressive spi- tions by Macrogen, USA. ders that reside in subterranean burrows with ‘‘simple’’ open Using a published ‘‘Calisoga longistarsis’’ mitochondrial genome entrances, similar to certain tarantulas. Based on statistical analy- (Masta and Boore, 2008), we modified universal primers to amplify ses of morphology, Bentzien (1976) hypothesized that there are a region of the cytochrome oxidase I gene (COI) comprising over only two species of Calisoga in California: the common, widespread 1200 base pairs (bp). In addition to the primers used in amplifica- ‘‘C. longitarsis’’ and the smaller, rare C. theveneti. However, he also tion, we sometimes used internal sequencing primers considered the possibility that spiders identified as C. theveneti (Supplementary Material). The COI fragment was targeted for all are merely ‘‘C. longitarsis’’ males that mature at an earlier age, Californian samples included in the study. Additionally, an approx- and that all populations represented a single species. Despite the imately 1000 bp mitochondrial region that contained partial 12S, fact that these results were never published, many arachnologists val-tRNA and 16S regions was sequenced for a subset of have come to use the combination ‘‘Calisoga longitarsis’’ to refer Californian specimens. to all Californian nemesiids (e.g., Ubick et al., 2005). California Based on preliminary analyses of mitochondrial data and geo- nemesiids are one of the more commonly encountered mygalo- graphic distribution, we collected nuclear data for forty individu- morphs in central and northern California, particularly during the als. We amplified the internal transcribed spacer (ITS) units 1 fall when wandering males enter houses in the San Francisco Bay and 2 using published primers (Ji et al., 2003). Additional nuclear area. That the binomial most commonly applied to this taxon – loci were developed from genomic resources generated specifically ‘‘Calisoga longitarsis’’ – remains unpublished and technically for Californian samples using comparative transcriptomic data (see unavailable (World Spider Catalog, 2015) underscores the need Thomson et al., 2010) derived from next-generation sequencing. A for systematic attention. In this study we employ multi-locus transcriptome was characterized for a spider from Panoche Hills DNA sequence data to examine the phylogenetic relationship of (Fresno Co.), and compared to an already published transcriptome California nemesiids relative to Old World Brachythele and to infer from Jasper Ridge (San Mateo Co., SRX652491; Bond et al., 2014). both species boundaries and phylogenetic relationships within the Methodological details (including phasing of data) are similar to California radiation. This robust phylogenetic hypothesis provides those used in Derkarabetian and Hedin (2014) and are further out- the framework for future biogeographic studies and formal mor- lined in the Supplementary Material. From this effort we selected phological taxonomic revision. seven exonic loci that amplified consistently across a geographic test panel. We performed PCR reactions with typical reagents, and primers are available in Supplementary Material. Purified 2. Methods nuclear loci were Sanger sequenced using forward and reverse pri- mers by Macrogen USA. 2.1. Taxon sampling and data collection 2.3. Higher-level alignment and phylogenetic analyses We tested the distinctiveness of Californian nemesiids relative to the Palearctic Brachythele by performing molecular phylogenetic An initial alignment of EF-1c was performed in Sequencher and analyses of available nemesiid taxa. Using published data and manually adjusted after translating codons into amino acids. To newly sampled spiders, we compiled a data set including multiple align the ribosomal 28S fragment, we used the option Q-INS-i in outgroup taxa representing the families Cyrtaucheniidae and MAFFT v7 (Katoh et al., 2005) as it considers RNA secondary struc- Ctenizidae as well as 21 nemesiids and allied taxa ture during alignment. The default settings include a gap-opening (Microstigmatidae and ‘‘Cyrtaucheniidae’’). This sample also penalty of 1.53 and the offset value equal to 0.0. We used the pro- included multiple representatives of both European Brachythele gram GBLOCKS v0.91b (Castresana, 2000) to identify and exclude and Californian spiders currently referred to as ‘‘Calisoga longitar- regions that could not be reliably aligned. Settings for this analysis sis’’ (see Supplementary Material for specimen information). allowed gap positions within the final blocks as well as smaller To investigate species diversity within Californian nemesiids, final blocks. EF-1c and 28S data were concatenated for phyloge- we sampled spiders from throughout the known distribution in netic analyses. The program jModeltest (Posada, 2008) was used central and northern California (following Bentzien, 1976). Our to aid in selection of nucleotide models for these and subsequent sampling effort also included original collections that extended phylogenetic analyses. We performed a maximum-likelihood 58 D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67
Fig. 1. Map of central and northern California showing collecting localities included in this study. Colors and clade names correspond to ten major biogeographic clades recovered in mitochondrial gene tree analyses. Localities where divergent mitochondrial and nuclear lineages were found in sympatry are indicated in white. Black x’s designate collection locations for CAS male specimens. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) analysis using the RAxML-HPC BlackBox 8.0.24 (Stamatakis et al., partitioning scheme and nucleotide models (GTR + G), except 2008) on the CIPRES Science Gateway (Miller et al., 2011). A HKY + G models were used for the three EF-1c partitions to achieve GTRgamma model was used for each of four partitions: three for convergence in certain estimated parameters. This analysis corresponding codon positions within EF-1c and a fourth for the employed a lognormal relaxed clock for each of the two gene 28S fragment. The sufficient number of bootstrap replicates was regions and a tree prior of birth–death with incomplete sampling. automatically detected by the analysis. We consider bootstrap val- The BEAST analysis was run for 100 million generations saving ues >70 as evidence for high support (Hillis and Bull, 1993; Alfaro every 4000th. We used TRACER v.1.5 (Rambaut and Drummond, et al., 2003). For a Bayesian BEAST analysis, we used the same 2007) to plot likelihood scores in order to assess convergence, to D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67 59 identify the appropriate burn-in length, and to verify that all effec- genetically distinctive but morphologically cryptic lineages tive sample size (ESS) values were well over 200. (Satler et al., 2013). We evaluated nuclear data separately from mitochondrial evi- 2.4. Mitochondrial gene tree, species delimitation and DNA barcoding dence to provide an independent test of putative species identified from mtDNA barcoding. We performed a species tree analysis Mitochondrial COI data were translated into amino acids to ver- where each of the forty individuals with sampled nuclear data ify an open reading frame. With no insertions or deletions, the COI was treated as a distinct OTU. Identified mtDNA barcode species alignment was trivial and performed in Sequencher. For the riboso- are considered validated by the nuclear species tree analysis when mal 12S/16S data, we performed analyses with MAFFT and individuals are recovered in a clade with other members of that GBLOCKS v0.91b with the same settings as outlined above. To infer species (when multiple individuals sampled), with non-trivial the mitochondrial gene tree, we concatenated the two fragments. branch lengths separating a clade/individual from their respective Similar to higher-level phylogenetic analyses, likelihood and sister clade. Species tree inference was performed using *BEAST Bayesian analyses contained four partitions: three for the COI with BEAST 1.8. Because initial analyses using more complex GTR codon positions and a fourth for the ribosomal fragment. We per- models failed to converge, all eight phased nuclear loci were ana- formed maximum-likelihood analysis in RAxML with the model lyzed with HKY + G models (Grummer et al., 2014) and a lognormal GTRgamma for each partition and automatic bootstrapping. For relaxed clock. The ITS rate was set to 1.0 and the rates of other loci the BEAST analysis, the use of HKY + G models for each of the four were estimated relative to that of ITS. An inverse gamma shape was partitions was necessary to achieve convergence. A strict clock was used for the species population mean prior, and an exponential used for the COI fragment and a lognormal clock for the 12S frag- shape was used for the Yule birth–death prior. Analyses were run ment, with the 12S rate estimated relative to the COI rate of 1.0 for 200 million generations with every 5000th generation saved. substitution per site. We used a birth–death and incomplete sam- pling tree prior. 2.6. Combined species tree analyses We used three different approaches to DNA barcoding to iden- tify putative species limits. The first method was the traditional We performed a combined species tree analysis of the nuclear barcoding approach (Hebert et al., 2003a, 2003b) where genetic and mitochondrial data similar to the *BEAST analysis of the distances are calculated and a threshold value that designates dis- nuclear-only data. OTUs for this analysis were mtDNA barcode spe- tinct species status is assigned. The divergences among relevant cies identified at the 9.5% threshold. In addition, in three instances sister clades recovered from the BEAST mtDNA gene tree were cal- individuals from divergent clades just under this threshold (9%) culated using the uncorrected p-distance similar to recently pub- were treated as distinct taxa (species Q was divided into Q1 and lished barcoding studies of mygalomorphs (Castalanelli et al., Q2, H into clades H and I, and F into F1 and F2). Mitochondrial bar- 2014; Hamilton et al., 2014). To be able to directly compare the code species that lacked nuclear data were excluded in this analy- results of this system to the aforementioned studies, we identified sis. As with other *BEAST analyses of these data, all loci were putative species using the threshold values of both 9.5% analyzed with an HKY + G model of sequence evolution. The COI (Castalanelli et al., 2014) and 5% (Hamilton et al., 2014). The 9.5% fragment was partitioned by codon position, and a strict clock value was used to characterize species diversity of mygalomorphs was employed for this gene region alone. Other gene regions used within the Pilbara region of Australia (the majority of which lognormal relaxed clocks whose rates were estimated relative to belonged to the family Nemesiidae), and the 5% cutoff was used that of COI. Other priors were the same as the nuclear-only analy- for species discovery within the North American tarantula genus sis, with analyses run for 200 million generations with every Aphonopelma sampled primarily from the United States. 5000th generation saved. The third barcoding approach used was the Automatic Barcode Gap Discovery (ABGD) method (Puillandre et al., 2012), imple- 2.7. Morphological data mented on the online ABGD server (http://www.abi.snv.jussieu. fr/public/abgd/). This analysis uses the ordered, ranked genetic dis- The morphology of adult male pedipalps and first legs (which tances from COI sequences to identify marked shifts from low possess mating spurs) is often used as a primary character for spe- intraspecific distances to higher interspecific distances. These cies delimitation in mygalomorph spiders, including nemesiids observed transitions in genetic distances represent barcode gaps. (e.g., Goloboff, 1995; Decae and Cardoso, 2005; Decae et al., Gap detection is an iterative process that assigns sampled individ- 2014). To assess qualitative morphological divergence across uals into increasingly partitioned groups. We used the same Californian populations, adult males (N = 9) were borrowed from parameters employed in the tarantula study (Hamilton et al., the California Academy of Sciences (the same males studied by 2014) for comparative purposes, as follows: Pmin = 0.0001, Bentzien, 1976), and augmented with males (N = 9) from our Pmax = 0.200, Steps = 10, X = 1, Nb bins = 20. Hamilton et al. own collections. Also, mature spermathecal organs for all adult (2014) found that species delimitation with this approach appears females included in our molecular study were dissected and exam- to be fairly robust to varying parameter settings. ined (N = 103). Specimens were imaged using a Visionary Digital BK Plus system (http://www.visionarydigital.com), including a 2.5. Nuclear gene genealogical concordance Canon 5D digital camera, Infinity Optics Long Distance Microscope, P-51 camera controller, and FX2 lighting system. Species delimitation using a single mitochondrial marker can be Individual images were combined into a composite image using misled due to various processes, including both incomplete lineage Helicon Focus V5.3, and then edited using Adobe Photoshop CS6. sorting and introgression. Additionally, deep mitochondrial struc- ture is not always accompanied with correspondingly deep nuclear differentiation (Ogden and Thorpe, 2002), and dispersal-limited 3. Results taxa (e.g., mygalomorph spiders) in particular may show spurious mtDNA breaks (Irwin, 2002). On the other hand, genealogical con- 3.1. Higher-level phylogenetic placement cordance among multiple sampled loci can provide convincing evi- dence for genetic boundaries (Avise and Ball, 1990; Kuo and Avise, The EF-1c data included for phylogenetic analysis consists of 2005), and thus multi-locus data can validate the presence of 924 aligned base pairs. The MAFFT alignment of the 28S fragment 60 D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67 resulted in 1964 aligned bp, and the GBLOCKS analysis reduced analyses consisted of 986 bp; 519 characters were variable with this length to 1812 bp. All sampled taxa were represented by the 433 of those being parsimony informative. EF-1c fragment; three individuals lacked the 28S fragment but Likelihood and Bayesian analyses recovered similar gene tree were closely related to taxa with complete data. Of the total topologies; the Bayesian BEAST topology is depicted in Fig. 3. The 2736 aligned characters, 665 were variable and 440 were parsi- partitioned HKY + G model with the strict molecular clock as mony informative. employed in BEAST indicates that deepest corrected divergences Maximum-likelihood and Bayesian BEAST analyses recovered among sampled California specimens are 22%. The mtDNA gene similar topologies, differing only in the placement of the clade con- tree is composed of divergent, geographically cohesive clades that taining Hermacha sp. and the undescribed taxon from Ngome are allopatric or parapatric to neighboring clades with few excep- Forest, South Africa. The topology recovered from the BEAST anal- tions. We arbitrarily name ten major clades with reference to their ysis is reported in Fig. 2. The sampled Californian spiders are recov- geographical distribution (Fig. 1, Supplementary Material). These ered with high support in a clade containing representatives of two clades include: Bay Area (locations 1–29), Sacramento Valley Mediterranean genera, Iberesia and Ambylocarenum. In the RAxML (30–36), Northern Sierra (37–53), Calaveras (50, 54), Tuolumne analysis, the Ambylocarenum sp. sample from Spain is recovered (55–58), Coulterville (59–63), Central Sierra (60, 64–74), Southern as sister to Californian spiders with a high support (BS = 94); this Sierra (75–78), Monterey (1, 79–101), and Panoche (l02–106). relationship is also recovered in the Bayesian BEAST analysis but We found sympatry of divergent mitochondrial lineages at with lower support (PP = 0.66). The European Brachythele is weakly three localities (Fig. 1). Bay Area and Monterey clades were found recovered as sister to Fufius sp., and this clade is recovered as sister at locality 1 (Coalinga Road), Northern Sierra and Calaveras clades to the aforementioned Iberesia + Amblyocarenum + Californian at locality 50 (west of Sheep Ranch), and Coulterville and Central clade, also with weak support. These genera are separated by Sierra clades at locality 61 (west of Coulterville). At almost all other branch lengths that are long relative to crown Nemesiidae (Fig. 2). localities with multiple sampled individuals, COI sequences were either identical or minimally divergent. An exception is locality 45 (east of Mount Aukum), where the two sampled COI sequences 3.2. Mitochondrial gene tree and DNA barcoding were just over 6% divergent. Using the traditional barcoding approach with a 9.5% threshold, The COI matrix included data for 194 spiders that we sampled 26 putative species are identified (Fig. 3, Supplementary Material). (Supplementary Material), plus COI and ribosomal mtDNA from a Most of the major geographic clades previously referenced are published mitochondrial genome (Masta and Boore, 2008). The composed of multiple species at this cutoff, but Tuolumne, aligned length of the COI fragment was 1263 base pairs; 560 posi- Calaveras and Coulterville clades each correspond to a single spe- tions were variable, of which 515 were parsimony informative. The cies (Fig. 3). Using the 5% threshold (similar to Hamilton et al., mitochondrial ribosomal fragment was obtained for 55 spiders. 2014), the number of species identified more than doubles to 59 The mtDNA ribosomal alignment after MAFFT and GBLOCKS species (Fig. 3, Supplementary Material). Of the aforementioned
Fig. 2. Maximum clade credibility tree from the BEAST analysis of concatenated EF-1c and 28S data. Clade of sampled Nemesiidae and allied taxa is indicated. Support values at nodes represent Bayesian posterior probabilities first, followed by ML bootstrap values. D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67 61
Fig. 3. Maximum clade credibility tree from the mtDNA BEAST analysis inferred from concatenated COI and ribosomal fragments. Nodal support is shown, with Bayesian posterior probabilities first and ML bootstraps second. For simplicity, individual haplotypes have been collapsed into clades corresponding to putative species at the 5% threshold. Clades are also labeled according to recovered species at the 9.5% threshold. The sampled localities comprising each clade are listed. Major geographic clades are also labeled (see Fig. 1).
Table 1 Nuclear loci summary information.
Primer combo Annotation # Seq. Length bp # Inform. # Uninform B1_B2 Orthologous to Ixodes ISCW003568 (conserved hypothetical protein) 40 945 44 3 D1_D3 Orthologous to Ixodes ISCW012830 (60S ribosomal protein L3) 38 924 87 10 D6_D7 Orthologous to Ixodes ISCW013467 (conserved hypothetical protein) 39 876 38 9 E10_E11 Orthologous to Ixodes ISCW019432 (ccaat enhanced binding protein) 40 603 25 10 F9_F10 Orthologous to Ixodes ISCW020841 (zinc finger protein) 37 578 56 14 G9_G10 Orthologous to Ixodes ISCW024803 (60S ribosomal protein L23) 40 496 36 7 H1_H2 Orthologous to Ixodes ISCW024894 (acetylcholinesterase) 40 585 41 7 CAS18sF1 CAS5p8sB1d ITS ribosomal 34 992 69 16
Notes: # Seq. = the number of individuals sequenced, # Inform. = the number of parsimony informative sites, # Uninform = the number of variable but parsimony unin- formative sites. geographic clades, only Coulterville corresponds to a single species instances individuals belonging to a single species were recovered at this cutoff. The Automatic Barcode Gap Discovery (ABGD) together in a clade. The exception was species H, as one member of method results in a much higher number of putative species. A H was not recovered with the other two (Fig. 4). However, these minimum of 95 species are inferred with this approach spiders belong to two divergent mitochondrial clades that are near (Supplementary Material), with most of these restricted to a single the cutoff threshold (9.1%). sampled geographic location. At the three localities with sympatric divergent mitochondrial clades, a specimen from each clade was sampled for nuclear data. 3.3. Species validation using nuclear-only data In these three instances all eight nuclear loci were informative in regards to distinguishing species membership (Fig. 4). In each case Gene annotation and patterns of nuclear gene variation are not only did the combined nuclear data substantiate the mtDNA summarized in Table 1. The eight nuclear loci totaled 5999 bp with clade membership, but also each of the nuclear loci individually 393 parsimony informative sites. The nuclear-only *BEAST analysis supported the mitochondrial species membership. with forty individuals treated as OTUs recovers a topology that dis- In every case the species identified at both the 9.5% and 5% cut- plays a great deal of topological concordance with the mtDNA gene offs are recovered in the same major clades with the nuclear-only tree, both in terms of species membership as well as major clade species tree as they are with the mitochondrial gene tree. membership (Fig. 4). Using the 9.5% threshold, 23 of 26 candidate Relationships among major clades from the nuclear species tree species were sampled for nuclear data. Of those 23, ten species had and the mtDNA gene tree show a large degree of congruence, but nuclear data collected for multiple individuals. In nine out of ten there are some differences. However, in none of these cases is a 62 D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67
Fig. 4. Maximum clade credibility tree from the *BEAST analysis of nuclear data with Bayesian posterior probabilities at each node. Individuals are labeled according to putative species at the 9.5% threshold, major geographic clade and voucher number. Sympatric specimens are indicated by a square (locality 50), a circle (locality 61) and a star (locality 1). Inset is adult female from Hare Canyon Road (Monterey County). strongly supported node (0.95 PP or above) from the nuclear spe- (Supplementary Material) and an obviously smaller percentage of cies tree in conflict with a strongly supported node in the mito- putative molecular species. This sample includes the CAS speci- chondrial gene tree. mens (without DNA data), most of which could be confidently placed into larger geographic clades based on geographic location (Supplementary Material, Fig. 1). Comparison of palpal and leg I 3.4. Combined data species tree clasper morphology among individuals from primary clades indicates limited morphological divergence, with only very subtle The species tree from the combined nuclear and mitochondrial differences in spination, bulb shape, and relative length of leg data is depicted in Fig. 5. Nodes that are in common with the elements (Fig. 6). Obviously, an important task will now be targeted nuclear-only species tree either maintain high support values or geographic sampling of adult males, informed by the barcoding generally received increased support, indicating that the mito- results. Our sample of females with mature spermathecal organs chondrial gene tree has not been misled by introgression and can is comprehensive, including all primary geographic clades, and all appropriately be analyzed with the nuclear loci in a combined spe- 26 conservative barcode species (Supplementary Material). cies tree analysis. In the recovered phylogeny, the three northern- However, female variation showed no obvious trends relative to most clades (Sacramento Valley, North Sierra and Bay Area) are clade or species membership, with morphological asymmetry often recovered as sister to southerly-distributed clades. Among these apparent within individuals, and variation among individuals from southern clades, the Central Sierra is sister to and deeply divergent a single location often greater than divergence among individuals from a clade containing the Tuolumne, Coulterville, Calaveras, from different primary clades (Supplementary Material). Southern Sierra, Monterey and Panoche clades. Relationships among these clades are not well supported except for the sister relationship between the Monterey and Panoche clades, both 4. Discussion distributed west of the Central Valley. 4.1. Taxonomic validity and composition of Calisoga 3.5. Morphological variation The combined results of our higher-level phylogenetic study Our sample of adult males is geographically biased, as males are and our population-level sampling confirm that California neme- only available for five of the ten primary geographic clades siid diversity consists of a single radiation that is divergent from D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67 63
Fig. 5. Maximum clade credibility tree from the *BEAST analysis of the combined mitochondrial and nDNA data with Bayesian posterior probabilities at each node. Putative species were designated based on the 9.5% threshold, and the comprising localities are listed. Major geographic clades are also labeled. Sympatric localities are indicated by a square (locality 50), a circle (locality 61) and a star (locality 1). Scale bar represents species tree branch lengths estimated using a rate of 1 substitution/site for the COI fragment.
(and not sister to) European Brachythele and should instead be related to sampled spiders). Due to their morphological similarity placed in the distinct genus Calisoga Chamberlin 1937. This conclu- to the type, the majority of the populations sampled in this study sion was also reached by Bentzien (1976) in his unpublished dis- (including the those in the higher-level analysis) have historically sertation based on observed morphological variation. The long been considered longitarsis. branches separating European Brachythele from Calisoga indicate Calisoga anomala (Schenkel 1950) comb. n. ancient divergences. Brachythele anomala Schenkel 1950. While the five named taxa from California were originally allo- Type locality: Wald um Guerneville und Monte Rio am Russian cated to three different families, Raven (1985) transferred them all River. to Nemesiidae. He further recognized that they belonged in the Remarks: The type specimen of C. anomala is an immature ani- genus Calisoga rather than Brachythele, but he neglected to transfer mal in poor condition (Bentzien, 1976). However, the type locality Brachythele longitarsis Simon 1891 and B. anomala Schenkel 1950 is perhaps the most specific of named Calisoga: forest around (Ubick and Ledford, 2005). Thus, we recommend taxonomic Guerneville and Monte Rio along the Russian River (Sonoma changes to make the two following names available. County, CA). Calisoga longitarsis (Simon 1891) comb. n. In addition to the two taxa above, the following species names Brachythele longitarsis Simon 1891. are also available for the genus Calisoga: C. sacra Chamberlin, 1937 Type locality: ‘‘California meridionalis’’. from Sacramento, CA, the type of genus; C. theveneti (Simon, 1891) Remarks: The type of longitarsis was reported as being collected from Mariposa, CA; and C. centronetha (Chamberlin and Ivie, 1939), in ‘‘southern California’’. The type specimen is a mature male and is from Mayfield (Palo Alto), CA. Thus, five names are available for located in the M.N.H.N. in Paris. Bentzien (1976) examined the California nemesiid diversity. In his unpublished dissertation, specimen and provided measurements, a description, and partial Bentzien (1976) considered Calisoga longitarsis, C. sacra, C. cen- illustrations. In the ‘‘1890’’ paper describing a female specimen tronetha, and C. anomala as synonyms. of longitarsis (published after the 1891 description of the type), With at least 26 molecular species and five available names, Simon lists a number of other regions where longitarsis specimens taxonomy within Calisoga clearly needs further attention. The occur; some of these are clearly in error (e.g., Idaho and Texas). small body size and the geographic distribution of ‘‘species C’’ indi- While the specific geographic origin of the type of longitarsis is cur- cate that it should tentatively be considered C. theveneti. The sam- rently unknown, it can be reasonably assumed that this species is pled population that is closest to the type locality of C. sacra represented in our comprehensive sampling (or is at least closely (Sacramento) is Deer Valley Rd. and belongs to ‘‘species Q1’’. The 64 D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67
Bay_Loc21_MY1514
noSier_Loc44 _GMY187
CentSier_ Loc65_MY4603
Coulter_Loc60 _MY 4658
Fig. 6. Digital images of palpal and leg I clasper morphology from male spiders representing four primary geographic clades. Spiders are labeled with primary clade, locality and voucher number. All views prolateral, scale bars = 1 mm. Arrows point to minor differences in spination and relative leg length observed in the sample. type locality of C. anomala falls within the distribution of ‘‘species cutoff used in the Pilbara study (Castalanelli et al., 2014), we find S’’, and the type of C. centronetha falls within that of ‘‘species U’’. a similar number of molecular species per sampled spider: about Unfortunately, the type locality of C. longitarsis is currently 1 species per 7 spiders sampled (7.5 in this study, 7 [1134 spi- unknown; should its origin be determined, this older name would ders/161 lineages] in Castalanelli et al., 2014). Even within the have priority if synonymous with one of the three latter named more conspicuous genus Aphonopelma, mitochondrial barcoding taxa. identified previously unknown species diversity with perhaps nine morphologically cryptic species recovered (Hamilton et al., 2014). 4.2. Molecular species diversity Of course, the number of ‘‘real species’’ represented in this study remains unknown. How much divergence is needed to correctly Given the results of other molecular studies of California myga- identify distinct species is a looming question in the DNA barcod- lomorph spiders (e.g., Bond and Stockman, 2008; Hedin et al., ing literature (Hamilton et al., 2014) and is likely taxon specific. In 2013; Satler et al., 2013), it is not surprising that the this study system, the 9.5% threshold does not appear to result in morphology-based ‘‘two species’’ hypothesis of species diversity an overestimate of putative species, as the nuclear data support within Calisoga is an underestimate. Using the conservative 9.5% genetic distinctiveness at this cutoff. This divergence threshold D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67 65 may in fact be conservative, and in a few instances the recovered in a clade using nuclear-only data, and we evaluated nuclear-only topology led us to treat clades just under the thresh- whether the branch lengths separating different species or individ- old (9%) as distinct OTUs. uals were consistent with distinct species status. We did not per- On the other hand, the approaches of species delimitation using form statistical evaluations of branch lengths, but rather methods such as ABGD and GMYC (Pons et al., 2006), which do not considered proportional length relative to overall tree length. use an arbitrary threshold, have been shown to return unrealistic Methods have been developed to statistically test species bound- numbers of species in other mygalomorph studies (e.g., Hamilton aries using species trees. For example, Bayes factor delimitation et al., 2014). In a study of the California trapdoor spider species (BFD) (Grummer et al., 2014) evaluates likelihood scores of differ- Aliatypus thompsoni, bGMYC identifies 61–79 species at a 95% con- ent species models that either split or group individuals (or reas- fidence interval with mitochondrial data, whereas the authors pre- signs them), and a related approach has been developed with the fer a conservative three species hypothesis based on analyses of program DISSECT (Jones et al., 2015) that allows more flexibility multilocus variation (Satler et al., 2013). In the current study, in testing split or lumped arrangements. The use of DISSECT ABGD infers a minimum of 95 species from 106 sampled locations appears to have the potential to over-split species with phylogeo- – almost one species per locality. The overestimation of species graphic structure (Jones et al., 2015), however, which is a concern using these methods is due to structured genetic patterns emerg- that likely extends to methods like BFD as well. Where the cutoff ing from the viscous nature of mygalomorph populations (e.g., between interspecific structure and species boundaries lies in low Hedin et al., in press). The biology of burrow-dwelling mygalo- dispersing organisms will continue to be an interesting question morphs may be particularly prone to generating extreme mito- as relative information content increases with genomic datasets. chondrial structuring. In species not characterized by balloon The observed concordance of nuclear and mitochondrial lineage dispersal, spiderlings generally establish burrows in favorable sub- boundaries provides convincing verification that Calisoga contains strate near the maternal burrow. Female mygalomorphs usually not one or two species but likely over two dozen. Particularly com- remain in the same burrow throughout their entire lives. Males, pelling evidence that the lineages recovered in this study represent on the other hand, are known to disperse from their burrow once ‘‘good species’’ is the discovery of three instances of sympatry, each they reach reproductive maturity as they seek opportunities to involving different species pairs (Figs. 1, 4 and 5). In all three mate with receptive females. Female philopatry and male-based instances, reproductive isolation appears to be quite high with no dispersal could create situations where mitochondrial data overes- detected evidence for gene flow among the sampled nuclear loci. timate isolation and the number of distinct lineages. Reproductive isolation in sympatry is considered the most con- The boundary between intraspecific variation and interspecific vincing evidence of distinct species boundaries (Mayr, 1942), and variation appears to be better defined in Aphonopelma tarantulas has been observed in morphologically cryptic diatoms (Amato (Hamilton et al., 2014) than in Calisoga. Some tarantula clades et al., 2007), lichens (Leavitt et al., 2011), earthworms (Donnelly show patterns of shallow intraspecific mtDNA divergence, indica- et al., 2013) and lizards (Barley et al., 2013). Certainly the extent tive of either recent population expansion or a nonviscous genetic of sympatry among Calisoga species is greater than that shown structure (Hamilton et al., 2011). This includes a recent phylogeo- here since we sampled relatively few spiders per locality. graphic analysis of tarantulas from central California (Wilson et al., Combining future fine-scale geographic sampling with larger sam- 2013) where the sampled area overlaps a great deal with the area ple sizes, to discover and study additional cases of sympatry, will sampled in this study. Because of some ecological similarities to constitute one of the strongest tests of species limits in Calisoga. tarantulas, we expected Calisoga to share similar phylogeographic patterns. However, the extreme level of mitochondrial structuring 4.3. Phylogenetic relationships and taxonomy observed within Calisoga is more similar to that seen in trapdoor spiders (Satler et al., 2013; Stockman and Bond, 2007; Cooper Many of the phylogenetic relationships recovered from the et al., 2011) and turret spiders (Starrett and Hedin, 2007; Hedin combined data species tree analysis are well supported, including et al., 2013). In these systems, mtDNA clades are narrowly dis- those deepest in the tree (Fig. 5). Additional data is needed to solid- tributed, often to a single sampled locality, and genetic divergence ify relationships among lineages within the Northern Sierra clade to neighboring clades can be surprisingly deep. Identifying the as well as those among the geographically restricted clades in boundary between intraspecific phylogeographic structure versus the central Sierran region, however. As seen in the mtDNA gene species boundaries is challenging in low-dispersing, spatially vis- tree and nuclear-only species tree, the combined phylogeny shows cous organisms (Bond and Stockman, 2008; Keith and Hedin, that sympatrically occurring species are distantly related, with two 2012; Schuchert, 2014). In an example from California, mitochon- of the three instances spanning the deepest divergence within drial lineages of the slender salamander Batrachoseps attenuatus Calisoga (Fig. 5). are narrowly distributed, leading to the suggestion that this taxon Among the Australian mygalomorphs from the Pilbara, most comprises 39 extant lineages (Highton, 2014), whereas the initial sampled males were morphologically distinct at the 9.5% threshold researchers that generated the data (Martínez-Solano et al., (Castalanelli et al., 2014). The exception was the Aname mellosa 2007) consider this taxon to represent a single species with deep complex, where Castalanelli et al. recovered ten lineages at that phylogeographic structure. threshold that were not obviously morphologically distinguishable. Because species boundaries based on mitochondrial data can be To what extent are the molecularly identified species of Calisoga misled by processes like introgression, incomplete lineage sorting morphologically identifiable? Surveying the morphology of female and/or phylogeographic structure unaccompanied by nuclear dif- genitalic characters, we observed no obvious species-specific or ferentiation, the need for nuclear data is well appreciated. Given clade specific morphology, even though variation among females the biology of these spiders, the possibility for discordance between was clearly evident (Supplementary Material). An inspection of the mitochondrial genome and the rest of the genome seemed con- male morphology reveals limited morphological divergence, with siderable. But rather than reveal potential pitfalls of a only subtle differentiation at the 9.5% cutoff (Fig. 6). Much more mitochondrial-only analysis, the nuclear-only species tree analysis extensive sampling of male specimens is needed to establish provided strong validation for the mtDNA-identified species-level whether these minor differences are taxonomically useful. diversity. Our nuclear-only approach to validating mitochondrial Bentzien (1976) considered most of the Californian populations species boundaries took a qualitative approach. We tested whether to belong to a single widespread species, but he did observe mor- individuals belonging to the same mtDNA-identified species were phological variation among these populations, including differing 66 D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67 average body sizes. While our sample sizes are small, we also References observed some size variation among different populations. Additional sampling is needed to determine whether differently Alfaro, M.E., Zoller, S., Lutzoni, F., 2003. Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling sized populations specifically mentioned by Bentzien (1976) repre- and bootstrapping in assessing phylogenetic confidence. Mol. Biol. Evol. 20, sent distinct species. For example, the males he observed at Indian 255–266. Valley, Sierra Co. were among the smallest in his study. The Amato, A., Kooistra, W.H., Levialdi Ghiron, J.H., Mann, D.G., Pröschold, T., Montresor, Calisoga specimens we found from the northern Sierra region were M., 2007. Reproductive isolation among sympatric cryptic species in marine diatoms. Protist 158, 193–207. often very large, however, suggesting the Indian Valley spiders Arnedo, M.A., Ferrández, M.A., 2007. Mitochondrial markers reveal deep population may be a distinct, unsampled species. Additionally, Bentzien found subdivision in the European protected spider Macrothele calpeiana (Walckenaer, both small (<7 mm) and large mature males (>10 mm) at Hopland 1805) (Araneae, Hexathelidae). Conserv. Genet. 8, 1147–1162. Avise, J.C., Ball, R.M., 1990. Principles of genealogical concordance in species Field Station, Mendocino Co., which suggests the possibility of concepts and biological taxonomy. Oxford Surv. Evol. Biol. 7, 45–67. sympatric species. Analyses of morphology have helped shed light Ayoub, N.A., Garb, J.E., Hedin, M., Hayashi, C.Y., 2007. Utility of the nuclear protein- on the taxonomic status of the Nemesiidae genera Ambylocarenum coding gene, elongation factor-1 gamma (EF-1c) for spider systematics, emphasizing family level relationships of tarantulas and their kin (Aranaea: (Decae et al., 2014) and Iberesia (Decae and Cardoso, 2005); a Mygalomorphae). Mol. Phylogenet. Evol. 42, 394–409. molecular perspective will undoubtedly provide additional clarifi- Barley, A.J., White, J., Diesmos, A.C., Brown, R.M., 2013. The challenge of species cation by revealing genetically unique lineages in these genera as delimitation at the extremes: diversification without morphological change in Philippine sun skinks. Evolution 67, 3556–3572. they have in Calisoga. Bentzien, M.M., 1976. The Biosystematics of the Spider Genus Brachythele Ausserer (Araneidae: Dipluridae). PhD Dissertation, UC Berkeley, CA. 4.4. Conclusions Bond, J.E., Stockman, A.K., 2008. An integrative method for delimiting cohesion species: finding the population-species interface in a group of Californian trapdoor spiders with extreme genetic divergence and geographic structuring. The results of our study reveal that the California genus Calisoga Syst. Biol. 57, 628–646. is distinct from the European genus Brachythele, and that Calisoga Bond, J.E., Beamer, D.A., Lamb, T., Hedin, M., 2006. Combining genetic and geospatial represents a species rich radiation, rather than just one or two spe- analyses to infer population extinction in mygalomorph spiders endemic to the Los Angeles region. Anim. Conserv. 9, 145–157. cies. A correct understanding of species boundaries is requisite for Bond, J.E., Hendrixson, B.E., Hamilton, C.A., Hedin, M., 2012. A reconsideration of the understanding evolutionary history as well as ecological relation- classification of the spider infraorder Mygalomorphae based on three nuclear ships (e.g., Pante et al., 2015). Additionally, the genetic data pro- genes and morphology (Arachnida: Araneae). PLoS One 7, e38753. Bond, J.E., Garrison, N.L., Hamilton, C.A., Godwin, R.L., Hedin, M., Agnarsson, I., 2014. vides an important perspective in conservation planning (e.g., Phylogenomics resolves a spider backbone phylogeny and rejects a prevailing Hedin, 2015) and habitat preservation (Moritz and Faith, 1998), paradigm for orb web evolution. Curr. Biol. 24, 1765–1771. and the inclusion of endemic arthropods has proven to be of high Castalanelli, M.A., Teale, R., Rix, M.G., Kennington, W.J., Harvey, M.S., 2014. Barcoding of mygalomorph spiders (Araneae: Mygalomorphae) in the Pilbara utility (Redak, 2000; Bond et al., 2006; Arnedo and Ferrández, bioregion of Western Australia reveals a highly diverse biota. Invertebr. Syst. 28, 2007; Harvey et al., 2011). Species discovery in organisms that 375–385. are narrowly distributed is an iterative process (Hedin et al., Castresana, J., 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Phylogenet. Evol. 17, 540–552. 2013), and further geographic sampling of Calisoga is needed to Chamberlin, R.V., Ivie, W., 1939. New tarantulas from the southwestern states. Bull. test hypotheses derived from this study and to reveal additional Univ. Utah 29, 1–17. diversity. While much remains to be done regarding systematic Cooper, S.J., Harvey, M.S., Saint, K.M., Main, B.Y., 2011. Deep phylogeographic structuring of populations of the trapdoor spider Moggridgea tingle (Migidae) work in this genus, this study sets the stage for investigating the from southwestern Australia: evidence for long-term refugia within refugia. temporal and spatial aspects of diversification of Calisoga in the Mol. Ecol. 20, 3219–3236. geologically dynamic region of central and northern California. Decae, A., Cardoso, P., 2005. Iberesia, a new genus of trapdoor spiders (Araneae, Other recent biogeographic studies of California mygalomorphs Nemesiidae) from Portugal and Spain. Rev. Ibérica Aracnol. 12, 3–11. Decae, A., Colombo, M., Manunza, B., 2014. Species diversity in the supposedly have revealed both shared patterns with other organisms as well monotypic genus Amblyocarenum Simon, 1892, with the description of a new as genuine surprises (Hedin et al., 2013). species from Sardinia (Araneae, Mygalomorphae, Cyrtaucheniidae). Arachnology 16, 228–240. Derkarabetian, S., Hedin, M., 2014. Integrative taxonomy and species delimitation in Funding harvestmen: A revision of the western North American genus Sclerobunus (Opiliones: Laniatores: Travunioidea). PLoS One 9, e104982. Donnelly, R.K., Harper, G.L., Morgan, A.J., Orozco-Terwengel, P., Pinto-Juma, G.A., This work was supported by a Theodore Roosevelt Memorial Bruford, M.W., 2013. Nuclear DNA recapitulates the cryptic mitochondrial Grant from the American Museum of Natural History awarded to lineages of Lumbricus rubellus and suggests the existence of cryptic species in an Dean Leavitt and funding from the Bureau of Land Management. ecotoxological soil sentinel. Biol. J. Linn. Soc. 110, 780–795. Goloboff, P.A., 1995. A revision of the South American spiders of the family Nemesiidae (Araneae, Mygalomorphae). Part I: species from Peru, Chile, Acknowledgments Argentina, and Uruguay. Bull. Am. Mus. Nat. Hist. 224, 1–189. Graham, M.R., Hendrixson, B.E., Hamilton, C.A., Bond, J.E., 2015. Miocene extensional tectonics explain ancient patterns of diversification among turret- For help in specimen collection we would like to thank Bob building tarantulas (Aphonopelma mojave group) in the Mojave and Sonoran Thomson, Levi Gray, Peter Scott, Anna Bennett, Eileen Bennett, deserts. J. Biogeogr. 42, 1052–1065. Chris Hamilton, Jordan Satler, Erika Garcia, Robin Hedin, Bob Grummer, J.A., Bryson Jr., R.W., Reeder, T.W., 2014. Species delimitation using Bayes Keith, Steven Thomas, Ryan Fitch, Dave Carlson, Axel Schönhofer, factors: simulations and application to the Sceloporus scalaris species group (Squamata: Phrynosomatidae). Syst. Biol. 63, 119–133. and Lars Hedin. Jason Bond provided assembled transcriptomes Hamilton, C.A., Formanowicz, D.R., Bond, J.E., 2011. Species delimitation and for the Jasper Ridge Calisoga, and Pionothele. Charles Griswold phylogeography of Aphonopelma hentzi (Araneae, Mygalomorphae, and Darrell Ubick facilitated the specimen loan from the Theraphosidae): cryptic diversity in North American tarantulas. PLoS One 6, e26207. California Academy of Sciences. Cheryl Hayashi provided lab space Hamilton, C.A., Hendrixson, B.E., Brewer, M.S., Bond, J.E., 2014. An evaluation of and other resources for RNA extraction work. sampling effects on multiple DNA barcoding methods leads to an integrative approach for delimiting species: a case study of the North American tarantula genus Aphonopelma (Araneae, Mygalomorphae, Theraphosidae). Mol. Supplementary material Phylogenet. Evol. 71, 79–93. Harrington, R.C., Near, T.J., 2012. Phylogenetic and coalescent strategies of species delimitation in snubnose darters (Percidae: Etheostoma). Syst. Biol. Supplementary data associated with this article can be found, in 61, 63–79. the online version, at http://dx.doi.org/10.1016/j.ympev.2015.05. Harvey, M.S., Rix, M.G., Framenau, V.W., Hamilton, Z.R., Johnson, M.S., Teale, R.J., 016. Humphreys, G., Humphreys, W.F., 2011. Protecting the innocent: studying D.H. Leavitt et al. / Molecular Phylogenetics and Evolution 91 (2015) 56–67 67
short-range endemic taxa enhances conservation outcomes. Invertebr. Syst. 25, Moritz, C., Faith, D.P., 1998. Comparative phylogeography and the identification of 1–10. genetically divergent areas for conservation. Mol. Ecol. 7, 419–429. Hebert, P., Cywinska, A., Ball, S., DeWaard, J., 2003a. Biological identifications Ogden, R., Thorpe, R.S., 2002. Molecular evidence for ecological speciation in through DNA barcodes. Philos. Trans. R. Soc. Lond. B 270, 313–321. tropical habitats. Proc. Natl. Acad. Sci. 99, 13612–13615. Hebert, P., Ratnasingham, S., de Waard, J.R., 2003b. Barcoding animal life: Opatova, V., Arnedo, M.A., 2014a. From Gondwana to Europe: inferring the origins cytochrome c oxidase subunit 1 divergences among closely related species. of Mediterranean Macrothele spiders (Araneae: Hexathelidae) and the limits of Philos. Trans. R. Soc. B 270, S96–S99. the family Hexathelidae. Invertebr. Syst. 28, 361–374. Hedin, M., 2015. High-stakes species delimitation in eyeless cave spiders (Cicurina, Opatova, V., Arnedo, M.A., 2014b. Spiders on a hot volcanic roof: colonisation Dictynidae, Araneae) from central Texas. Mol. Ecol. 24, 346–361. pathways and phylogeography of the Canary Islands endemic trap-door spider Hedin, M., Bond, J.E., 2006. Molecular phylogenetics of the spider infraorder Titanidiops canariensis (Araneae, Idiopidae). PLoS One 9, e115078. Mygalomorphae using nuclear rRNA genes (18S and 28S): conflict and Opatova, V., Bond, J.E., Arnedo, M.A., 2013. Ancient origins of the Mediterranean agreement with the current system of classification. Mol. Phylogenet. Evol. trap-door spiders of the family Ctenizidae (Araneae, Mygalomorphae). Mol. 41, 454–471. Phylogenet. Evol. 69, 1135–1145. Hedin, M., Carlson, D., 2011. A new trapdoor spider species from the southern Coast Pante, E., Puillandre, N., Viricel, A., Arnaud-Haond, S., Aurelle, D., Castelin, M., Ranges of California (Mygalomorphae, Antrodiaetidae, Aliatypus coylei, nov sp), Chenuil, A., Destombe, C., Forcioli, D., Valero, M., Viard, F., Samadi, S., 2015. including consideration of mitochondrial phylogeographic structuring. Zootaxa Species are hypotheses: avoid connectivity assessments based on pillars of 2963, 55–68. sand. Mol. Ecol. 24, 525–544. Hedin, M., Starrett, J., Hayashi, C., 2013. Crossing the uncrossable: novel trans-valley Pons, J., Barraclough, T.G., Gomez-Zurita, J., Cardoso, A., Duran, D.P., Hazell, S., biogeographic patterns revealed in the genetic history of low dispersal Kamoun, S., Sumlin, W.D., Vogler, A.P., 2006. Sequence-based species mygalomorph spiders (Antrodiaetidae, Antrodiaetus) from California. Mol. delimitation for the DNA taxonomy of undescribed insects. Syst. Biol. 55, Ecol. 22, 508–526. 595–609. Hedin, M., Carlson, D., Coyle, F., in press. Sky island diversification meets the Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, multispecies coalescent: divergence in the spruce-fir moss spider (Microhexura 1253–1256. montivaga) on the highest peaks of southern Appalachia. Mol. Ecol. Puillandre, N., Lambert, A., Brouillet, S., Achaz, G., 2012. ABGD, Automatic Barcode Hendrixson, B.E., DeRussy, B.M., Hamilton, C.A., Bond, J.E., 2013. An exploration of Gap Discovery for primary species delimitation. Mol. Ecol. 21, 1864–1877. species boundaries in turret-building tarantulas of the Mojave Desert (Araneae, Rambaut, A., Drummond, A.J., 2007. Tracer v1.5.